Pathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) - The calm before the cytokine storm

Abstract

Pathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) -
The calm before the cytokine storm

Full text

Faculty of Pharmaceutical,
Biomedical and Veterinary Sciences
Department of Biomedical Sciences
Pathogenesis of tuberculosis-associated immune
reconstitution inflammatory syndrome (TB-IRIS)
The calm before the cytokine storm
Dissertation submitted in partial fulfilment of the degree of Doctor in
Biomedical Sciences at the University of Antwerp to be defended by
Odin Goovaerts
Promotor: Prof. dr. Luc Kestens Antwerp 2015

Faculteit Farmaceutische, Biomediche
en Diergeneeskundige
Wetenschappen
Departement Biomedische
Wetenschappen
De pathogenese van tuberculose-geassocieerd immuun
reconstitutie inflammatoir syndroom (TB-IRIS)
De kalmte voor de cytokine storm
Proefschrift voorgelegd tot het behalen van de graad van Doctor in de
Biomedische Wetenschappen aan de Universiteit Antwerpen te verdedigen door
Odin Goovaerts
Promotor: Prof. dr. Luc Kestens Antwerp 2015

I
The research described in this dissertation was performed at the laboratory of
immunology at the Institute of Tropical Medicine (Antwerp). Odin Goovaerts was
funded by a doctoral fellowship of the Institute for Science and Technology Flanders
(IWT, ref. nr. 093166).
Cover picture: The calm before the storm. http://www.morguefile.com/

II
Doctoral Commitee
Composition of the jury:
Promotor: Prof. dr. Luc Kestens (University of Antwerp / Institute of Tropical Medicine)
Chairman: Prof. dr. Louis Maes (University of Antwerp)
Members: Prof. dr. Guido Vanham (University of Antwerp / Institute of Tropical Medicine)
Prof. dr. Ingrid De Meester (University of Antwerp)
Prof. dr. Dominique Schols (Rega Institute)
Dr. Katalin Wilkinson (University of Cape Town)

III
Table of Contents
Dankwoord – Acknowledgements........................................................................... VII
List of figures ............................................................................................................. XI
List of Tables ............................................................................................................ XII
List of abbreviations ................................................................................................ XIII
Samenvatting .......................................................................................................... XV
Summary .............................................................................................................. XVIII
Chapter I: General introduction to the immune system ............................................. 1
1.1 The innate immune system – the ancient guardian ............................................ 2
1.1.1 Cells of the innate immune system .............................................................. 2
1.1.2 Innate antigen recognition .......................................................................... 3
1.1.3 Cytokines .................................................................................................... 3
1.1.4 The typical inflammatory response ............................................................. 6
1.2 The adaptive immune system – more recent, more specific .............................. 8
1.2.1 Cells of the adaptive immune system .......................................................... 8
1.2.2 T cells - Antigen recognition, activation and proliferation. ........................... 9
1.2.3 Maturation of T cells ................................................................................. 10
1.2.4 Delayed type hypersensitivity response ..................................................... 12
Chapter II: The HIV-TB conundrum ........................................................................... 15
2.1 Tuberculosis – an ancient bacterium in a new world ........................................ 16
2.1.1 Epidemiology ............................................................................................ 16
2.1.2 Clinical presentation ................................................................................. 17
2.1.3 TB-infection and the immune system ........................................................ 17
2.1.4 Diagnosis .................................................................................................. 18
2.1.5 Therapy .................................................................................................... 19
2.2 HIV – the plague of the 20th and 21st century ................................................... 21
2.2.1 Epidemiology ............................................................................................ 22
2.2.2 Viral structure and lifecycle ....................................................................... 22
2.2.3 Clinical presentation ................................................................................. 23
2.2.4 HIV and the immune system ..................................................................... 24
2.2.5 Diagnosis and therapy .............................................................................. 25
2.3 HIV–TB co-infection – a lethal partnership ....................................................... 29

IV
2.3.1 Epidemiology ............................................................................................ 29
2.3.2 The immune system in HIV-TB co-infection ............................................... 30
2.3.3 HIV-TB therapy ......................................................................................... 32
Chapter III: Immune Reconstitution Inflammatory Syndrome ................................. 35
3.1 The many faces of IRIS .................................................................................... 36
3.1.1 What is IRIS .............................................................................................. 36
3.1.2 Epidemiology of IRIS ................................................................................. 37
3.2 Clinical presentation of TB-IRIS ........................................................................ 38
3.2.1 Consensus definition ................................................................................. 38
3.2.2 Risk factors and prevention ...................................................................... 40
3.2.3 Disease outcome and treatment ............................................................... 41
3.3 Immunopathogenesis of TB-IRIS ...................................................................... 42
3.3.1 Antigen load ............................................................................................. 42
3.3.2 T lymphocytes – the prime suspect ........................................................... 42
3.3.3 The cytokine storm ................................................................................... 45
3.3.4 Innate immunity – the unlikely suspect ..................................................... 46
3.3.5 Concluding remarks .................................................................................. 47
Chapter IV: Objectives ............................................................................................. 49
Chapter V: Lower pre-treatment T cell activation in early- and late-onset
tuberculosis-associated immune reconstitution inflammatory syndrome ............... 53
5.1 Abstract .......................................................................................................... 53
5.2 Introduction .................................................................................................... 54
5.3 Materials and methods ................................................................................... 55
5.3.1 Study population ...................................................................................... 55
5.3.2 Definitions ................................................................................................ 56
5.3.3 Patient selection and matching ................................................................. 56
5.3.4 Lymphocyte immunophenotyping ............................................................. 57
5.3.5 Ethical considerations ............................................................................... 58
5.3.6 Statistical analysis .................................................................................... 58
5.4 Results ............................................................................................................ 58
5.4.1 Study population ...................................................................................... 58
5.4.2 Decreased immune activation in early- and late-onset TB-IRIS prior to ART60

V
5.4.3 Memory-effector CD8 T cell shift during late- but not early-onset TB-IRIS .. 60
5.4.4 Memory-effector CD4 T cell shift during late- but not early-onset TB-IRIS .. 61
5.5 Discussion ....................................................................................................... 64
Chapter VI: Antigen-specific interferon-gamma responses and innate cytokine
balance in TB-IRIS ..................................................................................................... 67
6.1 Abstract .......................................................................................................... 67
6.2 Introduction .................................................................................................... 68
6.3 Materials and methods ................................................................................... 69
6.3.1 Study population ...................................................................................... 69
6.3.2 Patient selection and matching ................................................................. 69
6.3.3 Definitions ................................................................................................ 70
6.3.4 ELISpot assays .......................................................................................... 70
6.3.5 Cytokine multiplex assay ........................................................................... 71
6.3.6 Ethical considerations ............................................................................... 71
6.3.7 Statistical analysis .................................................................................... 71
6.4 Results ............................................................................................................ 72
6.4.1 Study population ...................................................................................... 72
6.4.2 Antigen-specific IFNγ ELISpot responses in HIV-TB patients developing TB-
IRIS.................................................................................................................... 72
6.4.3 Innate cytokine production upon stimulation with CMV, PPD and LPS ....... 73
6.5 Discussion ....................................................................................................... 78
Chapter VII: LPS-binding protein and IL-6 mark paradoxical tuberculosis immune
reconstitution inflammatory syndrome in HIV patients ........................................... 81
7.1 Abstract .......................................................................................................... 81
7.2 Introduction .................................................................................................... 82
7.3 Materials and methods ................................................................................... 83
7.3.1 Study population ...................................................................................... 83
7.3.2 Patient selection and matching ................................................................. 84
7.3.3 Definitions ................................................................................................ 84
7.3.4 Plasma analysis ........................................................................................ 84
7.3.5 Ethical considerations ............................................................................... 85
7.3.6 Statistical analysis .................................................................................... 85
7.4 Results ............................................................................................................ 86

VI
7.4.1 Study population ...................................................................................... 86
7.4.2 Markers of a leaky gut prior to and during ART ......................................... 87
7.4.3 PAMP binding plasma proteins prior to and during ART ............................ 87
7.4.4 Cytokine levels prior to and during ART ..................................................... 88
7.4.5 Central role of IL-6 during TB-IRIS ............................................................. 93
7.5 Discussion ....................................................................................................... 93
Chapter VIII: General Discussion .............................................................................. 97
8.1 Early- versus late-onset TB-IRIS – yet another face of IRIS................................ 97
8.2 T cells in TB-IRIS – wrongfully accused? ........................................................... 99
8.3 The innate immune system – pulling the strings behind TB-IRIS? ................... 100
8.4 Final conclusions ........................................................................................... 103
8.5 Food for thought ........................................................................................... 104
Curriculum Vitae and publication list ..................................................................... 109
Appendix I .............................................................................................................. 112
Appendix II ............................................................................................................. 113
Appendix III ............................................................................................................ 116
Appendix IV............................................................................................................ 117
Reference List ........................................................................................................ 118

VII
Dankwoord – Acknowledgements
Ik zal eerlijk zijn ... Ik had het aardig wat moeite om nog de juiste woorden te vinden
voor dit stukje tekst. Het moest iets “origineels” zijn, liefst met een paar memorabele
quotes en vooral mocht ik niemand vergeten te bedanken terwijl ik terug blik op 5
jaar als ITG’er. Ik had het brilliante idee om een wetenschappelijke tabel te maken
waar ik iedereen in kon vermelden; Naam | Bijdrage | Graad van dankbaarheid. Maar
uiteindelijk leek dit me toch net iets te pragmatisch (en ik wist trouwens niet welke
eenheden ik moest gebruiken voor “dankbaarheid”)...Tekst dan maar. Maar hoe zeg
je in godsnaam dankjewel voor zoiets als dit? Het gaat hier eigenlijk over veel meer
dan enkel dit boekje. Vijf jaar op het ITG, da’s zo wat de helft van mijn volwassen
leven. Een “ITG tijdperk” als het ware, vol met oude bekenden en nieuwe gezichten
(waarvan een groot aantal inmiddels ook al oude bekenden zijn geworden). Ik ben in
die periode zowel op professioneel vlak als op persoonlijk vlak enorm veel gegroeid
en al deze mensen (inclusief degenen die ik hier niet specifiek vermeld) hebben daar
hun steentje aan bijgedragen. Of het nu een rotsblok was of een heel klein kiezeltje, al
die positieve invloeden wegen samen echt wel door. Dus in plaats van enkel een
“dankjewel, zonder jullie had ik deze thesis niet gehaald”, zou ik jullie allemaal willen
bedanken voor iets belangrijkers; hetgene dat ik nu als mijn identiteit ervaar.
Mijn verhaal is eigenlijk niet zo uniek ... Ik was een laatstejaars studentje op de
Universiteit van Antwerpen. Student tropische biomedishe wetenschappen, op zoek
naar een plaats voor mijn masterthesis. Ik weet nog goed hoe zenuwachtig ik was
toen ik aan de eerste dag van mijn masterstage begon. Alles leek zoveel grootser
toen... Ik kwam terecht bij mijn promotor, Luc, op het labo immunologie van het ITG.
Onder de beschermende vleugel van mijn begleidster, Pascale, kon ik dus naar
hartelust “wetenschap beginnen doen”. Ik ben altijd geïnteresseerd geweest in
wetenschap, maar vanaf dat moment werd laboratoriumonderzoek voor mij eindelijk
tastbaar. Elk labo heeft trouwens zo zijn tradities. Elke vrijdag voormiddag bracht er
wel iemand koffiekoeken mee. Een gezellig labo met toffe collega’s en
koffiekoeken...reden genoeg om nadien te blijven plakken voor een doctoraat me
dunkt. Van die keuze heb ik nooit spijt gehad en ik kan me geen betere collega’s
wensen dan de mensen in (en rond) ons labo. Luc (Kestens), ik kan je niet genoeg
bedanken voor alle kansen die je me gegeven hebt! Ik apprecieer het vertrouwen die je
me gegeven hebt enorm. De vrijheid en zelfstandigheid die ik terug vond onder jou
leiding hebben me enorm veel bijgeleerd, ook op persoonlijk vlak. Pascale, thank you
for all your help, motivation, honesty...basically, thanks for teaching me how to be a
scientist! I remember feeling a bit insecure about my PhD when you left our lab, but
this thesis proves that you gave me all the basics I needed to make it. Wim, het in
haast onvoorstelbaar hoe je me keer op keer belangeloos te hulp schoot. Artikels
schrijven, statistiek, afdwalen over wetenschappelijke en niet wetenschappelijke
onderwerpen aan de koffietafel, noem maar op. Ik viel niet echt onder je

VIII
verantwoordelijkheid, maar toch stond je altijd klaar met advies. “Nen dikke merci”,
voor alles! Ann, ik ga mijn bureaugenoot en onze bijhorende gesprekjes enorm missen.
Van alle collega’s heb jij mijn goede (en vooral minder goede) momenten van het
dichtst bij meegemaakt. Viel dat een beetje mee? Enorm bedankt voor je luisterend
oor! Chris en Luc (Boel), bedankt om me de fijnere kneepjes van het vak bij te
brengen! Al die grote machines in het labo hebben dankzij jullie veel minder geheimen
voor mij! Géraldine, thanks for the advice when I needed it. Also thanks for showing
me around Paris that one time! Justine, my “partner in crime”, this has been quite the
journey for us, hasn’t it? Take care of our little IRIS project! Kim, mijn andere
bureaugenoot. Ik heb nooit iemand gekend die harder kon werken dan jij. Misschien
besef je het niet, maar het inspireerde mij om ook 200% te geven bij het werk!
Annette en Jef, ik ben blij dat ik nog lang genoeg gebleven ben om jullie te leren
kennen! Vikki, ik ben stiekem een beetje trots dat ik zelf een thesisstudent onder mijn
vleugels mocht nemen. Bedankt voor de toffe ervaring! Jeroen, ik ben je al een tijdje
niet meer komen bezoeken in de cryo. Jammer, want mijn PBMC’s werden altijd
geserveerd met een glimlach en een fijne babbel! Rafael, bedankt voor de veelvuldige
brainstorming sessies tijdens mijn vroege doctoraat! Een boomhut delen in Uganda
blijft ook één van mijn favoriete herinneringen! Huyen, I’m glad I got to know you! I
hope you and you’re family are doing well. Sandra, jij gaf me mijn eerste “echte”
labojas en wist op zicht de juiste maat te selecteren. Het klinkt misschien als een raar
detail, maar het gaf me een welkom gevoel, iets wat ik me 5 jaar later nog steeds
herinner! Ana-Marie, ik ben altijd blij geweest dat er nog een andere vroege vogel
was om een praatje mee te slaan! En ook bedankt om mijn rommelige bureau steeds
door de vingers te zien! Nancy, Nadine, Ciska, Karin, Yvette, ik ben absoluut geen held
als het op administratie aan komt. Ik mag dus “mijn twee pollen kussen” dat de
meiden van ons secretariaat er zijn. Geen enkele vraag was jullie te veel, jullie zijn mijn
administratieve heldinnen! Of course, this section wouldn’t be complete without a
well-deserved thank you to the (inter)national colleagues and partners that made all
my work possible. Margueritte, William, Harriet, Bob, Violette, Francoise M., Camille
L. and everyone else from the TB-IRIS study team, thank you so much for all your
hard work and advice! Ken, you deserve a special thank you for your kindness and
hospitality the first time I came to Uganda!
De “mentale gezondheid” van een PhD student durft al wel eens in het gedrang
komen ... Daarom is het o zo belangrijk om (naast een dagelijkse dosis cafeïne) een
gezonde dosis gelijkgezinde vrienden te hebben in het dagelijkse leven. Ik heb enorm
veel deugd gehad van de semi-dagelijkse lunches, after-work drankjes en zo veel
meer met de andere “jonge wetenschappers” onder ons en ik ben erg aan jullie
gehecht geraakt. Anali, you’ve always been like a big sister to me. I bet Elea will grow
up to be just as awesome as her mother is! Liselotte en Céline S., zonder jullie was ik
al lang zot geworden. Mailtjes, lunches, ijsjes, drankjes, lachen, klagen, steunen,

IX
vertellen, luisteren...zowel binnen als buiten het ITG. Kortom, jullie zijn absoluut
geweldig! Jasna en Irith, subliem hoe oprecht geïneteresseerd jullie altijd zijn in wat ik
en anderen te vertellen hebben! Tine and Jordan, you guys are one of a kind (or two of
a kind in this case)! Seeing your happy smiles always made me happy as well. Lotte, ik
ben blij dat jij er was om me gezelschap te houden op die congressen! Geen zorgen,
zo’n doctoraat is voorbij voor je het beseft. (Is dat nu motiverend of net niet?)
Eigenlijk zit het ITG vol met fijne mensen ... Doorheen de jaren ben ik enorm veel
mensen tegen het lijf gelopen en elk van hen vond ik leuk en interessant. Natacha,
Montserrat, Séverine, Philippe, Djibril, Gertjan, Michèle D., Maren, Nele, Winni,
Katrijn, Céline M., Derek, Jo, Betty, Kevin, Ellen, Guido, Leo, Sandra C., Katleen,
Sunita, Leen R., Deirdre, Wim, Miriam, Bouke, Linda, Manu, Eliane, Vincent, Fahdi,
Catherine, Pierre, Ermias, Taha, Maaike, Fiona, Kim V., Yvonne, Mike, Lieve, Tim,
Kirsten, Jessy, Vicky J., Sabrina, Tania, Katrien F., Vicky C., Marianne, Greet,
Benedicte, Valerie, Annelies, Mirriam, Sergio, Said, Hilde, Maarten, Patrick, Wendy,
Marleen, Marina, Kurt, Michèle K. Joris, Jos, Eric, An D.M., ... ik kan blijven
doorgaan. Of het nu een vriendelijke goeiedag was in de wandelgangen, een lunch,
een meeting, een vraag, een drankje na het werk, een onverwacht bureaubezoekje of
wat dan ook, het maakte me steeds vrolijk. Ik ben dan ook oprecht blij (en ook wel een
beetje trots) om jullie te kennen!
Er zijn ook een aantal niet-ITG vrienden die heel waardevol zijn voor mij ... Soms was
ik op zoek naar advies, soms naar afleiding en (heel soms?) wilde ik gewoon uitbundig
onnozel doen. Bij hen kon ik altijd terecht. Geert, mijn rots in de branding, je hebt me
nu zelfs al voorgedaan hoe je een doctoraat aflegt. Bedankt om keer op keer mijn
gezaag te aanhoren en me steeds weer gerust te stellen! Leen J., meer dan de zin
“great minds think alike” moet ik hier eigenlijk niet schrijven. Nieuwjaarsfeestjes, sushi
avonturen, wakeboarden (auw!), er is nooit een saai moment! Caroline, bedankt voor
de talloze smsjes met onvoorwaardelijke steun (ook die met inside jokes)! Oh, en
dankjewel voor de madeleintjes wanneer het écht nodig was. Katrien, ik vind het
geweldig hoe blij je altijd bent. Maar ik vind het nog geweldiger dat Thomas en ik op
dezelfde dag verjaren! Een mooier verjaardagskado kon je me niet geven. Rebekka,
bedankt om orde te scheppen in mijn warrige organisatie! Ik waardeer je vriendschap
echt. Joran, je weet dat ik een absoluut stresskonijn ben. Bedankt voor de vele
stressrelieve momenten! Peter, een vriendschap van 20 jaar is niet niets. Of we nu op
retraite waren in de Ardennen of gewoon een kebab aten bij u thuis, hillarische
absurditeiten zijn steeds verzekerd. Er zullen er maar weinig zijn die dit begrijpen,
maar: Wuuh!
Dan is er ook nog het thuisfront ... Ik word al wel eens als “raar” omschreven, meestal
op een positieve manier (hoop ik toch). Ik veronderstel dat de invloed van mijn familie
daar toch wel voor iets tussen zit. Liefste mama, liefste papa, ik ga hier geen

X
nieuwjaarsbrief opstellen. Ik denk dat het voor zich spreekt hoeveel respect en
dankbaarheid iemand voor zijn ouders heeft. Jullie zijn altijd trots op mij geweest, ook
wanneer ik faalde. Nooit was er gebrek aan steun, hulp en interesse ... ik ben jullie
eeuwig dankbaar voor alles. Freyja, Bart en Achiel, ik ben er eindelijk geraakt! Merci
voor de onvoorwaardelijke interesse en steun. Ik heb misschien maar één zus, maar
het is een geweldige zus. Grizzly ... ik mis je.
Tot slot ook nog een verdiende dankjewel aan de jury en alle geinteresseerden voor
hun aanwezigheid op mijn verdediging. Tevens een dankjewel aan Evy Pluym voor de
administratieve logistiek achter de thesis. Er zijn zo veel mensen die ik wilde
bedanken, dat de kans bestaat dat ik iemand vergeten ben te vermelden. (Als dit het
geval is; mezelf kennende ga ik me daar nog weken lang schuldig door voelen).
Daarom nog een algemene dankjewel aan iedereen!
Dankbaarheid alom dus ... ik kan eigenlijk niet geloven dat dit de laatste zinnen zijn
die ik in deze thesis neerschrijf. Waar blijft die memorabele quote? Misschien kan ik
hier iets neerzetten over de belangrijke gebeurtenissen uit de afgelopen jaren, zoals
mijn eerste publicatie. Langs de andere kant is het misschien wel leuk om nog wat
fijne herinneringen van tijdens mijn doctoraat boven te halen zoals mijn eerste
bezoek (ooit!) aan Afrika, of een sneeuwballengevecht met mijn collega’s in de tuin
van het ITG. Ik kan natuurlijk ook op een komische noot eindigen. Zoals op de ochtend
van de BMW (teambuilding) dagen, toen een dronken man me aansprak en zei dat ik
“schoon benen” had. (Mooi compliment, niet waar?) Er zijn veel te veel mooie
herinneringen om op te sommen eigenlijk. Hoe dan ook, ik zal mijn geweldige tijd als
doctoraatsstudent nooit vergeten en dat is vooral dankzij iedereen om mij
heen...”The End”.

XI
List of figures
Figure 1. Interactions between LPS and the innate immune system ............................ 7
Figure 2. Models of memory T cell differentiation ..................................................... 11
Figure 3. Maturation of CD4 and CD8 T cells ............................................................. 12
Figure 4. TB incidence rates, estimated by the WHO ................................................. 16
Figure 5. The number of people living with HIV worldwide, estimated by the WHO .. 22
Figure 6. Simplified graphical representation of an HIV virion ................................... 23
Figure 7. Generalised course of HIV infection ............................................................ 24
Figure 8. Generalized HIV lifecycle ............................................................................ 27
Figure 9. HIV prevalence in new TB cases, estimated by the WHO. ............................ 29
Figure 10. Model of immune activation and T cell maturation in HIV ......................... 32
Figure 11. Frequency of TB-IRIS during ART in a Ugandan cohort .............................. 57
Figure 12. Percentage of activated CD8+ cells in early- and late-onset TB-IRIS ........... 60
Figure 13. Percentage of CD8 maturation sub-stages in early-onset TB-IRIS patients . 62
Figure 14. Percentage of CD4 maturation sub-stages in early-onset TB-IRIS patients . 63
Figure 15. Antigen-specific IFNγ responses in TB-IRIS patients and controls............... 75
Figure 16. Pro- to anti-inflammatory ratios of innate cytokine production in TB-IRIS . 76
Figure 17. Correlation of cytokine ratios to IFNγ responses ....................................... 77
Figure 18. Markers of a leaky gut in TB-IRIS patients and controls during follow-up .. 89
Figure 19. LBP and sCD14 plasma levels in TB-IRIS patients and controls during ........ 90

XII
List of Tables
Table 1. Functional groups of selected cytokines......................................................... 5
Table 2. Summary of pathogens associated to IRIS and their incidence. .................... 37
Table 3. Consensus definition for paradoxical tuberculosis-associated IRIS. .............. 39
Table 4. Case definition for ART-associated TB and unmasking TB-associated IRIS. .... 40
Table 5. The frequency of clinical characteristics among patients with TB-IRIS .......... 40
Table 6. Characteristics of TB-IRIS patients and matched controls in chapter V. ........ 59
Table 7. Characteristics of TB-IRIS patients and matched HIV+TB+IRIS- controls. ......... 72
Table 8. In vitro cytokine production after CMV, PPD or LPS stimulation. ................. 74
Table 9. Characteristics of TB-IRIS patients and matched HIV+TB+IRIS- controls. ......... 86
Table 10. Cytokines and other plasma markers in TB-IRIS patients and controls. ....... 91
Table 11. Cytokines in HIV-TB- controls compared to TB-IRIS patients and controls ... 92

XIII
List of abbreviations
Listed in alphabetical order.
AIDS Acquired immune deficiency syndrome
a.k.a. Also known as
APC Antigen presenting cell
APP Acute phase protein
APR Acute phase response
BCG Bacillus Calmette–Guérin
CCL Chemokine (C-C motif) ligand
CCR7 C-C chemokine receptor type 7
CD Cluster of differentiation
CDC Centers for disease control and prevention
CFP-10 10 kDa culture filtrate antigen CFP-10
CMV Cytomegalovirus
CRP C-reactive protein
CTL Cytotoxic lymphocyte
CXCL Chemokine (C-X-C motif) ligand
DC Dendritic cell
DNA Deoxyribonucleic acid
DTH Delayed type hypersensitivity response
e.g. Exempli gratia – ‘for example’
ELISA Enzyme-linked immunosorbent assay
ELISpot Enzyme-linked immunoSpot assay
EndoCab Endotoxin core antibodies (LPS antibodies)
ESAT-6 6 kDa early secretory antigenic target
G-CSF Granulocyte-colony stimulating factor
GM-CSF Granulocyte-macrophage colony-stimulating factor
HIV+TB+IRIS- HIV-TB co-infected patients who did not experience TB-IRIS during
ART
HIV-TB+ HIV-uninfected patients receiving treatment for active TB-infection
HIV-TB- HIV & TB un-infected control patients
i.e. id est – ‘in other words’
I-FABP Intestinal fatty-acid binding protein
IFN α,β,γ Interferon alpha, beta, gamma
IL-1,2... Interleukin 1,2...

XIV
LAM Lipoarabinomannan
LBP LPS-binding protein
LOD Limit of detection
LPS Lipopolysaccharide
MHC I, II Major histocompatibility complex I, II
Mφ Macrophage
Mo Monocyte
NK cell Natural killer cell
NRTI Nucleoside-analogue reverse transcriptase inhibitors
NNRTI Non-nucleoside reverse transcriptase inhibitors
OI Opportunistic infection
PAMP Pathogen associated molecular pattern
PBMCs Peripheral blood mononuclear cells
RNA Ribonucleic acid
RT Reverse transcriptase
sCD14 Soluble CD14
TB-IRIS Tuberculosis-associated immune reconstitution inflammatory
syndrome
TGFβ Transforming growth factor beta
Th 1,2,17 T helper 1,2,17 cell
TLR Toll-like receptor
TNFα Tumor necrosis factor alpha
Treg Regulatory T cell
WHO World health organisation

XV
Samenvatting
Ondanks een goede respons op de behandeling tegen tuberculose, zullen tot 25% van
de patienten met een HIV-tuberculose (TB) coïnfectie een paradoxale verergering
meemaken van hun TB symptomen tijdens de eerste maanden van succesvolle
antiretrovirale therapie (ART). Deze complicatie wordt ”paradoxaal tuberculose-
geassocieerd immuun reconstitutie inflammatoir syndroom” (TB-IRIS) genoemd. Het
syndroom wordt gekenmerkt door ernstige ontstekingsverschijnselen die in 30% van
de gevallen kunnen leiden tot hospitalisatie en extra ontstekingsremmende therapie.
Het merendeel van de TB-IRIS gevallen wordt tijdens de eerste maand na het starten
van ART geïdentificeerd (zogenaamd vroege TB-IRIS). Desalniettemin kan TB-IRIS zich
ook op latere tijdstippen ontwikkelen (zogenaamd late TB-IRIS), zelfs tot 4 jaar na het
starten van ART. Aangezien de diagnose voornamelijk gebeurt op basis van klinische
tekens en symptomen, is het niet eenvoudig om deze aandoening te onderscheiden
van andere complicaties. Daarom is er een hoge nood om de pathogenese van TB-IRIS
beter te begrijpen en om bruikbare laboratorium-merkers te identificeren die kunnen
gebruikt worden voor de diagnose van TB-IRIS en mogelijk ook als voorspellende
merker.
Hoewel de pathogenese van TB-IRIS nog niet opgehelderd is, hebben een aanzienlijk
aantal studies samen de basis gelegd om de mechanismen die schuilen achter TB-IRIS
beter te begrijpen. Zo wordt het algemeen aanvaard dat IRIS het gevolg is van een
atypisch herstel van pathogeen-specifieke immuunreacties tijdens ART. Meer en meer
gegevens wijzen in de richting van een belangrijke rol voor zowel het adaptieve als
het aangeboren immuun systeem in TB-IRIS. Hoewel de rol van het aangeboren
immuunsysteem steeds meer ondersteund wordt in de literatuur, blijft het voorlopig
onduidelijk waar het precies fout loopt met het immuun systeem in TB-IRIS patiënten.
Bovendien bemoeilijkt de heterogeniteit van TB-IRIS de huidige studies en is het niet
duidelijk of vroege en late vormen van TB-IRIS dezelfde pathogenese omvatten.
Daarom is het nodig om verder na te gaan welke antigenen, antigeen-specifieke
responsen, cytokines en andere plasma proteïnen centraal staan in de inflammatoire
chaos die TB-IRIS omringd en dit binnen een goed gecontroleerde studie-populatie.
Met het oog op de uitgesproken rol van T cellen in de immunologie van HIV en
tuberculose, was onze initiële hypothese dat een onevenwichtig herstel van het T cel
compartiment een fundamentele bijdrage kon leveren aan de ontwikkeling van TB-
IRIS. Om deze verschillende aspecten van het immuunsysteem te bestuderen in TB-
IRIS, werd een grote prospectieve cohorte opgezet van Ugandese HIV-TB patienten
die ART startten. De studiepopulatie die hieruit voortkwam gaf ons de kans om TB-
IRIS patienten strikt te matchen met niet-IRIS controle patiënten volgens hun klinische

XVI
kenmerken, om zodoende rekening te houden met de heterogeniteit van TB-IRIS
(inclusief de duur van ART alvorens TB-IRIS zich ontwikkelde). Aansluitend bij onze
hypothese, werden drie verschillende onderzoeksplatforms gebruikt om de rol van T
cellen in TB-IRIS te bepalen, namelijk; flowcytometrie, IFNγ ELISpot analyse en
proteine bepaling in plasma en supernatants van ELISpots (ELISA/Luminex). Tevens
onderzochten we een aantal andere factoren om een mogelijke rol van het
aangeboren immuunsysteem na te gaan.
Ons eerste manuscript beschrijft de expressie van activatie- en maturatiemerkers op T
cellen in vers vol bloed van zowel vroege als late TB-IRIS patiënten. Zowel vroege als
late TB-IRIS patiënten toonden een lager niveau van T cel activatie vóór het starten
van ART. Een verschuiving in de T cel maturatiestadia kon echter enkel vastgesteld
worden bij late TB-IRIS patiënten. Aangezien dit betekent dat de duur van ART een
mogelijke impact kan hebben op immunologische metingen, werden de volgende
experimenten voornamelijk gericht op het bestuderen van een homogene selectie
van vroege TB-IRIS patiënten. Ons tweede manuscript beschrijft zowel IFNγ ELISpot
responsen als de aangeboren cytokine balans na stimulatie met verschillende
antigenen in deze patiënten. Verrassend genoeg werden er tijdens TB-IRIS in
vergelijking met controles geen verhoogde T cel responsen gemeten tegen TB-
antigenen, maar eerder een verlaging van de responsen tegen CMV en LPS. In het
geval van LPS, was deze verlaging geassocieerd met een pro-inflammatoire shift in de
aangeboren cytokinebalans. In ons derde en laatste manuscript, onderzochten we het
plasma van vroege TB-IRIS patiënten op de aanwezigheid van merkers voor de
zogenaamde “lekke darm” (zoals LPS en I-FABP), proteïnen die LPS binden (waaronder
LBP) en een breed spectrum aan cytokines van het adaptieve en aangeboren
immuunsysteem (waaronder IL-6 en G-CSF). TB-IRIS kon niet worden geassocieerd
met kenmerken van een lekke darm. Het plasma gehalte aan I-FABP bleek zelfs lager
te zijn in TB-IRIS patienten tijdens een opvolging van 6 maanden na het starten van
ART. Vóór het starten van ART konden een lager gehalte aan LBP, G-CSF en IL-6
vastgesteld worden in vergelijking met controles. Tijdens TB-IRIS zelf echter, werd een
hoger gehalte aan LBP en verscheidene cytokines aangetroffen (voornamelijk van het
aangeboren immuunsysteem). Enkel IL-6 toonde een onafhankelijk effect in
multivariaat modellen met deze cytokines, zowel voor als na het starten van ART.
Doorheen onze drie studies zijn we er in geslaagd om resultaten te genereren die de
aanpak van huidige TB-IRIS studies mogelijk helpen herörienteren. Enerzijds
suggereren onze resultaten dat de factoren die leiden tot vroege en late vormen van
TB-IRIS mogelijk gelijkaardig zijn, maar dat de kenmerken van het T cel compartiment
tijdens het optreden van TB-IRIS verschillend is. Hiermee wordt een mogelijke

XVII
systematische fout aangehaald, namelijk de duur van ART, waarmee toekomstige
studies rekening moeten houden. Anderzijds, en in tegenstelling tot onze hypothese,
illustreren onze bevindingen hoe een overgeactiveerd T cel compartiment dat
reageert tegen TB antigenen vermoedelijk niet de drijvende kracht achter TB-IRIS is.
Onze gegevens dragen daarom bij aan de steeds groter wordende hoeveelheid data
die een fundamentele rol van het aangeboren immuunsysteem in TB-IRIS
ondersteunt. Uit onze gegevens kunnen we concluderen dat paradoxale TB-IRIS
“paradoxaal” wordt voorafgegaan door een verminderde immuunrespons vóór het
starten van ART, gevolgd door een piek van cytokines van het aangeboren
immuunsysteem-tijdens ART. Als het ware een immunologische stilte voor de
cytokinestorm.

XVIII
Summary
During successful antiretroviral therapy (ART), up to 25% of HIV-patients co-infected
with tuberculosis (TB) develop worsening symptoms of TB, despite effective initial
response to concurrent TB treatment. This complication has been named paradoxical
tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS). The
syndrome is marked by excessive inflammation and about 30% of patients are in need
of hospitalisation or anti-inflammatory treatment. The majority of TB-IRIS cases
develop within the first month after starting ART (early-onset TB-IRIS). However, TB-
IRIS has been described after longer periods of ART as well (late-onset TB-IRIS), up to
4 years after starting ART. It is difficult to distinguish TB-IRIS from other
complications, since its diagnosis relies heavily on clinical examinations. Therefore,
there is an urgent need to understand the pathogenesis of TB-IRIS and to identify
reliable laboratory markers to predict and identify TB-IRIS.
Although the pathogenesis of TB-IRIS is still not well understood, studies on TB-IRIS
have succeeded in laying important foundations to start uncovering the mechanisms
behind this disease. The concept that IRIS involves an atypical restoration of
pathogen-specific immune responses during ART has gained acceptance. By exploring
the broad spectrum of the immune system, more and more evidence is emerging that
supports the roles of both the adaptive and the innate immune system in TB-IRIS.
Although evidence supporting the latter is accumulating, it is still unclear where
exactly the immune system derails to cause such inflammation. In addition, TB-IRIS’
heterogeneity has proven to be one of the challenges in TB-IRIS research and it is still
unknown whether the immunopathogenesis differs between early- and late-onset TB-
IRIS patients. Therefore, a need exists to explore which antigens, antigen-specific
responses, cytokines and other plasma proteins hold a central role in the
inflammatory chaos that marks TB-IRIS and this within a well-controlled study
population.
Given the distinct role of T cells in TB and HIV immunology, our initial hypothesis was
that an unbalanced reconstitution of the T cell compartment contributes to the
development of TB-IRIS. Focussing on the development of TB-IRIS within a population
of HIV-TB patients starting ART in Uganda, a large prospective study was set up to
explore different aspects of the immune system in TB-IRIS. The resulting study
population allowed us to account for TB-IRIS’ heterogeneity by strictly matching our
TB-IRIS patients to HIV+TB+IRIS- control patients according to their clinical
characteristics (including the duration of ART before TB-IRIS developed). In
accordance with our hypothesis, we made use of three different platforms; flow
cytometry, IFNγ ELISpot analysis and protein analysis of plasma and ELISpot

XIX
supernatants (ELISA/Luminex), in order to assess the role of T cells in TB-IRIS. In
addition, we explored a number of innate factors to address the potential
involvement of the innate immune system.
Our first manuscript describes the expression of activation and maturation markers
on T cells in fresh whole blood from both early- and late-onset TB-IRIS patients.
Whereas both presentations of TB-IRIS showed lower T cell activation prior to ART
compared to HIV+TB+IRIS- controls, only late-onset TB-IRIS patients experienced a
maturational shift in their T cell compartment when IRIS occurred. Given this possible
impact of ART duration on immunological measurements, we next focussed our
research on a homogeneous selection of early-onset TB-IRIS patients. Our second
manuscript describes IFNγ ELISpot responses as well as the innate cytokine balance in
response to several antigens in these patients. Surprisingly, we observed no elevated
T cell responses to TB-antigens, but rather a decreased response to CMV and LPS
compared to HIV+TB+IRIS- controls. For LPS, this decrease was associated with a pro-
inflammatory shift in the innate cytokine balance. In our third and final manuscript,
we explored markers of a leaky gut (including LPS and I-FABP), proteins that bind LPS
(including LBP) and a broad range of adaptive and innate cytokines (including IL-6 and
G-CSF) in early-onset TB-IRIS patients. TB-IRIS was not associated with a leaky gut. In
fact I-FABP levels were lower in TB-IRIS patients during 6 months after starting ART.
We observed lower levels of LBP, G-CSF and IL-6 prior to ART compared to
HIV+TB+IRIS- controls, but higher levels of LBP and several (mostly innate) cytokines
during TB-IRIS. Interestingly, only IL-6 showed an independent effect in multivariate
models containing cytokines associated with TB-IRIS prior to ART or during TB-IRIS.
Across our three studies, we managed to generate results that could help redirect the
focus of current TB-IRIS research towards the innate immune system. On one hand,
our findings suggest that early- and late-onset presentations of TB-IRIS may share
common predisposing factors, yet appear to be set apart by different T cell
characteristics at the time of the disease. This identifies the duration of ART as a
potential confounder in current studies, which should be taken into account in further
research. On the other hand and in contrast to our main hypothesis, our findings
show how an over-activated T cell compartment, responding to TB antigens, is
probably not the main driving mechanism behind TB-IRIS. In fact, our research adds to
the growing body of evidence that supports a fundamental role of the innate immune
system in TB-IRIS. Together, our results suggest that paradoxical TB-IRIS is
“paradoxically” preceded by diminished immune activation prior to art, followed by a
burst of innate cytokines upon ART initiation. In other words, TB-IRIS is marked by a
proverbial calm before the cytokine storm.

General introduction to the immune system
1
Chapter I: General introduction to the immune system
The human immune system is as diverse and complex as it is important. A myriad of
organs, tissues, cells and proteins all work in concert to defend the human body
against external threats. Even our largest organ, the skin, is charged with an
important barrier function against the daily bombardment with micro-organisms we
face. Though the majority of these organisms are benign (and some even necessary),
an important subset of micro-organisms is pathogenic in nature. These pathogens
come in many forms, but all of them try to invade our bodies, multiply, spread and
cause diverse forms of disease in the process. Luckily, our blood and tissues are filled
with immune cells of all shapes and sizes, each performing specific functions in
defending against different aspects of these external dangers.
As necessary as the immune system is to keep us safe and healthy, there is a darker
side to it as well. Ranging from peanut-allergies to auto-immune diseases like Lupus,
there are plenty of examples of an immune system gone haywire. Chronic
inflammatory conditions, regardless of whether or not they are caused by external
pathogens, often cause serious collateral tissue damage. TB-associated immune
reconstitution inflammatory syndrome (TB-IRIS) seems to fit tightly within this niche of
undesirable immune responses. As the name states, TB-IRIS’ main feature is tissue
destructive inflammation. Therefore, this thesis will attempt to shine an
immunological light on the pathogenesis of this syndrome.
In this chapter, we discuss the very basics of inflammation and the immune system
that are relevant to this dissertation. The experienced immunologist may want to skip
ahead to Chapter II, where we discuss the basics of HIV-TB co-infection (page 15) or to
Chapter III, where we review what is currently known about TB-IRIS (page 35).

General introduction to the immune system
2
1.1 The innate immune system – the ancient guardian
As the archetype of immune responses, the innate immune system provides non-
specific protection against pathogens. Consisting of physical barriers, different cell-
types and anti-microbial proteins, this system has been around since before we
evolved from invertebrates1. Cells of the innate immune system provide immediate
reaction to foreign objects in the body. They are capable of detecting molecular
patterns that are associated to pathogens or even recognize other cells that have
been compromised by viruses. Once a threat has been recognized, cells of the innate
immune system will initiate a broad range of signals and actions that lead to
inflammation, ultimately designed to clear the pathogen.
1.1.1 Cells of the innate immune system
The innate immune system generates a number of cell types that have adapted to
fight infections in different ways2,3. Perhaps the most well-known type of innate cells
belongs to the monocyte-macrophage lineage, the textbook example of phagocytes.
Macrophages evolve from cells in the bloodstream called monocytes, which have
migrated through the vascular endothelium to peripheral tissues. One of their main
functions in clearing infections is a process called phagocytosis. This process involves
“engulfment” of foreign objects and micro-organisms by the cell, thereby enveloping
the object in vesicles created by the cell membrane. These phagosomes fuse with
lysosomes, which contain digestive enzymes and peptides designed to break down
the object or pathogen in question.
Not all pathogens are extracellular threats, however. Viruses for example hide from
the immune system inside infected cells. The part of the innate immune response that
specializes in this kind of infection is a type of cytotoxic cell called the natural killer
(NK) cell4. These cells specialize in the “killing” of cells compromised by viral, bacterial
and protozoan infections and even malignant host-cells. Killing of these cells is a two-
signal process. NK cells have specific “killing-activating” receptors, which allow them
to initiate an attack once stimulated by molecules on the target cell. However, NK
cells also express “killing-inhibiting” receptors which recognize MHC class I expression
on healthy cells. The signal from these receptors overwrites the killing signal,
preventing NK cells from attacking uninfected tissue. Infected cells often
downregulate the MHC-I expression, allowing NK cells to attach to them and release
granules containing proteins that will kill the infected cell.
Apart from directly combatting infections, a number of cells such as dendritic cells
and macrophages (when activated by interferon-gamma (IFNγ)) have an important

General introduction to the immune system
3
function as antigen presenting cells (APC)5. These cells are able to bind digested
peptides of pathogenic origin to major histocompatibility complex proteins (MHC),
which are then expressed on the cell surface. In this way, the peptides are ‘presented’
to T cells, thereby creating a bridge between the innate and the adaptive immune
system. All these cells, together with other cells which are not described here
(neutrophils, basophils, eosinophils and so on) make up an arsenal of innate immune
cells to provide a rapid and robust response when a threat has been detected.
1.1.2 Innate antigen recognition
Evidently, physical barriers alone aren’t enough to protect our body against infection.
Once a pathogen gets past this first line of defense, the cells of the innate immune
system will need to recognize this threat. But how does this happen? How does the
body know these organisms aren’t supposed to be there? The answer partially lies
within certain molecules on these pathogens that are not present on cells of the host
system. These highly structurally conserved pathogen-associated molecular patterns
(PAMPs) can be recognized by an important family of receptors called toll-like
receptors (TLR), expressed by most cells of the immune system (but especially on cells
of the innate immune system)6. A set of 13 different TLRs has been identified in
humans, able to recognize a wide array of PAMPs7. Among the most well-known;
lipopolysaccharide (LPS) from bacterial cell walls is recognized by TLR4 and
lipoarabinomannan (LAM) from Mycobacterium tuberculosis is recognized by TLR28-10.
Viral single-stranded ribonucleic acid (RNA) on the other hand (from HIV for example)
is known to trigger TLR7/8 pathways11,12. Recognition of a pathogen by these TLRs will
lead to significant activation of the immune system and induces a cascade of
cytokines that will help mediate the immune reaction.
1.1.3 Cytokines
The collective term “cytokine” encompasses a large spectrum of signaling proteins
that have been grouped under several families; interleukins, colony-stimulating
factors, interferons, tumor necrosis factors, chemokines and growth factors13.
Cytokines from all these families are produced by a large collection of cells that
reaches far wider than just cells of the immune system. In fact, they serve as a means
of communication between these cells, in order to coordinate their many functions.
Once secreted, cytokines can bind to receptors on the cell that produced it, acting as
a feedback loop in an autocrine manner. More importantly, cytokines signal to
neighboring cells in a paracrine manner or even in an endocrine manner, by entering
the bloodstream and signal to cells that are far away.

General introduction to the immune system
4
Collectively, these cytokines regulate the different functions of cells of the immune
system14. Table 1 summarizes the origins and functions of a few of the more
prominent cytokines. Many of them are not exclusively produced by just one cell type
and they often have overlapping functions. Describing every function separately
would therefore take us too far. Nonetheless, it is important to state that cells are
exposed to a large mix of cytokines, derived from a large number of sources. The cells
in our body are therefore never exposed to just one signal and it is the sum of
synergistic and antagonistic effects of these signals that decides the eventual
behavior of the stimulated cells.
Cytokines thus offer a wide range of signals that can influence cellular proliferation,
differentiation, migration and function. Of particular interest are cytokines that are
pivotal in the induction of inflammation (i.e. pro-inflammatory cytokines) or the
reduction of inflammation (i.e. anti-inflammatory cytokines). Fighting off an infection
is usually accompanied by rise in pro-inflammatory cytokines. This rise needs to be
subsequently counterbalanced with anti-inflammatory cytokines, in order to avoid
unnecessary inflammation and tissue damage15. It doesn’t take much imagination to
conclude that the balance between these opposing forces could be particularly
important in inflammatory diseases.

General introduction to the immune system
5
Table 1. Functional groups of selected cytokines.
Cytokine
Mainly secreted by
Other Targets and effects
Pro-inflammatory
Interleukin 1
(IL-1)
Mφa/Mob
Vasculature (inflammation); hypot halam us (fever); liv er
(induction of APPc)
Interleukin 5
(IL-5)
Th2d cells
Eosinophil activation, role in allergies
Interleukin 6
(IL-6)
Mφ /Mo, endothelial
cells
Liver (in duces APP); ada ptive immunity (promotes Th17,
antibody secretion of B cells)
Interleukin 12
(IL-12)
Mφ /Mo, APCe, NKf cells
Influenc es adaptive immunity (promotes T h1)
Interleukin 17
(IL-17)
T helper 17 cells
Induces cytoki ne in many other cell types
Interleukin 18
(IL-18)
Mφ /Mo, APC Influences adaptive immunity (promotes Th1)
Interferon α/β
(IFNα/β)
Mφ /Mo, fibroblasts
Induces a n antiviral state in most nucleated cells ; increases
MHCg class I expression; activates NK cells
Interferon-γ
(IFNγ)
Th1 cells, CD8 T cells, NK
cells
Activates Mφ; increases expression MHC class I and class II
molecules; increases anti gen pres entation
Tumor N ecrosis
Factor-α (TNFα)
Mφ /Mo, Th1 cells, CD8
T cells
Vasculature (inflammation); liver (induces APP); induces
cell death; activates neutrophils
Anti-inflammatory
Interleukin 4
(IL-4)
Th2 cells, mast cells
Promotes Th2 differentiation. Counteracts Th1
differentiation.
Interleukin 10
(IL-10)
Mφ /Mo, regulatory T
cells
Down-regulates: pro-inflammatory cytokine production
and the expression of MHC class II and co-stimulatory
molecul es on Mφ
Transforming growth
factor β (TGFβ)
Mφ /Mo, T cells Inhibits: T cell proliferation and effector functi ons, B cell
proliferation, Mφ
Homeostasis and Chemotaxis
Interleukin 2
(IL-2)
T cells T cell proliferation; NK cell activation and proliferation; B
cell proliferation
Interleukin 7
(IL-7)
Stromal & epithelial
cells, APC
Important for B a nd T cell development
Interleukin 8
(IL-8)
Mφ /Mo, epithelial cells
Attracts immune cells
RANTESh
(CCL-5)
T cells
Attracts T cells, eosinophils and basophils
EOTAXIN
(CCL-11)
Epithelial cells,
fibroblasts…
Recruits eosinophils
IP-10
(CXCL-10)
Mφ /Mo, endothelial
cells, fibroblasts
Attracts leukocytes; produc ed in response to IFNγ
MIP-1βi
(CCL4)
CD8 T cells
Attracts NK cells, Monocytes and other cells.
G-CSF
j
Mφ /monocytes,
endothelium
Stimulates survival, proliferation, differentiation, and
function of leuk ocytes
a
Macrophages;
b
Monocytes;
c
Acute phase proteins;
d
T helper cell (1, 2, 17);
e
Antigen presenting cell (e.g.
dendritic cells); fNatural killer cells; gMajor histocompatibility complex; h Regulated on activation, normal T
cell expressed and secreted; iMacrophage inflammatory protein-1β; jGranulocyte-colony stimulating factor.
(Adapted from Immunology – Richard A14).

General introduction to the immune system
6
1.1.4 The typical inflammatory response
1.1.4.1 Local inflammation
When infection occurs, inflammation is one of the first responses to be noticed3,16.
We have all experienced the redness, heat, swelling and pain that accompany the
inflammatory response. In severe cases it can even lead to dysfunction of the organs
or tissues involved. Nevertheless, it is this process that functions as the initial
protection against pathogens that have made their way into the body. Let’s say the
skin was punctured and a number of pathogens have entered the body. The damaged
cells will release a number of chemical factors that activate and attract cells to the
site of invasion. Local innate immune cells (likely macrophages and neutrophils) will
recognise the invading pathogens through PAMP-TLR binding. In addition to directly
trying to kill the pathogen, they will engage a process to attract more immune cells
and soluble factors from the bloodstream to help fight off the threat.
Once activated by the invading pathogen, tissue-macrophages will start producing
several cytokines such as TNFα, IL-1 and IL-8 in addition to a broad spectrum of other
proteins. By creating a chemotactic gradient, these cytokines and chemokines will
attract leukocytes (white blood cells) to the site of infection. Among these leukocytes
are: neutrophils, macrophages, NK-cells and T cells. These cells will migrate through
the walls of nearby blood vessels and start producing chemotactic factors of their
own, which eventually leads to the accumulation of immune cells in the tissue. This
entire process usually leads to the clearing of the pathogen by phagocytosis and
oxidative burst among other mechanisms, which often coincides with tissue damage
(explaining the redness and pain that accompany inflammation). If successful, the
infection is stopped then and there. However, this is not always the case.
1.1.4.2 The Acute Phase Response
The acute phase response (APR) is a systemic version of local inflammation17,18. When
the local inflammatory response is insufficient to confine the pathogen, the continued
effort of the immune cells will spill over into the blood. Cytokines will reach other
organs, eventually leading to a systemic immune response marked by fever, anorexia
and somnolence. These behavioral changes help to conserve energy to be able to
fight the infection.
The increased levels of IL-1, IL-
6 and TNFα work in synergy to mediate several
systemic effects in response to infection. IL-6 and TNFα act on macrophages and
vascular endothelial cells to induce the production of colony-stimulating factors (G-
CSF, GM-CSF…). These factors stimulate the production of additional leukocytes.
Within 12 to 24 hours of the onset of an acute-phase response, the combined effect

General introduction to the immune system
7
of increased blood-cytokine levels induce the production of acute phase proteins
(APP) by the liver. APPs are usually expressed at low levels in healthy individuals, but
can reach a 1000x increase during infection. The most well-known acute phase
proteins are C-reactive protein (CRP) and Serum Amyloid A and components of the
complement system19,20. All these proteins perform different functions in response to
systemic inflammation. CRP for example plays an important role in the opsonitation
of pathogens, a process which helps phagocytosis.
A typical example of an acute phase response occurs during sepsis, where bacterial
PAMPs, such as lipopolysaccharide (LPS) enter the bloodstream. Evidently, LPS will be
recognized by TLRs on immune cells, leading to the production of IL-6 and other
cytokines, which induces the APR21. One of the acute phase proteins that have
important functions in sepsis is LPS-binding protein (LBP). As the name implies, it
functions by binding LPS and thus inhibiting excessive reactions to a massive presence
of LPS22. Other factors, like soluble CD14 (sCD14) produced by macrophages perform
similar functions. Both these proteins are needed for the binding of LPS to TLRs and
the modulation of reactions to LPS23. Figure 1 describes this process in more detail.
Bacteria
LPS
Monocyte
Macrophage
Hepatocyte
IL-6
TNFα
IL-1
LBP
Acute Phase Proteins
CRP
sCD14
LBP Lipoproteins
sCD14
LBP
mCD14
LBP
TLR4
AB
Figure 1. Interactions between LPS and the
innate immune system.
(A) Regulation of hepatic acute phase protein
synthesis by inflammatory mediators (adapted
from Heinrich PC et al. 199021
) and (B)
interactions of sCD14 and LBP with LPS-TLR4
binding (adapted from Kitchens RL et al. 200523)

General introduction to the immune system
8
1.2 The adaptive immune system – more recent, more specific
While the innate immune system is capable of mounting an almost immediate
response to infection, it is not perfect. In the evolutionary arms race between
pathogens and hosts, pathogens have evolved ways of avoiding the innate immune
response. When the infection cannot immediately be cleared, the adaptive immune
system takes action1. This part of the immune system, which largely consists of T- and
B-lymphocytes and their products, is able to recognize infections in a much more
specific way. Although it takes more time to mount a response, it is much more
efficient at clearing the infection. In addition, it is capable of storing a memory of
previous infections, which allows them to react more vigorously upon a secondary
encounter.
1.2.1 Cells of the adaptive immune system
Functionally speaking, the adaptive immune system can be split into two parts; the
humoral and the cell mediated immune system24. The humoral immunity focusses on
extracellular organisms and is largely mediated by plasma cells, derived from B-
lymphocytes. They aid in clearing the infection by releasing antibodies, directed at
specific antigens, in bodily fluids. Antibodies hereby bind to the pathogens in
question, either clumping them together and immobilising them or preparing them
for easier phagocytosis (a.k.a. opsonisation). The cell mediated immunity on the other
hand, depends on the cooperation of different immune cells and their direct
interaction with pathogens in order to defend the body. Instrumental in this defence
are the T-lymphocytes. Like all lymphocytes, T-lymphocytes originate in the bone
marrow. Unlike B-lymphocytes, which mature there, T-lymphocyte precursors migrate
towards the thymus, where they mature and where tolerance to auto-antigens is
induced (in order to avoid auto-immunity). Two main subtypes of T cells can be
identified, based on the expression of the co-receptors CD8 or CD4 on their
membranes.
1.2.1.1 Cytotoxic T cells
T cells expressing the co-receptor CD8 on their membranes are called cytotoxic T cells
(CTL) and are specialized in targeting virus-infected host-cells. Upon contact with an
infected cell, CD8 T cells release cytotoxins which they have stored in intracellular
granules. These toxins will perforate the cell and induce a process of apoptosis in the
target cell, leading to its death. Another effector function of these CTL is by cell-cell
contact, mediated through FAS ligands on the cell surface, which also induces
apoptosis. In addition to direct killing of infected cells, CTLs are also capable of
producing large amounts of the pro-inflammatory cytokine IFNγ, which in turn is
capable of stimulating and activating cells of the innate immune system.

General introduction to the immune system
9
1.2.1.2 T helper cells
T cells expressing the co-receptor CD4 are called T helper cells25. These cells are the
main orchestrators of the adaptive immune response. As their name says, they “help”
other cells of the immune system by stimulating their functions. Without the help of
these cells, the immune system becomes severely deficient, as seen in the acquired
immune deficiency syndrome (AIDS). T helper cells can be further differentiated into
different subpopulations, which can be identified by the primary cytokines they then
produce25,26. The most well-known subpopulations are T helper 1 (Th1) cells and T
helper 2 (Th2) cells. In addition, two more recent additions have been made to this
paradigm; Th17 cells and regulatory T cells (Tregs).
Th1 cells have historically been associated with promotion of the cellular immune
response by “helping” cytotoxic CD8 T cells, NK cells and macrophages in their
functions to clear infections. They typically differentiate in response to IL-12 and IL-18
stimulation from monocytes and mainly produce IFNγ. Th2 cells on the other hand
promote the antibody-mediated response of B-cells. They typically differentiate in
response to IL-4 and mainly produce IL-4, IL-5 and IL-13. Th17 cells are powerful pro-
inflammatory cells that have an important function in mucosal immunity and in
clearing extracellular pathogens such as bacteria and fungi. They usually differentiate
upon IL-6 stimulation and mainly produce IL-17. They are also capable of recruiting
monocytes and neutrophils to the site of infection. Finally, regulatory T cells are
important in moderating inflammation. They differentiate in response to TGFβ
stimulation. By producing IL-10, they are capable of tempering the pro-inflammatory
response after an infection has been cleared, thus maintaining the pro- and anti-
inflammatory balance.
1.2.2 T cells - Antigen recognition, activation and proliferation.
Before these T cells can perform their effector functions of cytokine production and
cytotoxicity, they need to recognize the threat, become activated and proliferate in
order to fight it. Much like the TLR of the innate immune system, the T cell
compartment has receptors called T cell receptors (TCR, often referred to as CD3). In
contrast to TLRs, each T cell line expresses only one TCR clone, directed at one specific
antigen. In addition, T-lymphocytes cannot recognize soluble antigens. Antigens
therefore need to be presented to T cells by APCs (e.g. macrophages or dendritic
cells).
The process of antigen presentation requires the antigens to be broken down to
peptides and attached to MHC molecules27 . Linked to the function of CD8 and CD4 T
cells, antigen recognition by these cells respectively happens through MHC class I or

General introduction to the immune system
10
class II antigen presentation. The key difference here is that MHC-I molecules are
expressed on virtually all cells of the body (with some exceptions such as red blood
cells and neurons) and present peptides from digested intracellular pathogens such as
viruses. In contrast, MCH-II molecules are expressed on APCs and are used to present
antigens from extracellular organisms that have gone through phagocytosis. Binding
of the TCR to viral antigens thus requires the CD8 molecule of CTLs to bind to MHC-I
before the antigen can be recognized. Similarly, recognition of extracellular antigens
requires binding of the CD4 molecule on T helper cells to MHC-II.
Once an antigen has been recognized, the next step in the T cell response is cellular
activation. Once activated, T cells will express activation markers such as HLA-DR and
CD38, which can be used as a marker of infection28,29. In addition to TCR-antigen
binding, a second signal is required for activation. T cells express co-signaling
molecules on their membrane, for example CD27 or CD28, which need to bind to their
ligand on antigen presenting cells, CD70 and B7 respectively. Another important
factor in T cell activation is cytokine signaling30. A large number of cytokines, often
produced by innate cells and T cells themselves, can stimulate and activate T cells.
These signals will further determine the behavior of T lymphocytes and their
differentiation into several subtypes29. After the initial activation, T cells will undergo
a process of differentiation and proliferation in order to perform their effector
functions. Each T cell line recognizes only one antigen and together, all T cells form a
broad repertoire of antigen specific TCRs, recognizing a similarly broad repertoire of
antigens. Each activated ancestral T cell will thus need to undergo clonal expansion to
form an army of active ‘effector T cells’ directed at a certain pathogen. Once the
infection is cleared, the lion share of these effector cells will die. However, a portion
of these antigen specific cells will be retained as “memory cells” (figure 2), which can
mediate a faster reaction upon re-exposure to the antigen.
1.2.3 Maturation of T cells
As described in figure 2, T lymphocytes that have not yet been exposed to antigens
are called naïve T cells. Once a naïve T cell recognizes an antigen, it will either mature
into effector T cells that immediately act upon the present infection or memory T cells
that will quickly proliferate upon secondary infection. Several models have been
proposed to explain the different steps of maturation undertaken by maturing T
cells31. Figure 2 shows 3 basic models of memory T cell differentiation after antigenic
stimulation. However, the most widely used model of differentiation for both CD4
and CD8 cells is slightly more complex, as shown in figure 329,32,33.

General introduction to the immune system
11
Supported by data on telomere length, this last linear model assumes the linear
progression from naïve T cells (Tn) to central memory T cells (Tcm), effector memory
T cells (Tem), terminal effector memory cells (Ttem) and terminal effector T cells
(Teff). Though it is unclear whether these steps are reversible, it is generally accepted
that Tcm and terminally differentiated cells do not revert to their previous step.
‘Late’ Effector T cell
Naïve T cell
Effector T cell
Memory T cell
+Ag
Naïve T cell Effector T cell Memory T cell
+Ag
(no Ag)
Naïve T cell ‘Early’ Effector T cell
+Ag +Ag
‘Central’ memory T cell ‘Effector’ memory T cell
(no Ag) (no Ag)
CCR7+ CCR7-
(no Ag)
A
C
B
Figure 2. Models of memory T cell differentiation
.
Model (A) describes a divergent pathway of
memory T cell formation, separating effector and memory cells as soon as an antigen (Ag) has been
recognised. Model (B) shows a linear model, where effector cells either die or give rise to memory
cells after the infection has passed. In model (C) a short duration of antigenic (Ag) stimulation favors
the development of central memory cells whereas a longer duration favors differentiation to effector
memory T cells. (Adapted from Kaech SM et al. 200231).

General introduction to the immune system
12
Different maturation steps can be determined by the expression of memory and
maturation markers such as CD45RO, CCR7 and CD27. These membrane molecules
have different functions that can be associated with the function of each maturation
step. While CD45RO’s function is still unclear, it has been used for decades to identify
cells that have been exposed to an antigen, a.k.a. ‘memory’ T cells. CCR7 on the other
hand is a chemokine receptor which receives signals that command T cells to migrate
towards (or stay in) lymphnodes. These receptors are therefore mostly expressed on
less mature T cells that are still within the central lymphoid system (Tn and Tcm).
CD27 on the other hand performs an important co-signal function during T cell
activation by APCs and will remain expressed for a longer period during maturation
(Tn, Tcm, Tem and Ttem).
As T cells mature, their functional priorities will also mature. Their main functions will
thus depend on the stage of maturation. T cells in the early memory-stages of
development will focus on cell-survival and proliferation by producing homeostatic
cytokines (e.g. IL-2). More terminally differentiated effector T cells on the other hand,
will focus on their effector role against infection by producing pro-inflammatory
cytokines (e.g. IFNγ).
1.2.4 Delayed type hypersensitivity response
Arguably, macrophages are quite efficient in clearing infections from our body.
However, pathogens have evolved many ways of surviving phagocytosis. Because of
these defence mechanisms, they can actively hide from the immune system inside the
macrophages. This is where the delayed type hypersensitivity response (DTH) comes
Tn Ttem
Tcm
+Ag
Tem Tef f
Proliferation
Inflammation
CD45RO-
CCR7+
CD27+
CD45RO+
CCR7-
CD27+
CD45RO-
CCR7-
CD27-
CD45RO+
CCR7+
CD27+
CD45RO+
CCR7-
CD27+/-
Figure 3. Maturation of CD4 and CD8 T cells. According to this model, T cells evolve through a linear
progression from naïve T cells (Tn) to central memory T cells (Tcm), effector memory T cells (Tem),
terminal effector memory cells (Ttem) and terminal effector T cells (Teff). During this evolution, the
markers of T cell mem
ory (CD45RO), T cell migration (CCR7) and T cell activation (CD27) are
transiently expressed. CD4 T cells lose CD27 when maturing from Tem to Ttem, while CD8 T cells lose
CD27 near the end of the Ttem stage. Early stages of maturation focus on cell survival and
proliferation, while more terminally differentiated cells focus on the production of (pro-inflammatory)
cytokines (Adapted from Appay V et al. 200833).

General introduction to the immune system
13
in1. The DTH is orchestrated by CD4 T helper cells and is aimed at recruiting and
activating monocytes/macrophages at sites of infection34.
Upon infection, antigens will be picked up by professional APCs (DCs) who will then
migrate to the lymph nodes draining the site of infection. Here, the antigen will be
presented to CD4 T cells, which will then become activated T helper cells. By now, the
endothelium of local blood vessels has started to express adhesion molecules, which
aid in the migration of both monocytes and T cells to the infected tissue. Once
arrived, T helper cells and monocytes will engage in an amplification loop which
enhances the magnitude of the DTH response. By producing IFNγ, T cells will activate
local macrophages and increase its anti-pathogenic capacity35. This results in a more
efficient clearing of the pathogens hiding inside these macrophages. In turn, these
macrophages will increase the expression of MHC-II, thus further stimulating the
cytokine production by T helper cells. Once the infection has been dealt with and
there are no more antigens to present, the response will gradually subside.
As with all forms of inflammation, DTHs carry a certain risk. Excessive stimulation of
macrophages will lead to tissue damage due to overproduction of reactive chemicals
and proteolytic enzymes. Moreover, the DTH is not always sufficient to eliminate the
infection. When this happens, the response can become chronic, leading to the
formation of granulomas. A typical example of this can be seen in active
Mycobacterium tuberculosis infection, discussed in the next chapter.

14

The HIV-TB conundrum
15
Chapter II: The HIV-TB conundrum
Mycobacterium tuberculosis (TB) and Human Immunodeficiency Virus (HIV) sing a
dangerous duet in the world of infectious diseases. Both infectious agents are already
formidable pathogens by themselves, one causing fatal respiratory problems and the
other causing acquired immune deficiency syndrome (AIDS). Since the emergence of
the HIV in the early 1980’s, TB has become a closely related opportunistic co-infection,
claiming the lives of millions of people on a yearly basis.
To this very day, the management of HIV-TB co-infected patients remains a challenge
for clinicians, especially in developing countries. Co-infection with TB and HIV
demands multiple drug regimens and a high level of adherence to treatment. Even
with perfect adherence, parallel treatment for TB and HIV sets the scene for several
complications. One of them is TB-IRIS, the subject of this thesis. This chapter will
therefore discuss the backgrounds of TB and HIV, their co-infection and associated
complications.

The HIV-TB conundrum
16
2.1 Tuberculosis – an ancient bacterium in a new world
Mycobacterium tuberculosis (MTB) can be considered a truly ancient microorganism.
MTB has been around since antiquity and its DNA has even been discovered in bison
remains of 17.000 years old36. Though most TB infections remain latent, about one in
ten infections become clinically active, killing about 50-60% of untreated patients37.
For a long time, the only TB-deterrent was vitamin D, acquired through sunlight and
cod-liver oil. Despite the discovery of anti-mycobacterial drugs, the emergence of HIV
and drug resistant TB strains have complicated TB management in the recent years. In
fact, the TB incidence continues to rise in specific settings, particularly in Africa,
where its incidence is related to poverty and immunosuppresion38.
2.1.1 Epidemiology
In most literature to date, one can read the statement that “over one third of the
world’s population is infected with TB”. A total of 1.86 billion individuals worldwide
(32% of the world population) are estimated to be infected with TB by the WHO39. In
2012, the worldwide incidence of active tuberculosis (TB) was 8.6 million. Figure 4
shows the TB incidence rates in 2012, estimated by the WHO.
Figure 4. TB incidence rates, estimated by the WHO.

The HIV-TB conundrum
17
2.1.2 Clinical presentation
In most cases, patients remain latently infected with TB because the immune system
is capable of suppressing the infection. In an estimated 10% of cases however, the TB
infection becomes active and patients will become infectious to others38.
Transmission of TB usually occurs through the formation of aerosols (small sputum
droplets) after coughing. For these reasons, individuals that live in close proximity of
TB patients are considered at higher risk of developing TB, especially if these persons
have a compromised immunity as seen in HIV infection.
TB disease can occur in different sites in the body. The most common location,
however, is the lung. Patients with active pulmonary TB develop a number of
symptoms, which include: cough (≥ 3 weeks), coughing up blood, fever, chills, night
sweats, lethargy, weight loss, loss of appetite and chest pains. In patients with
immunosuppression, pulmonary TB is often accompanied by extrapulmonary forms of
TB. This is a clinical scenario where other locations than the lungs, such as the lymph
nodes, lung pleura, the brain and the kidneys become infected. In rare cases, TB bacilli
can enter the blood and spread across the body. This severe form of TB will cause
disease at multiple sites of the body and is called “miliary TB” since the chest x-ray
has the appearance of millet seeds scattered throughout the lung. If not accompanied
by pulmonary TB, these non-pulmonary presentations of TB are generally considered
to be less infectious by the centers for disease control and prevention (CDC).
2.1.3 TB-infection and the immune system
Mycobacterium tuberculosis is a small acid-fast bacterium with an intracellular
lifestyle. Their preferential habitats are resting alveolar macrophages which reside in
the pulmonary interstitium and local lymph nodes. During phagocytosis of TB,
macrophages will attempt to digest the engulfed microorganism. However, TB has
developed ways of avoiding the lysis process within these phagocytes by inhibiting
fusion between phagosomes and lysosomes which contain digestive enzymes. This
protection mechanism allows TB to safely replicate within these cells. During
infection, TB will encounter a whole army of different immune cells and the final
outcome of TB infection depends on the balance between this immune response and
the virulence of TB.
During initial infection, local macrophages will attempt to kill TB by phagocytosis.
Recognition of TB-PAMPs (such as LAM) by TLRs will trigger a local inflammatory
response, which attracts neutrophils and monocytes from the blood. Since TB is able
to hide inside the cells, the infection will persist and give rise to a DTH response. After
2-3 weeks of TB replication, the bacterial load will trigger a local adaptive immune

The HIV-TB conundrum
18
response through the draining lymph nodes. An increasing number of immune cells
will be attracted to the site of infection, regulated by a plethora of cytokines40.
Monocytes, CD4 and CD8 T cells, neutrophils and more will take position around the
site of infection to form nodules called granulomas41. T cells have an important
function in protecting against TB, which is demonstrated by the synergy between HIV
and TB infection. By producing IFNγ, Th1 cells, cytotoxic T cells and NK cells activate
infected macrophages to continue digesting already phagocytized mycobacteria.
Cytotoxic T cells also play an important role in killing intracellular TB and/or TB
infected macrophages42. Although this immune response more often than not fails to
completely eradicate the infection, it usually is able to confine the infection to the
existing granuloma as so called latent TB. However, when dealing with highly virulent
strains or in cases of severe immunosuppression or even an overly vigorous immune
response to TB, the TB infection can become active and start to spread.
2.1.4 Diagnosis
Diagnosis can be done at several levels; on the basis of clinical signs, detection of TB
bacilli themselves or by detecting TB related immune reactions. All these different
methods of detection vary in sensitivity and specificity. More importantly, a lot of
techniques require sophisticated lab equipment which is not abundant in resource
limited settings. Therefore, there is a constant trade-off between the sensitivity of the
test and the applicability in the field.
Perhaps the most accessible way of identifying a patient with active tuberculosis is
the clinical diagnosis. Especially when dealing with individuals at high risk of
developing TB disease, a patient presenting with classical TB symptoms like coughing,
fever and lethargy can be considered a possible TB-case. Further clinical examinations
can be done, like detection of TB granulomas by chest x-ray.
In resource limited settings, the most widely used method of detection is by sputum
Ziehl-Nielsen smear microscopy, despite its relatively low sensitivity (50-80%)43.
Although this method can confirm the presence of TB bacteria in the lungs, it is not
very useful when dealing with extrapulmonary TB or cases of smear negative TB. This
diagnostic test can be additionally confirmed with mycobacterial culture. However,
these cultures take a long time to grow and require access to laboratory facilities.
More recent improvements on these techniques include fluorescence microscopy and
automated liquid culture. A final way of directly detecting the presence of TB is by
polymerase chain reaction tests (e.g. GeneXpert44) which have a very high sensitivity.
Although there is an added benefit of detecting drug resistant TB strains, this

The HIV-TB conundrum
19
technique requires a well-equipped lab and implementation in the field is therefore
limited.
Thanks to the immune responses that coincide with TB exposure and infection, a
number of other detection methods have been developed. These techniques rely on
the use of TB-associated antigens. The best-known is the tuberculin skin test (TST),
where a purified protein derivative of mycobacteria (PPD) is injected subcutaneously.
In patients previously exposed to TB this will result in a DTH response, which causes
swelling. TB exposure can be determined by the size of this swelling, although this
DTH response can be severely diminished in the context of HIV infection. The TST is
however not specific for TB and will also react upon exposure to other mycobacteria,
including vaccination with bacille Calmette-Guérin (BCG). In contrast, the recently
developed IFNγ release assays provide a higher specificity by using antigens specific to
M. tuberculosis45. 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture
filtrate antigen (CFP-10) are two antigenic molecules which are secreted by live TB
bacilli. Adding these antigens to a sample of whole blood from patients exposed to TB
induces a strong IFNγ response which can be measured. Finally, TB also expresses
non-protein antigens which are part of the mycobacterial cell wall.
Lipoarabinomannan (LAM), for example, is a TB-associated PAMP that is recognized
by TLR2. Recently an experimental but interesting way of detecting (latent)
tuberculosis has been discovered which involves the detection of LAM in morning
urine of patients46.
2.1.5 Therapy
Since ancient times, the only treatment for TB was cod-liver oil and exposure to
sunshine. These regimens had a positive effect on the patients’ vitamin D levels,
which in turn bolstered their immune system against TB. Even so, it was up to the
patients’ immune systems to bring the infection under control. Nowadays, the use of
antibiotics remains an efficient way of dealing with TB infection. However, the
increasing emergence of multi-drug resistant and extensively drug-resistant TB
continues to cause serious problems in health care systems.
The WHO recommends a combination of bacteriostatics and bactericidal drugs for
first line treatment of TB. A standard treatment regimen requires an intensive phase
of two months with daily doses of Isoniazid, Rifampicin, Pyrazinamide and
Ethambutol. This phase is then followed by a consolidation phase of four months with
Isoniazid and Rifampicin47. In latent infections where no active disease can be
diagnosed, a six month course of Isoniazid is sufficient. The simultaneous
administration of these drugs is necessary to avoid drug resistance, given the

The HIV-TB conundrum
20
tendency of TB to develop resistance against treatments. Currently the WHO
recommends a hierarchical selection of four second line drugs for simultaneous
administration in cases of drug resistant TB48.

The HIV-TB conundrum
21
2.2 HIV – the plague of the 20th and 21st century
Compared to TB, HIV can be considered a very young and recent pathogen. HIV made
its first documented appearance in 1981, when certain groups of people began to
show a cluster of unusual diseases49. It soon became clear that the appearance of
diseases such as Kaposi’s sarcoma in homosexuals and intravenous drug users was
related to a drop in the number of CD4 T cells. In 1982 the term acquired immune
deficiency syndrome (AIDS) was first used by the CDC50. One year later, in 1983, the
virus was isolated from a lymph node of an infected individual in France and named
human immunodeficiency syndrome (HIV)51,52. By now it has become clear that HIV
originates from simian immunodeficiency viruses, transmitted from monkeys to
humans most probably through the hunt and consumption of bush-meat in
Cameroon. The virus was able to get a foothold in humans and mutate into the HIV
virus that we know today. As HIV can be transmitted through blood-blood or sexual
contact, promiscuous sexual practices in Kinshasa (at that time the largest hub for
trading, transport and travel in central Africa) created a suitable environment for HIV
to start its march across the globe53.
What makes HIV so relentless is the fact that it has a marked ability for immune
evasion. HIV possesses a very strong capacity for mutations. In fact, the genetic
diversity of HIV within only one individual is about the same as that of Influenza
across the entire world population at any given year54. HIV cannot be eradicated from
the body due to its reservoir in resting CD4 cells, where it remains hidden from the
immune system as a pro-virus. Possible target sites for antibodies are also difficult to
access, making it hard to neutralize HIV. These abilities have allowed HIV to elude
several efforts to find an effective cure, even today.

The HIV-TB conundrum
22
2.2.1 Epidemiology
According to the world health organization (WHO) and UNAIDS, a total of 35.3 million
people worldwide were infected with HIV in 201255. There were 2.3 million new HIV
infections globally with 1.6 million AIDS deaths in 2012. The lion share of infections
(24.9 million) can be found in sub-Saharan Africa. Figure 5 shows the number of
people living with HIV in 2012, estimated by the WHO.
2.2.2 Viral structure and lifecycle
There are different species, strains, subtypes and variants of HIV. A complete
description of these types would be outside the scope of this thesis. We focused on
the most common species; HIV-1, from here on referred to as HIV. As a lentivirus, HIV
has a relatively simple structure (figure 6)56. The viral particle consists of a lipid
envelope with surface molecules gp120 and gp41. Gp120 binds to CD4 molecules on T
cells, monocytes and dendritic cells57. Gp41 then initiates fusion of HIV with the target
cell’s membrane. The viral particle contains a protein capsid that surrounds the viral
ribonucleic acid (RNA) as well as HIV’s 3 main enzymes which are released into the
cytoplasm upon fusion. The viral reverse transcriptase (RT) enzyme converts the viral
RNA into double-stranded deoxyribonucleic acid (DNA). This DNA is then integrated
into the host genome by the viral integrase enzyme. The viral RNA (and DNA) contains
the genetic information required to produce all HIV-proteins, including the enzymes.
Figure 5. The number of people living with HIV worldwide, estimated by the WHO.

The HIV-TB conundrum
23
HIV takes advantage of the host-cells natural transcription process to produce more
viral RNA as well as the required proteins to form new HIV particles. These proteins
are cut down to size by the viral protease enzyme before being assembled, after
which the assembled particles leave the host-cell.
2.2.3 Clinical presentation
HIV infects cells that express the
CD4 molecule, making CD4 T cells its
prime target58. A healthy individual
normally harbors more than 800
CD4 T cells per µl blood. This
drastically changes through the
course of HIV infection (figure 7). In
the first weeks after infection with
HIV, patients experience flu-like
symptoms in the so called “acute
phase” of HIV infection. The acute
phase is marked by a temporary
peak in the number of viral copies in
the blood, while the number of CD4
T cells steeply declines. The immune
system rapidly counterbalances this
overload of HIV particles by
mounting a strong cellular immune response. The number of HIV-specific CTL rapidly
rises, accompanied by a CD4 T cell response. This immune response is aimed at
reducing the viral load and results in a temporary recovery of the number of CD4 T
cells. After a few weeks, seroconversion takes place. HIV patients will produce
antibodies against viral antigens (e.g. gag p24), which can be detected in the process
of HIV diagnosis. Usually, this is followed by a transition into an asymptomatic phase.
The asymptomatic or ‘chronic’ phase can last up to 10 years. This period can be
described as a kind of status quo between viral replication and the immune response.
An active balance is maintained, where new CD4 T cells are constantly replacing the
ones destroyed either directly or indirectly by HIV infection. This kind of balance
cannot be maintained indefinitely. Eventually the immune system will become
exhausted and the number of CD4 T cells will start to decline again; the prelude to the
symptomatic phase. HIV will gradually start to get the upper hand and once again the
gp41 gp120
envelope glycoproteins
envelope
lipid-membrane
reverse
transcriptase
single-stranded
RNA
matrix protein p17
major structural
core protein p24
Figure 6. Simplified graphical representation of an
HIV virion (adapted from Karlsson Hedestam GB et
al. 200856).

The HIV-TB conundrum
24
viral load increases. CD4 T cells become anergic, their numbers decline and a number
of symptoms; fever, weight loss, diarrhea, etc. are soon to follow.
The final stage is defined as ‘AIDS’ by the CDC when an HIV-positive patient reaches a
threshold of <200 CD4 T cells per µl blood or develops AIDS defining opportunistic
infections (OIs). AIDS patients will experience sequential episodes of OIs. These are
caused by microorganisms to which a healthy individual can mount an effective
cellular immune response. Since HIV seriously affects this response, AIDS patients lack
a sufficient defense against new infections or infections that were previously under
control by the immune system. A good example is Mycobacterium tuberculosis, which
is considered one of the AIDS defining illnesses. If left untreated, AIDS patients will
eventually succumb to these infections. In the absence of HIV infection, the immune
response that tries to contain TB may be highly inflammatory with devastating
physiological effects. However, in (untreated) patients with an advanced stage of
AIDS, the balance is shifted from inflammation towards TB growth and dissemination,
resulting in less inflammatory pathology59.
2.2.4 HIV and the immune system
HIV specifically infects cells that are pivotal in the immune system which is supposed
to protect against infection, explaining the large impact HIV has on a patient’s health.
HIV RNA Copies per ml Plasma
10²
10
7
10³
10
4
10
5
10
6
1200
0
100
200
300
400
500
600
700
800
900
1000
1100
CD4+ T lymphocyte count (cells/mm³)
0113 6 9 12 1234567 8910
YearsWeeks
Primary
Infection
Acute HIV syndrome
Wide dessimation of virus
Seeding of lymhoid organs
Clinical Latency
Constitutional
Symproms
Opportunistic
Diseases
Death
Figure 7. Generalised course of HIV infection. The evolution of the CD4 T cell count (blue) and viral
load (red) during acute infection, clinical latency and AIDS phase (reproduced from Fauci AS et al.
1996
58
).

The HIV-TB conundrum
25
Progressive depletion of the peripheral CD4 T cell pool is the most fundamental event
in the pathogenesis of HIV infection and the progression to AIDS. In addition, a
massive CD4 T cell depletion also takes place in mucosal tissues throughout all stages
of HIV infection60-62. Cells infected with HIV will either die as a direct result of the high
rate of HIV replication (osmotic cell lysis) or when targeted by antibody mediated
phagocytosis and cytotoxic apoptosis. What’s more, a large number of HIV uninfected
CD4 T cells die as a result of indirect bystander mechanisms63. This results in a
severely weakened adaptive immune response to infections.
Despite the gradual loss of the immune system’s primary orchestrator and contrary to
initial belief, a strong immune response is mounted during the acute phase. As one
might expect, the innate immune system is the first to respond during acute HIV
infection. It has been shown that HIV ssRNA encodes for multiple TLR7/8 ligands that
can mediate immune activation64. This TLR stimulation leads to a cascade of cytokine
production in the periphery65,66. IFNα in particular has been shown to be up-regulated
during acute HIV infection, followed by secondary TNFα, inducible protein 10 and IL-
18 secretion. This rise in pro-inflammatory cytokines during acute HIV infection
(secreted by DCs and monocytes) is accompanied by activation and expansion of NK
cells, even before the development of any detectable antibody responses67. Apart
from their role in eliminating HIV-infected cells, NK cells might also be involved in
killing uninfected CD4 T cells, thus contributing to the CD4 T cell decline during HIV
disease progression68,69. Finally, IL-10 and IFNγ production is also up-regulated, which
has been associated with the subsequent rise in the HIV-specific adaptive immune
response. Since HIV is an intracellular pathogen, it induces a strong CTL response to
HIV peptides presented by MHC-I molecules. In addition, HIV infection induces a
broad humoral response with production of HIV specific antibodies. For almost any
other infection, this defense should be enough to eradicate the virus. Nevertheless,
these immune responses are insufficient to fully eliminate HIV and eventually the
battle is lost.
2.2.5 Diagnosis and therapy
HIV-treatment has seen a long and tedious road in scientific development. Diagnosis
of HIV-infection used to mean nothing less than a gradual death of the patient. When
HIV was first discovered, scientists were confident that a cure would quickly be found.
However, 30 years later an actual cure still eludes the top scientific minds of this
world, though promising progress has been made. Though we may not yet be able to
fully cure HIV infection, the discovery of HIV inhibiting compounds has managed to
greatly improve survival of HIV-patients. Antiretroviral therapy (ART) is currently
being distributed worldwide in order to combat the HIV pandemic. Continuing

The HIV-TB conundrum
26
endeavors to improve the drugs have also helped to decrease the number of side
effects during treatment, though the real long term effects on the immune system
remain to be seen. The WHO recommends administration of ART when CD4 counts
have reached a threshold of 350 cells/µl, although there is debate on the earlier start
of ART70.
Several types of drugs exist that interact with different steps in the cycle of HIV-
replication (figure 8). Binding-inhibitors such as Maraviroc compete with HIV for
binding to co-receptors on a cell’s surface. Fusion-inhibitors like Enfurvitide on the
other hand, inhibit the merging of an HIV particle with the cellular membrane by
binding to gp41. These drugs hereby effectively block HIV RNA from entering the cells.
If HIV somehow still gets into the cell, the reverse transcriptase inhibitors come into
play. Both nucleoside-analogue reverse transcriptase inhibitors (NRTI) and their non-
nucleoside counterpart (NNRTI) inhibit the reverse transcription of viral RNA to DNA,
which is a vital step for the reproduction of HIV particles. Next, the integration of this
reverse transcribed DNA into the host genome can also be blocked by integrase
inhibitors, Raltegravir for example. Finally, cells that have already been infected with
HIV are infected for life because of the integrated viral DNA in the genome.
Nevertheless, production of new HIV particles can be halted at the step where viral
protease cleaves viral poly-proteins (Gag and Pol) into structural and enzymatic
proteins. Protease inhibitors such as Ritonavir are able to neutralize this protease.
However, none of these drugs are able to completely clear HIV from its reservoirs.
Therefore ART only succeeds in bringing viral loads back to undetectable levels.

The HIV-TB conundrum
27
2.2.5.1 Immunological response to ART
Once a patient has started ART, the immune system gradually starts to recover. The
increase of CD4 T cells upon ART initiation has been described as a 3 phase process71 .
T cells upon ART initiation has been described as a 3 phase process64. The highest rate
of reconstitution is observed during the first six months of ART, with 20 - 30 cells/µl
monthly. The second phase has a CD4 recovery rate of 5 to 10 cells/µl per month and
lasts until the end of the second year of therapy. Finally, the third phase lasts until at
least the seventh year of therapy and has a recovery rate of about 2 to 5 cells/µl per
month.
During the first weeks after starting ART, T cell reconstitution follows a biphasic
pattern72. The most rapid reconstitution is seen in CD4 T cell numbers during the first
three weeks after starting ART, followed by rapid reconstitution of the CD8 T cell
compartment during the first 6 weeks. This early T cell recovery has been shown to
mainly involve a redistribution of memory cells from lymphoid tissues towards the
peripheral blood or expansion of already circulating T cell clones73-75. Followed by this
initial release of memory cells, various mechanisms contribute to a rise in mostly
naïve T cells during the later phases; de novo production of T lymphocytes by the
HIV with 2 copies
of RNA genome
Env -CD4 binding
CD4
Env
CCR5/CXCR4
Fusion with
host-cell
Uncoating
Reverse transcription
of viral genome
Nuclear import
of proviral DNA
Integration into host genome DNA
Transcription
of viral RNA
Translation of
viral proteins Virion
assembly
Budding of virus
1
2
3
4
5
Protease step
Figure 8. Generalized HIV lifecycle. This figure represents the replication process of HIV and indicates
interaction sites of binding-inhibitors (1), fusion-inhibitors (2), (N)NRTI’s (3), integrase-inhibitors (4)
and protease-inhibitors (5) with HIV’s lifecycle. (Adapted from Rambaut A et al. 2004
57
).

The HIV-TB conundrum
28
thymus, homeostatic proliferation of the residual T cells and extension of T cell half-
life76. This process generally leads to partial recovery of an HIV-infected individual,
where the immune system is once again capable of defending against other
pathogens. However, recovery as a result of ART is not perfect. AIDS is associated
with a number of immune defects, as discussed in the next section, which can persist
even during effective ART.

The HIV-TB conundrum
29
2.3 HIV–TB co-infection – a lethal partnership
Co-infection with HIV and TB represents a partnership between 2 of the 3 most lethal
singular infectious diseases on the planet77. When infected with both diseases at the
same time, there seems to be a mutual amplification in their systemic effects. This
interplay between HIV and TB, as well as social and demographic factors can explain
why both infections often occur simultaneously78. Thanks to the deleterious synergy
between them, HIV and TB co-infection has a serious impact on healthcare systems
worldwide. Management of this co-pandemic thus remains a significant challenge for
health care providers, owning to atypical clinical presentations, drug interactions and
diagnostic complications.
2.3.1 Epidemiology
HIV infection increases the risk of active TB about 34-fold, ranging from 20 to 37
according to some sources37,79. Among all opportunistic infections worldwide, TB is
the leading cause of HIV-related mortality. TB accounts for an estimated 25% of HIV-
related deaths with an annual death-burden of about 430.00080,81. The largest
prevalence of HIV-TB co-infection can be found in Sub-Saharan Africa, particularly in
South-Africa (figure 9). In 2008, there were an estimated 1.4 million new cases of
tuberculosis (TB) among persons with HIV infection79. Among all TB cases worldwide,
the incidence of HIV positive cases was 1.1 million (12%) in 2012.
Figure 9. HIV prevalence in new TB cases, estimated by the WHO.

The HIV-TB conundrum
30
2.3.2 The immune system in HIV-TB co-infection
The relationship between HIV and TB goes beyond that of normal opportunistic
infections that occur during progression to AIDS. There is a bidirectional synergy
between them, which is partly due to their immunology. The depletion of CD4 T cells
caused by HIV is one of the primary factors contributing to the increased risk of active
TB infection. However, HIV also induces functional changes in T cells and
macrophages, which influences their ability to contain TB78,82,83. Co-infection with TB,
in turn, accelerates HIV disease progression and the development of other OIs in AIDS
patients84. HIV preferentially infects and depletes TB-specific T cells, lowering the
defensive capabilities against TB even further85. These events can be attributed to the
enhancing effect TB has on the already elevated level of immune activation in HIV-
patients.
2.3.2.1 Immune activation in HIV-TB co-infection
During the course of HIV infection, the human body is literally flooded with HIV
particles. This chronic exposure, together with reactivation of a number of underlying
infections (such as cytomegalovirus) and exposure to opportunistic infections such as
TB, provide a continuous source of antigenic stimulation. Though it is a fundamental
hallmark of AIDS progression, both HIV and TB (and especially the co-infection) are
marked by a high level of immune activation. This is a rather broad phenomenon that
involves different cellular and molecular aspects of the immune system.
HIV-TB co-infection is characterized by elevated expression of activation- and
apoptosis markers on CD8 and CD4 T cells, NK cells, monocytes and so on86-89.
Moreover, high levels of pro-inflammatory cytokines and chemokines from both
innate and adaptive origins can also be observed in co-infected patients90-93. Under
normal circumstances (for example in singular TB infection), this activation reflects a
protective response against infection. However, there is a paradoxical association
between the level of T cell activation in HIV-patients (measured by the expression of
HLA-DR and CD38 on CD8 T cells) and progression towards AIDS94-96. In fact, negative
correlations have been described between peripheral CD4 counts and the level of CD8
T cell activation as well as HIV viral load97,98. A direct consequence of T cell activation
is the enhancement of cellular transcription processes. This does not only enhance
the production of cellular proteins (such as cytokines) but also the transcription of
viral proteins if the cell is infected with HIV. This evokes a vicious circle, whereby the
immune activation perpetuates itself through the synergistic action of T cell activation
and cytokine production, as well as the spread of and stimulation by HIV99. In this way,
TB-induced immune activation contributes to the spread of HIV and the apoptotic
pressure on the T cell compartment in HIV patients.

The HIV-TB conundrum
31
Next to antigenic stimulation of the T cell compartment, an important source of the
HIV- and TB-induced immune activation can also be found in the innate immune
system. HIV induces massive depletion in mucosal CD4 T cells which leads to the
destruction of the intestinal barrier function and translocation of bacteria and their
products into the bloodstream100. Therefore, HIV infection is associated with elevated
plasma levels of the gram-negative bacterial cell wall component LPS and intestinal
fatty-acid binding protein (I-FABP, a marker of intestinal cell damage)101. Similar to
LPS, the cell wall of TB contains lipoarabinomannan (LAM), which can be detected in
the patient’s urine102. Both LAM and LPS are well-known pathogen associated
molecular patterns (PAMPS) and are strong activators of monocytes by binding to
Toll-like receptors 2 and 4 respectively. T cells can thus be activated directly by
microbial products or indirectly by inflammatory cytokines derived from macrophages
and DC103,104. Associated with the increased levels of LPS and LAM, a corresponding
increase in LPS binding protein (LBP) and sCD14 is described in HIV, TB and HIV-TB co-
infected patients105-107.
2.3.1.3 HIV and T cell maturation
The sustained level of immune activation in HIV(-TB) infection is a major driving factor
of T cell proliferation and differentiation, giving rise to high numbers of antigen-
experienced cells. This continuous proliferation may be one of the factors that
eventually lead to the exhaustion of the T cell compartment in HIV-TB patients.
Although a drop in absolute CD4 and CD8 T cell counts has been reported during
active TB infection without HIV, the balance between maturational T cell stages
seems to remain undisturbed108,109. HIV infection, however, is associated with a
deregulation of haematopoiesis, a reduced thymic output and fibrosis of lymphatic
tissue110-115, which may prevent normal T cell homeostasis. Therefore, HIV patients
experience a decline in their general T cell renewal capabilities. This results in a
reduced ability to replenish the naïve T cell pool and to continually replace the
terminally differentiated and depleted CD4 and CD8 T cells. Over time, this will lead to
the accumulation of older, more differentiated CD4 and CD8 T cells116,117, which have
a decreased capacity to proliferate and a stronger focus on the production of pro-
inflammatory cytokines118,119. This all happens in a systemic environment that is more
focused on the production of pro-inflammatory (rather than homeostatic) cytokines
and is marked by an increased apoptosis-induced cell death. Together, all these
changes reflect a general shift of the T cell population towards more differentiated
and senescent, antigen-experienced cell populations that fill the immunological
space. Figure 10 shows a model of the process of immune activation and T cell
maturation, leading to immune exhaustion in HIV-infection.

The HIV-TB conundrum
32
2.3.3 HIV-TB therapy
The treatment modality for TB is the same for HIV-infected as for HIV-uninfected
patients, with an intensive phase of 2 months, followed by a 4 month regimen. TB
treatment always takes precedence over ART and the timing of subsequent ART-
initiation has to be decided on an individual basis, after assessing the short-term risk
of disease progression and death, based on CD4 count and type of TB120. This is
particularly important in developing countries, where HIV-TB patients often show up
at the clinic with very low CD4 counts (<50 cells/µl). Despite the increased incidence
of complications, ART initiation after 2 weeks of TB treatment has been shown to
reduce mortality in these patients121-123.
Currently, the WHO recommends immediate treatment for TB in patients co-infected
with HIV and TB. The timing of ART-initiation thus depends on the number of CD4 T
cells/µl the patient currently has. If there are no other indications to start ART and if
the patient has over 350 CD4 T cells/µl, ART can be postponed until TB treatment is
completed. With CD4 count between 200 and 350 cells/µl, ART could be postponed
until the intensive phase of treatment is finished. In HIV-TB patients with CD4 counts
below 200 cells/µl, ART is administered as soon as the TB treatment is tolerated by
the patient (usually 2 weeks to 2 months)124.
Shift to terminally
differentiated T cells
Systemic Immune
Activation
↓ CD4+ T cells
Bacterial translocation
(LPS,...)
Disease reactivation
and OI ’s
Immune response to
HIV
↑ Pro-inflammatory
cytokines
Activation induced
apoptosis
Exhaustion and collapse of the immune system
↓ T cell proliferative
capacity & homeostasis
HIV infection
Figure 10. Model of immune activation and T cell maturation in HIV. (Adapted from Appay et al.
2008
99
).

The HIV-TB conundrum
33
2.3.3.1 Complications in HIV-TB therapy
The increasing availability of ART in countries where TB is endemic has allowed
substantial improvement in the survival of HIV-TB patients. However, combination
therapy of anti-tubercular drugs and ART faces a number of challenges. One major
problem in the treatment of TB is the development of resistant TB strains. It is
possible that HIV contributes to the development of drug resistant TB strains, due to
the intestinal effects of HIV (and its treatment) and consequently the malabsorption
of TB drugs. On the other hand, the damaged immune system of HIV patients might
provide a more suitable environment for the less virulent MDR strains125,126.
Another important challenge in dual TB and HIV treatment are the substantial
pharmacokinetic interactions which can occur and which might provide a reason for
postponing ART when possible. Such interactions have especially been observed when
treating with Rifampicin, for example; Rifampicin is known to interact with NNRTIs
(such as Nevirapine) and PIs, leading to sub-therapeutic levels of these drugs and
subsequent treatment failure127. The antiretroviral drug regimen should therefore be
carefully chosen to minimize drug-interactions with anti-TB treatment, with Efavirenz
being the drug of choice128,129.
2.3.3.2 Persistent immune defects during immune reconstitution
Although information on the recovery of the immune system during concomitant HIV
and TB treatment is limited, the immune reconstitution of HIV patients on ART has
been extensively studied. The appropriate treatment regimen usually leads to
recovery of the patient, marked by a drastic increase in the patient’s CD4 count and
clearing of TB symptoms. However, compared to healthy individuals, persistent
deficits in absolute CD4 T cell numbers and subsets have been reported, even after 14
years of successful ART130. Thus, despite the quantitative CD4 T cell recovery, certain
qualitative immune defects may persist during ART. These defects are especially
prominent in the T cell compartment, while the effects on cells of the innate immune
system are less well defined131-135.
During the course of ART, the typical AIDS-associated immune activation usually
declines. However, several studies have reported elevated levels of T cell activation
after ART initiation, which could be associated with a slower CD4 T cell recovery and
higher mortality rates130,136. Persistence of immune defects could be due to several
factors, including: age, genetic predisposition, undetectable HIV replication,
subclinical opportunistic infections (or a persistent antigen load) and bacterial
translocation due to the partially irreversible intestinal damage caused by HIV71.
These defects and their possible causes are often aggravated in HIV patients that

The HIV-TB conundrum
34
received ART in a very late stage of AIDS. In fact, one study showed that patients who
started ART while having less than 200 CD4 T cells/µl had higher levels of CD8 T cell
activation and higher levels of IFNγ, IL-2, IL-6 and IL-10 up until 2 months on ART136. It
thus seems that the severe depletion of CD4 T cells and the associated complications
might have a (longer) lasting effect on the recovery of the immune system. Moreover,
HIV-TB patients starting ART at late stages of AIDS progression are at high risk of
developing an inflammatory complication during treatment called TB-IRIS.

Immune Reconstitution Inflammatory Syndrome
35
Chapter III: Immune Reconstitution Inflammatory
Syndrome
Imagine you have been infected with HIV. You are living in a resource limited setting,
with limited accessibility to ART. The chances are high that ART won’t become
available to you until you start to develop AIDS related symptoms, TB-infection being
one of them. You start to become ill, eventually ending up with full blown AIDS and
tuberculosis symptoms.
Luckily, you live close to a hospital, where you are now receiving treatment. First, the
doctors prescribe an intensive treatment for TB. Quite soon after that, you need to
start ART because your immune system is practically gone. Things are looking good.
You are responding well to both treatments and pretty soon you’ll be able to get on
with your life.
But suddenly, only 2 weeks after starting ART, you start to feel sicker again. Coughing,
fever, inflammation, it’s like tuberculosis has unexpectedly returned and with a
vengeance. Doctors are having a hard time determining what’s wrong with you. Is it a
resistant strain of tuberculosis? Is the treatment failing after all? Eventually, you are
informed that you are probably experiencing something called “immune reconstitution
inflammatory syndrome (IRIS)”. Depending on how severe the inflammation is, you
might even be in need of hospitalization, additional drugs and there’s even a very
slight chance of death.

Immune Reconstitution Inflammatory Syndrome
36
3.1 The many faces of IRIS
3.1.1 What is IRIS
A subset of HIV patients who initiate ART are at risk of developing a complication
during treatment called immune reconstitution inflammatory syndrome, also known
as immune reconstitution disease (IRD) or immune reconstitution syndrome (IRS). IRIS
was first described in 1992 in a patient who developed localized forms of
Mycobacterium avium complex disease during Zidovudine treatment137. Generally
speaking, IRIS can be described as a clinical deterioration of HIV patients in the first
months after starting ART, marked by excessive inflammation. At least three
conditions need to be present for an HIV patient to be at risk of developing IRIS;
severe immune suppression, an underlying opportunistic infection (or antigenic
stimulus) and initiation of ART as the trigger. Despite these basic facts, IRIS embodies
a heterogeneous collection of clinical manifestations.
This heterogeneity already becomes apparent with the fact that IRIS can occur as 2
clinical forms. The “unmasking” form of IRIS presents when an HIV patient is infected
with an undiagnosed (usually subclinical) OI before initiating ART. During ART, the
patient’s immune recovery can result in exaggerated immune responses, thus
“unmasking” the hidden OI. “Paradoxical” IRIS on the other hand occurs in HIV
patients that have been diagnosed with an OI and are being treated for it. During ART,
the patient can paradoxically develop excessive inflammatory reactions and
symptoms consistent with the underlying OI, despite successful treatment against it.
Next to the above mentioned clinical scenarios, IRIS has many presentations which
are largely dependent on the OI involved. A broad range of opportunistic infections
have been associated to IRIS, including non-TB mycobacteria, hepatitis B and C
viruses, herpes viruses and parasites such as toxoplasma and schistosoma. IRIS has
even been associated with malignancies such as Kaposi’s sarcoma and non-aids-
defining inflammatory conditions such as sarcoidosis, rheumatoid arthritis and grave’s
disease138. The frequency and morbidity of these different presentations varies. IRIS is
particularly frequent, however, among HIV patients co-infected with Mycobacterium
tuberculosis, cryptococci and cytomegalovirus (CMV)139,140.
All these different forms of IRIS illustrate the high degree of clinical variation in IRIS
and explain why studies often focus on IRIS related to one specific OI. However, even
within a pathogen-specific form of IRIS such as TB-IRIS, a large degree of
heterogeneity exists. Patients who experience TB-IRIS may develop a heterogeneous
array of symptoms, with varying degrees of severity. With the increasing worldwide

Immune Reconstitution Inflammatory Syndrome
37
distribution of ART in areas where TB is endemic, the risk of TB-associated immune
reconstitution syndrome (TB-IRIS) will also increase. Up to 25% of patients with an
HIV-TB co-infection will develop TB-IRIS during successful ART. This work will
therefore focus on the TB-specific development of IRIS.
3.1.2 Epidemiology of IRIS
Cryptococcal-IRIS is viewed as the most lethal version of IRIS, with an estimated
mortality rate of 20.8%140. TB-IRIS on the other hand has a mortality rate of an
estimated 3.2%, with deaths mostly due to neurological complications. Variable
incidences have been reported across studies. Table 2 summarizes the reported
incidences and clinical presentations of IRIS to a wide range of pathogens.
Table 2. Summary of pathogens associated to IRIS and their incidence.
Opportunistic Infection
Pooled incidence, %
(95% CI, when available)
Clinical presentation (exacerbation of…)
Most documented pathogens associated to IRIS
Mycobacterium t uberc ulosis 15.7 (9.7-24.5) Inflammation at different sites,
particularly the lymph nodes
Cryptoc occus 19.5 (6.7-44.8) Cryptococcal meningitis
Cytomegalovirus retinitis 37.7 (26.6-49.4) CMV retinitis
Other pathogens associated to IRIS
Non-TB Mycobacteria
avium complex 3.5%
141
Lymphadenopat hy, pulmonary disease, …
BCG (Bacillus Calmette–
Guérin)
6-15%
in children with HIV
142
“unmasking” BCG-infection, regional
adenitis.
Parasite
Toxoplasma 1.5-9.5% 143-145 Cerebral toxoplasma – confusion,
headache …
Schistos oma 3 reported cases136,146,147 Hepat osplenomegaly, abdominal pain,
schistosomal colonic polyposis, …
Strongyloides stercoralis 4 reported cases Cough, pneumonitis, eosi nophilia ,fever,
hepatitis …
Leishmaniasis 25 reported cases
148
Post-kala-azar dermal leishmaniasis,
visceral leishmaniasis, uveitis
Viruses
Herpes Zoster 12.2 (6.8-19.6) (Multi-)dermatomal Zoster Myelitis or
Ramsay Hunt syndrome (rarely)
Hepatitis B/C 22.2 149 / 18% 150 Hepatitis flares, rise in liver enzyme levels
Kaposi’s sarcoma herpes virus 6.4 (1.2-24.7) Uncontrolled Kaposi-Sarcoma
develop ment
John Cunningham poliomavir us 16.7 (2.3-50.7) Progressive multifocal
leukoenc ephalopathy
(Adapted from Lawn SD et al. 2011
151
and Muller M et al. 2010
140
).

Immune Reconstitution Inflammatory Syndrome
38
3.2 Clinical presentation of TB-IRIS
3.2.1 Consensus definition
Since its debut in the clinical world, TB-IRIS has caused a lot of confusion among
physicians. TB-IRIS presents itself as a clinical worsening of TB symptoms during ART
and it is therefore difficult to distinguish from other complications such as treatment
failure, resistant TB strain infection, drug-toxicity and so on. One major hurdle in TB-
IRIS diagnosis and early research was the lack of a clear definition of TB-IRIS. In 2006,
an international meeting of researchers working in the field of HIV and TB infection
convened in Uganda to address the issue of a consensus definition. By combining
common features among existing case definitions and weighing their practical use in
resource limited settings, a consensus was reached152. The consensus definition on
paradoxical TB-IRIS is summarized in table 3. In contrast to the more frequent
paradoxical form of TB-IRIS, the unmasking form of TB-IRIS is less well-defined. Under
the general classification of “ART-associated TB”, a provisional consensus definition
was proposed for unmasking TB-IRIS, summarized in table 4. Because of the clearer
and more frequent diagnosis, this work focusses on paradoxical TB-IRIS, from here on
referred to as “TB-IRIS”.
One look at table 3 already illustrates the degree of clinical variation which surrounds
TB-IRIS, judging by the wide array of symptoms a TB-IRIS patient may (or may not)
develop. The frequency of these symptoms has previously been studied in a Ugandan
cohort, on which this thesis is based (table 5, see also Appendix I for more details on
overlapping patients between chapters))153. Despite the relatively large frequency
(65%) of pulmonary TB prior to ART among patients destined to develop TB-IRIS in
this cohort, the majority (70%) of the clinical presentations of TB-IRIS during ART were
consistent with extrapulmonary TB. Twenty-five percent of TB-IRIS presentations
were consistent with pulmonary TB and an overlapping portion of these patients (19%
of total) had both pulmonary and extrapulmonary presentations. Interestingly, 24% of
TB-IRIS patients were diagnosed with only minor INSHI criteria and did not fit either
category. This latter group of patients further emphasizes the varying degree of
severity in which TB-IRIS may manifest itself. Thus, even with the consensus
definition, diagnosis of TB-IRIS mostly relies on its clinical characteristics. Detection of
TB-IRIS remains a diagnosis per exclusionem, meaning that all other possible
explanations need to be excluded before TB-IRIS can be diagnosed. Therefore, there is
an urgent need for reliable laboratory markers for the diagnosis of the syndrome.

Immune Reconstitution Inflammatory Syndrome
39
Table 3. Consensus definition for paradoxical tuberculosis-associated IRIS.
There are three components to this case definition:
(A) Antecedent requirements
Both of the two following requirements must be met:
• Diagnosis of tuberculosis: the tuberculosis diagnosis was made before starting ART and this
should fulfil WHO criteria for diagnosis of smear-positive pulmonary tuberculosis, smear-
negative pulmonary tuberculosis, or extra-pulmonary tuberculosis
• Initial response to tuberculosis treatment: the patient’s condition should have stabilised or
improved on appropriate tuberculosis treatment before ART initiation (This does not apply to
patients starting ART within 2 weeks of starting tuberculosis treatment)
(B) Clinical criteria
The onset of tuberculosis-associated IRIS manifestations should be within 3 months of ART initiation,
reinitiation, or regimen change because of treatment failure.
Of the following, at least one major criterion or two minor clinical criteria are required:
Major criteria
• New or enlarging lymph nodes, cold abscesses, or other focal tissue involvement
• New or worsening radiological features of tuberculosis
• New or worsening CNS tuberculosis
• New or worsening serositis
Minor criteria
• New or worsening constitutional symptoms (fever, night sweats, or weight loss)
• New or worsening respiratory symptoms (cough, dyspnoea, or stridor)
• New or worsening abdominal pain accompanied by peritonitis, hepatomegaly, splenomegaly, or
abdominal adenopathy
(C) Alternative explanations for clinical deterioration must be excluded if possible
• Failure of tuberculosis treatment because of tuberculosis drug resistance
• Poor adherence to tuberculosis treatment
• Drug toxicity or reaction
• Another opportunistic infection or neoplasm (it is particularly important to exclude an alternative
diagnosis in patients with smear-negative pulmonary tuberculosis and extra-pulmonary
tuberculosis where the initial tuberculosis diagnosis has not been microbiologically confirmed)
(From: Meintjes et al. 2008152).

Immune Reconstitution Inflammatory Syndrome
40
Table 4. Case definition for ART-associated TB and unmasking TB-associated IRIS.
Table 5. The frequency of clinical characteristics among patients with TB-IRIS in Uganda.
3.2.2 Risk factors and prevention
Although the (immuno-)pathogenesis of TB-IRIS is not well understood and effective
diagnostic markers are yet to be discovered, several risk factors of TB-IRIS have been
identified. The most prominent risk factor is undoubtedly a very low CD4 count, the
trademark of advanced AIDS related immunosuppression. Patients who initiate ART
with CD4 counts <200 cells/µl, especially those below 50 cells/µl have been shown to
There are two definitions to consider:
ART-associated tuberculosis
ART-associated tuberculosis (all cases of tuberculosis diagnosed during ART) should be defined as
• Patient is not receiving treatment for tuberculosis when ART is initiated
• Active tuberculosis is diagnosed after initiation of ART and was screened negative for TB by
sputum culture
• The diagnosis of tuberculosis should fulfil WHO criteria for smear-positive pulmonary
tuberculosis, smear-negative pulmonary tuberculosis, or extra-pulmonary tuberculosis
Unmasking tuberculosis-associated IRIS (provisional)
The following could suggest a diagnosis of unmasking tuberculosis- associated IRIS:
• Patient is not receiving treatment for tuberculosis when ART is initiated and then presents with
active tuberculosis within 3 months of starting ART
and one of the following criteria must be met:
• Heightened intensity of clinical manifestations, particularly if there is evidence of a marked
inflammatory component to the presentation
• Once established on tuberculosis treatment, a clinical course that is complicated by a
paradoxical reaction
(From: Meintjes et al. 2008152).
Major criteria
Frequency
Minor criteria
Frequency
n (%)a
n (%)a
Increasing lymphadenopathy
28 (53)
Fever
45 (85)
Worsening abdominal ultrasound
fi di
16 (30)
Weight loss
30 (57)
Worsening chest X-ray features
13 (25)
Night sweats
26 (49)
Abscesses
3 (6)
Cough
36 (68)
Other focal tissue swelling 2 (4) Dyspnea 29 (55)
Tuberculous meningitis
0 (0)
Stridor
1 (2)
Tuberculous ascites
3 (6)
Peritonitis
9 (17)
Tuberculous pericarditis
1 (2)
Abdominal pain and
l h d th
8 (15)
Tuberculous pleural effusion
0 (0)
Abdominal pain and
ht l
6 (11)
Joint effusion
0 (0)
Abdominal pain and
ll
3 (6)
an = 53 (From: Worodria W et al. 2012153).

Immune Reconstitution Inflammatory Syndrome
41
have an increased risk of TB-IRIS153,154. At these advanced stages of AIDS, patients do
not have the luxury to postpone ART. Therefore, the second major risk factor for TB-
IRIS is a shorter treatment interval between TB-treatment and subsequent ART.
Patients initiating ART within 2 months of starting TB-treatment have an up to 10 fold
increased risk of TB-IRIS.
Arguably, these risk factors also increase the antigenic burden of TB in HIV-TB patients
prior to ART. This higher antigen load is therefore viewed as a third risk factor for TB-
IRIS. Indeed, patients with disseminated or extra-pulmonary TB have been
documented to have an increased risk of TB-IRIS 155. Less well established risk factors
include the rapid recovery of the immune system upon ART and the associated rapid
decline in viral load155-157. These conditions likely reflect strong response to treatment
and could be associated to a stronger reaction to the underlying antigenic stimulation.
The most efficient way to prevent TB-IRIS development, in fact, would be to prevent
the conditions that provide an increased risk. An early start of ART (>350 cells/µl) as
recommended by the WHO would for example prevent most TB-IRIS cases. However,
this is not always feasible in resource limited settings. In developing countries, many
patients still present at the clinic with very low CD4 counts (< 50 cells/µl), which is
likely to remain an important issue for the time being. In patients with CD4 counts
>200 cells/µl, ART initiation could be postponed until after the intensive phase of
treatment to reduce the risk of TB-IRIS. However, in patients with very low CD4
counts, postponing ART is associated with increased death122,158. Therefore, by
choosing the lesser of 2 evils, ART is still initiated as soon as TB-treatment is tolerated
by the patient (between 2 weeks to 2 months).
3.2.3 Disease outcome and treatment
Despite its relatively low mortality rate, TB-IRIS still causes serious morbidity in HIV-TB
patients. Though the symptoms are usually self-limiting, about 30% of patients
experience severe symptoms and are in need of hospitalization or additional
treatment to provide relief159. This not only increases the cost of patient care, but the
increased pill burden can have adverse effects on treatment-adherence as well, which
in turn adds to the risk of resistance development.
Depending on how advanced the TB-IRIS case is, a number of symptomatic
treatments are available. Mild-to-moderate cases can be managed by the
administration of non-steroidal anti-inflammatory drugs160. Severe reactions, on the
other hand, might be modulated by the use of corticosteroids; in a double-blind
placebo-controlled randomized clinical trial, prednisone administration was shown to
reduce the need for hospitalization without an excess in treatment side-effects or

Immune Reconstitution Inflammatory Syndrome
42
severe infections159. In some cases, surgery can provide more comfort, for example by
needle aspiration of cold abscesses which develop during TB-IRIS161. Finally, as a last
resort in life-threatening situations, the cessation of ART can be considered. However,
ART interruption could be associated with antiretroviral resistance and should
therefore not be considered lightly. Moreover, TB-IRIS patients re-initiating ART could
still develop a new episode of TB-IRIS.
3.3 Immunopathogenesis of TB-IRIS
Over the last two decades since IRIS was first described, many study groups have
undertaken research to elucidate the pathogenesis behind IRIS. Ranging from small
retrospective studies to research in large prospective cohorts that spanned years,
significant progress in understanding TB-IRIS has been made. Given the
heterogeneous nature of TB-IRIS, studying it is not as straight forward as it may seem.
IRIS studies have been faced with a number of challenges, including; the initial lack of
a consensus definition, limited sample sizes and the adequate selection of (and
matching to) control patients. Although the collective scientific effort has led to the
identification of several soluble and cellular factors that are associated with TB-IRIS,
the syndrome thus still remains a puzzling complication to this very day.
3.3.1 Antigen load
Due to the severe immunosuppression and shorter treatment interval in TB-IRIS
patients, a high antigen load has been proposed as a risk factor for TB-IRIS. However,
surprisingly little is known about the actual levels of TB-antigens in TB-IRIS patients,
though some progress has been made in this respect. One study monitored the
presence of TB-specific antibodies as an indirect measurement for M. tuberculosis
antigen load and reported higher levels of antibodies against phenolic glycolipid (a TB-
antigen) prior to ART initiation162. However, only 23% of patients showed a humoral
response to the TB-specific antigens CFP-10 and ESAT-6, while no significant
difference could be observed in the level of LAM-antigens between IRIS patients and
controls. In contrast, higher levels of LAM have been detected in the urine of TB-IRIS
patients prior to ART initiation compared to HIV+TB+IRIS- controls163. In a review on
TB-IRIS, LPS has also been proposed as a possible contributor to the development of
TB-IRIS164. These findings indicate that a high antigen load might indeed be implicated
in TB-IRIS pathogenesis. However, it still remains unclear if or which specific antigens
might be involved and which parts of the immune system react to them.
3.3.2 T lymphocytes – the prime suspect
As described in the previous chapters, T cells have a pivotal role in HIV and TB
immunity. To date, the strongest predictive factor for TB-IRIS is still a low CD4 count

Immune Reconstitution Inflammatory Syndrome
43
prior to ART. Given this prerequisite and the fact that TB-IRIS only develops after ART
initiation, a restoring CD4 T cell compartment thus seems to be a necessity. Most
studies have therefore focused on the role of an unbalanced recovery of the T cell
compartment as a cause of TB-IRIS, with varying results.
3.3.2.2 T cell activation in TB-IRIS
Low CD4 counts in progressive HIV infection are typically associated with high levels
of T cell activation97,98,136,165,166, which may persist during ART. Such a persistent level
of T cell activation during successful ART suggests an incomplete recovery of the
immune system167 or could be associated with a reaction to persisting underlying
opportunistic infections such as TB or their residual antigens136,165,168,169. A high level
of immune activation prior to or during ART might thus provide a suitable biomarker
for TB-IRIS and might even be a possible driving mechanism behind it.
The relationship between these high levels of T cell activation and IRIS has been
studied by research groups focussing on non-pathogen specific forms of IRIS. One of
these studies reported elevated expression of the activation marker PD-1 on CD4T
cells prior to ART as well as during ART. While elevated immune activation prior to
ART could not be confirmed, two other non-pathogen specific IRIS studies reported
increased immune activation during IRIS on both CD4 and CD8 T cells17 0, or CD8 T cells
exclusively17 1.
Although these studies could provide insight in common mechanisms between forms
of IRIS associated to different pathogens, the heterogeneity of these patients could
potentially mask TB-specific mechanisms that lead to TB-IRIS. A more recent study
compared immune activation between TB-specific IRIS and a number of non-TB IRIS
cases172. Interestingly, activation of CD8 T cells was associated with TB-IRIS but not
with the other forms of IRIS, illustrating the importance of homogeneous study
populations. Two smaller studies, on 3 and 11 TB-IRIS patients respectively, also
revealed elevated HLA-DR expression on CD4 T cells during the IRIS event173,174.
Together, these findings provide the first steps in elucidating the possible contribution
of excessive T cell activation in both TB-specific IRIS and IRIS in general. The elevated
levels of T cell activation observed in these studies could be indicative of underlying
antigenic stimulation in TB-IRIS patients. However, a longitudinal study in 10 TB-IRIS
patients did not observe elevated expression of these markers, which illustrates the
inconsistencies in current literature on TB-IRIS175.

Immune Reconstitution Inflammatory Syndrome
44
3.3.2.1 T cell maturation in TB-IRIS
As discussed previously in section 2.3.2 of this thesis, T cell activation is a major
driving factor behind the maturation of T cells through several memory and effector
stages. AIDS progression is therefore marked by accumulation of terminally
differentiated T cells, which have a predominantly pro-inflammatory character119.
Although an increased redistribution of these pro-inflammatory subtypes during
immune reconstitution could drive TB-IRIS, little is known about these T cell
maturation profiles in TB-specific IRIS. However, there are again some things we can
learn from studies on non-pathogen specific forms of IRIS. One such study, in fact,
reported a shift from CD8 and CD4 central memory T cells to more terminal subtypes
during IRIS in a cohort where 40% of IRIS cases were related to TB171. Nonetheless,
such shifts have only sporadically been observed elsewhere176, or not at all170. The
role of such maturational shifts in the T cell compartment thus remains largely
unexplored.
3.3.2.3 Antigen-specific responses in TB-IRIS
Upon ART, the initial CD4 T cell recovery in HIV-patients is largely mediated by the
redistribution of T cell memory subpopulations, which is strongly associated with a
rapid improvement of the DTH response. Incidentally, the first IRIS studies described a
stronger restoration of the DTH to purified protein derivative (PPD) in HIV-TB patients
after they developed IRIS compared to patients who had not developed IRIS137,177. This
led to the hypothesis that an excessive restoration of TB-specific T cell responses
might be driving TB-IRIS. A number of studies were therefore undertaken to
investigate the TB antigen-specific IFNγ reactions in TB-IRIS.
One of the earlier TB-IRIS studies, performed on 7 patients experiencing paradoxical
TB-IRIS, described a sharp increase in PPD-specific IFNγ producing Th1 cells in concert
with elevated production of Th1 type cytokines (IL-2, IL-12, IFNγ, IP-10 and MIG) when
compared to HIV+TB+IRIS- controls who did not develop TB-IRIS178. However, only 3/7
patients displayed a Th1 response to ESAT-6. Two studies later confirmed the
increased Th1 reactivity to PPD on 3 and 7 paradoxical TB-IRIS patients respectively,
although only the latter one observed a concomitant increase in ESAT-6
reactivity173,179. Two more studies reported similar results after performing PPD
stimulated interferon-gamma release assays in TB-IRIS patients180,181.
In line with these findings, a retrospective study by Meintjes et al.175 reported an
elevated frequency of IFNγ secreting T cells recognizing ESAT-6, α-crystallins 1 and 2,
and PPD in TB-IRIS patients, compared to HIV+TB+IRIS- controls. In stark contrast
however, their subsequent longitudinal analysis revealed highly similar IFNγ reactions

Immune Reconstitution Inflammatory Syndrome
45
in TB-IRIS patients and controls, casting doubt on the instrumental role of T cells in
TB-IRIS. While there is evidence for increased PPD-specific responses, it thus remains
unclear whether there is a causal link between TB-IRIS and these IFNγ-responses, as it
is for other TB-specific antigens.
3.3.2.4 Other T cell subsets in TB-IRIS
So far, most T cell focused TB-IRIS studies mainly investigated markers related to the
Th1 subtype, due to their importance in HIV and TB. However, there is some evidence
in support of a role for other T cell subtypes in TB-IRIS. Thanks to their role in bringing
inflammatory reactions under control, regulatory T cells (Tregs) might have an
important role in managing TB-IRIS related inflammation. Two studies uncovered an
expansion of (FOXp3+) Tregs in TB-IRIS patients154,173 , although only one of them
observed a lowered capacity for IL-10 production of these regulatory T cells. In
contrast, two other studies did not observe any differences in Treg expansion175,176.
The precise contribution of Tregs to TB-IRIS (if any) thus still remains to be discovered.
Finally, one study reported higher numbers of killer Ig like receptor negative (KIR-) γδ+
T cells in TB-IRIS patients, while reporting lower numbers of KIR+ γδ+ T cells before and
after ART initiation. Since over 90% of these cells are reported to be TB-specific, this
might provide a driving mechanism behind TB-IRIS182.Though results are limited, these
studies provide interesting alternatives to the mainstream Th1 hypothesis currently
used in TB-IRIS studies which should be further explored.
3.3.3 The cytokine storm
There is more to the immunology of HIV and TB than just the T cell compartment. All
cells of the immune system communicate with each other through the production of
cytokines. Given the fact that TB-IRIS’ main feature is tissue destructive inflammation,
the association of TB-IRIS to a plethora of cytokines is perhaps not that surprising. As
discussed, an early in vitro study on TB-IRIS patients observed an explosive production
of Th1 cytokines upon PPD stimulation. In parallel, production of a number of non-
specific pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, TNFα, RANTES and
MCP-1) was also increased178. Following these results, a later correspondence argued
that TB-IRIS is likely associated to an increase in a broad range of cytokines, or in
other words; “a cytokine storm”183.
Subsequent studies have since then confirmed the existence of a cytokine storm
during TB-IRIS. Increased in vitro production of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p40,
IFNγ, granulocyte-macrophage colony-stimulating factor and TNFα upon stimulation
with heat-killed TB was reported in 22 TB-IRIS patients compared to HIV+TB+IRIS-
controls184. Further studies examining plasma cytokine levels reported elevations in

Immune Reconstitution Inflammatory Syndrome
46
an even larger selection of cytokines during TB-IRIS170,185-191 . Although not all
cytokines have been consistently reported across these publications, increased
plasma levels of IFNγ, TNFα and in particular IL-6 seem to be a recurring feature.
Ample evidence thus exists to support a peaking cytokine environment during TB-IRIS.
However, blood cytokine levels are subject to rapid fluctuations and measurements
during the IRIS event could therefore just be a snapshot of the current inflammation.
While the cytokine storm might be particularly relevant during ongoing TB-IRIS
related inflammation, only a limited number of observations describe variances in
blood cytokine levels in TB-IRIS patients prior to ART. One study reported higher pre-
ART levels of IL-6 to be predictive of TB-IRIS, although these patients also had lower
CD4 counts than HIV+TB+IRIS- controls192. In contrast, another study reported lower
pre- ART MCP-1 levels in TB-IRIS patients187, while some reported no differences at
all186.
Thanks to these findings, the occurrence of a cytokine storm during TB-IRIS has been
generally accepted. However, it remains unclear which pre-ART mechanisms might
predispose patients to such a drastic rise in cytokine levels. Moreover, the
inconsistencies across studies make it difficult to pinpoint which cytokines in
particular, if any, hold a central role in TB-IRIS inflammation. Of interest, a
randomized placebo-controlled clinical trial for the treatment of TB-IRIS showed
beneficial effects of prednisone via suppression of predominantly innate pro-
inflammatory cytokine responses, not via a reduction of the numbers of antigen-
specific T cells in peripheral blood. Moreover, elevations in IL-6 and TNFα are
consistently reported across studies. These findings therefore support the growing
attention for the role of the innate immune system in TB-IRIS.
3.3.4 Innate immunity – the unlikely suspect
Though most studies to date have focussed on the role of T cells in TB-IRIS, there have
been doubts about the causal role of the adaptive immune system175,184 . Arguably, the
development of TB-IRIS only a few days after ART initiation suggests that TB-IRIS can
develop without a substantial recovery of the T cell compartment. When we take into
account that a large portion of the cytokines which peak during IRIS are of myeloid or
dual myeloid-lymphoid origin and given the essential role of macrophages in HIV and
TB immunology, a role for the innate immune system becomes increasingly apparent.
As an alternative driving mechanism behind TB-IRIS, Van den Bergh et al. proposed a
potential role for cells from the monocyte lineage by suggesting that TB-IRIS could
result from hyperactivation of previously dysfunctional macrophages in response to
TB antigens when ART is initiated193. Other attractive theories have also been

Immune Reconstitution Inflammatory Syndrome
47
proposed, suggesting an uncoupling of the adaptive and the innate immune system in
TB-IRIS patients194. An impaired ability to respond to the pre-ART antigen load could
then lead to priming of the innate immune system, followed by an inflammatory burst
when ART is initiated and T cell “help” ensues195.
So far, the number of studies investigating the innate immune factors in TB-IRIS is
limited, however there is some evidence to support these theories. One study on 3
paradoxical TB-IRIS patients revealed a higher expression of TLR2 on monocytes in TB-
IRIS patients prior to and during ART. In addition, stimulation of cultured PBMC’s with
LAM caused an increase in TNFα production, without a corresponding increase of IL-
10196. These findings are supported by a recent gene-expression study, which
reported a pro-inflammatory monocyte-gene expression profile that was also
perturbed in pattern recognition receptor pathways197. In addition to macrophages,
other innate immune factors have also been implicated in TB-IRIS. Indeed, high levels
of CRP have been reported in TB-IRIS patients before and during ART175,198 , as well as
high pre-ART plasma-levels of complement factor C1q and C1-inhibitor199. Moreover,
one study reported an increased degranulation capacity of NK-cells prior to ART in TB-
IRIS patients200.
Together, these findings provide the groundwork for an interesting new approach to
TB-IRIS research. Indeed, monocytes, NK-cells and even neutrophils (well-known
effector cells in tissue inflammation) could be major players in TB-IRIS development.
Exploring the contribution of the innate immune system to TB-IRIS, especially prior to
ART, might be a valuable step in the direction of elucidating the pathogenesis behind
TB-IRIS.
3.3.5 Concluding remarks
Collectively, studies focussing on general and on TB-specific IRIS have succeeded in
laying important foundations to uncover the mechanisms behind TB-IRIS’
pathogenesis. By exploring the broad spectrum of the immune system, evidence is
emerging that support the roles of both the T cell compartment and the innate
immune system in TB-IRIS. Although evidence supporting the latter is increasing
exponentially, it is still unclear where exactly the immune system derails to cause
such inflammation. Perhaps both adaptive and innate immune factors are important
players in TB-IRIS and the answer to solving the puzzle may lie somewhere in between
them. Pooling all evidence together, one could surmise that TB-IRIS originates from an
exaggerated general immune response to TB-bacilli or their residual antigens. Fittingly
named ‘the cytokine storm’, a peak in a plethora of cytokines seems to mark this
response.

Immune Reconstitution Inflammatory Syndrome
48
While these characteristics of TB-IRIS have become widely accepted, the pre-ART
mechanisms leading up to this event are still a mystery and reliable laboratory
markers to predict and identify this syndrome have yet to be found. One major hurdle
in most studies to date has probably been the abundant heterogeneity that
characterizes IRIS. A particular issue that has been overlooked in most studies is the
fact that TB-IRIS development is not restricted to the 3 month period delimited in the
INSHI definition. In fact, TB-IRIS can develop at variable intervals during ART and has
even been observed as late as 4 years after the start of ART201. Given the
immunological turbulence in the first months after starting ART, it may be prudent to
take the duration of ART into account when selecting TB-IRIS patients in a study.
Furthermore, there seems to be a need to further explore which antigens, antigen-
specific responses, cytokines and other plasma proteins hold a central role in the
inflammatory chaos that marks TB-IRIS, in particular those related to innate
pathways.
Focussing on the development of TB-specific IRIS within a population of HIV-TB
patients starting ART in Uganda, a large prospective study was set up to address
several of these issues. The clinical characteristics of this study population are briefly
summarized in Appendix I. The following chapters provide a detailed overview of the
work we completed in the hope of adding key elements to the TB-IRIS puzzle.

Objectives
49
Chapter IV: Objectives
Our study’s main objective was to investigate the immunopathogenesis of TB-IRIS
while exploring potential biomarkers for prediction and diagnosis. Our initial
investigation left from the hypothesis that an unbalanced reconstitution of the T cell
compartment made a fundamental contribution to TB-IRIS. As illustrated by 3
manuscripts/publications, our research involved the following 3 steps:
4.1 To investigate the role of CD4 and CD8 lymphocyte
populations in early- and late-onset TB-IRIS
The putative role of T cells in HIV and TB immunology has made them prime targets
for unraveling the immunopathogenesis behind TB-IRIS. In the previous chapters, we
summarized relevant literature on the activation and maturation state of the T cell
compartment and their conceivable role in TB-IRIS pathogenesis. Although the INSHI
definition covers TB-IRIS cases which occur within 3 months on ART, TB-IRIS can
develop at different intervals during ART until up to 4 years after treatment initiation.
Given the immunological turbulence in the first months of ART, it is unclear if so
called early and late presentations of TB-IRIS involve a separate immunpathogenesis.
In the subsequent manuscript, we propose that the role of excessive T cell activation
in TB-IRIS might be less elaborate than initially believed. In fact, our data provide
evidence of reduced CD8 T cell activation prior to ART leading to early- and late-onset
TB-IRIS and do not suggest the presence of an over-activated CD8 T cell compartment
at TB-IRIS event. In addition, we provide the first indications of heterogeneous T cell
maturation between early-onset and late-onset TB-IRIS. We hereby show that early-
and late-onset TB-IRIS may share common predisposing factors, yet appear to be set
apart by a different pathogenesis at the time of the disease.
Odin GOOVAERTS, Wim JENNES, Marguerite MASSINGA-LOEMBE, Ann CEULEMANS,
Chris VEREECKEN, William WORODRIA, Harriet MAYANJA-KIZZA, Robert
COLEBUNDERS, Luc KESTENS, the TB-IRIS Study Group. Lower immune activation prior
to ART in early- and late-onset tuberculosis-associated immune reconstitution
inflammatory syndrome. (Under review)

Objectives
50
4.2 To assess antigen specific IFN-γ responses and innate
cytokine production in early-onset TB-IRIS
The general principle that TB-IRIS results from an exaggerated immune response to
antigens, possibly TB-antigens, has gained acceptance. We reviewed literature
concerning antigen-specific IFNγ responses in TB-IRIS in the previous chapter, which
provided some evidence of an explosive Th1 response driving TB-IRIS. Nonetheless, it
is still unclear if and which antigen-specific responses contribute to TB-IRIS and little is
known about the innate cytokine responses in TB-IRIS patients after TLR stimulation.
Taking the lessons learnt from the previous objective, the following manuscript
focused on patients who developed TB-IRIS within one month on ART, thus providing
a more homogeneous study population. We argue that TB-IRIS is marked by a
disturbed recovery of antigen-specific IFNγ responses (to CMV and LPS), rather than
an excessive TB-driven IFNγ response. For LPS, this was linked with a pro-
inflammatory shift in the innate cytokine balance. Together, our data provide
evidence for a prominent role for innate immune inflammation and possibly
monocyte dysfunction in TB-IRIS pathogenesis.
Odin GOOVAERTS, Wim JENNES, Marguerite MASSINGA-LOEMBE, Ann CEULEMANS,
William WORODRIA, Harriet MAYANJA-KIZZA, Robert COLEBUNDERS, Luc KESTENS,
the TB-IRIS Study Group (2014). Antigen-specific interferon-gamma responses and
innate cytokine balance in TB-IRIS (Published; PLoS One 9: e113101)
4.3 To determine levels of PAMP-binding proteins and cytokines
in plasma from TB-IRIS patients
The occurrence of a cytokine storm has become known as one of the more prominent
characteristics of TB-IRIS and involves a chaotic systemic increase in cytokines from
different origins. Although an increasing amount of evidence is suggesting a role for
the innate immune system in TB-IRIS, it is currently unknown which cytokines hold a
central role amidst this chaos. Moreover, the involvement of intestinal LPS and PAMP-
binding proteins remains largely unexplored, even though TLR stimulation is one of
the fundamental mechanisms in innate immunity.
The previous objectives provided little evidence for an excessive T cell response in TB-
IRIS, but rather indicate a potential involvement of the TLR pathway. The subsequent
publication therefore aimed to gain further insight in the innate immune response by
exploring bacterial translocation, PAMP-binding proteins and cytokines in plasma
from TB-IRIS patients. We identified IL-6 as a central molecule in the cytokine storm
and suggest IL-6, LBP and I-FABP as potential biomarkers for TB-IRIS. We hereby

Objectives
51
provide further evidence of the involvement of the innate immune system in TB-IRIS
immunopathogenesis.
Goovaerts O, Jennes W, Massinga-Loembe M, Ceulemans A, Worodria W, Mayanja-
Kizza H, Colebunders R, Kestens L the TB-IRIS Study Group (2013). LPS-binding protein
and IL-6 mark paradoxical tuberculosis immune reconstitution inflammatory
syndrome in HIV patients. (Published; PLoS One 8: e81856).

52

T cell activation and maturation
53
Chapter V: Lower pre-treatment T cell activation in early-
and late-onset tuberculosis-associated immune
reconstitution inflammatory syndrome
Manuscript currently under review
5.1 Abstract
Objective: Tuberculosis-associated immune reconstitution inflammatory syndrome
(TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving
antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not
well understood. We investigated T cell activation and maturation profiles in patients
who developed TB-IRIS at different intervals during ART.
Design: Case-control study within a cohort of HIV-TB patients initiating ART in Uganda
(n=251).
Methods: Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who
developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal
numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was
performed on fresh blood, drawn before and after ART initiation and during TB-IRIS
events. T cell activation and maturation was measured on CD4 and CD8 T cells using
CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies.
Results: CD8 T cell activation before ART was decreased in both early-onset (77% vs.
82%, p=0.014) and late-onset (71% vs. 83%, p=0.012) TB-IRIS patients compared to
HIV+TB+IRIS- controls. After ART initiation, the observed differences in T cell activation
disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing
from memory to terminal effector CD4 and CD8 T cell populations (p≤0.028).
Conclusion: Our data provide evidence of reduced CD8 T cell activation before ART as
a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-
IRIS itself was not marked by an over-activated CD8 T cell compartment. Late- but not
early-onset TB-IRIS was characterized by a more terminally differentiated T cell
phenotype.

T cell activation and maturation
54
5.2 Introduction
Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome
(TB-IRIS) is a complication that arises during successful antiretroviral therapy (ART) in
HIV-tuberculosis (TB) co-infected patients receiving TB-treatment152. TB-IRIS presents
in up to 25% of HIV-TB patients as worsening symptoms of TB during ART, despite a
favourable response to TB-treatment (hence the name “paradoxical TB-IRIS”)140. The
syndrome poses a significant diagnostic challenge to physicians and it may require
hospitalisation or additional therapy202,203. In the majority of patients, TB-IRIS occurs
within the first few weeks of ART (early-onset TB-IRIS)204. Nevertheless, about 15% of
TB-IRIS cases develop later than 3 months and even up to 4 years after starting
ART153,201. This heterogeneity in time between ART initiation and TB-IRIS contributes
significantly to the diagnostic confusion that is already surrounding the syndrome and
it is unknown which common and differentiating factors drive these early and late
presentations of the disease.
Although the pathogenesis of TB-IRIS is not well understood, the idea that IRIS involves
an atypical restoration of pathogen-specific immune responses during ART has gained
acceptance151,152,170. Known risk factors of TB-IRIS include a high TB-antigen burden and
a short interval between initiation of TB treatment and ART. The strongest predictor
for developing TB-IRIS, however, is a low CD4 T cell count prior to ART initiation163,205.
Low CD4 counts in progressive HIV infection are typically associated with high levels of
T cell activation97,98,136,165,166, which may persist during ART. Persistent T cell activation
during successful ART, as measured by expression of CD38 and HLA-DR, suggests an
incomplete recovery of the immune system167 and could be associated with a reaction
to persisting underlying opportunistic infections such as TB or their residual
antigens136,165,168,169. This distinct role of T cells in TB and HIV immunology has led to
the hypothesis that an unbalanced reconstitution of the T cell compartment
contributes to the development of TB-IRIS206.
Studies of non-pathogen specific IRIS have reported elevated expression of activation
markers during IRIS event on either all T cells170 or exclusively on CD8 T cells171 or CD4
T cells176. Although these studies reported no differences in the expression of CD38
and HLA-DR prior to ART, one study reported elevated pre-ART PD-1 expression on
CD4 T cells in IRIS patients176. One previous TB-specific IRIS study found no differences
in CD8 or CD4 T cell activation either before or during ART175. Yet in contrast, increased
CD8 T cell activation was recently reported to be specifically relevant during TB-IRIS
compared to non-pathogen specific IRIS172, illustrating the inconsistencies between
studies. Although T cell activation is a major driving factor behind T cell maturation,
little is known about T cell maturation profiles in TB-specific IRIS. Nevertheless, an

T cell activation and maturation
55
unbalanced redistribution during ART of memory T cells with a pro-inflammatory
phenotype (e.g. terminally differentiated T cells119) could drive IRIS inflammation. A
shift from CD8 and CD4 central memory T cells to more terminal subtypes has been
reported during non-pathogen specific IRIS171. However, such shifts have only been
sporadically observed elsewhere176 or not at all170.
The role of T cell phenotypes in TB-IRIS thus still remains unclear. Importantly,
published IRIS studies either did not differentiate between early- and late-onset TB-
IRIS or entirely focussed on early-onset TB-IRIS, leaving T cell dynamics in late-onset
TB-IRIS largely unexplored. In this study, we therefore compared T cell activation and
maturation markers in early- and late-onset TB-IRIS patients with those in carefully
matched controls from a large prospective study in Uganda1 53. Both early- and late-
onset TB-IRIS patients showed decreased immune activation prior to ART compared to
HIV+TB+IRIS- controls. We also report a maturational shift in late-onset TB-IRIS patients
towards more terminal T cell subtypes, which we did not observe in early-onset TB-
IRIS.
5.3 Materials and methods
5.3.1 Study population
The clinical spectrum of HIV-TB IRIS was studied in a prospective observational study
at Mulago Hospital, Kampala, Uganda between 2007-2011153,163,207. HIV-TB co-infected
adults treated for active TB infection for less than 2 months and eligible for ART were
enrolled in the study. All patients were started on a non-nucleoside reverse
transcriptase inhibitor-based ART according to Ugandan national guidelines. The
median interval from starting TB-treatment to starting ART was 6 weeks. Patients
were followed up for a period of 10 months to monitor paradoxical TB-IRIS
development. Sixty (24%) out of 254 HIV-TB co-infected patients developed TB-IRIS.
Patients who did not develop IRIS-related symptoms served as HIV+TB+IRIS- controls.
Fresh blood samples were collected when patients were diagnosed with inflammatory
symptoms consistent with TB-IRIS and at predetermined intervals; before initiation of
ART (baseline) and at 1 month, 2 months, 6 months and 9 months after starting ART.
In this study, samples taken at baseline and during TB-IRIS or corresponding control
time point were analysed. In addition, two groups of HIV-uninfected subjects were
recruited. One group was receiving treatment for active TB for less than 4 months
(HIV-TB+ controls) while the other had no clinical signs of active TB (HIV-TB- controls).
HIV-uninfected subjects had samples taken only once.

T cell activation and maturation
56
5.3.2 Definitions
Mycobacterium tuberculosis infection was diagnosed according to the TB/HIV WHO
guidelines20 8. Investigations to confirm TB infection included: clinical examination,
chest X-rays and abdominal ultrasounds, sputum smear microscopy for acid-fast
bacilli and mycobacterial culture of sputum, aspirate or effusion if available. TB-IRIS
cases were classified by a committee of two co-authors (RC and WW) after reviewing
all suspected TB-IRIS cases evaluated by the study physicians according to the
International Network for the Study of HIV-associated IRIS (INSHI) clinical case-
definition152. TB-IRIS was diagnosed and sampled when patients presented with at
least 1 major criterion (e.g. enlarged lymph nodes) or 2 minor criteria (e.g. fever and
cough).
5.3.3 Patient selection and matching
The INSHI definition of TB-IRIS currently states that patients can be diagnosed with
TB-IRIS when symptoms occur within the first 3 months of ART, which includes the
majority of TB-IRIS patients152. In our cohort, 77% of all TB-IRIS patients developed
symptoms within the first month on ART, but additional cases with similar symptoms
were diagnosed until 10 months after initiating ART without treatment
interruption153. To limit TB-IRIS heterogeneity, we classified TB-IRIS patients as early-
onset TB-IRIS patients (range 4 - 28 days on ART) and as late-onset TB-IRIS patients
(range 42 - 307 days on ART, see figure 11). Study eligibility criteria included: adult
(>18 years), confirmed HIV-TB infection according to WHO guidelines (when
applicable) and eligible for ART. Exclusion criteria included: pregnancy, prior use of
ART, Grade 3 renal or liver abnormalities and haemoglobin concentration < 8g/100ml.
Patients and controls did not have clinical signs of other opportunistic infections at
the time of the study, except for one late-onset TB-IRIS patient who developed genital
herpes during ART. None of the TB-IRIS patients or controls were receiving anti-
inflammatory treatment when pre-ART or TB-IRIS events were sampled. All TB-IRIS
patients were matched 1 by 1 with HIV+TB+IRIS- controls for sex, age (≤ 10 years
difference) and baseline CD4 count (+/-15 CD4 T cells/mm³), these patients were
therefore all at a similar stage of HIV-disease progression. The IRIS event from each
TB-IRIS patient was paired with the closest available HIV+TB+IRIS- control time point.
Early-onset IRIS events were paired with control samples taken at 1 month on ART,
while late-onset IRIS events were paired with control samples taken at 1 month (n =
1), 2 months (n = 5), 6 months (n = 1) or 9 months (n = 2) on ART.

T cell activation and maturation
57
5.3.4 Lymphocyte immunophenotyping
Fresh whole blood was collected and processed locally within 6 hours of collection.
Without antigenic stimulation, whole blood was stained with fluorescently labelled
antibodies, lysed, washed with PBS and fixed with 1% paraformaldehyde in PBS
before measuring with a FACSCalibur four-color flow cytometer (Becton Dickinson
(BD)). Three antibody panels were used to determine lymphocyte activation and
maturation; panel 1: CD45RO-FITC (Dako), CD38-PE (BD), CD8-PerCP (BD), HLA-DR-
APC (BD); panel 2: CD45RO-FITC, CCR7-PE (BD-pharmingen), CD8-PerCP, CD27-APC
(eBioscience); panel 3: CD45RO-FITC, CCR7-PE, CD4-PerCP (BD), CD27-APC. Data were
analysed with FlowJo software (v9.7 Tree Star), using the following gating strategy.
First, lymphocytes were gated on a forward scatter area versus side scatter area
dotplot. Next, CD4 bright and CD8 bright lymphocytes were gated for further
determination of subpopulations. Activation of memory CD8 T cells (panel 1) was
determined by gating CD8 bright lymphocytes for CD45RO+ events and subsequently
determining the percentage of CD38 and HLA-DR double positive CD8 memory
lymphocytes. Maturational T cell subpopulations were identified by gating CD8 bright
(panel 2) or CD4 bright (panel 3) lymphocytes for CD45RO+ and CD45RO- events and
subsequently analysing the (co-)expression of CCR7 and CD27, as described
previously33,209 . Analysed subpopulations were expressed as frequencies of total CD8
bright or CD4 bright lymphocytes and included: naïve (Tn; CD45RO-CCR7+CD27+),
Figure 11. Frequency of TB-IRIS during ART in a Ugandan cohort. This figure shows a graphical
representation of the frequency of TB-IRIS development in our cohort across a year of follow-up. Here
it can clearly be seen that ~75% of patients who developed TB-IRIS in our cohort, did so within the first
month. This is the period after ART initiation when T cell recovery is the most turbulent. By only
including these clustered patients in the early-onset TB-
IRIS group (red) and thus limiting the possible
effect of ART duration, we provide a much more homogeneous study population. The combination of
the later presentations of TB-IRIS (striped pattern) as a separate “late-onset TB-IRIS” group allowed us
to observe the possible effects of ART duration on the immunopathogenesis during TB-IRIS.
Frequency of TB-IRIS during ART
0 30 60 90 120150180210240270300330
0
5
10
15
Early TB-IRIS
"Late" TB-IRIS
Days on ART
# of TB-IRIS cases

T cell activation and maturation
58
central memory (Tcm; CD45RO+CCR7+CD27+), effector memory (Tem;
CD45RO+CCR7-CD27+), terminal effector memory (Ttem; CD45RO+CCR7-CD27-), early
effector (Tearly eff; CD45RO-CCR7-CD27+) and effector (Teff; CD45RO-CCR7-CD27-) T
cells.
5.3.5 Ethical considerations
The study was approved by: the Research Committee of the Infectious Diseases
Institute (IDI), the ethical review board of Makerere University, the Uganda National
Council of Science and Technology, the institutional review board of the Institute of
Tropical Medicine of Antwerp and the Ethics Committees of the Faculties of Medicine
of the University of Antwerp. Written informed consent was obtained from all study
participants.
5.3.6 Statistical analysis
Paired comparisons between TB-IRIS patients and HIV+TB+IRIS- controls were done
using the Wilcoxon signed-rank test. Unpaired comparisons between early- and late-
onset TB-IRIS patients and with HIV-TB+ and HIV-TB- controls were done using the
Mann-Whitney U test. Statistics were performed using SPSS software (version 17.0),
GraphPad Prism (version 5) or R (version 2.15.3) with significance level set at p < 0.05.
Because of the hypothesis driven nature of this study, no correction for multiple
testing was applied210 211.
5.4 Results
5.4.1 Study population
Twenty-two early-onset TB-IRIS patients were selected, of whom 18 had flow
cytometry data available prior to ART initiation and 16 had data available during IRIS
event. In addition, 10 late-onset TB-IRIS patients were selected, of whom 8 had data
available prior to ART initiation and 9 had data available during IRIS event. TB-IRIS
patients were paired at each time point with an equal number of HIV+TB+IRIS-
controls. Prior to treatment, 14/22 early-onset TB-IRIS patients and 13/22 HIV+TB+IRIS-
controls had pulmonary TB, while 8/22 early-onset TB-IRIS patients and 9/22 controls
had extrapulmonary TB. For both late-onset TB-IRIS patients and controls, 8/10 had
pulmonary TB and 2/10 had extrapulmonary TB. Early-onset TB-IRIS patients and their
HIV+TB+IRIS- controls did not differ for age, sex, baseline CD4 count, baseline viral load
or TB treatment duration prior to ART (table 6). Baseline characteristics of late-onset
TB-IRIS patients and HIV+TB+IRIS- controls were also similar, except for age. Though
within matching criteria, late-onset TB-IRIS patients were a median of 5 years older
than their HIV+TB+IRIS- controls (p = 0.028). No differences in baseline characteristics
were observed between early-onset and late-onset TB-IRIS patients. Early-onset TB-

T cell activation and maturation
59
IRIS occurred a median 15 (14-27) days after starting ART, compared to 98 (60-196)
days for late-onset TB-IRIS patients (p < 0.001). Compared to their matched
HIV+TB+IRIS- control time points, early-onset TB-IRIS events occurred a median of 14
days (interquartile range (IQR): 4 - 16) earlier (p = 0.002) during ART. Late-onset TB-
IRIS events occurred a median of 29 (IQR: 10 - 55) days later (p = 0.022) during ART
than their respective HIV+TB+IRIS- control time points. An additional group of 16 HIV
and TB negative subjects (HIV-TB- controls) was also analysed, who did not differ
significantly for age or sex from TB-IRIS patients (data not shown). Twenty-seven HIV
uninfected controls were included in the study, of whom 11 were being treated for TB
(HIV-TB+ controls) and 16 did not have symptoms of TB (HIV-TB- controls). Neither HIV-
TB+ nor HIV-TB- controls differed from TB-IRIS patients or HIV+TB+IRIS- controls
regarding age or sex.
Table 6. Characteristics of TB-IRIS patients and matched controls in chapter V.
Early TB-IRIS Late TB-IRIS Early vs
Late
Variables
TB-IRIS
(n=22)
HIV+TB+IRIS-
(n=22)
Pa
TB-IRIS
(n=10)
HIV+TB+IRIS-
(n=10)
Pa
Pb
Characteristics Baseline
Sex male n (%)c
12 (55)
12 (55)
1.000
7 (70)
7 ( 70)
1.000
0.409
Age (years) 40 (34-43) 39 (35-43) 0.822 40 (36-49) 35 (33-42) 0.028 0.568
CD4 (cell/mm³) 25 (12-59) 30 (17-61) 0.398
51 (21-128) 50 (21-111) 0.343 0.230
TB treatment duration
prior to ART (days)
42 (25-63) 37 (25-60) 0.394 55 (28-73) 40 (29-51) 0.285 0.360
Viral Load
(Log copies/ml)d
5.7 (5.3-5.7) 5.6 (5.2-5.8) 0.918 5.3 (2.9-6.9) 5.5 (5.2-5.7) 0.593 0.688
Characteristics TB-IRIS event
Days since start of ART e
15 (14-27)
29 (28-30)
0.002
98 (60-196)
56 (54-152)d
0.022
<0.001
Values are shown as median values with IQR. Median age difference in years between late-onset TB-IRIS
patients and HIV+TB+IRIS- controls was 5 (IQR: 1.75-7.5). aWiloxon signed-rank test. b Mann-Whitney U test.
cChi square fishers exact test for binominal data. dFor early TB-IRIS cases and HIV+TB+IRIS- controls; n = 16,
viral loads were only availa ble for 3 late-onset TB-IRIS patients. eFor early TB-IRIS cases and HIV+TB+IRIS-
controls; n = 16, for late TB-IRIS cases and HIV+TB+IRIS- controls; n = 9. Median time d ifference between IRIS
event and corresponding HIV+TB+IRIS- control time point was 14 (4-16) days for early-onset and 29 (10-55) days
for late-onset TB-IRIS.

T cell activation and maturation
60
5.4.2 Decreased immune activation in early- and late-onset TB-IRIS prior to
ART
To assess the putative role of an overly-activated T cell compartment in TB-IRIS, the
percentage of activated (HLA-DR+/CD38+) memory CD8 T lymphocytes in fresh
peripheral whole blood samples was compared between TB-IRIS patients and
HIV+TB+IRIS- controls (figure 12). We observed a lower percentage of activated CD8 T
cells in early-onset TB-IRIS patients prior to ART (77% vs. 82%, p = 0.014) compared to
HIV+TB+IRIS- controls, but no differences during IRIS event. Similarly, we observed a
lower percentage of activated CD8 T cells prior to ART in late-onset TB-IRIS patients
compared to HIV+TB+IRIS- controls (71% vs. 83%, p = 0.012), but no differences during
IRIS event. As
expected, both early-
and late-onset TB-IRIS
patients showed
significantly elevated
percentages of
activated CD8 T cells
compared to HIV-TB+
and HIV-TB- controls
at any given time
point (p ≤ 0.030).
5.4.3 Memory-effector CD8 T cell shift during late-onset but not early-onset
TB-IRIS
To investigate whether TB-IRIS is associated with maturation abnormalities of the CD8
T cell subset, maturation stages of CD8 T cells were studied in peripheral blood of TB-
IRIS patients and controls (figure 13). Based on the expression of CD45RO, CCR7 and
CD27, T cell subsets were subdivided in naïve (Tn), central memory (Tcm), effector
memory (Tem), terminal effector memory (Ttem), early effector (Tearly eff) and
effector (Teff) T cells. Prior to ART, no differences were observed between early- or
late-onset IRIS patients and their HIV+TB+IRIS-controls. During IRIS event, early-onset
Figure 12. Percentage of activated CD8+ cells in early- and late-onset TB-IRIS. This box and Tukey
whisker plot represents median percentages and IQR of HLA-DR+/CD38+ cells within CD8+/CD45RO+ T
cells for early- and late-onset TB-IRIS patients (red) compared to HIV+TB+IRIS- (blue) controls. Median
values and IQR for 16 HIV-TB- (yellow) and 11 HIV-TB+ (green) controls are also represented. Full lines
above indicate significant differences between paired patients (Wilcoxon signed-rank test). The level
of significance was set to P < 0.05 for all tests. Number of patients (and paired HIV+TB+IRIS- controls)
prior to ART were; 18 for early-onset TB-IRIS and 8 for late-onset TB-IRIS. Number of patients (and
paired HIV+TB+IRIS- controls) during IRIS event or corresponding control time point were; 16 for early-
onset TB-IRIS and 9 for late-on set TB-IRIS.
0
20
40
60
80
100 p = 0.014 p = 0.012
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% HLA-DR+ CD38+
in me mory CD8+ T cells
1
10
HIV
+
TB
+
IRIS
-
TB-IRIS
HIV
-
TB
-
HIV
-
TB
+

T cell activation and maturation
61
TB-IRIS patients showed a slightly lower percentage of CD8 Tcm cells compared to
HIV+TB+IRIS- controls (1.0% vs. 1.9%, p = 0.026). At this time point, late-onset TB-IRIS
patients showed markedly lower percentages of CD8 Tem cells (14% vs. 23%, p =
0.008) and markedly higher percentages of CD8 Teff cells (42% vs. 27%, p = 0.021)
compared to HIV+TB+IRIS- controls. This shift was not observed in early-onset TB-IRIS
patients. In fact, late-onset IRIS events showed trends towards lower CD8 Tem (p =
0.051) and higher CD8 Teff (p = 0.066) frequencies compared to early-onset IRIS
events. At both time points, early- and late-onset TB-IRIS patients showed significantly
lower percentages of CD8 Tn and Tcm subsets compared to HIV-TB- controls (p ≤
0.031) and lower percentages of CD8 Tn compared to HIV-TB+ controls (p ≤ 0.003).
5.4.4 Memory-effector CD4 T cell shift during late-onset but not early-onset
TB-IRIS
Finally, we explored the distribution of maturation stages (Tn, Tcm, Tem, Ttem, Tearly
eff and Teff) in the CD4 T cell compartment (figure 14). Similar to the CD8 T cell
compartment, we observed no differences pre-ART. During early-onset IRIS event, we
observed slightly lower proportions of CD4 Tearly eff cells (0.9% vs. 1.8%, p = 0.044)
and Teff cells (0.6% vs. 1.0%, p = 0.004) compared to HIV+TB+IRIS- controls.
Proportions of CD4 Teff cells at early-onset IRIS event were also lower compared to
those at late-onset IRIS event (p = 0.044). In contrast, late-onset IRIS events again
showed a shift in maturation steps: proportions of CD4 Tem cells were lower (25% vs.
43%, p = 0.011) and proportions of Ttem cells were higher (27% vs. 14%, p = 0.028)
compared to HIV+TB+IRIS- controls. At both time points, early- and late-onset TB-IRIS
patients showed significantly lower percentages of CD4 Tn and Tcm subsets compared
to HIV-TB- controls (p ≤ 0.027) and lower percentages of CD4 Tn compared to HIV-TB+
controls (p ≤ 0.001).

T cell activation and maturation
62
Figure 13. Percentage of CD8 maturation sub-stages in early-onset TB-IRIS patients. These box and
Tukey whisker plots represent median percentages and IQR of; A, naïve cells (Tn); B, central memory
cells (Tcm); C, early effector cells (Tearly eff); D effector memory cells (Tem); E, effector cells (Teff); F,
terminal effector memory cells (Ttem) within CD8+ T cells for early- and late-onset TB -IRIS patients
(red) and HIV+TB+IRIS- controls (blue), 16 HIV-TB- (yellow) and 11 HIV-TB+ (green) controls. Full lines
above indicate significant differences between paired patients (Wilcoxon signed-rank test). Dashed
lines above indicate significant differences between unpaired patient groups (Mann-Whitney U test).
The level of significance was set to P < 0.05 for all tests. Number of patients (and paired HIV+TB+IRIS-
controls) prior to ART were; 17 for early-onset TB-IRIS and 8 for late-onset TB-IRIS. Number of patients
(and paired HIV+TB+IRIS- controls) during IRIS event or corresponding HIV+TB+IRIS- control time point
were; 16 for early-onset TB-IRIS and 9 for late-onset T B-IRIS.
0
20
40
60
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD8 Tn
0
5
10
15
p = 0.026
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD8 Tcm
0
10
20
30
40
50
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD8 Tearly eff
0
20
40
60
80
p = 0.008
p = 0.051
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD8 Tem
0
20
40
60
80
p = 0.021
p = 0.066
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD8 Teff
0
10
20
30
40
50
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno A RT
% CD8 Ttem
A B
C D
E F
CCR7+/CD27+CCR7-/CD27+
CCR7-/CD27-
CD45RO +CD45RO -
1
10
HIV
+
TB
+
IRIS
-
TB-IR IS
HIV
-
TB
-
HIV
-
TB
+

T cell activation and maturation
63
0
20
40
60
80
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Tn
0
20
40
60
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Tcm
0
5
10
15
20
25
p = 0.044
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Tearly eff
0
20
40
60
80
p = 0.011
Late TB-IRISHIV
-
Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Tem
0
5
10
15
20
p = 0.004
p = 0.044
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Teff
0
20
40
60
80
100
p = 0.028
Late TB-IRISHIV - Early TB-IRIS
pre-ART IRIS pre-ART IRISno ART
% CD4 Ttem
A B
CD
E F
CCR7+/CD27+CCR7-/CD27+CCR7-/CD27-
CD45RO +CD45RO -
1
10
HIV
+
TB
+
IRIS
-
TB-IRIS
HIV
-
TB
-
HIV
-
TB
+
Figure 14. Percentage of CD4 maturation sub-stages in early-onset TB-IRIS patients. These box and
Tukey whisker plots represent median percentages and IQR of; A, naïve cells (Tn); B, central memory
cells (Tcm); C, early effector cells (Tearly eff); D effector memory
cells (Tem); E, effector cells (Teff); F,
terminal effector memory cells (Ttem) within CD4+ T cells for early- and late-onset TB -IRIS patients
(red) and HIV+TB+IRIS- controls (blue), 16 HIV-TB- (yellow) and 11 HIV-TB+ (green) controls. Full lines
above indicate significant differences between paired patients (Wilcoxon signed-rank test). Dashed
lines above indicate significant differences between unpaired patient groups (Mann-Whitney U test).
The level of significance was set to P < 0.05. Number of patients (and paired HIV+TB+IRIS- controls)
prior to ART were; 17 for early-onset TB-IRIS and 6 for late-onset TB -IRIS. Number of patients (and
paired HIV+TB+IRIS-controls) during IRIS event or corresponding HIV+TB+IRIS- control time point were;
16 for early-onset TB-IRIS and 9 for late-onset TB-IRIS.

T cell activation and maturation
64
5.5 Discussion
HIV-TB patients with low CD4 counts who start ART are at high risk of developing TB-
IRIS153,212. Although the immunopathogenesis of TB-IRIS is still not completely
understood, the explosive restoration of T cell function is believed to play a distinct
role171,176,178,206. In the present study, we compared T cell activation and maturation in
fresh whole blood samples between Ugandan TB-IRIS patients and matched
HIV+TB+IRIS- controls before ART initiation and at IRIS event. Approximately 75% of
TB-IRIS patients in our cohort developed TB-IRIS early (< 1 month) during ART153.
Approximately 25% of TB-IRIS patients developed symptoms at later intervals (> 1
month) during ART, although with otherwise similar clinical symptoms. Since late-
onset TB-IRIS has never been studied as a separate group, we decided to compare
late-onset TB-IRIS with early-onset TB-IRIS. Our data show lower CD8 T cell activation
levels prior to ART initiation in both early- and late-onset TB-IRIS patients compared
to HIV+TB+IRIS- controls. During IRIS event, however, the observed difference in T cell
activation disappeared. Instead, late-onset but not early-onset TB-IRIS patients
developed a shift towards terminal effector T cell subpopulations when TB-IRIS
occurred.
We report lower levels of T cell activation in both early-onset and late-onset TB-IRIS
patients prior to ART, suggesting common pre-ART mechanisms leading to early- and
late-onset TB-IRIS. On one hand, such mechanisms could involve a lowered cytotoxic
function as well as reduced local production of interferon-gamma by the CD8 T cell
compartment. However, previous studies175,181 as well as our own findings213 suggest
that interferon-gamma responses to TB-antigens are similar between TB-IRIS patients
and HIV+TB+IRIS- controls prior to ART. On the other hand, we previously observed
lower pre-ART IL-6 and lipopolysaccharide-binding protein levels in plasma from TB-
IRIS patients from our cohort214, which is in line with the lower level of CD8 T cell
activation observed here. We believe that these lower cytokine levels reflect the
inability of the innate immune system to mount an effective response to the pre-ART
TB antigen load. T cell activation is dependent on antigen presenting cells215 and IL-6
has been shown to induce CD8 T cell activation216,217. Therefore we hypothesize that
the lower pre-ART CD8 T cell activation levels in TB-IRIS patients could be a
downstream consequence of this diminished innate response, rather than a sign of
diminished CD8 T cell function. Interestingly, it was previously suggested that an
impaired innate ability to respond to the pre-ART antigen load could lead to priming
of the innate immune system, followed by an inflammatory burst when ART is
initiated195. Our data thereby provide further evidence of an impaired immune
response prior to ART leading to early- and late-onset TB-IRIS. During TB-IRIS event,

T cell activation and maturation
65
the observed difference in T cell activation disappeared and we did not observe
elevated T cell activation, in contrast to previous studies171,176,178. Interestingly, we
have previously shown that the cytokine storm during TB-IRIS is dominated by innate
factors214. Moreover, the causal role of excessive T cell responses in TB-IRIS has
previously been questioned175. Although it is not clear why CD8 T cell activation did
not rise in parallel with this cytokine storm, our data thus do not support the
presence of an over-activated CD8 T cell compartment in TB-IRIS patients.
Persistent immune activation during HIV infection typically coincides with a depletion
of the naïve T cell pool218-220. However, little is known about T cell maturation profiles
in TB-specific IRIS. We observed slightly lower percentages of CD8 Tcm cells during
early IRIS event and a subtle decrease in CD4 effector populations. One possible
explanation would be that these subsets migrated to tissue in response to the local
inflammation during early-IRIS event. In contrast to early-onset IRIS, late-onset IRIS
was characterized by a much more pronounced shift from memory to effector T cell
subpopulations, resembling the one observed in a previous non-pathogen specific IRIS
study171. This study had a large proportion (41%) of TB-IRIS cases and reported a shift
from CD8 and CD4 central memory T cells to a more terminally differentiated subtype
during IRIS. The median time to IRIS was 38 (IQR, 24-56) days on ART, a time frame in
between that of our early- and late-onset TB-IRIS patients. The phenotypic maturation
of T cells is believed to be dependent on antigen-load and cytokine environment221-223.
Since TB-IRIS is associated with a high antigen load and a cytokine storm183,224,22 5, one
could argue that the exposure to this inflammatory environment induces a
maturational shift during the redistribution of the T cell compartment. A longer
period on ART allows for a greater redistribution of the memory T cell pool and
possibly a longer exposure to this environment. It is therefore tempting to speculate
that for these reasons, the maturational shift was more pronounced in patients who
developed TB-IRIS at a later time during ART. Future studies should investigate the
possible involvement of persisting antigen loads and elevated cytokine environments
in late-onset TB-IRIS development.
Since all our TB-IRIS patients were closely matched HIV+TB+IRIS- controls for CD4 count
and viral load, we do not expect a bias due to differences in disease stage. However,
the unpredictability of TB-IRIS poses a serious challenge for prospective studies to
adequately match control time points to IRIS events. As a result, early control time
points were a median of 2 weeks later than their matched early-onset TB-IRIS
patients. Since this bias probably underestimated residual T cell activation in controls,
it likely has not affected our conclusion of a lack of increased T cell activation at early-
onset TB-IRIS. Late control time points were a median of 1 month earlier than late-

T cell activation and maturation
66
onset TB-IRIS cases. This bias probably overestimated terminal effector T cell
populations in controls, thus actually underestimating the observed shift to terminal
subtypes in late-onset TB-IRIS patients. Nevertheless, given the small number of early-
and late-onset TB-IRIS patients and the relatively large age difference between late-
onset TB-IRIS patients and controls, further studies differentiating between early- and
late-onset TB-IRIS are required to confirm our findings.
Taken together, our data provide evidence of reduced CD8 T cell activation prior to
ART leading to early- and late-onset TB-IRIS and do not suggest the presence of an
over-activated CD8 T cell compartment at TB-IRIS event. In addition, we provide the
first indications of heterogeneous T cell maturation between early-onset and late-
onset TB-IRIS, with late-onset TB-IRIS patients experiencing a more terminally
differentiated maturation profile. Our study hereby shows that early- and late-onset
TB-IRIS may share common predisposing factors, yet appear to be set apart by a
different pathogenesis at the time of the disease. These presentations of TB-IRIS
should therefore be studied separately in the future. A better understanding of the
immune responses before and during ART in TB-IRIS patients could lead to novel
markers for the detection and prevention of this important complication.

Antigen specific responses
67
Chapter VI: Antigen-specific interferon-gamma responses
and innate cytokine balance in TB-IRIS
(Published in November 2014; PLoS One 9, with minor adjustments)
6.1 Abstract
Background: Tuberculosis-associated immune reconstitution inflammatory syndrome
(TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving
antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune
response to TB-antigens. We compared the recovery of IFNγ responses to TB- and
non-TB antigens and explored in vitro innate cytokine production in TB-IRIS patients.
Methods: In a prospective cohort study of HIV-TB co-infected patients treated for TB
before ART initiation, we compared 18 patients who developed TB-IRIS with 18
HIV+TB+IRIS- controls matched for age, sex and CD4 count. We analyzed IFNγ ELISpot
responses to CMV, influenza, TB and LPS before ART and during TB-IRIS. CMV and LPS
stimulated ELISpot supernatants were subsequently evaluated for production of IL-
12p70, IL-6, TNFα and IL-10 by Luminex.
Results: Before ART, all responses were similar between TB-IRIS patients and
HIV+TB+IRIS- controls. During TB-IRIS, IFNγ responses to TB and influenza antigens
were comparable between TB-IRIS patients and HIV+TB+IRIS- controls, but responses
to CMV and LPS remained significantly lower in TB-IRIS patients. Production of innate
cytokines was similar between TB-IRIS patients and HIV+TB+IRIS- controls. However,
upon LPS stimulation, IL-6/IL-10 and TNFα/IL-10 ratios were increased in TB-IRIS
patients compared to HIV+TB+IRIS- controls.
Conclusion: TB-IRIS patients did not display excessive IFNγ responses to TB-antigens.
In contrast, the reconstitution of CMV and LPS responses was delayed in the TB-IRIS
group. For LPS, this was linked with a pro-inflammatory shift in the innate cytokine
balance. These data are in support of a prominent role of the innate immune system
in TB-IRIS.

Antigen specific responses
68
6.2 Introduction
Together with the HIV pandemic there has been a global increase in the number of
tuberculosis (TB) infections120. An estimated 14 million individuals are dually infected
with HIV and TB worldwide79. Despite recent WHO recommendations for early
antiretroviral therapy (ART)226, treatment is started at late stages of HIV infection in
many developing countries227. This puts patients at increased risk of developing
tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS)
during ART152 . TB-IRIS presents in up to 25% of HIV-TB patients as worsening
symptoms of TB during ART, despite a favourable response to TB-treatment (hence
the name “paradoxical TB-IRIS”)1 40. This complication typically occurs within the first 2
months after starting ART, with the majority occurring within the first few weeks204.
TB-IRIS poses a significant diagnostic challenge to physicians and reliable laboratory
markers to help detect this syndrome are urgently needed202.
Known risk factors of TB-IRIS include a low CD4 count, high TB-antigen burden and
short interval between initiation of TB treatment and ART163,205 . The pathogenesis of
TB-IRIS remains largely unclear, although there are clear signs of tissue-destructive
inflammation during immune reconstitution (reviewed in194,195). This process could
involve an amplified immune response to TB bacilli or their residual antigens195,228.
Early research suggested that TB-IRIS development was linked to elevated T-helper
type 1 (Th1) responses to TB-antigens. Indeed, studies in TB-IRIS patients reported
elevated IFNγ responses to several TB-associated antigenic compounds such as
purified protein derivative (PPD), 6 kDa early secretory antigenic target (ESAT-6) and
10 kDa culture filtrate antigen (CFP-10)173,177-179,196. However, elevated IFNγ responses
to TB-antigens are often also seen in HIV-TB patients who do not develop TB-
IRIS175,180,181, casting doubt on the causal role of Th1 cells in TB-IRIS pathogenesis. It
has been suggested that disturbances in the innate immune system187 or in the
interplay between the innate and adaptive immune system195 could drive TB-IRIS
pathogenesis. This is supported by repeated findings of elevated levels of IL-6 and
TNFα, among other innate cytokines, during TB-IRIS170,178,184,186,196,225. Interestingly,
elevated innate cytokine production has also been reported in TB-IRIS patients after
toll-like receptor (TLR) 2 stimulation with lipomannan, a TB pathogen-associated
molecular pattern (PAMP)229. Put together, there is evidence that responses to
antigenic stimulation are unbalanced in TB-IRIS, both in the innate and the adaptive
arm of the immune system. However, the roles of antigen-specific IFNγ production
and TLR stimulation in TB-IRIS remain to be completely elucidated.
In this study, we aimed to assess the ART related recovery of IFNγ responses in TB-IRIS
patients. To this end, we determined specific responses to a number of TB and non-TB

Antigen specific responses
69
antigens in a well matched selection of TB-IRIS patients and controls from a large
prospective cohort207. In addition, we explored the possible innate component of TB-
IRIS by studying cytokine production upon TLR4 stimulation with lipopolysaccharide
(LPS). We report a disturbed reconstitution of the IFNγ response to CMV and LPS,
without an excessive IFNγ response to TB-antigens. In addition, we observed a pro-
inflammatory shift in the innate cytokine balance upon LPS stimulation during TB-IRIS,
providing evidence of the involvement of the innate immune system in TB-IRIS.
6.3 Materials and methods
6.3.1 Study population
Patients were recruited in a prospective observational study on paradoxical TB-IRIS at
Mulago Hospital, Kampala, Uganda, between January 2008 and July 2010 as described
previously153,163,207. The present study is based on HIV-TB co-infected adults who were
being treated for active TB infection and put on ART within 2 months after starting TB
treatment (median 6 weeks prior to ART). All HIV-patients received non-nucleoside
reverse transcriptase inhibitor-based ART according to Ugandan national guidelines
and were monitored for paradoxical TB-IRIS development during at least 3 months.
Blood samples were taken before ART initiation (pre-ART) and when patients were
diagnosed for TB-IRIS (IRIS event). Patients without IRIS-related symptoms were used
as HIV+TB+IRIS- controls and had samples taken pre-ART, at 2 weeks and 1 month on
ART. To compare with the expected immunocompetent antigen responses, two
additional groups of HIV-uninfected subjects were recruited. One group was receiving
treatment for active TB for less than 4 months (HIV-TB+ controls) while the other had
no clinical signs of active TB (HIV-TB- controls). HIV-uninfected subjects had samples
taken only once. HIV-TB-
6.3.2 Patient selection and matching
A large majority of patients from our cohort developed TB-IRIS within 1 month after
ART initiation. To limit heterogeneity among TB-IRIS patients, we included only
patients who developed TB-IRIS within one month on ART. This stricter selection of
patients reduces potential bias due to differences in kinetics and immunopathology
between early- and late-onset TB-IRIS. Selected TB-IRIS patients with PBMCs available
pre-ART and at occurrence of IRIS were matched 1 by 1 with HIV+TB+IRIS- controls for
sex, baseline CD4 count (+/- 15 CD4 cells/mm³) and age (≤ 10 years difference).
Samples from HIV+TB+IRIS- controls, serving as a control time point for their paired
IRIS event, were selected at either 2 weeks or 1 month on ART. This selective pairing
allowed for a matched time on ART between TB-IRIS patients and HIV+TB+IRIS-
controls.

Antigen specific responses
70
6.3.3 Definitions
Mycobacterium tuberculosis infection was diagnosed according to the TB/HIV WHO
guidelines208. The diagnostic evaluation for TB included: clinical examination, chest X-
rays and abdominal ultrasounds, sputum smear microscopy for acid-fast bacilli and
mycobacterial culture of sputum, aspirate or effusion if available. TB-IRIS cases were
classified by a committee of two co-authors (RC and WW) after reviewing all
suspected TB-IRIS cases evaluated according to the International Network for the
Study of HIV-associated IRIS (INSHI) clinical case-definition for resource limited
settings152. This evaluation included: a symptom questionnaire, detailed physical
examination to confirm TB-IRIS and exclude alternative causes and comparison of a
second chest X-ray and abdominal ultrasound scan to the patient’s baseline
examination. Biological TB-IRIS samples collected when patients developed new or
worsening symptoms following initiation of ART. These symptoms included at least 1
major criterion, such as enlarged lymph nodes, or 2 minor criteria, such as fever and
cough.
6.3.4 ELISpot assays
PBMCs were collected from TB-IRIS patients and cryopreserved in liquid nitrogen.
Samples were consequently thawed and IFNγ responses to antigens were measured
by ELISpot (Diaclone SAS, Besançon Cedex, France) according to the manufacturer's
instructions. In brief, PBMCs were cultured overnight in an antibody-coated ELISpot
filter plate at 200,000 cells/200µl in RMPI+2.5% human serum in the presence of
either 10 µg/ml cytomegalovirus (CMV) lysate (Institut Virion\Serion GmbH,
Würzburg, Germany), 5 µg/ml whole influenza virus antigen (H3N2 A/Sydney/5/97,
National Institute for Biological Standards and Control, Hertfordshire, Great Britain),
10 µg/ml PPD (Statens Serum Institute, Copenhagen, Denmark), 10 µg/ml
recombinant ESAT-6 (Statens Serum Institute, Copenhagen, Denmark), and 10 µg/ml
recombinant CFP-10 (a kind gift from Lionex Diagnostics and Therapeutics,
Braunschweig, Germany). In addition, 100 ng/ml LPS (E. coli O55:B5, Sigma-Aldrich
BVBA, Diegem, Belgium) was included separately as a strong inducer of innate
cytokines. Medium only and 5 µg/ml staphylococcal enterotoxin B (SEB, Sigma-Aldrich
BVBA, Diegem, Belgium) were used as negative and positive controls, respectively.
The number of IFNγ spot-forming cells (SFC) per 106 PBMCs was determined using the
ELISpot Reader and ELISpot Reader software (AID, Strassberg, Germany). Data from
unstimulated medium controls was subtracted before reporting the number of SFC.
Because we directly compared quantitative responses between patient groups, no
criteria were used for defining positive responses.

Antigen specific responses
71
6.3.5 Cytokine multiplex assay
ELISpot supernatants from PBMCs stimulated with CMV and LPS were stored at -80°C
until further use. Samples were thawed within 1 hour prior to analysing cytokine
levels by using the Bio-Plex™ human cytokine assay kits (Bio-Rad Laboratories NV-SA,
Nazareth, Belgium) according to the manufacturer’s instructions. We measured in
vitro levels of IL-6, TNFα and IL-10 as a representation of innate pro- and anti-
inflammatory cytokine production. In addition, IL-12p70 was measured as a link
between changes in IFNγ responses and monocyte function. Samples were diluted 2x
for TNFα and IL-10 and 10x for IL-6 to allow optimal detection of both high and low
cytokine concentrations. The LOD (pg/ml) for each cytokine was; 0.5 (IL-12p70), 1.8
(IL-6), 0.2 (IL-10) and 1.1 (TNFα).
6.3.6 Ethical considerations
The study was approved by the Research Committee of the Infectious Diseases
Institute (IDI), the ethical review board of Makerere University School of Medicine,
the Uganda National Council of Science and Technology and by the institutional
review board of the Institute of Tropical Medicine of Antwerp and the Ethics
Committees of the Faculties of Medicine of the University of Antwerp. Written
informed consent was obtained from all study participants.
6.3.7 Statistical analysis
Differences between paired patients and changes in concentration over time for each
patient were analysed using the Wilcoxon signed-rank test for paired data.
Differences between HIV-infected and HIV-uninfected patients were analysed using
the Mann-Whitney U test. Correlations were calculated using the Spearman's rank
correlation. Statistics were performed using SPSS software (version 17.0) or GraphPad
Prism (version 5) with significance level set at p < 0.05. Because of the hypothesis
driven nature of this study, no correction for multiple testing was applied210,211.

Antigen specific responses
72
6.4 Results
6.4.1 Study population
A total of 18 TB-IRIS patients were paired with 18 HIV+TB+IRIS- controls (table 7). TB-
IRIS patients and HIV+TB+IRIS- controls did not differ regarding baseline viral load, TB
treatment duration prior to ART, or ART duration prior to IRIS event or corresponding
control time point. Twenty-two HIV uninfected controls were included in the study, of
whom 9 were being treated for TB (HIV-TB+ controls) and 13 did not have symptoms of
TB (HIV-TB- controls). Neither HIV-TB+ nor HIV-TB- controls differed from TB-IRIS
patients or HIV+TB+IRIS- controls regarding age or sex.
Table 7. Characteristics of TB-IRIS patients and matched HIV+TB+IRIS- controls.
6.4.2 Antigen-specific IFNγ ELISpot responses in HIV-TB patients developing
TB-IRIS
We examined IFNγ responses to TB-antigens, CMV, Influenza and LPS (a well-known
TLR4 antagonist) in order to assess the antigen-specific and non-specific immunity in
TB-IRIS patients compared to HIV+TB+IRIS-, HIV-TB+ and HIV-TB- controls (figure 15). At
pre-ART, we observed no difference in IFNγ responses between TB-IRIS patients and
HIV+TB+IRIS- controls for any of the antigens tested. Both groups showed a diminished
IFNγ response to PPD, CMV, Influenza and LPS when compared to HIV-TB+ controls (p
≤ 0.027) and to LPS and influenza when compared to HIV-TB- controls (p ≤ 0.015).
IFNγ responses to the different TB-antigens were also similar between TB-IRIS
patients and HIV+TB+IRIS- controls at IRIS event (figure 15). Both groups showed signs
of higher PPD responses compared to before ART. This increase was statistically
significant for HIV+TB+IRIS- controls (p=0.031), while TB-IRIS patients showed a trend
(p=0.109). Both groups also showed signs of higher PPD responses compared HIV-TB-
Characteristics
TB-IRIS (n=18)
HIV+TB+IRIS- (n=18)
pa
Prior to ART
Male sex, n (%)
8 (44)
8 (44)
0.815b
Age (years)
34 (33-43)
40 (31-44)
0.760
CD4 (cell/mm³)
19 (11-119)
23 (8-93)
0.477
TB treatment duration prior to
ART (days)
36 (23-56) 46 (23-58) 0.663
Viral load (log copies/ml)d
5.4 (5.3-5.8)
5.53 (5.1-5.6)
0.646
During ART
Days between start of ART and
TB-IRIS/control event
14 (12-22) 16 (14-28) 0.297
Values are shown as median values with interquartile range. TB-IRIS patients were matched to HIV+TB+IRIS-
controls for baseline CD4 count, age and sex. The level of significance was set to P < 0.05 for all tests.
aWilcoxon signed-rank test. bMc Nemar test for binominal data. dn = 10.

Antigen specific responses
73
controls, yielding significant results for TB-IRIS patients only (p = 0.007). In contrast,
IFNγ responses to CMV and LPS were significantly lower in TB-IRIS patients compared
to HIV+TB+IRIS- controls (p = 0.039 and p = 0.016, respectively). TB-IRIS patients also
showed lower CMV responses compared to HIV-TB+ controls (p = 0.027), and lower
LPS responses compared to HIV-TB+ and HIV-TB- controls (p < 0.001 and p = 0.001,
respectively). In contrast to TB-IRIS patients, HIV+TB+IRIS- controls showed a
significantly recovered response to LPS compared to pre-ART (p = 0.015), which was
still lower compared to HIV-TB+ controls (p = 0.034). Responses to influenza were not
significantly different between TB-IRIS patients and HIV+TB+IRIS- controls. Responses
to influenza did not recover within the first weeks of ART for either TB-IRIS patients or
for HIV+TB+IRIS- controls compared to those in HIV-TB+(p ≤ 0.003) and HIV-TB- controls
(p ≤ 0.015) at every time point. Additional figures can be found in Appendix III.
6.4.3 Innate cytokine production upon stimulation with CMV, PPD and LPS
We next explored whether production of innate cytokines could provide an
explanation for the delayed reconstitution of CMV and LPS responses in TB-IRIS
patients. To this end, we analysed levels of the pro-inflammatory cytokines IL-12,
TNFα and IL-6 and of the anti-inflammatory cytokine IL-10 in ELISpot supernatants
after stimulation with CMV, PPD and LPS, but found no differences between TB-IRIS
patients and HIV+TB+IRIS- controls (table 8). Systemic inflammation can be marked by
increases in pro-inflammatory cytokines as well as by decreases in anti-inflammatory
cytokines and their net balance has been shown to determine the clinical outcome of
inflammation230-234. Accordingly, we explored IL-6/IL-10 and TNFα/IL-10 ratios in TB-
IRIS patients and HIV+TB+IRIS-controls (figure 16). After LPS stimulation, we observed
significantly higher IL-6/IL-10 (p = 0.025) and TNFα/IL-10 (p = 0.047) ratios in TB-IRIS
patients compared to HIV+TB+IRIS- controls (figure 16A). These differences
corresponded to significant increases of IL-6/IL-10 (p = 0.005) and TNFα/IL-10 (p =
0.004) ratios from pre-ART to IRIS event in TB-IRIS patients but not in HIV+TB+IRIS-
controls. Interestingly, IL-6/IL-10 and TNFα/IL-10 ratios correlated inversely with IFNγ
ELISpot responses to LPS in the total HIV+TB+ patient population (figure 17).
Stimulation with CMV or PPD did not yield significant differences in cytokine ratios
between patient groups (figure 16B&C) nor resulted in significant correlations
between IFNγ ELISpot responses and cytokine ratios (figure 17B&C). Additional figures
can be found in Appendix IV.

Antigen specific responses
74
Table 8. In vitro cytokine production after CMV, PPD or LPS stimulation.
Pre-ART
IRIS event
Change over time (pa)
TB-IRIS
HIV+TB+IRIS-
pa
TB-IRIS
HIV+TB+IRIS-
pa
TB-IRIS
HIV+TB+IRIS-
CMV stimulationb
IL-12p70 1.3 (0.6 - 2.4) 0.9 (0.6 - 2.4) 0.866 2.6 (1.1 - 3.1) 2.2 (1.1 - 3.1) 0.725 0.225 0.107
IL-6 207.9 (64.1 - 1633.8) 103.3 (32.2 - 523.6) 0.735 1491.5 (118.9 - 2538.4) 933.7 (295.1 - 3126.7) 0.311 0.237 0.028
TNFα 135.4 (37.9 - 338.8) 49.2 (2.1 - 145.3) 0.753 279.5 (52.4 - 354.0) 159.8 (100.7 - 380.1) 0.462 0.091 0.091
IL-10 0.6 (0.5 - 1.0) 0.2 (0.2 - 1.2) 0.237 1.4 (0.4 - 1.9) 1.6 (0.45 - 4.2) 1.000 0.753 0.043
PPD stimulationc
IL-6 (ng/ml) 16.3(9.0 - 35.9) 15.9 (8.9 - 19.1) 0.345 54.2 (11.1 - 71.1) 33.1 (27.7 - 40.6) 0.594 0.128 0.116
TNFα (ng/m l) 1.3 (0.7 - 2.0) 1.0 (0.6 - 2.5) 0.221 3.8 (1.2 - 9.1) 3.4 (1.0 - 6.2) 0.953 0.043 0.463
IL-10 16.8 (10.5 - 47.0) 17.3 (8.5 - 30.8) 0.152 16.3
(7.8 - 47.5)
23.5 (8.8 - 30.1) 0.859 0.735 0.463
LPS stimulationd
IL-12p70 4.8 (2.3-7.6) 3.9 (3.0 - 5.9) 0.453 4.7 (3.1 - 9.5) 5.4 (3.0 - 7.5) 0.277 0.315 0.900
IL-6 (ng/ml) 25.7 (10.9 - 58.6) 22.2 (10.2 - 36.6) 0.215 22.9 (12.2 - 106.8) 25.6 (13.7 - 42.6) 0.352 0.156 0.245
TNFα (ng/m l) 4.9 (1.8 - 9.3) 4.2 (2.0 - 7.6) 0.352 5.0 (2.0 - 25) 7.5 (3.0 - 13.4) 0.469 0.307 0.363
IL-10 72.6 (31.4 - 304.6) 60.0 (23.2 - 215.2) 0.408 47.1 (18.9 - 121.6) 97.8 (41.2 - 207.7) 0.215 0.334 0.683
Values are shown as median values with interquartile range in pg/ml unless stated otherwise. The level of significance was set to p < 0.05 for all tests.
a
Wilcoxon
signed-rank test. Due to limited availability of PBMCs, the number of patients differed; b7 at pre-ART and 8 during I RIS event, c 13 at pre-ART and 9 during IRIS event,
d 16 at pre-ART and 16 during IRIS event.

Antigen specific responses
75
CMV
0
500
1000
1500
2000
p = 0.039
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
p = 0.893
p = 0.156
p = 0.297
#SFC/10
6
PBMC
Influenza
0
100
200
300
400
500
500
1000
p = 1.000p = 0.195
p = 0.125
p = 0.875
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
#SFC/10
6
PBMC
PPD
0
1000
2000
3000
4000
p = 0.496
p = 0.116
p = 0.031
p = 0.109
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
#SFC/10
6
PBMC
ESAT-6
0
100
200
300
400
500
500
1000
1500
p = 1.000p = 0.695
p = 0.563
p = 0.313
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
#SFC/10
6
PBMC
CFP-10
0
100
200
300
400
500
p = 0.496
p = 0.826
p = 1.000
p = 0.310
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
#SFC/10
6
PBMC
LPS
0
100
200
300
400
p = 0.016
p = 0.426
p = 0.015
p = 0.191
IRIS
-
TB
+
TB
-
IRIS
+
HIV
-
IRIS
-
IRIS
+
pre-ART IRIS event
HIV
+
TB
+
#SFC/10
6
PBMC
A
B
C
D
E
F
Figure 15. Antigen-specific IFNγ responses in TB-IRIS patients and controls. Dots on these graphs
represent IFNγ spot-forming cells per 106 PBMCs in TB-IRIS patients (IRIS+) and HIV+TB+IRIS-controls
(IRIS-) after stimulation with CMV lysate (A), influenza antigen A (B), LPS (C), PPD (D), ESAT-6 (E) and
CFP-
10 (F). Dots connected with full lines represent matched patient pairs. Horizontal lines represent
median values for HIV-TB+ controls and HIV-TB- controls. Horizontal capped lines represent statistical
comparisons between matched patients or between time points. The level of significance was set to p
< 0.05. A Wilcoxon signed-rank test was used to calculate p values between matched HIV-patients and
time points. Due to limited avai
lability of PBMCs and pairwise exclusion, the number of patients across
antigens and time points differed. Number of patients pre-ART were; 13 (A), 10 (B), 16 (C), 14 (D), 14
(E) and 14 (F). Number of patients during IRIS event were; 8 (A), 6 (B), 16 (C), 9 (D), 9 (E) and 9 (F).

Antigen specific responses
76
100
1000
10000
IRIS
-
IRIS
+
pre-ART IRIS event
IRIS
-
IRIS
+
p = 0.109
p = 0.461
p = 1.000
p = 0.031
IL-6 / IL-10
1
10
100
1000
p = 0.813
p = 0.313
p = 0.219
p = 0.938
IRIS-IRIS+
pre-ART IRIS event
IRIS-IR IS+
TNFα / IL-10
100
1000
10000
p = 0.219
p = 0.910
p = 0.455
p = 0.562
IRIS
-
IRIS
+
pre-ART IRIS event
IRIS
-
IRIS
+
IL-6 / IL-10
10
100
1000
p = 0.219
p = 0.570
p = 0.685
p = 0.844
IRIS
-
IRIS
+
pre-ART IRIS event
IRIS
-
IRIS
+
TNFα / IL-10
10
100
1000
10000
p = 0.005
p = 0.025
p = 0.897
p = 0.426
IRIS
-
IRIS
+
pre-ART IRIS event
IRIS
-
IRIS
+
IL-6 / IL-10
10
100
1000
p = 0.004
p = 0.047
p = 0.587
p = 0.091
IRIS
-
IRIS
+
pre-ART IRIS event
IRIS
-
IRIS
+
TNFα / IL-10
A-LPS-stimulated
B-CMV-stimulated
C -PPD-stimulated
Figure 16. Pro- to anti-inflammatory ratios of innate cytokine production in TB-IRIS. Dots
represent cytokine ratios in PBMC supernatants after stimulation with LPS (A), CMV (B) or
PPD (C). Dots connected with full lines represent matched pairs of TB-IRIS patients (IRIS+)
with HIV+TB+IRIS- controls (IRIS-
). Horizontal capped lines represent comparisons between
matched patients or between time points. A, pre-ART n = 16, IRIS event n = 16; B, pre-ART n
= 7, IRIS event n = 8; C, pre-ART n=13, IRIS event n = 9. A Wilcoxon signed-rank test was used
to calculate p values between HIV patients. The level of significance was set to p < 0.05.

Antigen specific responses
77
0100200300
10
100
1000
10000
0.002
-0.520
p =
R =
# SFC/106PMBC (IFN )
IL-6 / IL-10
0100200300
10
100
1000
p =
R =
-0.510
0.003
# SFC/106PMBC (IFN )
TNF / IL-10
A
- LPS-stimulated
B
- CMV-stimulated
050010001500
100
1000
10000
p = 0.121
R = 0.404
# SFC/10
6
PMBC (IFN )
IL-6 / IL-10
050010001500
10
100
1000
p = 0.349
R = 0.251
# SFC/10
6
PMBC (IFN )
TNF / IL-10
C
- PPD-stimulated
01000200030004000
100
1000
10000
p = 0.565
R = 0.146
# SFC/106PMBC (IFN )
IL-6 / IL-10
01000200030004000
10
100
1000
p = 0.798
R = 0.065
# SFC/106PMBC (IFN )
TNF / IL-10
Figure 17. Correlation of cytokine ratios to IFNγ responses after antigenic stimulation. Graphs
represent the correlation between IFNγ responses and IL-6/IL-10 or TNFα/IL-10 ratios after stimulation
with LPS (A), CMV (B) or PPD (C). Dots represent both TB-IRIS patients and HIV+TB+IRIS- controls during
TB-IRIS or corresponding control time point. The level of significance was set to p < 0.05.

Antigen specific responses
78
6.5 Discussion
TB-IRIS could involve an amplified immune response to TB bacilli or their residual
antigens195,228. Here, we aimed to study the recovery of antigen-specific responses of
HIV-TB patients who developed TB-IRIS during ART. To that end, we compared IFNγ
responses of PBMCs to a panel of TB-associated antigens and non-TB antigens
between TB-IRIS patients and HIV+TB+IRIS- controls, matched for CD4 count, age and
sex. In addition, we explored innate cytokine responses to CMV, PPD and LPS. We
report a disturbed reconstitution of the IFNγ response to CMV and LPS during TB-IRIS,
without an excessive IFNγ response to TB-antigens. In addition, we observed a pro-
inflammatory shift in the innate cytokine balance upon LPS stimulation during TB-IRIS.
TB-IRIS patients showed lower IFNγ responses to the antigen CMV and to LPS during
TB-IRIS, compared to HIV+TB+IRIS- controls. Unlike CMV and LPS, however, we did not
observe significantly increased or decreased IFNγ responses to any of the TB-antigens,
nor to Influenza in TB-IRIS patients. These results are in line with a number of
previous studies175,178,180,181. However, our findings are not in agreement with previous
reports of elevated PPD-responses during TB-IRIS173,177-179,196 . The magnitude of IFNγ
ELISPOT responses to TB-antigens has been associated to the antigen-load, duration
of TB-treatment and the extent of immune-suppression in HIV patients235-237. All three
of these factors have been identified as risk-factors for TB-IRIS which could potentially
influence immunological measurements, consequently leading to discrepancies across
studies. In the present study, we minimize this potential bias by directly comparing
(time-)matched patients under very similar clinical conditions, possibly explaining the
discrepancy with studies that observed elevations in TB-associated responses.
Together, our results suggest that the conditions associated with TB-IRIS, such as a
high TB-antigen load and persistent inflammation176,212,238,239, could disturb the
reconstitution of the response to CMV), rather than cause an excessive TB-specific
IFNγ response. In addition, the lowered response to LPS could point towards a role of
the innate immune system.
Since we previously reported a significant rise in IL-6 plasma levels from pre-ART to
TB-IRIS event, which lead to higher IL-6 levels compared to HIV+TB+IRIS- controls225, we
next hypothesised that TB-IRIS might result from an aberrant innate immune
response. In contrast to our previous in vivo measurements, we found no significant
differences in the in vitro production of pro- and anti-inflammatory cytokines
between matched TB-IRIS patients and HIV+TB+IRIS- controls after exposure to CMV,
PPD and LPS. In addition to absolute levels, however, the balance between pro-
inflammatory and anti-inflammatory cytokine levels has been shown to drive systemic

Antigen specific responses
79
inflammation230-234. Accordingly, we found that TB-IRIS was associated with a pro-
inflammatory shift in the IL-6/IL-10 and TNFα/IL-10 ratios after stimulation with LPS,
but not CMV or PPD. An increase in the IL-6/IL-10 ratio, caused by a decrease in IL-10,
has previously been associated to the severity of systemic inflammatory response
syndrome in patients with sepsis231. Of note, the IL-10 levels upon LPS stimulation in
the current study were also somewhat lower during TB-IRIS. Although this difference
did not reach statistical significance, it could have shifted the cytokine balance
towards the pro-inflammatory side. In line with our findings, TB-IRIS patients from our
cohort have previously been shown to have a pro-inflammatory monocyte-gene
expression profile that is also perturbed in pattern recognition receptor pathways197.
Another study previously reported elevated TNFα production during IRIS upon TLR2
stimulation with lipomannan, without an equivalent rise in IL-10196. In the present
study, we report a similar cytokine imbalance in the TLR4 branch of innate cytokine
production. One could therefore argue that a disturbed equilibrium between pro- and
anti-inflammatory cytokine-production upon TLR stimulation is implicated in the high
degree of inflammation seen in TB-IRIS. This preferential involvement of TLRs in TB-
IRIS could also explain why no cytokine shifts were observed after CMV- or PPD-
stimulation, since these antigens preferentially induce an adaptive response via the
major histocompatibility complex class II/T cell receptor pathway.
Intriguingly, the unbalanced cytokine ratios were inversely correlated to the LPS-
induced IFNγ responses. This finding is somewhat contradictory, given the fact that IL-
6, TNFα and IFNγ are all pro-inflammatory cytokines. However, IL-6 and TNFα are
directly produced by monocytes after LPS stimulation, while IFNγ is not. Rather, LPS-
induced IFNγ originates from T cells and NK cells in response to monocyte derived
cytokines240,241. We hypothesise that the cytokine ratio shifts result from aberrant
monocyte behaviour in these patients, given the association of monocyte dysfunction
with chronic HIV infection242,243. In fact, aberrant monocyte behaviour has previously
been suggested to play a role in TB-IRIS193 and is in line with our hypothesis on the
role of TLRs in TB-IRIS. Since the balance between monocyte-derived cytokines seems
to be disturbed upon TLR stimulation, we speculate that this negatively affected the
subsequent induction of IFNγ.
One limitation of our study was that patients and controls were not tested for CMV
and influenza infection status, potentially complicating the interpretation of the CMV
and influenza ELISPOT responses that we measured. This may not have been a major
problem for CMV, which reaches a high seroprevalence in sub Saharan Africa244,245.
While influenza is clearly present in sub Saharan Africa246,247, limited availability of

Antigen specific responses
80
epidemiological data make it difficult to assess the expected recall response of our
patients to this antigen.
Taken together, our data provide no evidence of an excessive IFNγ response to TB-
associated antigens or other common antigens during TB-IRIS. In fact, TB-IRIS was
associated with a disturbed reconstitution of the IFNγ responses to CMV and LPS. For
LPS, this was linked with a pro-inflammatory shift in the innate cytokine balance.
Together, our data provide evidence for a prominent role for innate immune
inflammation and possibly monocyte dysfunction in TB-IRIS pathogenesis. Resolving
the immune responses leading to TB-IRIS pathogenesis could provide novel targets for
treatment and prevention.

Plasma markers of TB-IRIS
81
Chapter VII: LPS-binding protein and IL-6 mark paradoxical
tuberculosis immune reconstitution inflammatory
syndrome in HIV patients
(Published in November 2013; PLoS One 8, with minor adjustments).
7.1 Abstract
Background: Tuberculosis-associated immune reconstitution inflammatory syndrome
(TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients
initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS
is becoming increasingly apparent, however the potential involvement in TB-IRIS of a
leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not
been investigated before. Here we aimed to investigate the innate nature of the
cytokine response in TB-IRIS and to identify novel potential biomarkers.
Methods: From a large prospective cohort of HIV-TB co-infected patients receiving TB
treatment, we compared 40 patients who developed TB-IRIS during the first month of
ART with 40 patients matched for age, sex and baseline CD4 count who did not. We
analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14,
endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and
anti-inflammatory cytokines before and during ART.
Results: We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and
LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-
1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013)
compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in
multivariate models containing significant cytokines from pre-ART (p = 0.039) and
during TB-IRIS (p = 0.034).
Conclusion: We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP
levels during IRIS event as potential biomarkers in TB-IRIS. Our results show no
evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6
holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS.
Future clinical studies should investigate the importance and clinical relevance of
these markers for the diagnosis and treatment of TB-IRIS.

Plasma markers of TB-IRIS
82
7.2 Introduction
Over one third of the 33 million people living with HIV are co-infected with
tuberculosis (TB)248. While the rollout of antiretroviral therapy (ART) has increasingly
contributed to halt HIV disease progression and to reduce the risk of opportunistic
infections, complications still occur. During successful ART, up to 25% of HIV-TB co-
infected patients paradoxically develop worsening symptoms of TB, despite effective
initial response to concurrent TB treatment140. This complication has been named
paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome
(TB-IRIS)152 and may require hospitalization or additional immune suppressive
therapy203. TB-IRIS typically develops within the first 2 months after starting ART (early-
onset TB-IRIS), with the majority of cases occurring before 1 month204. As TB-IRIS is
often difficult to distinguish from other complications, there is an urgent need for
reliable laboratory markers to predict and identify this syndrome202.
Although several risk factors such as low CD4 count, high TB-antigen burden and short
interval between initiation of TB treatment and ART are well established163,205, the
actual pathogenesis of TB-IRIS remains to be elucidated. TB-IRIS is characterized by
tissue-destructive inflammation when CD4 T cells are being replenished195 and might
thus involve an amplified immune reaction to TB bacilli or their residual antigens228.
Many studies have therefore focused on the potential role of T cells and a polarized T-
helper 1 cell response in TB-IRIS170,176,183. In contrast, recent opinions have arisen that
implicate cells of the innate immune system in TB-IRIS pathogenesis193 or indeed
disturbances in the interplay between the innate and adaptive immune system19 5
which could lead to inflammatory conditions upon ART initiation.
Inflammation in TB-IRIS coincides with elevations in a plethora of cytokines, referred
to as the cytokine storm since it was first observed183. Most studies of IRIS to date
have focused on pro- and anti-inflammatory cytokines during the IRIS event170,176,184-
186. Interestingly, several studies noted a role for cytokines of myeloid or dual
myeloid/lymphoid origin, like IL-6 and TNFα, in the ongoing inflammation184. Other
plasma markers were less consistently reported across studies, possibly as a result of
variations in size and origin of the study populations, comparisons that are
unmatched for baseline CD4 count, or inclusion of IRIS cases related to other, non-TB
pathogens. It is therefore still unclear which cells and cytokines lie at the basis of TB-
IRIS and its inflammation.
The immune reaction and inflammation in response to TB typically involves
stimulation of toll-like receptors (TLR) by antigens such as lipoarabinomannan (LAM)
grouped under the name pathogen associated molecular patterns (PAMPs).

Plasma markers of TB-IRIS
83
Interestingly, a higher level of pre-ART urinary LAM has been reported in TB-IRIS
patients163. This finding, along with reports of elevated C-reactive protein (CRP) prior
to ART192,249 and during IRIS event175,198 , supports a possible role for the TLR-pathway
in TB-IRIS. However, the potential involvement of gut derived bacterial PAMPs such as
LPS and proteins that regulate TLR stimulation by binding PAMPs remains largely
unexplored. LPS enters the blood through a phenomenon known as a “leaky gut”,
characterized by increased intestinal permeability, which is associated with immune
activation in HIV disease250. LPS-binding protein (LBP) and soluble CD14 (sCD14) are
able to regulate this antigenic stimulation by binding LPS and other
PAMPs22,23,106,107,251 and could therefore influence TB-IRIS inflammation.
In this study we aimed to find biomarkers for early-onset TB-IRIS in one of the largest
TB-IRIS cohorts described to date. We hypothesized that a more pronounced
presence of PAMPs from a leaky gut such as LPS could contribute to the cytokine
storm in TB-IRIS in a non-TB related manner and that this effect is regulated by
plasma proteins that bind PAMPs, which have not been investigated in TB-IRIS before.
We report lower plasma levels of LBP before ART initiation but higher levels of LBP
during IRIS event. Similarly, the innate cytokine profile showed lower levels before
ART and higher levels during TB-IRIS, with IL-6 holding a dominant role. Our results
support the theory that dysfunctions in the innate immune system make a large
contribution to TB-IRIS pathogenesis.
7.3 Materials and methods
7.3.1 Study population
Patients from a prospective observational study at Mulago Hospital, Kampala,
Uganda, were studied as described previously153,163,207. We recruited HIV-TB co-
infected adults who were receiving treatment for active TB infection for no more than
2 months and were eligible for ART. After enrollment, all patients were started on a
non-nucleoside reverse transcriptase inhibitor-based ART according to Ugandan
national guidelines. Including adherence preparation, which took 2 to 3 visits to the
clinic, the median interval from starting TB treatment to starting ART for all patients
was 6 weeks. Patients were followed up for a minimum of 3 months to monitor
paradoxical TB-IRIS development. Patients who did not develop IRIS-related
symptoms for a minimum of 3 months served as HIV+TB+IRIS- controls. Plasma
samples were collected before initiation of ART (baseline) and at 2 weeks, 4 weeks, 3
months, and 6 months after starting ART. Patients who developed TB-IRIS had an
extra blood sample taken when diagnosed with inflammatory symptoms during
ongoing IRIS event.

Plasma markers of TB-IRIS
84
7.3.2 Patient selection and matching
The large majority of TB-IRIS patients from our cohort developed an IRIS event within
the first month of ART. To limit heterogeneity amongst TB-IRIS patients, we excluded
all patients who developed TB-IRIS later than 1 month. This homogeneous selection of
patients reduces potential bias due to different cytokine kinetics and
immunopathology between early- and late-onset TB-IRIS. TB-IRIS patients included in
this study developed IRIS between 4 and 28 days on ART and were paired with
controls at their closest corresponding time points. We thus matched IRIS events,
sampled during ongoing inflammation, from 4 to 20 days on ART with HIV+TB+IRIS-
control samples taken after 2 weeks on ART and IRIS events from 21 to 28 days on
ART with HIV+TB+IRIS- control samples taken after 1 month on ART. TB-IRIS patients
were also matched 1 by 1 with HIV+TB+IRIS- controls for sex, baseline CD4 count and
age. After matching for sex, we secondly prioritized matching patients with +/- 15 CD4
cells/mm³. Thirdly, patients were matched for age with ≤ 10 years difference.
7.3.3 Definitions
Mycobacterium tuberculosis infection was diagnosed according to the TB/HIV WHO
guidelines208. Investigations to confirm TB infection included: clinical examination,
chest X-rays and abdominal ultrasounds, sputum smear microscopy for acid-fast
bacilli and mycobacterial culture of sputum, aspirate or effusion if available. TB-IRIS
cases were classified by a committee of two co-authors (RC and WW) after reviewing
all suspected TB-IRIS cases evaluated by the study physicians according to the
International Network for the Study of HIV-associated IRIS (INSHI) clinical case-
definition152. TB-IRIS was diagnosed and patients were subsequently sampled when
they presented with at least 1 major criterion, such as enlarged lymph nodes, or 2
minor criteria, such as fever and cough. Patients who developed TB-unrelated types
of IRIS were excluded from the analysis.
7.3.4 Plasma analysis
Venous blood was drawn into EDTA tubes and plasma was separated from cells by
centrifugation and stored at -80°C. Plasma concentrations of LBP, sCD14 and markers
of a leaky gut, i.e. endotoxin-core antibody IgG (EndoCab) and intestinal fatty-acid-
binding protein (I-FABP), were determined by ELISA (HyCult biotechnology BV, Uden,
The Netherlands). Dilutions were 2000x (LBP), 100x (sCD14), 100x (EndoCab) and 2x
(I-FABP) for each protein respectively. When enough plasma was available,
lipopolysaccharide (LPS) levels were determined by using the kinetic limulus
amebocyte lysate assay (Kinetic-QCL, Lonza) according to the manufacturer’s
instructions (see Appendix II).

Plasma markers of TB-IRIS
85
Plasma concentrations of 18 cytokines and chemokines were determined in duplicate
using Bio-Plex™ human cytokine assay kits (Bio-Rad Laboratories NV-SA, Nazareth,
Belgium) according to the manufacturer’s instructions. A range of cytokines from
myeloid or lymphoid origin that could play a role in TB-IRIS were selected. These
included IL-1RA, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-17, interferon gamma
(IFNγ),granulocyte-colony stimulating factor (G-CSF), granulocyte-monocyte colony
stimulating factor (GM-CSF), Eotaxin-1 (CCL11), macrophage inflammatory protein 1β
(CCL4), RANTES (CCL5), tumor necrosis factor alpha (TNFα) and interferon gamma-
induced protein 10 (CXCL10). The Bio-Plex™ array reader and Manager 5.0 software
were used to analyze cytokine concentrations using a weighted five-parameter
logistic curve-fitting method. Results below limit of detection (LOD) were given the
value LOD/2. The respective LOD (in pg/ml)for each cytokine was: 6.7 (IL-1RA); 2.0 (IL-
2); 0.4 (IL-4); 2.6 (IL-6); 2.9 (IL-7); 2.6 (IL-8); 2.8 (IL-9); 2.4 (IL-10); 4.3 (IL-12p70); 2.4
(IL-17); 9.0 (IFNγ); 3.4 (G-CSF); 20.4 (GM-CSF); 40.2 (CCL11); 1.7 (CCL4); 10.5 (CCL5);
8.9 (TNFα) and 15.0 (CXCL10).
7.3.5 Ethical considerations
The study was approved by the institutional review board of the Institute of Tropical
Medicine of Antwerp, the Ethics Committees of the Faculties of Medicine of the
University of Antwerp and the University of Makerere, the Research Committee of the
Mulago Hospital and the Uganda National Council of Science and Technology. Written
informed consent was obtained from all study participants.
7.3.6 Statistical analysis
Statistics were performed using SPSS software (version 17.0), R software (version
2.15.0) or GraphPad Prism (version 5) with significance level set at p <0.05.
Differences between paired patients and changes in concentration over time within
each patient were analyzed using the Wilcoxon signed-rank test for paired data.
Differences between HIV-infected and HIV-uninfected patients were analysed using
the Mann-Whitney U test. Variables with >70% undetectable measurements were
transformed into dichotomous variables and analyzed with a McNemar test for
nominal data. No correction for multiple testing was applied as this is a hypothesis
driven study with the aim to generate new biomarkers of TB-IRIS for further
research210,211. Conditional multivariate logistic regression was used to determine the
strongest cytokine predictors. Cytokines were included in multivariate models if p
values from Wilcoxon signed-rank or McNemar tests were <0.05 and stepwise
elimination were applied to determine significant independent effects.

Plasma markers of TB-IRIS
86
7.4 Results
7.4.1 Study population
From a prospective study population of 254 HIV-TB co-infected patients who were
started on ART at Mulago Hospital in Kampala in Uganda, 53 patients developed
paradoxical TB-IRIS. Thirteen TB-IRIS patients were excluded, including 6 who
developed IRIS later than 1 month (range: 42-84 days) on ART, 5 who had inadequate
samples and 2 who presented with other co-infections in addition to TB. The
remaining 40 TB-IRIS patients were paired with 40 HIV+TB+IRIS- controls (who did not
develop TB-IRIS), matched for sex, baseline CD4 count and age (table 9). A median
difference of 1 day (interquartile range (IQR): 0 - 4) was observed for time on ART
prior to sampling of IRIS event or corresponding time point between patients and
HIV+TB+IRIS- controls, which was statistically significant. TB-IRIS patients and
HIV+TB+IRIS- controls did not significantly differ with regards to baseline levels of CRP
or interval between TB- and HIV-treatment initiation. All patients with available HIV
viral loads showed a significant decrease in HIV viral load upon ART and no significant
differences at either baseline or IRIS event were observed between the two groups.
Thirty-seven HIV uninfected controls were included in the study, of whom 18 were
being treated for TB (HIV-TB+ controls) and 19 did not have symptoms of TB (HIV-TB-
controls). Neither HIV-TB+ nor HIV-TB- controls differed from TB-IRIS patients or
HIV+TB+IRIS- controls regarding sex (data not shown). HIV-TB+ controls were younger
compared to TB-IRIS patients and HIV+TB+IRIS- controls (p < 0.001 for both).
Table 9. Characteristics of TB-IRIS patients and matched HIV+TB+IRIS- controls.
Variables
TB-IRIS (n=40)
HIV+TB+IRIS- (n=40)
pa
Characteristics Baseline
Sex male n (%) 23 (58) 23 (58) 1.000
Age (years)
35 (31-42)
38 (32-42)
0.320
CD4 (cell/mm³)
21 (10-54)
24 (15-54)
0.549
Days between TB t reatm ent and ARTb
31 (24-58)
46 (30-62)
0.291
CRP (mg/ L) 10.9 (5.62-28.80) 21.14 (5.77-27.39) 0.614
Temperature (°C) 36.3 (36.0 – 36.7) 36.4 (35.8 – 36.6) 0.742
Viral Load (Log copies/ml)
5.6 (5.3-5.8)
5.5 (5.2-5.9)
0.719
Characteristics TB-IRIS event
Days since start of ART
14 (12-14)
14 (14-18)
<0.001
Viral Load (Log copies/ml)
c
3.4 (3.1-3.4) 3.4 (3.3-3.8) 0.158
Temperature (°C) 37.8 (37.1-38.2) 36.3 (35.8-36.5) <0.001
Weight gain sinc e start ART (kg)
0.0 (-2.5-+1.0)
0.5 (-1.0-+2.0)
0.194
Values are shown as median values with interquartile range. Due to m issing data and pairwise exclusion the
number of patients may vary between 30 and 40 unless stated otherwise. Level of significance was set to p <
0.05. aWilcoxon signed-rank test. bNumber of days between the start of TB therapy and initiation of ART.
cn = 14.

Plasma markers of TB-IRIS
87
7.4.2 Markers of a leaky gut prior to and during ART
Increased intestinal permeability could lead to a more pronounced presence of
PAMPs in the bloodstream. We assessed presence of LPS and damage to the intestinal
epithelium by analyzing plasma levels of EndoCab and I-FABP in TB-IRIS patients and
HIV+TB+IRIS- controls at baseline, IRIS event or corresponding time point and at 3 and
6 months on ART (figure 18A & B). Additional comparisons were performed with HIV-
TB+ and HIV-TB- controls. When enough plasma was available, we analyzed LPS levels
in TB-IRIS patients and HIV+TB+IRIS- controls at baseline and IRIS event or
corresponding time point (figure 18C). We observed no differences pre-ART between
study groups. We did however observe a significant decline in I-FABP levels in TB-IRIS
patients between pre-ART and IRIS event (p = 0.019) but not in HIV+TB+IRIS- controls.
At IRIS event or corresponding time point, I-FABP levels were significantly lower in TB-
IRIS patients compared to HIV+TB+IRIS- controls (p = 0.013), and they remained so at
month 3 (p = 0.049) and month 6 (p = 0.002) on treatment. No differences between
groups were observed for LPS nor for its antibody EndoCab. A significant increase in
Endocab levels could be observed over time during follow-up in both groups. For TB-
IRIS patients, this increase occurred between IRIS event and 3 months of ART (p =
0.010). For HIV+TB+IRIS- controls, this increase occured between 3 and 6 months of
ART (p = 0.044). Comparisons with HIV-TB+ and HIV-TB- controls also did not yield
significant results. Comparisons with HIV-TB+ and HIV-TB- controls also did not yield
significant results.
7.4.3 PAMP binding plasma proteins prior to and during ART
Proteins that bind PAMPs have the ability to interfere with inflammatory processes.
To evaluate the possible importance of these proteins, we evaluated plasma samples
for LBP and sCD14 concentrations at baseline, IRIS event or corresponding time point
and at 3 and 6 months on treatment (figure 19). Additional comparisons were
performed with HIV-TB+ and HIV-TB- controls. We found significantly lower LBP values
in TB-IRIS patients than in HIV+TB+IRIS- controls at baseline (p = 0.016, figure 19A). In
contrast, TB-IRIS patients showed significantly higher levels of LBP during IRIS event
compared to HIV+TB+IRIS- controls (p = 0.010). In both groups, LBP levels increased
after initiating ART, but this was much more pronounced for TB-IRIS patients. Over
the next 6 months during ART, levels of LBP declined significantly in both groups and
no longer showed significant differences between these two groups. No significant
differences in sCD14 levels were apparent between TB-IRIS and HIV+TB+IRIS- controls
at any time point (figure 19B). Nevertheless, a significant increase of sCD14 was seen
in TB-IRIS patients after initiation of ART compared to baseline (p = 0.037). Compared
to HIV-TB+ controls, TB-IRIS patients showed significantly lower LBP levels prior to ART
(p = 0.009) and after 6 months of ART (p = 0.002), while HIV+TB+IRIS- controls had

Plasma markers of TB-IRIS
88
lower levels after 3 (p = 0.015) and 6 months of treatment (p = 0.002). Higher levels of
sCD14 could also be observed during IRIS event, compared to HIV-TB+ controls (p =
0.005). Compared to HIV-TB- controls, TB-IRIS patients showed significantly higher LBP
levels during IRIS event (p < 0.001), while HIV+TB+IRIS- controls had higher levels prior
to ART ( p = 0.003) and during IRIS event (p = 0.006). Both TB-IRIS patients and
HIV+TB+IRIS- controls had significantly higher levels of sCD14 compared to HIV-TB-
controls at every time point (p < 0.001 for all).
7.4.4 Cytokine levels prior to and during ART
The inflammation in TB-IRIS coincides with the production of a plethora of cytokines,
which could result from TLR stimulation. To elucidate which cytokines take part in the
development of TB-IRIS in our study, we analyzed plasma levels of 18 different
cytokines. We compared values at baseline and during IRIS event or corresponding
control time points between TB-IRIS patients and HIV+TB+IRIS- controls, as well as
changes between both time points for each patient group (table 10). Additional
comparisons were performed with HIV-TB- controls. At baseline, we observed
significantly lower cytokine levels in TB-IRIS patients than in HIV+TB+IRIS- controls for
IL-6 (p = 0.041) and G-CSF (p = 0.036). During IRIS event, we observed significantly
higher cytokine levels in TB-IRIS patients for IL-1RA, IL-4, IL-6, IL-7, IL-8 and G-CSF (p ≤
0.032). These cytokines were also significantly increased at IRIS event compared to
pre-ART in TB-IRIS patients. Additional increases over time were observed for IFNγ,
CCL4 and CCL5 in TB-IRIS patients, while CCL4 and CXCL10 decreased over time in
HIV+TB+IRIS- controls. Delta values (changes over time) of IL-6, IL-7, IL-8, G-CSF, CCL4,
CXCL10 and LBP all showed significant differences between TB-IRIS patients and
HIV+TB+IRIS- controls (p ≤ 0.028). IL-9 and IL-17 were below detection limit in >70% of
our study subjects. No significant differences in detectability were observed between
patient groups using dichotomous variables for both of these cytokines. IL-2, GM-CSF
and TNFα were undetectable in >90% of our samples and were excluded from the
analysis. Prior to ART, both TB-IRIS patients and HIV+TB+IRIS- controls had significantly
higher plasma levels of IL-1RA, IL-4 and IL-8 but significantly lower levels of G-CSF and
CXCL10, compared to HIV-TB- controls (p ≤ 0.032, table 11). Compared to HIV-TB-
controls, TB-IRIS patients had lower pre-ART levels of IL-7 and CCL4, while HIV+TB+IRIS-
controls had higher pre-ART levels of IL-6 and IL-10 (p ≤ 0.043). During IRIS event,
both TB-IRIS patients and HIV+TB+IRIS- controls had significantly higher plasma levels
of IL-4, IL-6 and IL-8 but significantly lower levels of G-CSF and CXCL10, compared to
HIV-TB- controls (p ≤ 0.032). Compared to HIV-TB- controls, TB-IRIS patients had higher
levels of IL-1RA and lower levels of IL-7 when TB-IRIS occurred (p ≤ 0.047).

Plasma markers of TB-IRIS
89
Figure 18. Markers of a leaky gut in TB-IRIS patients
and controls during follow-up.
Horizontal lines represent median plasma levels for
each patient group at each time point. A Wilcoxon
signed-
rank test was used to calculate p values. The
level of significance was set to p < 0.05. Dotted lin es
indicate significant changes over time per patient-
group and full lines indicate significant differences
between matched patients. The number of HIV-TB- and
HIV-TB+ controls is 19 and 18 respectively. (A) EndoCab.
Number of patients per group during consecutive time
points is 35, 35, 30 and 29 respectively. (B) I-FABP.
Number of patients per group during consecutive time
points is 38, 38, 32 and 31 respectively. (C) LPS.
Number of patients per group during consecutive time
points is 11 and 13 respectively. GMU, IgG median
units; EU, endotoxin units.
0
100
200
300
300
600
900
p = 0.010 p = 0.044
Pre-ART IRIS event Month 3 Month 6
TB
+
TB
-
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
HIV
+
TB
+
HIV
-
Endocab (GMU/ml)
0
2
4
6
p = 0.049 p = 0.002
p = 0.019
Pre-ART IRIS event Month 3 Month 6
TB+
TB-IRIS-
IRIS+IRIS-
IRIS+IRIS -
IRIS+IRIS -
IRIS+
HIV+TB+
HIV -
p = 0.013
I-FABP(ng/ml)
IRIS
-
IRIS
+
0
10
20
30
40
50
Pre-ART IRIS event
IRIS
-
IRIS
+
HIV
+
TB
+
LPS (EU/ml)
A
B
C

Plasma markers of TB-IRIS
90
0
20
40
60
80
100
p = 0.010
p = 0.016
p <0.001 p <0.001 p <0.001
p = 0.041 p <0.001 p = 0.001
Pre-ART IRIS event Month 3 Month 6
TB
+
TB
-
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
HIV
+
TB
+
HIV
-
LBP (µg/ml)
0
2
4
6
8
10
p = 0.037 p <0.001 p <0.001
p <0.001 p = 0.002
Pre-ART IRIS event Month 3 Month 6
TB
+
TB
-
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
IRIS
-
IRIS
+
HIV
+
TB
+
HIV
-
sCD14 (µg/ml)
A
B
Figure 19. LBP and sCD14 plasma levels in TB-IRIS patients and controls during. Horizontal lines
represent median levels of LBP (A) and sCD14 (B) for each patient group at each time point. A
Wilcoxon signed-
rank test was used to calculate p values. The level of significance was set to p < 0.05.
Dotted lines indicate significant changes over time per patient-group and full lines indicate significant
differences between matched patients. The number of HIV-TB- and HIV-TB+ controls is 19 and 18
respectively. The number of both TB-IRIS patients and HIV+TB+IRIS- controls during consecutive time
points is 38, 38, 32 and 31 respectively. P values calculated for HIV-negative controls are not shown.

Plasma markers of TB-IRIS
91
Table 10. Cytokines and other plasma markers in TB-IRIS patients and controls.
Pre-ART
IRIS event
Change over time (pb)
∆ change
TB-IRIS
(n=34
a
)
HIV+TB+IRIS-
(n=34
a
)
pb
TB-IRIS
(n=31
a
)
HIV+TB+IRIS-
(n=31
a
)
pb
TB-IRIS
(n=30
a
)
HIV+TB+IRIS-
(n=30
a
)
pb
Cytokines (pg/ml)
IL-1RA
56.7 (33.5-87.7)
54.1 (33.5-79.9)
0.831
89.3 (54.1-123.0)
46.6 (25.7-97.7)
0.009
0.021
0.558
0.054
IL-4
0.8 (0.2c-1.3)
1.1 (0.4-1.6)
0.217
1.2 (0.9-1.6)
1.0 (0.4-1.3)
0.032
0.042
0.882
0.094
IL-6
5.0 (2.9-8.3)
6.5 (3.7-13.0)
0.041
20.3 (10.4-43.7)
6.7 (3.0-11.6)
<0.001
<0.001
0.861
<0.001
IL-7 4.4 (3.5-5.1) 4.5 (3.5-5.3) 0.986 5.9 (3.8-7.4) 3.7 (3.1-5.8) 0.017 0.008 0.967 0.021
IL-8
7.7 (6.5-13.2)
9.0 (6.3-13.2)
0.614
10.4 (7-23.8)
7.3 (5.5-13.6)
0.008
0.012
0.171
0.001
IL-9
1.4 (1.4-1.4)c
1.4 (1.4-1.4)c
0.347
1.4 (1.4-1.4)c
1.4 (1.4-1.4)c
0.735
0.866
0.208
0.249
IL-10 2.4 (1.2
c
-6.0) 2.2 (1.2
c
-4.0) 0.302 4.5 (2.0-7.5) 1.2
c
(1.2
c
-6.5) 0.090 0.100 0.469 0.405
IL-12
2.2c (2.2c-4.4)
3.1 (2.2c-7.4)
0.355
4.4 (2.2c-7.9)
2.2c (2.2c-6.0)
0.236
0.126
0.614
0.501
IL-17
1.2 (1.2-1.2)c
1.2 (1.2-1.2)c
0.937
1.2c (1.2c-16.7)
1.2c (1.2c-24.5)
0.647
0.480
0.158
0.852
CCL11 79.7 (44.4-121.0) 102.9 (55.6-130.4) 0.122 72.5 (46.9-96.9) 75.1 (45.1-126.8) 0.614 0.770 0.050 0.465
G-CSF
29.7 (21.6-52.5)
40.7 (25.4-73.9)
0.036
45.6 (30.1-93.0)
44.3 (22.7-63.4)
0.032
<0.001
0.750
0.003
IFNγ 66.5 (29.9-103.0) 78.6 (46.9-132) 0.136 93.9 (58.8-129.3) 62.8 (39.1-138.4) 0.075
0.028
0.946 0.054
CCL4 47.9 (32.2-67.3) 66.6 (34.8-93) 0.092 71.8 (55.6-102.3) 65.4 (37.7-109.6) 0.456 <0.001 0.001 0.028
CCL5 (ng/ml)
4.4 (3.1-5.0)
4.3 (3.2-5.9)
0.437
5.5 (4.5-8.3)
5.8 (3.6-7.3)
0.170
0.007
0.271
0.178
CXCL10 (ng/ml) 8.4 (6.8-12.9) 10.3 (6.0-16.2) 0.726 11.4 (5.9-16.9) 5.5 (3.2-15.0) 0.057 0.125 0.009 0.007
Other plasma marke rs
LBP d (µg/ml)
29.7 (15.42-37.1)
42.3 (25.1-53.5)
0.016
75.5 (45.0-≥100.0)
42.7 (27.13-70.4)
0.010
<0.001
0.041
<0.001
sCD14
d
(µg/ml)
3.4 (2.5-3.9) 3.3 (2.5-4.3) 0.342 3.6 (3.0-4.3) 3.5 (2.6-4.3) 0.879 0.037 0.940 0.192
I-FABP d (ng/ml)
1.0 (0.5-1.5)
0.9 (0.5-1.7)
0.741
0.6 (0.3-1.0)
1.1 (0.7-1.9)
0.013
0.019
0.355
0.113
EndoCa be (GMU/ml)
28.4 (20.7-59.0)
34.3 (18.2-61.7)
0.600
32.6 (17.6-59.8)
37.8 (15.6-65.2)
0.909
0.091
0.957
0.343
LPS
f
(EU/ml) 16.2 (11.5-19.5) 17.7 (15.3-20.4) 0.424 13.3 (11.4-14.2) 12.7 (11.9-16.2) 0.814 0.131 0.148 0.477
Cytokine levels are shown as median values with inter-quartile range. Values were compared between matched patient pairs per time point and between time points per
study group. Level of significance was set to p < 0.05. Differences between patients for IL-9 and IL-17 were analyzed with a McNemar test. IL-2, GM-CSF and TNFα were
excluded from analysis. aSome patients were not included due to insufficient sample volume or pairwise exclusion. bWilcoxon signed-rank test unless stated otherwise.
cValues were below detection limit and represented as LOD/2. dBaseline n= 38, IRIS event n = 38. eBaseline n= 35, IRIS event n = 35. fBaseline n= 11, IRIS event n = 13.
P values in the final column represent the difference between delta (∆) values over time of TB-IRIS cases HIV+TB+IRIS- controls.

Plasma markers of TB-IRIS
92
Table 11. Cytokines in HIV-TB- controls compared to TB-IRIS patients and controls
Pre-ART
IRIS event
HIV-TB-
(n=10a)
TB-IRIS
(n=34a)
p
b
HIV+TB+IRIS-
(n=34a)
p
b
TB-IRIS
(n=31a)
p
b
HIV+TB+IRIS-
(n=31a)
p
b
Cytokines (pg/ml)
IL-1RA 25. (13.7-60.3) 56.7 (33.5-87.7) 0.032 54.1 (33.5-79.9) 0.001 89.3 (54.1-123.0) 0.032 46.6 (25.7-97.7) 0.148
IL-4
0.2 (0.2-0.2)c
0.8 (0.2c-1.3)
0.022
1.1 (0.4-1.6)
0.002
1.2 (0.9-1.6)
0.004
1.0 (0.4-1.3)
0.003
IL-6 2.8 (2.4-3.6) 5.0 (2.9-8.3) 0.060 6.5 (3.7-13.0) <0.001 20.3 (10.4-43.7) 0.003 6.7 (3.0-11.6) 0.027
IL-7 6.0 (4.3-8.1) 4.4 (3.5-5.1) 0.043 4.5 (3.5-5.3) 0.654 5.9 (3.8-7.4) 0.046 3.7 (3.1-5.8) 0.061
IL-8
3.8 (3.2-4.4)
7.7 (6.5-13.2)
0.003
9.0 (6.3-13.2)
0.001
10.4 (7-23.8)
0.001
7.3 (5.5-13.6)
0.003
IL-9
1.4 (1.4-1.4)c
1.4 (1.4-1.4)c
0.678
1.4 (1.4-1.4)c
0.509
1.4 (1.4-1.4)c
0.319
1.4 (1.4-1.4)c
0.577
IL-10
1.4 (1.2c-2.4)
2.4 (1.2c-6.0)
0.212
2.2 (1.2c-4.0)
0.012
4.5 (2.0-7.5)
0.217
1.2c (1.2c-6.5)
0.644
IL-12 3.3 (2.1-6.8) 2.2
c
(2.2
c
-4.4) 0.150 3.1 (2.2
c
-7.4) 0.778 4.4 (2.2
c
-7.9) 0.814 2.2
c
(2.2
c
-6.0) 0.518
IL-17
1.2c (1.2c-22.0)
1.2 (1.2-1.2)c
0.115
1.2 (1.2-1.2)c
0.609
1.2c (1.2c-16.7)
0.233
1.2c (1.2c-24.5)
0.756
CCL11
56.6 (35.7-118.0)
79.7 (44.4-121.0)
0.623
102.9 (55.6-130.4)
0.614
72.5 (46.9-96.9)
0.172
75.1 (45.1-126.8)
0.722
G-CSF
111.6 (76.8-142.9)
29.7 (21.6-52.5)
<0.001
40.7 (25.4-73.9)
0.010
45.6 (30.1-93.0)
0.002
44.3 (22.7-63.4)
0.001
IFNγ
1.1c (1.1c-173.4)
66.5 (29.9-103.0)
0.147
78.6 (46.9-132)
0.087
93.9 (58.8-129.3)
0.106
62.8 (39.1-138.4)
0.092
CCL4
109.2 (42.2-140.0)
47.9 (32.2-67.3)
0.038
66.6 (34.8-93)
0.275
71.8 (55.6-102.3)
0.181
65.4 (37.7-109.6)
0.288
CCL5 (ng/ml)
4.8 (3.7-10.0)
4.4 (3.1-5.0)
0.263
4.3 (3.2-5.9)
0.747
5.5 (4.5-8.3)
0.275
5.8 (3.6-7.3)
0.929
CXCL10 (ng/ml)
0.8 (0.7-1.0)
8.4 (6.8-12.9)
<0.001
10.3 (6.0-16.2)
<0.001
11.4 (5.9-16.9)
<0.001
5.5 (3.2-15.0)
<0.001
Cytokine levels are shown as median values with inter-quartile range. Values from HIV-TB- controls were compared to those of TB-IRIS patients and HIV+TB+IRIS- controls
at baseline and during IRIS event. Level of significance was set to p < 0.05. Differences between patients for IL-9 and IL-17 were analyzed with a Chi-square test. aSome
patients were not included due to insufficient sample volume or pairwise exclusion. bMann-Whitney U test unless stated otherwise. cValues were below detection limit
and represented as LOD/2. No cytokines were determined in HIV-TB+ controls, due to sample limitations.

Plasma markers of TB-IRIS
93
7.4.5 Central role of IL-6 during TB-IRIS
We next attempted to identify which of the cytokines that we established to be
associated with TB-IRIS could lie at the basis of this cytokine storm. We thus explored
which cytokines were interdependent or had independent effects. Plasma markers
that showed significant (p < 0.05) univariate effects either pre-ART (IL-6 and GCSF) or
during IRIS event (IL-1RA, IL-4, IL-6, IL-7, IL-8 and G-CSF) were included in 2 separate
multivariate conditional logistic regression models for paired patients. Only IL-6 was
retained after stepwise elimination of covariates in the pre-ART model (p = 0.039,
Odds Ratio: 0.87; 95% confidence interval: 0.77-0.99 per increase of 1 pg/ml) as well
as in the IRIS event model (p = 0.034, Odds Ratio: 1.40; 95% confidence interval: 1.03-
1.91 per increase of 1 pg/ml). We thus identified the innate cytokine IL-6 as the most
central molecule in the cytokine disturbances occurring before and during
inflammation in our TB-IRIS cohort.
7.5 Discussion
In this study, we analyzed plasma levels of markers of increased intestinal
permeability, PAMP-binding proteins as well as pro- and anti-inflammatory cytokines
before and during ART in one of the largest TB-IRIS cohorts described to date. The
role of myeloid cytokine patterns in TB-IRIS is becoming increasingly apparent184.
Furthermore, PAMPs are well known inducers of myeloid cytokine responses through
TLR stimulation252 and PAMP-binding proteins can interfere with this induction23.
Results from our study suggest that plasma IL-6, G-CSF and LBP levels prior to ART as
well as IL-6, LBP and I-FABP levels during the first weeks of treatment are potential
markers of TB-IRIS. In addition, we identified IL-6 as a key protein in the plethora of
cytokines and chemokines we found to be associated with TB-IRIS. Our data thus
support an important role for the innate immune system in TB-IRIS.
Disease progression in AIDS patients is associated with increased immune activation,
which has been linked to enterocyte damage and microbial translocation as a
consequence of HIV infection and can persist during ART101. We hypothesized that a
more pronounced presence of bacterial components such as LPS, which can enter the
blood as a result of impaired intestinal barrier function in HIV patients, could enhance
the inflammation in TB-IRIS patients. In this study, we report plasma LPS levels which
are higher compared to studies that used endpoint detection methods of LPS instead
of kinetic assays105. As comparing results across different platforms can be challenging
and we report LPS levels in a limited number of TB-IRIS patients, these measurements
should be confirmed in a larger cohort. Nevertheless, we observed no statistically
significant differences in LPS levels between our study groups. While contrary to our
hypothesis, this is in line with our findings on EndoCab and I-FABP. Our data therefore

Plasma markers of TB-IRIS
94
demonstrate no evidence that translocation of bacterial PAMPs through a leaky gut
could have contributed to TB-IRIS in our study. In fact, we observed significantly lower
levels of I-FABP in TB-IRIS patients during the 6 month follow-up period after ART
initiation. I-FABP is specifically released into the bloodstream by damaged
enterocytes and elevated plasma levels of I-FABP thus reflect damage to the intestinal
epithelium. Though data on I-FABP in HIV patients during treatment are limited,
higher I-FABP levels were found associated with impaired homing of T cells to the gut
(T-helper 17 cells in particular)253, while lower I-FABP levels associated with better
CD4 T cell recovery106. Lower I-FABP levels during the first 6 months of treatment
could thus reflect a stronger homing of T cells to the gut, possibly as a reaction to
elevated IL-6 levels which stimulate T helper 17 cell development. However, whether
there is a causal contribution to TB-IRIS remains unclear.
While a contribution of LPS to TB-IRIS thus seems unlikely, there is evidence of other
PAMPs being implicated in IRIS pathogenesis. Indeed, higher cryptococcal antigen
titers have been reported pre-ART in cryptococcal-IRIS239, though it is unclear whether
these are actual PAMPs. Moreover, the urinary concentration of LAM, a TB-associated
PAMP, was found to be elevated in TB-IRIS patients from our cohort prior to the start
of ART163. Mycobacterial PAMPs typically initiate the innate immune response to TB
infection through stimulation of TLRs254. This response can be modulated by sCD14
and LBP, both of which can regulate binding of mycobacterial PAMPs to TLRs255. We
thus hypothesized that these PAMP-binding proteins could regulate the inflammation
in TB-IRIS by interfering with TLR stimulation by mycobacterial PAMPs. LBP is a well-
known acute phase protein and as such we found it to be drastically upregulated
during the ongoing inflammation at the time of TB-IRIS. Strikingly however, we found
lower levels of LBP at baseline in TB-IRIS patients compared to HIV+TB+IRIS- controls. It
is tempting to speculate that these lower levels make TB-IRIS patients more sensitive
to circulating LBP-binding PAMPs. Indeed, lower levels of LBP may be an indication of
less antigen being cleared, leading to enhanced TLR stimulation and subsequent
inflammation upon ART. Alternatively, lower levels of LBP could be a consequence of
elevated mycobacterial PAMP concentrations, leading to the formation of antigen-LBP
complexes which are bound to and/or internalized by CD14+ innate immune cells23.
Thirdly, as LBP is produced by the liver in reaction to IL-6256, these findings could
suggest a below average reaction to circulating PAMPs by the innate immune system
in TB-IRIS patients before ART.
Upon TLR stimulation, innate immune cells generally start releasing a number of
cytokines with IL-6 as one of the first and central cytokines being produced257, which
eventually could lead to a phenomenon described as the cytokine storm during TB-

Plasma markers of TB-IRIS
95
IRIS183. We report elevated plasma levels of a number of cytokines commonly
associated with activation of myeloid cells, such as IL-1RA, IL-6, IL-8, G-CSF, in TB-IRIS
patients during the IRIS event. These results indicate a large contribution of the innate
immune system during the inflammation of TB-IRIS, possibly as a result of TLR
stimulation. Conversely, we observed lower IL-6 and G-CSF levels pre-ART, which
points to perturbations in the innate immune system before ART has started. When
including the fact that we observed lower LBP levels pre-ART, one could speculate
that these lower cytokine levels reflect the inability of innate immune cells such as
macrophages to mount a sufficient immune response to the presence of a large
amount of PAMPs, as has been suggested previously187,195,239. Intriguingly, our
multivariate models of cytokines that were significantly associated with TB-IRIS
rendered the same innate molecule as the strongest predictor at both time points,
narrowing down the plethora of cytokines that are associated with TB-IRIS to one
single protein, IL-6. Interestingly, elevated IL-6 seems to be a recurring feature in most
IRIS studies that included this cytokine170,184,186, emphasizing its possible importance.
Our findings during IRIS event are in agreement with two South African TB-IRIS
studies184,186, although these studies did not report pre-ART differences in TB-IRIS
patients. However, a recent study of an Indian TB-IRIS cohort reported pre-ART
elevations of IL-6 as a risk factor for TB-IRIS192. This difference can be explained by the
stringent matching for baseline CD4 count which we applied to our patient selection.
Furthermore, previous studies on TB-IRIS187 and cryptococcal IRIS239 reported lower
pre-ART levels of the innate cytokines CCL2 and both TNFα and G-CSF respectively,
which is in line with our findings, although TNFα was undetectable in most of our
samples.
Taken together, our results support the theory that a disturbed function of the innate
immune system during TB treatment sets the scene for TB-IRIS to occur after ART has
been initiated. It is conceivable that a high PAMP burden is created upon TB
treatment to which innate immune cells such as macrophages are not able to mount a
sufficient immune response. Our findings of lower IL-6, G-CSF and LBP levels pre-ART
are in line with this theory. Once ART has been initiated, the subsequent rise in CD4 T
cells could thus provide the needed assistance to these innate immune cells to initiate
a full blown acute phase response and give rise to TB-IRIS as was proposed
previously195. While the significant increase in IFNγ between pre-ART and IRIS event in
our study supports this, the fact that IFNγ levels during ART were not significantly
different between our study-groups still suggests that the innate immune system is
responsible for the bulk of the inflammation during TB-IRIS, as is reflected by elevated
IL-6 plasma concentrations among other innate cytokines.

Plasma markers of TB-IRIS
96
An important limitation to our study was the unpredictability of TB-IRIS, which poses
a serious challenge to prospective studies to adequately match IRIS events to control
time points. We matched IRIS events with samples from HIV+TB+IRIS- controls after 2
weeks or 1 month on ART with a margin of 1 week, yet still observed a statistically
significant difference in timing. This difference results from IRIS events consistently
occurring before matched HIV+TB+IRIS- control time points, with a median difference
of 1 day. Furthermore, it cannot be excluded that in these resource-limited settings,
patients presenting with IRIS symptoms close to their next scheduled visit of 14 days
might have waited a day or two before attending the clinic, introducing a potential
bias in our reported timing of TB-IRIS. Nevertheless, our findings during TB-IRIS were
overall consistent with other studies. Moreover, all IRIS events were diagnosed on the
basis of clear inflammatory symptoms and were subsequently sampled, thus truly
representing ongoing TB-IRIS related inflammation.
In conclusion, we report lower plasma levels of LBP before ART initiation but higher
levels of LBP during IRIS event. Similarly, the innate cytokine profile showed lower
levels before ART and higher levels during TB-IRIS, with IL-6 holding a dominant role.
Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS
and support the theory that dysfunctions in the innate immune system make a large
contribution to TB-IRIS pathogenesis. Consequently, plasma levels of IL-6, LBP and I-
FABP could be potential markers of TB-IRIS which should be validated in future clinical
studies for the diagnosis and treatment of TB-IRIS.

General Discussion
97
Chapter VIII: General Discussion
Tuberculosis-associated immune reconstitution inflammatory syndrome remains a
puzzling complication in HIV-TB patients who start ART. Marked by a high degree of
morbidity and abundant heterogeneity, TB-IRIS is in fact a disease with many faces.
Due to the difficult clinical diagnosis, there is an urgent need to understand the
mechanisms behind TB-IRIS and to identify potential biomarkers to improve the
diagnosis. In our research, we’ve added yet another face to TB-IRIS when we
described the possible difference between early- and late-onset TB-IRIS. Focusing on
early-onset TB-IRIS, we started off with the hypothesis that an unbalanced
reconstitution of the T cell compartment would make a fundamental contribution to
TB-IRIS. Across our three studies, however, we illustrate how the role of an over-
activated T cell compartment, responding to TB antigens, is probably not the main
driving mechanism behind TB-IRIS. We describe how TB-IRIS is preceded by
diminished immune activation prior to art, followed by a cytokine storm which seems
to be largely driven by innate factors.
8.1 Early- versus late-onset TB-IRIS – yet another face of IRIS.
IRIS develops as either unmasking or paradoxical forms, is associated to a large array
of pathogens and is accompanied by different symptoms that vary in type and
severity. The name ‘immune reconstitution inflammatory syndrome’ could thus be
viewed as a collective term for a set of complications during ART with similar, yet
highly variable clinical presentations. To these complex layers of IRIS presentations,
our study population adds yet another dimension; early- versus late-onset TB-IRIS.
Currently, the standard definition for TB-IRIS only includes patients who develop TB-
IRIS within a timeframe of 3 months after starting ART, which is when the majority TB-
IRIS cases usually develop. However, TB-IRIS has been diagnosed at later intervals as
well, even as late as 4 years after starting ART. Such so called ‘late-onset TB-IRIS
cases’ were also described up until 10 months of ART in our Ugandan cohort and
presented with similar but delayed IRIS symptoms compared to their early-onset
counterparts153. In this thesis, we have taken the first steps in investigating whether
or not these later presentations of TB-IRIS involve a different immunopathogenesis.
Taking a closer look at the timing of TB-IRIS in our study population, it became clear
that 75% of all TB-IRIS patients clustered together and developed TB-IRIS before 1
month of ART, while the other 25% developed at later intervals during ART.
Importantly, ART induces drastic changes in a patient’s immune system, especially
during the first months of treatment. In fact, the rate of T cell recovery upon ART is
the highest during the first 6 weeks on ART72. This means that the extent to which a
patient’s T cell compartment contributes to or is affected by TB-IRIS might be

General Discussion
98
dependent on the duration of ART, even after only 1 month of ART. With the
intention of improving the homogeneity of our patient selection, we therefore pooled
the first 75% of patients together as early-onset TB-IRIS patients. This minimized a
possible confounding effect of ART duration, given the immunological turbulence in
the first months of ART. On the other hand, we were able to pool the remaining 25%
as a new group of late-onset TB-IRIS patients. This in turn allowed us to investigate
the effect of ART duration in TB-IRIS patients who were clinically similar to early-onset
patients, yet as a group, developed symptoms at a significantly later time during ART.
In contrast to early-onset TB-IRIS, late-IRIS event was characterized by a much more
pronounced shift from memory to effector T cell subpopulations, whose function is
typically associated with the production of pro-inflammatory cytokines119. This makes
it tempting to speculate that late-onset TB-IRIS, more so than their early-onset
counterparts, is driven by this skewed T cell compartment. But perhaps it is a bit
presumptuous to declare them as diseases with a completely different immune
pathogenesis, given their clinical similarities. What if, instead of T cells driving late-
onset TB-IRIS, we turn things around and consider TB-IRIS as the driving mechanism
behind the observed shifts in T cell phenotype? This would mean that the effect of TB-
IRIS on the T cell compartment is dependent (at least in part) on the duration of ART.
This actually makes sense, because T cell maturation is believed to be dependent on
antigen-load and the cytokine environment221-223, which just so happen to be major
factors in TB-IRIS183,224,225. While this explains how TB-IRIS could drive T cell
maturation, it still does not explain why this effect is only visible in patients who
develop TB-IRIS at a later time during ART. For this we have to consider the fact that
shortly after starting ART, there might not be enough time for the still recovering T
cell compartment to make a large contribution to TB-IRIS, as was pointed out
previously193. It has been shown that during protective immune reconstitution,
central memory T cells expand first, followed but the effector T cell population258. A
longer period on ART allows for a greater redistribution and functional recovery of the
memory T cell pool and possibly a longer exposure to the antigen load. This might
make the T cell compartment more susceptible to the sudden cytokine storm when
TB-IRIS occurs. As a result, the inflammatory environment during TB-IRIS might only
skew T cell maturation (at least at detectable levels) when the T cell compartment has
had enough time to recover, hence only in late-onset TB-IRIS patients.
Together, all this suggests that the duration of ART before IRIS develops could
influence the T cell phenotype of TB-IRIS patients. Off course, these experiments were
performed in a small subpopulation of TB-IRIS patients with a cut off at 1 month of
ART which may only be applicable to our cohort. Nonetheless, given the scientific

General Discussion
99
attention that has been given to the role of T cells in TB-IRIS, these findings could
provide an important perspective for TB-IRIS research. In fact, this could be a partial
explanation as to why the importance of T cells in TB-IRIS seems to vary between
studies, since a difference in ART duration could influence the immunological
measurements. These findings arguably provide a reason to take the duration of ART
into account in future TB-IRIS studies, especially when studying the T cell
compartment.
8.2 T cells in TB-IRIS – wrongfully accused?
Due to the pivotal role of T cells in HIV and TB immunology and the fact that ART
initiation at low CD4 counts is a prerequisite for TB-IRIS, a significant theoretical role
has been attributed to T cells in TB-IRIS related inflammation153,212. An explosive
restoration of T cell function, possibly a TB-specific Th1 response, is believed to play a
distinct role171,176,178,195,206,228. However, our observations in late-onset TB-IRIS patients
suggest that the contribution of the T cell compartment in TB-IRIS might be
dependent on the duration of ART. This could also suggest that enhanced T cell
responses in late-onset TB-IRIS could be a consequence of other factors driving TB-
IRIS. Keeping this in mind, we thus focused the rest of our research on early-onset TB-
IRIS patients in order to understand the role of the T cell compartment in TB-IRIS
while minimizing the potential bias of ART duration. We initially hypothesised that an
unbalanced reconstitution of the T cell compartment made a fundamental
contribution to TB-IRIS and therefore investigated the significance of the T cell
compartment on three different levels; expression of activation markers, in vitro IFNγ
responses to a range of TB- and non-TB antigens and plasma cytokine levels.
In contrast to our hypothesis, we did not observe significantly increased IFNγ
responses to any of the TB-antigens in TB-IRIS patients before or during ART. Neither
did we observe distinctly higher plasma levels of IFNγ at these time points225, nor did
we encounter evidence of an over-activated CD8 T cell compartment in TB-IRIS
patients. Unlike TB responses, however, we found that responses to the antigen CMV
were lower during TB-IRIS compared to HIV+TB+IRIS- controls, which is in line with a
previous study178. These findings indicate that it is unlikely that an excessive Th1
response to TB-antigens is driving early-onset TB-IRIS in our cohort. Instead, it even
seems that the reconstitution of the response to CMV could be disturbed. This might
be due to the high TB-antigen load and persistent inflammation associated with TB-
IRIS176,212,238,239.
Our results are in agreement with a number of previous reports of similar T cell
activation and IFNγ responses between TB-IRIS patients and HIV+TB+IRIS-

General Discussion
100
controls175,180,181. Even so, they are not in line with previous reports of elevated PPD-
responses during TB-IRIS173,177-179,196. Our findings thereby contradict the generally
accepted concept of an explosive T cell restoration playing a distinct role in TB-IRIS. It
is important to note that immunological measurements in HIV-TB patients are
associated with the degree of immunosuppression, the TB-treatment – ART interval
and the TB-antigen load235-2 37. These factors just so happen to be risk factors of TB-
IRIS. In addition, we established the potential impact of ART duration on the
behaviour of the T cell compartment in TB-IRIS patients (described in 8.1). In other
words, TB-IRIS patients are subject to severe immunological turbulence after starting
ART, which could potentially bias adaptive immune responses when compared to
control subjects who are not experiencing a similar level of immunosuppression and
antigenic stimulation. This could in fact be one of the reasons for the large number of
inconsistencies across IRIS studies. In this thesis, we minimize this potential bias by
directly comparing (time-) matched patients under very similar clinical conditions,
possibly explaining the discrepancy with studies that observed elevations in TB-
associated responses. This highlights the importance of adequate selection of control
patients when studying TB-IRIS.
Collectively, the results we derived from our stringently matched patients and
controls thus provide evidence on three different levels that the role of T cells in
early-onset TB-IRIS may not be as elaborate as previously believed. Nonetheless, we
did observe a significant rise plasma IFNγ levels in TB-IRIS patients between the pre-
ART time point and the IRIS event. Given the prerequisite of ART initiation for TB-IRIS
to develop, one might still surmise that T cells provide a necessary trigger for TB-IRIS.
Nevertheless, the actual mechanism that drives early-onset TB-IRIS inflammation does
not seem to be located in the T cell compartment and might perhaps be found in
other parts of the immune system.
8.3 The innate immune system – pulling the strings behind TB-
IRIS?
By exploring the broad ranges of the immune system, the collective effort of previous
TB-IRIS studies has accumulated a large amount of evidence that support the roles in
TB-IRIS of both the T cell compartment and the innate immune system. Although it is
still unclear where exactly the immune system derails to cause TB-IRIS, evidence
implicating the innate immune system is increasing exponentially. On top of that, our
results have now established that an overly vigorous T cell response is probably not
the main feature in our TB-IRIS cohort. Looking past our initial hypothesis, we
therefore attempted to catch a glimpse of which other pre- and post-ART mechanisms
could lie behind TB-IRIS. We next directed our attention towards the possible

General Discussion
101
contribution of the innate immune system by further exploring the role of PAMPs and
the proteins that bind them, as well as the cytokine profiles in early-onset TB-IRIS
patients.
Given the generally accepted theory that TB-IRIS results from a high antigen load prior
to ART, it made sense to explore which antigens are actually involved. Because we
couldn’t observe any enhanced IFNγ responses in TB-IRIS patients prior to ART (or
during ART for that matter), we next speculated that the antigens involved may be of
innate origin. The well-known phenomenon of bacterial translocation in AIDS and the
consequential immune activation caused by LPS thus lead to our hypothesis that a
more pronounced presence of LPS could contribute to TB-IRIS. However, our results
showed no evidence of increased bacterial translocation in TB-IRIS patients. We
therefore concluded that the presence of LPS did not make a large contribution to TB-
IRIS development. Nonetheless, there is evidence of other PAMPs being involved in
TB-IRIS. Of particular interest is the observation of increased urinary LAM prior to ART
in TB-IRIS patients from our cohort163. The presence of an increased amount of TB-
associated PAMPs prior to ART as a predisposing factor for TB-IRIS therefore remains
a realistic possibility.
Assuming an increased presence of antigens, we expected a higher level of immune
activation prior to ART. Surprisingly however, we observed a lower level of CD8 T cell
activation and lower plasma levels of IL-6, G-CSF and LBP in our TB-IRIS patients
before ART was initiated, compared to HIV+TB+IRIS- controls. Although plasma
experiments were only performed in early-onset TB-IRIS, this decrease in CD8 T cell
activation was also observed in late-onset TB-IRIS patient, pointing to a common pre-
ART mechanism between these clinical presentations. Only a limited amount of
studies have reported pre-ART factors associated with TB-IRIS before. However, our
findings are in line with previous studies on TB-IRIS187 and cryptococcal IRIS239 which
reported lower pre-ART levels of the innate cytokines CCL2 and both TNFα and G-CSF
respectively. One interpretation of the hypo-activation observed in our studies might
be the presence of immune exhaustion, characterized by a progressive loss of T cell
function prior to ART. This is a widely accepted phenomenon in HIV-progression
which is associated to the expression of PD-1 on T cells (among other markers) and
deregulated cytokine production by both the adaptive and innate immune system259.
Elevated expression of PD-1 on CD4 T-cells prior to ART has in fact been reported in
patients who later developed different forms of IRIS176. It was suggested that this
finding might indicate a functional impairment that is reversed with ART, which could
be associated with a higher antigen load and higher frequencies of antigen-specific T
cells ready to get mobilized once immunosuppression reverses. Though it might be

General Discussion
102
informative to further explore markers of immune exhaustion (e.g. PD-1 and TGFβ),
our results did not indicate a strong association between T cells and TB-IRIS.
Furthermore, we observed similar pre-ART levels of IL-2 and IFNγ as well as a similar
distribution of T cell memory sub-types between (early-onset) TB-IRIS patients and
HIV+TB+IRIS- controls. Since T cell exhaustion in HIV is commonly associated with
perturbations in these factors, our results do not fully support the idea of this
exhaustion being more pronounced in TB-IRIS patients than in HIV+TB+IRIS- controls.
Perhaps a more likely explanation could be that the decreased CD8 T cell activation is
a consequence of a more general down regulation of the immune system, or possibly
innate immune dysfunction. Consider our observation of IL-6 as a central molecule in
the down regulated pre-ART cytokine levels of TB-IRIS patients. To that we can add
the fact that IL-6 levels are known to drive both CD8 T cell activation and production
of LBP216,217, which were both down regulated prior to ART as well. In addition, lower
levels of LBP could also indicate the presence of a large amount of PAMPs that have
formed complexes with LBP. Given the previous reports of increased pre-ART urinary
LAM in our cohort, one could thus speculate that these lower pre-ART cytokine levels
reflect the inability of innate immune cells such as macrophages to mount a sufficient
immune response to the presence of a large amount of PAMPs. A logical conclusion
would therefore be that TB-IRIS is preceded by a general perturbation in the innate
immune system before ART has started, compared to matched HIV+TB+IRIS- controls.
While the immune reaction seems to be toned down prior to ART, the opposite is true
during TB-IRIS as we observed a burst in a plethora of cytokines, similar to previous
reports184,186. In concert with this cytokine storm, we observed a pro-inflammatory
shift in IL-6 to IL-10 and TNFα to IL-10 ratios after in vitro stimulation with LPS of
PBMCs collected during TB-IRIS, notwithstanding the fact that the absolute cytokine
levels did not reach statistical significance. In accordance with these findings, TB-IRIS
patients from our cohort showed an increased pro-inflammatory monocyte-gene
expression profile that was also perturbed in pattern recognition receptor
pathways197. Furthermore, a similar imbalance was previously reported during IRIS
after in vitro TLR2 stimulation with lipomannan, demonstrated by elevated TNFα
production without an equivalent rise in IL-10196. Interestingly enough, most of the
immune factors we observed to be skewed both prior to ART and during TB-IRIS were
of innate origin. In fact, our results show that IL-6 holds a central role at both these
instances. Given the lack of evidence to support the role of an adaptive response, our
results therefore suggest a prominent contribution of the innate immune system in
TB-IRIS, possibly mediated by TLR pathways.

General Discussion
103
Together, these findings contribute to the increasing amount of evidence that
implicates the innate immune system in TB-IRIS. In line with a previous theory that
suggested the pre-ART priming and subsequent over-activation of the innate immune
system in TB-IRIS195, our results seem to suggest a certain duality between
immunological events before and after ART initiation. On one hand, the lower levels
of immune activation prior to ART could reflect a diminished ability of the innate
immune system to mount a sufficient immune response to clear the antigen load. This
process may thus contribute to the increased antigenic burden of TB-IRIS patients
prior to ART. On the other hand, once ART is initiated, the corresponding rise in IFNγ
could trigger the activation of cells of the innate immune system which respond more
strongly to this antigen load and produce a burst of cytokines. With IL-6 in the middle
of it all, this predominantly innate cytokine storm marks the rise of tissue destructive
inflammation during TB-IRIS.
8.4 Final conclusions
Considering all the facts reported in this thesis, we might be able to put together
some key pieces of the puzzle that is the immunopathogenesis of TB-IRIS. First and
foremost, we showed that early- and late-onset presentations of TB-IRIS may share
common predisposing factors, yet appear to be set apart by a different pathogenesis
at the time of the disease. It would therefore be prudent to study these presentations
separately in future studies. Second of all and in contrast to our main hypothesis, it
seems that the T cell compartment is not the main driving mechanism behind TB-IRIS.
Instead, fluctuations in the T cell compartment could rather be a consequence of TB-
IRIS itself and might perhaps be dependent on the duration of ART as well as the HIV-
patient’s disease status. In the end it seems that TB-IRIS is largely driven by innate
factors that are lowered prior to ART but increased during TB-IRIS. Paradoxical TB-IRIS
thus seems to be “paradoxically” preceded by lower immune activation prior to ART,
leading up to a burst of cytokines upon ART. In other words; a proverbial calm before
the cytokine storm.

General Discussion
104
8.5 Food for thought
Over 2 decennia of research have provided the foundations to help uncover the
mechanisms behind the pathogenesis of TB-IRIS. Even so, TB-IRIS has proven to be a
puzzling complication and the collective endeavor to understand TB-IRIS is far from
over. With what we know about TB-IRIS today, including the results portrayed in this
thesis, we can reflect on where this research is taking us, what the next steps should
be and what obstacles we may encounter along the way.
As we said before, the term “IRIS” implies a set of complications during ART with
similar, yet highly variable clinical presentations. This remains true, even when
focussed on IRIS specific to one single pathogen such as TB. Studies can compensate
for this heterogeneity, at least to some degree, by defining TB-specific IRIS patients
according to the INSHI definition and thoroughly matching them to controls.
Nonetheless, the clinical picture of TB-IRIS remains highly variable and may not be
uniform across different studies: unmasking vs. paradoxical TB-IRIS, early- vs. late-
onset TB-IRIS, pulmonary vs. extrapulmonary TB, major vs. minor symptoms, varying
degrees of severity, …. One cannot help but wonder if and how these different
presentations influence immunological measurements. This in turn raises the concern
that the collective body of results on TB-IRIS may become just as heterogeneous as
the disease itself.
A number of inconsistencies across studies could be ascribed to the inclusion of non-
pathogen specific IRIS cases (as discussed in section 3.3 of this thesis), but this is likely
not the only factor involved. Our results, for example, have already suggested that the
duration of ART before TB-IRIS develops as a potential confounding factor in studies
investigating the T cell compartment. As another example of potential confounders,
one study has shown that a lower ratio of IFNγ to IL-10 could differentiate TB-IRIS
patients infected with drug resistant TB from TB-IRIS patients with drug sensitive
TB191. Interestingly, IFNγ/IL-10 ratios are also thought to define disease severity of
pulmonary and extrapulmonary tuberculosis in HIV-negative patients260. Consider for
a moment that INSHI-defined TB-IRIS patients can present major symptoms consistent
with either pulmonary and/or extrapulmonary TB, or even no major symptoms at all
(only minor symptoms). Such different presentations and the accompanied difference
in bacterial/antigen burden could potentially influence the degree of antigenic
stimulation and thus the way the immune system reacts. It doesn’t seem illogical to
assume that the clinical diversity within TB-specific IRIS could be an additional source
of bias. After all, most IRIS studies to date have focussed on the role of cellular and
soluble markers of inflammation (e.g. T cell activation and production of IFNγ and
IL-6). We know for a fact that these markers increase during inflammatory processes.

General Discussion
105
It stands to reason that the detection of these markers may depend on the degree of
inflammation a TB-IRIS patient is experiencing, in other words; how severe the TB-IRIS
case is.
Does this mean these markers are useless as research tools? No, it doesn’t. However,
it is a valid argument which underscores the importance of accounting for disease
severity when studying TB-IRIS. We cannot yet exclude that factors which are thought
to be associated to TB-IRIS might be (more) suited as markers of disease severity.
After all, it is possible that some markers may only be picked up during severe TB-IRIS
but not during mild TB-IRIS, due to the difference in inflammation. Scoring disease
severity is not a new concept in clinical studies. A very early example of such a
classification system is the APACHE II score, which has been used to generally define
disease severity in hospitalized patients261. Interestingly, IL-6 has been associated with
disease severity and all-cause mortality during bacterial infection/sepsis using this
score system262, which already provides a link to TB-IRIS. Perhaps another example
more closely related to TB-IRIS is the current use of a TB-severity score or “TBscore”; a
simple tool for clinical monitoring of TB patients in low-resource settings263-265. Of
note, higher TB-severity scores are thought to be independently associated with
elevated matrix metalloproteinases (MMP) 1 & 3266. This provides yet another
interesting link to TB-IRIS, which has been associated with a distinct pattern of MMP
gene and protein activation (including MMP-1,-3 & -7)267. Based on these examples, a
severity score of TB-IRIS seems well within reach and might include both clinical
aspects as well as lab-markers such as IL-6 and MMP’s. In the interest of improving
comparability between different TB-IRIS studies, it might thus be a good idea to come
up with a system of scoring TB-IRIS severity during its diagnosis and perhaps even
include it in the INSHI definition. This might become a useful tool for the evaluation of
newly identified biomarkers within the clinical spectrum of TB-IRIS.
Since inflammatory markers may be dependent on the disease status of a patient, one
does have to be reserved when considering the usefulness of these factors as
biomarkers for TB-IRIS, even prior to ART. Let’s take a critical look at our results on
IL-6 for example: we observed lower levels of IL-6 prior to ART in patients that were
destined to develop TB-IRIS. This was in comparison with HIV+TB+IRIS- controls at a
similar level of CD4 depletion. Does this make IL-6 a candidate research tool for TB-
IRIS? Yes. Does this make “lower IL-6” a pre-ART risk factor for TB-IRIS? It depends. In
seeming contrast to our findings, a recent study of an Indian TB-IRIS cohort reported
pre-ART elevations of IL-6 as a risk factor for TB-IRIS192. This was in comparison with
HIV+TB+IRIS- controls who had significantly higher CD4 counts prior to ART. In this
apparent contradiction, the effect of pre-ART CD4 count (still the primary risk factor

General Discussion
106
for TB-IRIS) becomes clear, putting both findings in completely different contexts.
Having a higher plasma level of IL-6 might be a risk factor for TB-IRIS within the
general population (maybe as a surrogate marker of low CD4 counts), whereas a
lower IL-6 level may become a risk factor within a group of patients experiencing very
similar immunosuppression. When we consider the direct application of these results
as biomarkers, these contexts need to be taken into account. Although “lower IL-6”
prior to ART may be a CD4 count-independent characteristic of TB-IRIS, it may be hard
to observe without matched controls. Therefore its use as a predictive biomarker
outside the scope of clinical studies may be limited. From a general diagnostic
perspective (i.e. when identifying TB-IRIS patients within the general HIV-TB infected
population), “higher IL-6” prior to ART may therefore be a more practical addition to
the established risk factors of TB-IRIS.
Still, matched observations such as ours provide a lot of additional information
related to the underlying pathogenesis of TB-IRIS. It would have been interesting to
directly compare our results on IL-6 to the increased levels of urinary LAM that were
observed prior to ART in TB-IRIS patients from our cohort, although this was
determined compared to unmatched controls163. A negative correlation between
(lower) pre-ART IL-6 levels and (higher) levels of LAM could further support the idea
that TB-IRIS originates from a disturbed innate immune function. It would also have
been interesting to correlate the development of late-onset TB-IRIS with these pre-
ART LAM levels, since the spectrum of antigenic stimulation (antigen load) prior to
ART could potentially influence the time it takes for TB-IRIS to develop (early- vs. late-
onset TB-IRIS). Regrettably, we were unable to calculate a correlation due to limited
patient-overlap between our studies. Nonetheless, it is interesting to note that
detection of LAM for prediction of TB-IRIS did not provide an additional benefit over
baseline CD4 counts, whereas the presence of a lower pre-ART IL-6 level was CD4
count-independent. Arguably, discovering one single magic marker of TB-IRIS might
prove challenging and it is hard to say which markers would be better than others. If
we are to reliably predict the occurrence of TB-IRIS, a combination of multiple
markers that have been discovered so far will likely be necessary.
Our main findings contribute to the increasing body of evidence pointing towards a
role of the innate immune system in TB-IRIS. This is a viewpoint shared with results
from a monocyte gene-expression study performed in TB-IRIS patients from our
cohort, which revealed a potential role of monocytes in the disease197. Although these
experiments were performed in the same cohort (as was the case LAM the LAM study
mentioned above), sample restrictions lead to limited overlap in patients between
our studies which makes a collective statistical interpretation difficult. Even so it is

General Discussion
107
intriguing to see the same patterns popping up again and again, and not just in the
studies outlined here. Perturbations in pro-inflammatory cytokines and IL-6 levels in
particular, have become a repeating feature across TB-IRIS studies. This is supported
by observations made during TB-IRIS by Tran et al.197, who reported a loss of balance
between genes related to pro- and anti-inflammatory processes, including: IL-6
signalling, the complement system and the role of pattern recognition receptors
(PPR). This is an interesting finding indeed, given our similar observations of a pro-
inflammatory cytokine shift during TB-IRIS after in vitro TLR stimulation. Based on
these combined findings, it is tempting to start speculating on the role of TLR’s in TB-
IRIS. Given their potential to recognize a broad range of different PAMPs, TLRs could
provide a common mechanism between forms of IRIS associated to different
pathogens. Different degrees of TLR stimulation or variances in the response to these
stimuli could very well lead to the unbalanced inflammatory responses seen in (TB-)
IRIS. A very relevant characteristic of TLR’s is that the antigenic signal required to
induce the anti-inflammatory cytokine IL-10 is much higher than the one required for
pro-inflammatory cytokines such as TNFα268. One could therefore hypothesize that an
upstream or downstream disturbance in the TLR pathways (or PPRs in a broader
sense) could be a determining factor in TB-IRIS development. It would be interesting
to determine the cellular expression of different TLRs on different cells of the innate
immune system (e.g. monocytes and neutrophils) in TB-IRIS patients. In addition, it
might be very informative for TB-IRIS studies to explore the intracellular pro- and anti-
inflammatory pathways associated to TLR/PPR stimulation. Fully addressing this
research avenue, especially on a genetic level, could help elucidate the mechanisms
behind TB-IRIS.
In the end, new TB-IRIS studies will still be faced with the same difficulties as previous
studies. There is a continuous need for international cooperation in order to set up
large TB-IRIS cohort studies that are well-designed to deal with these challenges,
including TB-IRIS’ heterogeneous nature. Though TB-IRIS may be a puzzling
complication, the tireless collective effort of previous and ongoing TB-IRIS studies is
starting to paint a detailed picture of what could be going on behind the scenes of this
disease. It is up to future studies to build on these foundations and finally elucidate
the ultimate mechanisms behind the immunopathogenesis of TB-IRIS. The subjects
outlined above may provide some interesting food for thought for these future
studies to consider.

108

Addendum
109
Curriculum Vitae and publication list
Odin Goovaerts Tel: +32 (0) 472 65 29 50
Date of birth: 15th July 1985 E-mail:
Odin.Goovaerts@gmail.com
Academic Education
2010-2014: PhD in Biomedical Sciences University of Antwerp,
Belgium
Institute of Tropical Medicine
(ITM)
Grant: Agentschap voor Innovatie door Wetenschap en Technologie (IWT)
Dissertation: Pathogenesis of tuberculosis-associated immune reconstitution
inflammatory syndrome (TB-IRIS) – The calm before the cytokine
storm
Promotor: Prof. Dr. Luc Kestens
2003-2009: BSc/Msc in Biomedical Sciences University of Antwerp,
Belgium
Specialisation: Tropical Biomedical Sciences
Laboratory Animal Science for Scientists cat. C’ certificate
Dissertation: Characterisation of T-helper 17 cells during immune reconstitution in
HIV-patients receiving antiretroviral therapy.
Promotor: Prof. Dr. Luc Kestens
Supervisor: Dr. Pascale Ondoa
Minors: Communication
Pre-clinical drug research
Molecular cellbiology
Work Experience
Oct. - Dec. 2009: Scientific collaborator Institute of Tropical Medicine
Study: Vaginal micro flora and immunology in Belgian participants
Supervisors: Dr. Vicky Jespers
Prof. Dr. Guido Vanham

Addendum
110
Presentations and Courses
Conference Presentations:
• HIV Research For Prevention (HIV R4P) 2014, Cape Town – South Africa: Antigen-
specific interferon-gamma responses and innate cytokine balance in TB-IRIS. Odin
Goovaerts, Wim Jennes, Marguerite Massinga-Loembe, Ann Ceulemans, William
Worodria, Harriet Mayanja-Kizza, Robert Colebunders, Luc Kestens, the TB-IRIS Study
Group. (Poster presentation)
• International Conference on the Pathogenesis of Mycobacterial Infections 2014,
Stockholm – Sweden: Lower immune activation prior to ART in early- and late-onset
TB-IRIS. Odin Goovaerts, Wim Jennes, Marguerite Massinga-Loembe, Pascale Ondoa,
Ann Ceulemans, Chris Vereecken, William Worodria, Harriet Mayanja-Kizza, Robert
Colebunders, Luc Kestens, the TB-IRIS Study Group. (Oral Presentation)
• European Congress of Immunology 2012, Glasgow - United Kingdom: LPS-binding
protein and IL-6 mark paradoxical tuberculosis immune reconstitution inflammatory
syndrome in HIV patients. Odin Goovaerts, Wim Jennes, Marguerite Massinga-
Loembe, Ann Ceulemans, William Worodria, Harriet Mayanja-Kizza, Robert
Colebunders, Luc Kestens, the TB-IRIS Study Group. (Poster presentation)
Courses:
• State of the art lectures in tropical medicine – ITM, 2010-2014
• Leadership and Teamwork – University of Antwerp, 2014
• Academic English‐writing research papers – Linguapolis, 2013
• FTNLS WORKSHOP – Differential Network Medicine: a systems approach to unmask
sensors & drivers of human diseases – Flemish Institute for Biotechnoloy, 2013
• Cell Analysis Technology introduction – Becton Dickinson, 2012
• Reference Manager Course – ITM, 2012
• qPCR experiment design and data‐analysis course – Biogazelle, 2012
• Scientific Reasoning and Reporting – University of Antwerp, 2012
• Research data and document management course – ITM, 2012

Addendum
111
Peer reviewed publications:
TB-IRIS: a manifestation of adaptive or innate immunity? – The Lancet Infectious
Diseases (Comment in press)
Odin Goovaerts, Luc Kestens
Lower pre-treatment T cell activation in early- and late-onset tuberculosis-
associated immune reconstitution inflammatory syndrome. – (Under review)
Odin Goovaerts, Wim Jennes, Marguerite Massinga-Loembe, Pascale Ondoa, Ann
Ceulemans, Chris Vereecken, William Worodria, Harriet Mayanja-Kizza, Robert
Colebunders, Luc Kestens, the TB-IRIS Study Group
Antigen-specific interferon-gamma responses and innate cytokine balance in TB-
IRIS. – PLoS One 2014 Nov 21; 9(11)
Odin Goovaerts, Wim Jennes, Marguerite Massinga-Loembe, Ann Ceulemans, William
Worodria, Harriet Mayanja-Kizza, Robert Colebunders, Luc Kestens, the TB-IRIS Study
Group
LPS-binding protein and IL-6 mark paradoxical tuberculosis immune reconstitution
inflammatory syndrome in HIV patients. – PloS One 2013 Nov 28; 8(11)
Odin Goovaerts, Wim Jennes, Marguerite Massinga-Loembe, Ann Ceulemans, William
Worodria, Harriet Mayanja-Kizza, Robert Colebunders, Luc Kestens, the TB-IRIS Study
Group
Low prevalence of vitamin D deficiency in Ugandan HIV-infected patients with and
without tuberculosis - Int J Tuberc Lung Dis 2012 Nov 1; 16(11)
Anali Conesa-Botella, Odin Goovaerts, Marguerite Massinga Loembé, William
Worodria, Doreen Mazakpwe, Kenneth Luzinda, Harriet Mayanja-Kizza MBChB,
Robert Colebunders, Luc Kestens
Searching for lower female genital tract soluble and cellular biomarkers: defining
levels and predictors in a cohort of healthy Caucasian women - PLoS One 2012 Aug
31; 7(8)
Jordan K. Kyongo, Vicky Jespers, Goovaerts Odin, Jo Michiels, Joris Menten, Raina N.
Fichorova, Tania Crucitti, Guido Vanham, Kevin K. Ariën
Shifts in Mycobacterial Populations and Emerging Drug-Resistance in West and
Central Africa - PLoS One 2014 Dec 10; 9(12)
Gehre F, Ejo M, Fissette K, de Rijk P, Uwizeye C, Nduwamahoro E, Goovaerts O,
Affolabi D, Gninafon M, Lingoupou FM, Barry MD, Sow O, Merle C, Olliaro P, Ba F, Sarr
M, Piubello A, Noeske J, Antonio M, Rigouts L, de Jong BC.

Addendum
112
Appendix I
Baseline characteristics of the Ugandan cohort on which this study was based.
Characteristics
Total HIV+TB+ (including TB-IRIS)
TB-IRIS
Baseline characteristics of patients enrolled in the study
# patients enrolled
302
53
# patients who initiated ART
254
53
Male sex, n (%)
190 (56)
31 (58)
Age (years)
35
37 (33-44)
CD4 (cell/mm³)
52 (19-128)
23 (11-66)
TB treatment duration prior to ART (days)
44 (28-64)
37 (24-59)
# patients with pulmonary TB, n (%)
196 (77%)
34 (64%)
# patients with extra-pulmonary TB, n (%)
44 (17%)
16 (36%)
# patients with both pulmonary and extra-pulmonary TB, n (%)
14 (6%)
0 (0%)
Values are shown as absolute counts and percentage or median values with interquartile range.
Baseline characteristics of TB-IRIS patients and HIV+TB+IRIS-controls included in each chapter.
Characteristics
HIV+TB+IRIS-
TB-IRIS
Characteristics per chapter
V
VI
VII
V*
VI
VII
# patients selected
22
18
40
22
18
40
Sample type
Whole Blood
PBMCs
Plasma
Whole Blood
PBMCs
Plasma
Male sex, n (%)
12 (55)
8 (44)
23 (58)
12 (55)
8 (44)
23 (58)
Age (years)
39 (35-43)
40 (31-44)
38 (32-42)
39 (35-43)
40 (31-44)
38 (32-42)
Baseline CD4 (cell/mm³)
30 (17-61)
23 (8-93)
24 (15-54)
30 (17-61)
23 (8-93)
24 (15-54)
TB treatment duration prior to ART (days)
37 (25-60)
46 (23-58)
46 (30-62)
37 (25-60)
46 (23-58)
46 (30-62)
Patient overlap with chapter V
-
5
17
-
11
21
Patient overlap with chapter VI
5
-
10
11
-
18
Patient overlap with chapter VII
17
10
-
21
18
-
Values are shown as absolute counts and percentage or median values with interquartile range. Due to limited availability of different sample
types, not all patients and controls consistently overlapped between the chapters. Whole blood samples were analyzed locally, when still fresh.

Addendum
113
Proenzyme
Substrate + H2O
Endotoxin
Enzyme
Enzyme
Peptide + pNA
Appendix II
Lonza Kinetic-QCL Test (www.lonza.com/pharmabiotech)
Explanation of the test: Kinetic-QCL™ is a quantitative, kinetic assay for the detection
of Gram-negative bacterial endotoxin. A sample is mixed with the LAL/substrate
reagent, placed in an incubating microplate reader, and automatically monitored over
time for the appearance of a yellow colour. The time required before the appearance
of a yellow colour (Reaction Time) is inversely proportional to the amount of
endotoxin present. That is, in the presence of a large amount of endotoxin the
reaction occurs rapidly; in the presence of a smaller amount of endotoxin the
Reaction Time is increased. The concentration of endotoxin in unknown samples can
be calculated from a standard curve.
The use of LAL for the detection of endotoxin evolved from the observation by Banga
that a Gram-negative infection of Limulus polyphemus, the horseshoe crab, resulted
in fatal intravascular coagulation. Levin and Bangb,c later demonstrated that this
clotting was the result of a reaction between endotoxin and a clottable protein in the
circulating amebocytes of Limulus. Following the development of a suitable
anticoagulant for Limulus blood, Levin and Bangd prepared a lysate from washed
amebocytes, which was an extremely sensitive indicator of the presence of endotoxin.
Solume,f and Young, Levin, and Prendergastg have purified and characterized the
clottable protein from LAL and have shown the reaction with endotoxin to be
enzymatic.
The present LAL method utilizes the initial part of the LAL endotoxin reaction to
activate an enzyme, which in turn releases p-nitroaniline (pNA) from a synthetic
substrate, producing a yellow color.
Principle: Gram-negative bacterial endotoxin catalyses the activation of a proenzyme
in the LAL7. The initial rate of activation is determined by the concentration of
endotoxin present. The activated enzyme catalyses the release of pNA from the
colourless substrate Ac-Ile-Glu-Ala-Arg-pNA. The free pNA is measured
photometrically, at 405 nm continuously throughout the incubation period. The
concentration of endotoxin in a sample is calculated from its Reaction Time by
comparison to a standard curve.

Addendum
114
Experimental procedure
1. One plate can fit 21 samples. Prepare endotoxin-free glass tubes N201 (21 per
plate) and label them. Fill each tube with 900 µL of LAL water.
2. Transfer 100µL of plasma in the 900 µL of LAL water for a 1 in 10 dilution. Recap
the tubes, vortex thoroughly and inactivate the samples at 75°C for 30 min.
3. Label 21 endotoxin free dilution tubes for further sample dilution. Leave them in
the flow. Label 4 endotoxin free dilution tubes for standards dilution.
4. Reconstitute the endotoxin with the specified amount of water (indicated on the
data sheet included in the kit) and mix thoroughly for 15 min. The concentration
of the stock is 50EU/mL.
NB Switch on the plate reader and start up the included software. In the plate-reader
software: PPC = 0.5 EU/mL (standard) is the concentration of the spiked
endotoxin in control-wells. The amount of spiked endotoxin should be in the
middle of the standard curve. In case of high background, spike with 5EU/mL.
Click on a sample number and “edit”. Change each sample name according to the
specimen you are testing and once in is finished, do not forget to save. Exit and
your template is now ready to run the assay. The machine is now heating at 37°C,
and is ready to receive the plate later on.
5. Prepare standard dilution from the standard stock (50EU). 900mL of LAL water +
100µL of endotoxin stock. Vortex each tubes thoroughly for 1 minute, before
transferring the endotoxin into the next tube. S1= 0.005EU/mL, S2 = 0.05EU, S3 =
0.5 EU, S4 = 5EU, S5 = stock 50EU/mL
6. Transfer 100µL of standards in the well starting from the less diluted tube (S1).
Then spike the appropriate wells with 10µL of S4 (5EU; final concentration =
0.5EU) using a pipet of 10µL. Cover the plate.
7. Prepare the dilution of samples in Mag2Cl, typically 1 in 500 (this is used for the
TB samples) or 1 in 1000, using endotoxin free dilution tubes. First vortex the
capped tubes thoroughly after their inactivation and then proceed to the 1 in 500
dilution in dilution tubes. Vortex again each tube (2 by 2) containing the 1 in 500
diluted samples (for 30 sec).
8. Briefly vortex the tubes and transfer 100 of each diluted sample on the
corresponding well, starting from the non-spiked wells. At the end add 100 µl of
water on the blanked wells.

Addendum
115
9. Transfer the plate with its lid on the reader. Close the cover. Run the
“1strial101212”. The software will ask to put the plate in the reader, but it is
already done. The plate is shaken and the machine counts down 10 min during
which the plate is heated at 37° for10 min.
10. In the meantime, prepare the substrate and work fast. 4 vials of substrate are
need per plate. Substrate is reconstituted with 2.6 mL of LAL water. Shake gently
but thoroughly. Put the vial back in the box to protect them from light. Prepare
your multichannel pipette (100µL, 8 tips)
11. At the end of the 10 minutes pre heating, the program asks to add the substrate.
Rapidly transfer the reagent in a new and clean plastic reagent reservoir. Open
the reader, pipet 100µL of reagent using the multichannel and rapidly transfer on
the plate while it is still on the reader. NB: Do not take the plate out of the
reader.
12. Once all the substrate is loaded, leaver the plate without the lid and close the
cover of the reader. Run the assay. DO not open the cover while the assay is
running. Wait a few minutes to check that the readings are going fine. The assay
typically lasts for 1:15 h
a. Bang, F.B. A bacterial disease of Limulus polyphemus. Bull. Johns Hopkins Hosp. 98:325 (1956).
b. Levin, J. and F.B. Bang. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull.
Johns Hopkins Hosp. 115:265 (1964).
c. Levin, J. and F.B. Bang. A description of cellular coagulation in the Limulus. Bull Johns Hopkins Hosp.
115:337 (1964).
d. Levin, J. and F.B. Bang. Clottable protein in Limulu s: its localization and kinetics of its coagulation by
endotoxin. Thromb. Diath. Haemorrh. 19:186 (1968).
e. Solum, N.O. Some characteristics of the clottable protein of Limulus Polyphemus blood cells. Thromb.
Diath. Haemorrh. 23:170 (1970).
f. Solum, N.O. The coagulogen of Limulus polyphemus hemocytes. A comparison of the clotted and non-
clotted forms of the molecule. Thromb. Res. 2:55 (1973).
g. Young, N.S., J. Levin, and R.A. Prendergast. An invertebrate coagulation system activated by
endotoxin: Evidence for enzymatic mechanism. J. Clin. Invest. 51:1790 (1972).

Addendum
116
Appendix III
CMV
0
500
1000
1500
p = 0.156p = 0.297
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
#SFC/10
6
PBMC
Influenza
0
50
100
Pre-ART IRIS
IRIS+ IRIS-
Pre-ART W2/M1
p = 0.125p = 0.875
#SFC/10
6
PBMC
PPD
0
1000
2000
3000
4000
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
p = 0.031p = 0.109
#SFC/10
6
PBMC
ESAT-6
0
100
200
300
400
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
p = 0.563p = 0.313
#SFC/10
6
PBMC
CFP-10
0
100
200
300
400
500
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
p = 1.000
p = 0.310
#SFC/10
6
PBMC
LPS
0
100
200
300
400
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
p = 0.191
p = 0.015
#SFC/10
6
PBMC
A
B
C
D
E
F
Antigen-specific IFNγ responses in TB-IRIS patients and controls. Dots on these graphs represent IFNγ
spot-forming cells per 106 PBMCs in TB-IRIS patients (IRIS+) and HIV+TB+IRIS-controls (IRIS-) after
stimulation with CMV lysate (A), influenza antigen A (B), LPS (C), PPD (D), ESAT-6 (E) and CFP-
10 (F).
Dots connected with full lines represent matched time points within either TB-IRIS patients (IRIS+
) or
HIV+TB+IRIS- controls (IRIS. Horizontal capped lines represent statistical comparisons between time
points. Due to limited sample availability, the number of patients with both time points available was
limited. The number of patients represented per time point for each patient group were: A, n = 7; B, n =
4; C, n = 14; D, n = 7; E, n = 7; F, n = 7. A Wilcoxon signed-
rank test was used to calculate p values. The
level of sign ificance was set to p < 0.05.

Addendum
117
Appendix IV
100
1000
10000
p = 0.031p = 0.109
Pre-ART IRIS
IRIS+ IRIS-
Pre-ART W2/M1
IL-6 / IL-10
1
10
100
1000 p = 0.938p = 0.813
Pre-ART IRIS
IRIS+ IRIS-
Pre-ART W2/M1
TNFα / IL-10
100
1000
10000 p = 0.562p = 0.219
Pre-ART IRIS
IRIS+ IRIS-
Pre-ART W2/M1
IL-6 / IL-10
10
100
1000 p = 0.844p = 0.219
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2 /M1
TNFα / IL-10
10
100
1000
10000 p = 0.426p = 0.005
Pre-ART IRIS
IRIS+ IRIS-
Pre-ART W2/M1
IL-6 / IL-10
10
100
1000 p = 0.091
p = 0.004
Pre-ART IRIS
IRIS+ IR IS-
Pre-ART W2/M1
TNFα / IL-10
A-LPS-stimulated
B-CMV-stimulated
C
-PPD-stimulated
Pro- to anti-inflammatory ratios of innate cytokine production in TB-IRIS. Dots on these
graphs
represent cytokine ratios in PBMC supernatants after stimulation with LPS (A), CMV
(B) and PPD (C). Dots connected with full lines represent matched time points within either
TB-IRIS patients (IRIS+) or HIV+TB+IRIS- controls (IRIS-
). Horizontal capped lines represent
statistical comparisons between time points. Due to limited sample availability, the number
of patients with both time points available was limited. The number of patients represented
per time point for each patient group were: A, n = 14; B, n = 7; C, n = 6. A Wilcoxon signed-
rank test was used to calculate p values. The level of significance was set to p < 0.05.

Addendum
118
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