Early Depletion of Mycobacterium tuberculosis –Specific T Helper 1 Cell Responses after HIV‐1 Infection

ArticleinThe Journal of Infectious Diseases 198(11):1590-8 · January 2009with30 Reads
Impact Factor: 6.00 · DOI: 10.1086/593017 · Source: PubMed
Abstract

The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection. M. tuberculosis-specific T helper 1 (Th1) cells were studied in HIV negative (n=114) and chronically HIV infected (n=68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin (purified protein derivative) with interferon-gamma ELISPOT and intracellular cytokine staining. In a longitudinal study, the effect of acute HIV infection on M. tuberculosis-specific Th1 cells was determined by polychromatic flow cytometric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV. In tuberculosis (TB)-asymptomatic subjects (i.e., subjects with unknown TB status who did not show clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with detectable M. tuberculosis-specific Th1 cells (P< .001) a decrease which was not observed among subjects with active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis-specific Th1 cells in 4 subjects remained TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative (P< .01). Taken together, these data suggest a mechanism of rapid M. tuberculosis-specific Th1 cell depletion that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV infected.

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Available from: Michael Hoelscher
Early Depletion of Mycobacterium
tuberculosis–Specific T Helper 1 Cell Responses
after HIV-1 Infection
Christof Geldmacher,
1,a
Alexandra Schuetz,
2,a
Njabulo Ngwenyama,
1
Joseph P. Casazza,
1
Erica Sanga,
2
Elmar Saathoff,
4
Catharina Boehme,
4,7
Steffen Geis,
2,4
Leonard Maboko,
2
Mahavir Singh,
6
Fred Minja,
3
Andreas Meyerhans,
5
Richard A. Koup,
1
and Michael Hoelscher
4
1
Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland;
2
National Institute for Medical Research, Mbeya Medical Research Programme, Referral Hospital, and
3
Mbeya Regional Medical Office,
National Tuberculosis and Leprosy Programme, Mbeya, Tanzania;
4
Department of Infectious Diseases and Tropical Medicine, Klinikum of the
Ludwig-Maximilians-University of Munich,
5
Institute of Virology, University of Saarland, Homburg, and
6
Lionex Diagnostics and Therapeutics,
Braunschweig, Germany; and
7
Foundation for Innovative New Diagnostics, Cointrin, Switzerland
Background. The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immu-
nodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to
better understand the mechanism underlying M. tuberculosis–specific pathogenicity early after onset of HIV infection.
Methods. M. tuberculosis–specific T helper 1 (Th1) cells were studied in HIV negative (n 114)and chronically
HIV infected (n 68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin
(purified protein derivative) with interferon-
ELISPOT and intracellular cytokine staining. In a longitudinal study,
the effect of acute HIV infection on M. tuberculosis–specific Th1 cells was determined by polychromatic flow cyto-
metric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV.
Results. In tuberculosis (TB)–asymptomatic subjects (i.e., subjects with unknown TB status who did not show
clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with
detectable M. tuberculosis–specific Th1 cells (P .001), a decrease which was not observed among subjects with
active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis–specific Th1 cells in 4 subjects remained
TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative
(P .01).
Conclusions. Taken together, these data suggest a mechanism of rapid M. tuberculosis–specific Th1 cell depletion
that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV
infected.
HIV infection is characterized by a progressive depletion
of CD4
T cells that eventually leads to AIDS, as defined
by the onset of opportunistic infections. The mechanism
of the pathogenesis underlying HIV-associated immune
damage may differ between the acute and chronic phases
of infection. It is widely accepted that ongoing viral rep-
lication and virus-induced cell death are responsible for
the massive depletion of memory CD4
T cells from
mucosal sites during acute simian immunodeficiency vi-
rus (SIV) infection and HIV infection [1–5]. Yet, despite
this rapid depletion of memory CD4
T cells early dur-
ing HIV infection, most opportunistic infections typi-
cally cause complications only after extended periods of
HIV disease progression. Few pathogens cause disease
early after onset of HIV infection. One such pathogen of
high clinical relevance is the acid-fast bacillus (AFB) My-
cobacterium tuberculosis. During the first year of coinfec-
tion with HIV and M. tuberculosis, the risk of developing
active tuberculosis (TB) increases dramatically [6, 7]. TB
Received 2 May 2008; accepted 23 June 2008; electronically published 27
October 2008.
Potential conflicts of interest: none reported.
Financial support: European Commission, Directorate General XII, International
Cooperation–Developing Countries (grant ICA-CT-2002–10048 to M.H.).
a
Present affiliation: Henry M. Jackson Foundation for the Advancement of
Military Medicine, Walter Reed Program, Mbeya Referral Hospital, Mbeya, Tan-
zania (A.S.); Department of Infectious Diseases and Tropical Medicine, Klinikum
der Ludwig-Maximilians-Universitaet, Munich, Germany.
Reprints or correspondence: Dr. Christof Geldmacher, Department of Infectious
Diseases and Tropical Medicine, Klinikum der Ludwig-Maximilians-Universitaet,
Leopoldsstr. 5, 80802 Munich, Germany (geldmacher@lrz.uni-muenchen.de).
The Journal of Infectious Diseases 2008; 198:15908
© 2008 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2008/19811-0004$15.00
DOI: 10.1086/593017
MAJOR ARTICLE
1590
JID 2008:198 (1 December)
Geldmacher et al.
Page 1
disease occurs in HIV-infected persons at all CD4
T lympho
-
cyte counts [7], and especially in developing nations, pulmonary
TB frequently is the first manifestation of AIDS [7], suggesting
that the pathology of M. tuberculosis infection in HIV-positive
individuals may differ from that observed for most other oppor-
tunistic infections.
M. tuberculosis commonly causes latent infection in the lungs
that is tightly controlled by the M. tuberculosis–specific cellular im-
mune response in healthy individuals but results in disease during
periods of immunosuppression. Secretion of interferon (IFN)–
by
CD4
Th1 cells can activate M. tuberculosis–infected macrophages
and contribute to the containment of the intraphagosomal patho-
gen [8, 9]; hence, these cells are important in the control of M.
tuberculosis infection. Optimal activation of infected macrophages
also may require involvement of costimulatory cell-surface proteins
[10] and therefore is dependent on cellular contact with M. tuber-
culosis–specific lymphocytes. Interestingly, the same interactions
also contribute to maximal HIV-1 replication inside alveolar mac-
rophages [11] and may lead to efficient transmission of HIV from
macrophages coinfected with M. tuberculosis and HIV to M. tuber-
culosis–specific CD4
T cells. However, despite the fact that M. tu
-
berculosis–specific immunity is apparently greatly suppressed in in-
dividuals coinfected with both pathogens, the effects of HIV
infection on M. tuberculosis–specific CD4
T cells are not well un
-
derstood. M. tuberculosis–specific CD4
T cell responses are present
in subjects with pulmonary TB regardless of HIV infection status
[11, 12], yet HIV infection is associated with a reduced delayed-type
hypersensitivity reaction to the tuberculin skin test; the latter obser-
vation suggests that HIV affects M. tuberculosis–specific Th1 cell
responses in vivo.
The region of difference 1 (RD1) antigens, early secreted an-
tigenic target 6 (ESAT6) protein and culture filtrate protein 10
(CFP10), are more specific for M. tuberculosis than tuberculin
(purified protein derivative [PPD]) and have more recently been
used to identify M. tuberculosis–specific cellular immune re-
sponses [12–16]. The ESAT6 and CFP10 antigens, especially in
combination with PPD, are therefore suitable for more detailed
study of M. tuberculosis–specific CD4
T cell responses and the
effect of HIV on these responses.
To investigate the mechanisms underlying the early onset of pul-
monary TB often observed after HIV infection, we studied the effect
of chronic HIV infection on M. tuberculosis–specific Th1 cell re-
sponses in TB-asymptomatic subjects (i.e., subjects with unknown
TB status who did not show clinical signs suggestive of TB) and
subjects with active pulmonary TB. We further dissected the events
occurring early after HIV infection onset by following up 5 women
from a cohort of commercial sex workers in Tanzania who had
latent M. tuberculosis infection before they became infected with
HIV. We analyzed the M. tuberculosis–specific CD4
T cell response
during and up to 12 months after HIV seroconversion in these 5
women and determined their clinical outcome by means of a final
interview that took place 3 years after the last study follow-up visit.
SUBJECTS, MATERIALS, AND METHODS
Study subjects. For analysis of M. tuberculosis–specific Th1 cell
responses, volunteers were recruited from 3 ongoing studies that
have been described elsewhere [17–19]. This substudy received a
separate ethical clearance from the local and national institutional
review boards, and a new consent form was signed by the volunteers
in case additional specimens were obtained. For cross-sectional
analysis of M. tuberculosis–specific Th1 cell responses, 14 patients
who had positive AFB smear results and clinical symptoms of pul-
monary TB (defined as productive cough for 4 weeks, night
sweats, weight loss, and loss of appetite) were recruited from an
ongoing TB diagnostic trial [18]. During the study, all participants
received health care for acute medical problems. Patients with diag-
nosed TB were referred for TB treatment that accorded with World
Health Organization standards. During the course of this study, all
individuals were antiretroviral naive. TB was diagnosed on the basis
of positive AFB staining results for 3 independent sputum samples
and a clinical diagnosis. Latent infection with M. tuberculosis was
defined on the basis of cellular responses to RD1 antigens in the
absence of clinical symptoms suggestive of TB. HIV infection status
was determined by using 2 diagnostic HIV-specific enzyme-linked
immunoassays (Enzygnost Anti HIV1/2 Plus [Dade Behring] and
Determine HIV 1/2 [Abbott]). CD4
cell counts were determined
in fresh whole blood samples by use of the BD multitest kit (Becton
Dickinson).
Antigens. ESAT6 and CFP10 antigens (Lionex) and PPD for
in vitro testing (Statens Serum Institut) were used at a final con-
centration of 10
g/mL. Peptides that overlapped by 11 amino
acids were designed for ESAT6 (AF420491.1) and CFP10
(AAC83445) by using the PeptGen peptide generator from the
HIV Molecular Immunology Database (available at: http://
www.hiv.lanl.gov/content/immunology/).
IFN-
ELISPOT assays. Freshly isolated peripheral blood
mononuclear cells (PBMCs) were screened for responses spe-
cific for recombinant ESAT6 and PPD by stimulation of
2.5 10
5
PBMCs/well in duplicate overnight. The assay was
performed as previously described [20]. Responses with 20
spot-forming cells (sfc) per 10
6
PBMCs and 3 times the value
for the negative control sample were scored as positive. Discor-
dant findings, defined as results in which only 1 of the duplicate
wells was scored as positive, were excluded from analysis.
Conjugated antibodies for flow cytometric analysis. The
following antibodies were used: CD3-Cy7-allophycocyanin, IFN-
–fluorescein isothiocyanate, and CCR5-Cy7-phycoerythrin (PE)
(BD Biosciences); CD27-Cy5PE and CD45RO–Texas red–PE
(Beckman Coulter); and CD4-PECy5.5 (Caltag). The following an-
tibodies were conjugated in-house in accordance with standard
protocols (available at: http://drmr.com/abcon/index.html): CD8
quantum dot 655 and tumor necrosis factor (TNF)–
–Alexa680.
Stimulation and flow cytometric analysis of PBMCs. Cell
stimulation and staining were performed by using a modified
HIV and Loss of M. tuberculosis–Specific Th1 Cells
JID 2008:198 (1 December)
1591
Page 2
version of a method described elsewhere [21]. After6hofstim-
ulation, PBMCs were washed once with PBS and stained with
Vivid (Molecular Probes) [22] and anti-CCR5 for 10 min at
room temperature in the dark. After washing, surface proteins
were stained for 20 min. The cells were washed again and per-
meabilized using the Cytofix/Cytoperm kit (BD Biosciences).
After intracellular staining, cells were washed and fixed with 1%
paraformaldehyde. Cells were analyzed with a modified flow cy-
tometer (LSR II; BD Immunocytometry Systems). Electronic
compensation was conducted with antibody capture beads (BD
Biosciences) stained separately with the individual antibodies
used in the test samples. Flow cytometry data were analyzed us-
ing FlowJo (version 8.2; TreeStar).
Statistical analysis. Statistical data analyses were per-
formed using Prism software (GraphPad). The tests used for
statistical analysis are mentioned in the figure legends.
RESULTS
Study population. A total of 182 subjects were tested in a
cross-sectional study with an IFN-
ELISPOT assay for recogni-
tion of recombinant ESAT6 protein. Table 1 summarizes CD4
cell counts, the number of M. tuberculosis–specific CD4
T cells
detected, and the results of AFB staining for HIV-negative and
HIV-positive TB-asymptomatic subjects and for HIV-negative
and HIV-positive subjects with pulmonary TB.
HIV infection is associated with significantly decreased M.
tuberculosis–specific CD4
T cell responses in individuals
with no clinical signs of TB. HIV infection was associated
with a significant decrease in the frequency of detectable IFN-
ELISPOT responses to ESAT6 in patients with no clinical signs of
TB (P .001, by Fisher’s exact test). Whereas 50 (47%) of 106
HIV-negative, TB-asymptomatic subjects responded to ESAT6
(5 subjects were excluded because of discordant results between
duplicate wells), only 10 (19%) of 54 of HIV-positive, TB-
asymptomatic subjects had detectable responses (3 subjects were
excluded because of discordant results between duplicate wells).
Intracellular cytokine staining demonstrated that detectable
IFN-
responses were mediated by CD4
T cells (figure 1
).
Comparison of the overall median magnitude of responses for
106 HIV-negative subjects (18 sfc/10
6
PBMCs) with that for 54
HIV-positive subjects (2 sfc/10
6
PBMCs) revealed that HIV in
-
fection was associated with a 9-fold reduction in the number of
M. tuberculosis–specific CD4
T cells (P .01) (figure 2A
).
PPD-specific responses followed an identical trend (P .001)
(figure 2C). Contrary to our expectation, there was no linear
correlation between M. tuberculosis–specific CD4
T cells and
the total CD4
T cell count (figure 2B and 2D). Taken together,
these results demonstrate that at least half of the study popula-
tion from the Mbeya region probably had latent M. tuberculosis
infection, and HIV infection was greatly decreasing the M. tu-
berculosis–specific Th1 cell responses in these subjects.
In HIV-infected individuals, active TB is associated with de-
tectable ESAT6 responses. To study the differences between in-
dividuals with latent TB and those with active TB, we measured the
level of ESAT6-specific Th1 cells in HIV-negative and HIV-positive
individuals who had a positive AFB smear result. In line with the
results of a previous report [12], 10 (91%) of 11 HIV-positive pa-
tients with pulmonary TB had detectable ESAT6 responses, with a
median of 118 sfc/10
6
PBMCs (figure 2A). In agreement with our
previous observation, detection of M. tuberculosis–specific CD4
T
cell responses did not correspond to the total CD4
cell count.
Three (100%) of 3 HIV-negative patients with pulmonary TB re-
sponded to ESAT6. Taken together, our results suggest that the M.
tuberculosis–specific Th1 cells present during latent infection are
depleted after HIV infection and their level increases only after M.
tuberculosis reactivation or de novo infection, independent of the
total CD4
T cell count. We therefore hypothesize that detection of
M. tuberculosis–specific Th1 cells in HIV-positive individuals is a
marker for immune exposure to new M. tuberculosis antigen and
therefore indicates at least transient M. tuberculosis reactivation.
Table 1. Summary of laboratory findings for 4 groups of subjects stratified by HIV infection status and tuberculosis (TB) status.
Group
Subjects
enrolled,
no.
CD4
cell count
ESAT6 response PPD response
Subjects
positive for
AFB, no.
Median
(range),
cells/ mL
Subjects
evaluated,
no.
Median
(range)
a
Subjects
with
response, %
Median
(range)
a
Subjects
with
response, %
HIV negative,
TB asymptomatic 111 851 (529–1214) 29 17 (0–2584) 47 46 (0–2186) 68 ND
HIV positive,
TB asymptomatic 57 300 (20–905) 56 2 (0–1852) 19 4 (0–1518) 31 ND
HIV positive,
pulmonary TB 11 119 (64–501) 9 118 (18–2164) 91 280 (106–2746) 100 11
HIV negative,
pulmonary TB 3 815 (361–1401) 3 122 (52–128) 100 380 (192–996) 100 3
NOTE. AFB, acid-fast bacilli; ESAT6, early secreted antigenic target 6; PPD, purified protein derivative.
a
No. of spot-forming cells per 10
6
peripheral blood mononuclear cells.
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Geldmacher et al.
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High levels of HIV coreceptor CCR5 expression on M. tu-
berculosis–specific Th1 cells. Most newly transmitted HIV
viruses infect cells that express surface viral receptors CD4
and CCR5 [23]. To study whether M. tuberculosis–specific
Th1 cells are potentially susceptible to viral infection during
primary HIV infection, we next analyzed CCR5 expression in
9 HIV-negative subjects with latent M. tuberculosis infection
and compared these cells (gated in figure 3A) to different
CD4
T cell subsets defined by their expression of the T cell
memory markers CD27 and CD45RO (figure 3C). Naive
CD4
T cells (CD27
CD45RO
) that did not express CCR5
were used to determine cutoff values (figure 3B). As expected,
a higher fraction of CD27
CD45RO
CD4
T cells (median,
50.7%) expressed CCR5, compared with CD27
CD45RO
CD4
T cells (median, 18.7%). A median of 58.8% of M.
tuberculosis–specific Th1 cells (range, 29.4%–69.3%) ex-
pressed CCR5, constituting a 2-fold increase compared with
total memory CD4
T cells (median, 27.9%). The level of
CCR5 cell surface expression was also significantly increased
(P .01). Taken together, these data demonstrate that a large
fraction of M. tuberculosis–specific Th1 cells from subjects with
latent M. tuberculosis infection express the cellular receptors re-
quired for HIV entry for most newly transmitted viruses.
Rapid depletion of M. tuberculosis–specific Th1 cells dur-
ing early during HIV infection. Rapid depletion of memory
CD4
T cells is a hallmark of acute SIV and HIV infection
[1–5]. Despite this significant decrease of memory CD4
T
cells, Candida albicans–specific, CMV-specific, and tetanus
toxoid–specific CD4
T cells can usually still be detected early
in HIV infection [24, 25]. In particular, the lack of correlation
between M. tuberculosis–specific Th1 cells and the total CD4
cell count led us to hypothesize that M. tuberculosis–specific
CD4
T cells are depleted early in HIV infection. To clarify
this, we studied the dynamics of RD1-specific and PPD-
Figure 1. CD4
T cell interferon (IFN)–
responses detected after in vitro stimulation with purified protein derivative (PPD) and recombinant early
secreted antigenic target 6 (ESAT6) protein. Shown are representative dot plots from 1 study participant for the detection of IFN-
and activation marker
CD69 within the CD4
T cell (left panels) and CD8
T cell (right panels) subsets after6hofstimulation of whole blood and intracellular cytokine staining
for IFN-
.
HIV and Loss of M. tuberculosis–Specific Th1 Cells
JID 2008:198 (1 December)
1593
Page 4
specific CD4
T cell responses in 5 subjects with latent TB
who acquired HIV infection during the HIV Superinfection
Study [19].
Before HIV infection, Th1 cell responses targeting either
one of the RD1 peptide sets (ESAT6 or CFP10) and PPD were
detected in all 5 subjects (figure 4). As expected, during the
last HIV-seronegative follow-up visit, RD1-specific responses
(range, 0.09%– 0.21% of memory CD4
T cells; background
subtracted) and PPD-specific responses (range, 0.1%– 0.8%
of memory CD4
T cells) were of relatively low magnitude.
Although the RD1 peptide sets were specifically designed to
study M. tuberculosis–specific CD8
T cell responses, none
were detected in these or any other HIV-negative subjects
with latent M. tuberculosis infection who were tested through-
out this study. In 4 of 5 subjects with latent infection, M.
tuberculosis–specific responses were rapidly depleted within
the first year after HIV seroconversion. In contrast, M. tuber-
culosis–specific Th1 cell responses did not fluctuate notably in
subjects with latent M. tuberculosis infection, who remained
HIV seronegative (P .01 [data not shown]). Importantly,
none of these 4 subjects received a diagnosis of active TB within
4 years after HIV infection.
The fifth subject (H19) had the most dramatic decrease in CD4
cell counts (a 58% decrease from 810 cells/
L to 343 cells/
L) and a
high viral load continuously 100,000 RNA copies/mL. In this sub-
ject, M. tuberculosis–specific Th1 responses increased after HIV in-
fection and in particular, the population of PPD-specific CD4
T
cells expanded dramatically 1 year after HIV seroconversion (figure
4). In addition, a strong CD8
T cell response targeting CFP10
emerged after HIV infection, reaching 0.57% of CD8
T cells. Sub
-
Figure 2. Chronic HIV infection and Th1 cell responses to early secreted antigenic target 6 (ESAT6) and purified protein derivative (PPD) in
tuberculosis (TB)–asymptomatic subjects and subjects with pulmonary TB. Shown are the spot-forming cells per 10
6
peripheral blood mononuclear cells
(PBMCs) responding to recombinant ESAT6 protein (A) and PPD (C) for each subject and the corresponding total CD4
cell counts for HIV-infected
individuals in (B and D). Blue circles, HIV-negative, TB-asymptomatic subjects (n 110); red circles, HIV-negative subjects with pulmonary TB (n 3);
blue diamonds, HIV-infected, TB-asymptomatic subjects (n 55); red diamonds, HIV-positive subjects with pulmonary TB (n 11); horizontal black
lines, median values. The cutoff value was 20 sfc/10
6
PBMCs (red dotted line). Statistical analysis for panels A and C was performed by using the
Mann-Whitney test; for panels B and D, the Spearman rank test was used.
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Geldmacher et al.
Page 5
ject H19 received a diagnosis of active TB, and was treated for it,
within 15 months after HIV seroconversion.
DISCUSSION
The present study was primarily designed to dissect the under-
lying mechanism associated with the dramatic increase in the
risk of developing pulmonary TB shortly after HIV infection for
subjects who are latently infected with M. tuberculosis.Wead-
dressed the following 3 questions: (1) Does chronic HIV infec-
tion affect M. tuberculosis–specific cellular immunity in individ-
uals with latent M. tuberculosis infection? (2) Do these cells
express the HIV coreceptor CCR5? (3) How does acute HIV
infection affect the M. tuberculosis–specific cellular immune re-
sponses?
The high prevalence of ESAT6-specific responses observed
among HIV-negative, TB-asymptomatic subjects suggests
that at least half of the study population from the Mbeya
region was latently infected with M. tuberculosis. However,
the absence of these cells in many HIV-positive subjects does
not exclude latent M. tuberculosis infection, as demonstrated
by the detection of M. tuberculosis–specific Th1 cell responses
before HIV infection and their disappearance thereafter.
Taken together, these data suggest that typically, coinfected
individuals in TB-endemic regions are already latently in-
fected with M. tuberculosis before HIV infection. Although
not a sufficient cause, this may contribute to the early mani-
festation of TB associated with HIV infection.
Chronic HIV infection was associated with a reduced per-
centage of subjects who responded to M. tuberculosis antigen,
indicating that cellular immunity to M. tuberculosis was re-
duced by chronic HIV infection. The rapid depletion of M.
tuberculosis–specific responses early after HIV infection and
their presence in HIV-positive subjects with pulmonary TB
suggests that the population of these cells (re)expands after
ongoing or transient reactivation of mycobacterial growth or
de novo exposure. This interpretation is further supported by
2 recent studies [26, 27].
Our results support a scenario in which early M. tuberculosis
specific Th1 cell depletion could be caused by direct HIV infec-
tion. Active HIV viral replication indeed is a potent suppressor
of M. tuberculosis–specific Th1 cell responses, as demonstrated
by the dramatic expansion of this cell population observed after
initiation of antiretroviral therapy [28, 29]. High levels of surface
expression of the viral coreceptor CCR5 should contribute to
direct HIV infection. PPD antigen alone can trigger produc-
tive HIV-1 infection of CD4
T cells from HIV-1–positive
Figure 3. Proportion of Mycobacterium tuberculosis–specific Th1 cells expressing HIV coreceptor CCR5. Shown is the cell surface expression of CCR5
(B) on gated interferon (IFN)–
positive CD4
T cells (A) detected after6hofstimulation with region of difference 1 peptides. C and D, the proportion
of CCR5
CD4
T cells in different CD4
T cell subsets for 9 HIV-negative subjects who were latently infected with M. tuberculosis. Statistical analysis
was performed by using the Wilcoxon matched pairs test.
HIV and Loss of M. tuberculosis–Specific Th1 Cells
JID 2008:198 (1 December)
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Page 6
cocultured dendritic cells in vitro [30], and it is sufficient to
induce HIV replication and cell death in PBMCs from HIV-
positive subjects who have positive tuberculin skin test results
[31]. Alveolar macrophages are easily infected with HIV in
vitro and are also infected in vivo during the acute and final
phase of SIV infection [32]. In addition, anti–M. tuberculosis
cellular immunity maximizes HIV replication inside alveolar
macrophages [11], which could enhance M. tuberculosis
specific” HIV transmission to M. tuberculosis–specific Th1
cells. A state of complete metabolic quiescence during latent
M. tuberculosis infection may undermine this specific mode of
transmission. However, it has been suggested that rather than
being caused by metabolic quiescence, M. tuberculosis latency
is the result of a “continuous cross talk between the host im-
mune system and the persisting pathogens,” as indicated by
active sites of cell proliferation and follicle-like structures
within affected parts of the lungs from latently M. tuberculosis
infected individuals [33, p. 89]. The high viral load found in
bronchoalveolar lavage (BAL) fluid from sites of active TB
disease, but not from unaffected sites [34, 35], further sup-
Figure 4. Depletion of Mycobacterium tuberculosis–specific Th1 cells early during HIV infection. Shown are the frequencies of purified protein
derivative (PPD)–specific (upper panel) and region of difference 1–specific (lower panel) CD4
T cells as a percentage of memory CD4
T cells, for each
HIV -infected subject studied (H228, H590, H140, H80, and H19). The upper left set of panels show representative dot plots after stimulation with
medium alone or early secreted antigenic target 6 (ESAT6). All other panels show the percentage of M. tuberculosis–specific memory CD4
T cells
detected before and after HIV infection (red arrow) for 5 subjects who became HIV infected. The percentage of memory CD4
T cells was determined
by CD27 and CD45RO expression. Naive CD4
T cells (CD27
CD45
cells) were excluded from analysis of memory CD4
T cells. Interferon (IFN)–
positive memory CD4
T cells were detected after overnight stimulation with PPD, ESAT6, or culture filtrate protein 10 (CFP10). The background was
subtracted.
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Page 7
ports the hypothesis that the proinflammatory microenviro-
ment caused by local M. tuberculosis infection enhances HIV
transmission to M. tuberculosis–specific CD4
T cells in vivo.
The importance of Th1 cytokines in mediating protection from
TB is well documented [36, 37]. Rapid depletion of M. tuberculosis
specific Th1 cells early in HIV infection could therefore be key to the
tremendous increase in the risk of developing TB. In contrast,
CMV-specific CD4
T cells persist until the late stages of HIV in
-
fection [25], and typically, CMV-associated pathology does not oc-
cur until the very late stages of AIDS, further supporting our hy-
pothesis that rapid depletion of M. tuberculosis–specific CD4
T
cells is important in the specific pathologic interactions of M. tuber-
culosis and HIV. However, despite the depletion of this important T
cell subset, only a minority of coinfected subjects develop TB soon
after HIV infection. Specific CD8
T cells, nonconventional T cell
subsets, or alternative sources of IFN-
and TNF-
, such as NK
cells, may compensate for some of the effector functions of the M.
tuberculosis–specific Th1 cells [8, 38]. In addition, Th17 cells that do
not produce IFN-
or TNF-
have been shown to participate in M.
tuberculosis–specific immunity [39]. Such responses may still be
sufficient in most cases to control M. tuberculosis after depletion of
M. tuberculosis–specific Th1 cells. Alternatively, host genetic poly-
morphisms affecting anti–M. tuberculosis immunity could account
for differences in susceptibility to disease. Finally, differences in
pathogen load or virulence may also contribute to different TB dis-
ease outcomes in subjects coinfected with HIV and M. tuberculosis.
Interestingly, early in HIV infection the pulmonary and nondis-
seminated form of TB predominates, whereas in patients with AIDS
who have very low CD4
cell counts, disseminated and extrapul
-
monary TB disease is frequently observed, indicating that even in
such subjects there is residual anti–M. tuberculosis immunity left
after acute HIV infection.
In conclusion, our results demonstrate that acute HIV in-
fection is associated with the rapid loss of M. tuberculosis
specific Th1 cells in the peripheral blood. This contrasts with
the gradual decline of the total CD4
cell count during the
chronic phase of HIV infection and may be caused by direct
HIV infection of these cells. Taken together, these data sug-
gest a mechanism of rapid M. tuberculosis–specific Th1 cell
depletion that may contribute to the early onset of TB that is
often observed in latently infected individuals who become
HIV infected.
Acknowledgments
We thank all of the HIV Superinfection Study (HISIS) participants and
the excellent staff at the Mbeya Medical Research Programme that con-
ducted the HISIS study, especially Weston Assisya, Frowin Nichombe,
Clemence Konkamkula, and Vera Kleinfeldt. Furthermore we would espe-
cially like to thank Natanya Sandler, Philip Scheinberg, and Jason Brenchley
(Vaccine Research Center, National Institutes of Health) for many exciting
discussions during the writing of the manuscript.
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