Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ).
ABSTRACT Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: We have recently described the first true genome-wide screen for CD4(+) T-cell reactivity directed against Mycobacterium tuberculosis (MTB) in latent TB-infected individuals. The approach relied on predictions of HLA-binding capacity for a panel of DR, DP, and DQ alleles representative of those most commonly expressed in the general population, coupled with high throughput ELISPOT assays. The results identified hundreds of novel epitopes and antigens, and documented the novel observation that T cells in latent MTB infection are confined to the CXCR3(+)CCR6(+) phenotype and largely directed against three antigenic "islands" within the MTB genome. In parallel, we have made generally available to the scientific community the technical approaches and reagents developed in the process, such as motifs, algorithms, and binding assays for several common HLA class II alleles, and a panel of single allele HLA class II transfected cell lines representative of the most frequent specificities in the general population. Recent efforts have been focused on characterization of epitopes and antigens recognized by patients with active TB and individuals vaccinated with BCG, with the aim of providing the first systematic evaluation of the overlap between latent, active, and BCG cohorts. The definition of a broad range of epitopes restricted by common HLA molecules, will facilitate development of diagnostic reagents, allow a rigorous evaluation of T-cell responses associated with TB infection in humans, and enable the evaluation of the immunogenicity of different vaccine candidates. Furthermore, it might suggest new candidates for vaccine and diagnostic development.Frontiers in Immunology 01/2014; 5:124.
- [Show abstract] [Hide abstract]
ABSTRACT: In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4(+) T cells as important contributors. In this study we show that TB-specific CD4(+) T cells have a characteristic chemokine expression signature (CCR6(+)CXCR3(+)CCR4(-)), and that the overall number of these cells is significantly increased in LTBI donors compared with healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6(+)CXCR3(+)CCR4(-) cells and found significant differences to conventional Th1, Th17, and Th2 cells, but no major changes between healthy and LTBI donors. CCR6(+)CXCR3(+)CCR4(-) cells display lineage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program, including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6(+)CXCR3(+)CCR4(-) cells reveals characteristics important for controlling latent TB infections.The Journal of Immunology 08/2014; · 5.36 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Tuberculosis remains a major global health problem worldwide, and hence there is a need for novel vaccines that better induce cellular-mediated immunity (CMI). In search of a better vaccine target, the QuantiFERON-TB Gold In-Tube Test (QFT-GIT) and the interferon-γ ELISPOT assay (ELISPOT) were used to compare the magnitude of CMI in patients. Results of the ELISPOT assay led to the discovery of specific epitopes within the early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) proteins. Both peptides showed a strong association with several HLA class II DRB1 molecules in the Japanese population. Using ESAT-6-specific HLA class II tetramers, we determined that the expression of ESAT-6-specific CD4+ lymphocytes was significantly decreased in treated patients compared with active patients. In addition, programmed death-1 (PD-1)/killer cell lectin-like receptor G1 (KLRG-1) double positive cells were found only in treated patients and not in those with active TB. These data could provide clues for the development of novel tuberculosis vaccines.Journal of Immunology Research 01/2014; 2014:764028. · 2.93 Impact Factor
The Journal of Immunology
Dissecting Mechanisms of Immunodominance to the Common
Tuberculosis Antigens ESAT-6, CFP10, Rv2031c (hspX),
Rv2654c (TB7.7), and Rv1038c (EsxJ)
Cecilia S. Lindestam Arlehamn,* John Sidney,* Ryan Henderson,* Jason A. Greenbaum,*
Eddie A. James,†Magdalini Moutaftsi,‡Rhea Coler,‡Denise M. McKinney,* Daniel Park,x
Randy Taplitz,xWilliam W. Kwok,†Howard Grey,* Bjoern Peters,* and Alessandro Sette*
Diagnosis of tuberculosis often relies on the ex vivo IFN-g release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB.
However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we
investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic
target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were
tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in
prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early
secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate
patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity
was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence
in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective
restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent.
Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recog-
nizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results
highlight the feasibility of more clearly defined TB diagnostic reagent.
ported in 2010. The vast majority (90–98%) of infected individ-
uals are able to contain the infection asymptomatically, resulting
in latent infection. However, 2–10% will develop active TB in
their lifetime, resulting in further spread of the disease (1).
Current diagnosis of TB relies on the conventional immunologic
assay, the tuberculin skin test (TST). This test is primarily useful in
ruling out TB infection, but it cannot reliably distinguish active TB
infection from recovered TB, latent TB, Mycobacterium bovis
bacillus Calmette-Gue ´rin (BCG) vaccination, or infection with
nontuberculous mycobacteria. A major advance in distinguishing
prior or current infection with Mycobacterium tuberculosis from
The Journal of Immunology, 2012, 188: 5020–5031.
uberculosis (TB) is one of the major causes of death from
infectious disease worldwide, claiming 1.4 million lives in
2010. In addition, ∼9 million new cases of TB were re-
BCG vaccination and some of the nontuberculous mycobacte-
rial diseases occurred with the introduction of ex vivo analysis
of peripheral blood cells for responses to two M. tuberculosis-
specific Ags, early secreted antigenic target 6 kDa (ESAT-6)
(Rv3875) and culture filtrate protein 10 kDa (CFP10) (Rv3874).
These Ags are located in the region of difference 1, which is ab-
sent from M. bovis BCG and the majority of environmental my-
cobacteria, resulting in their diagnostic specificity (2–4). The
IFN-g release assays (IGRAs) T-SPOT.TB and QuantiFERON-TB
Gold In-Tube both use ESAT-6 and CFP10. These Ags have been
studied extensively (3, 5–16) and are often used as tools to ex-
amine M. tuberculosis-specific immune responses. In addition to
ESAT-6 and CFP10, QuantiFERON-TB Gold In-Tube also uses
the Rv2654c (TB7.7) Ag.
The goal of this study was to examine responses by latently
infected individuals to a set of TB Ags commonly used for di-
agnostic purposes. Importantly, we were also interested in deter-
If not, we wanted to examine the concept that inclusion of defined
epitopes derived from Ags present in IGRAs or other Ags might be
an avenue to enhance the performance of diagnostic tests reliant
on the use of these same common Ags. As representative Ags, we
chose Rv2031c (hspX, 16 kDa) and Rv1038c (EsxJ). On the basis
of the results of this initial study, we expected to provide a platform
for a much broader analysis with multiple Ags and even perform
a genome-wide screen. Several Ags encoded by the DosR regulon
have been described as preferentially recognized by individuals
with latent infection (17–20). One of these Ags, Rv2031c, has
been studied in detail, and several T cell epitopes have been de-
scribed previously (20–23). The Rv2031c Ag has been shown to
be predominantly expressed when M. tuberculosis is subjected to
*Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology,
La Jolla, CA 92037;†Benaroya Research Institute at Virginia Mason, Seattle, WA
98101;‡Infectious Disease Research Institute, Seattle, WA 98104; andxAntiviral
Research Center, University of California, San Diego, San Diego, CA 92103
Received for publication December 9, 2011. Accepted for publication March 17,
This work was supported by National Institutes of Health Contract N01-AI-900044C
Address correspondence and reprint requests to Dr. Alessandro Sette, Division of
Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena
Circle, La Jolla, CA 92037. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: CFP10, culture filtrate protein 10 kDa; ESAT-6,
early secreted antigenic target 6 kDa; IEDB, Immune Epitope Database; IGRA, IFN-
g release assay; LTBI, latent tuberculosis infection; SFC, spot-forming cell; TB,
tuberculosis; TST, tuberculin skin test.
oxygen deprivation in vitro (24, 25), conditions thought to be
encountered by M. tuberculosis in vivo when persisting in an
immunocompetent host (26).
Several other proteins from TB have been identified as Ags, in-
cluding members of the ESAT-6 protein family (27). One of these
Ags, Rv1038c (EsxJ), has been previously identified as an immu-
nodominant Ag (28, 29) and exhibits a high degree of homology
with other members of the ESAT-6 protein family such as Rv3620c
(EsxW), Rv2347c (EsxP), Rv1197 (EsxK), and Rv1792 (EsxM).
it possible to investigate responses ex vivo, precluding the need
for in vitro stimulation and expansion of the cells. The capacity
to study responses to Ags ex vivo affords probing and an under-
standing of the immunological mechanisms and specificity under-
lying the diagnostic use of the IGRAs.
In the current study, we report a side-by-side comparison of
ESAT-6, CFP10, Rv2031c, Rv2654c, and Rv1038c in latently
infected individuals. By combining an ex vivo T cell response
approach with HLA peptide binding assays and subject HLA
typing, we were able to identify and characterize the molecular
mechanisms influencing their recognition by human T cells. There
are striking differences in recognition when comparing these Ags,
with CFP10 and ESAT-6 being the most dominantly recognized.
Further characterization of dominant antigenic regions revealed
that the same minimal epitope core sequence is recognized by
multiple donors. Furthermore, depending on the specific epitope,
the mechanism of dominance could be attributed to either the
prevalence of a single specific HLA molecule or promiscuous
recognition. These data provide new insights into T cell responses
to commonly used TB Ags and highlight novel differences in im-
munodominance patterns and restrictions.
Materials and Methods
Leukapheresis samples were obtained from 40 adults, 22 with latent TB
infection (LTBI) and 18 control donors from the University of California,
San Diego, Antiviral Research Center clinic (age range 20–65 y). Ethnicity
of subjects studied can be found in Supplemental Table II. Subjects were
initially identified by having a history of a positive TST. Latent TB was
confirmed by a positive QuantiFERON-TB Gold In-Tube (Cellestis, Vic-
toria, Australia) as well as a history, physical examination, and/or chest
x-ray that was not consistent with active TB. None of the study subjects
had been vaccinated with BCG or had laboratory evidence of HIV or
hepatitis B. The control donors had a negative TST as well as a negative
QuantiFERON-TB Gold In-Tube. Approval for all procedures was ob-
tained from the Institutional Review Board (FWA#00000032).
Sets of peptides of 15 aa in length, overlapping by 10 residues, were
synthesized to cover the entire length of Rv3874 (CFP10), Rv3875 (ESAT-
were combined into pools of up to 10 peptides with any given pool con-
taining peptides from only one Ag. For mapping of minimal epitope core
sequences, sets of peptides of 15 aa in length, overlapping by 14 residues,
were synthesized to cover the antigenic region sequences. These peptides
were tested individually.
Peptides were purchased from Mimotopes (Clayton, Victoria, Australia)
and/or A and A (San Diego, CA) as crude material on a small (1 mg) scale.
Peptides used for tetramers were synthesized on a larger scale and purified
(.95%) by reversed-phase HPLC. The Immune Epitope Database (IEDB)
submission identification number for the peptides is 1000489.
MHC molecules were purified from EBV-transformed homozygous cell
lines by mAb-based affinity chromatography, as described in detail else-
where (30). HLA-DR, -DQ, and -DP molecules were captured by a re-
peated passage of lysates over LB3.1 (anti–HLA-DR), SPV-L3 (anti–HLA-
DQ), and B7/21 (anti–HLA-DP) columns.
MHC–peptide binding assays
are based on the inhibitionof binding of a high-affinityradiolabeled peptide
to purified MHC molecules and have been described in detail elsewhere
(30). Briefly, 0.1–1 nM radiolabeled peptide was coincubated at room
temperature or 37˚C with 1 mM to 1 nM purified MHC in the presence of
a mixture of protease inhibitors. Following a 2- to 4-d incubation, the
percentage of MHC-bound radioactivity was determined by capturing
MHC/peptide complexes on LB3.1 (DR), L243 (DR), HB180 (DR/DP/
DQ), SPV-L3 (DQ), or B7/21 (DP) Ab-coated Optiplates (Packard In-
strument, Meriden, CT), and bound counts per minute was measured using
the TopCount (Packard Instrument) microscintillation counter. Under the
conditions used, where [label],[MHC] and IC50$ [MHC], the measured
IC50values are reasonable approximations of the true Kdvalues (31, 32).
PBMC isolation and HLA typing
PBMC were obtained by density gradient centrifugation (Ficoll-Hypaque;
Amersham Biosciences, Uppsala, Sweden) from 100 ml leukapheresis
sample, according to the manufacturer’s instructions. Cells were suspended
in FBS (Gemini Bio-products, Sacramento, CA) containing 10% DMSO,
and cryopreserved in liquid nitrogen for further analysis.
Genomic DNA isolated from PBMC of the study subjects by standard
techniques (QIAmp; Qiagen, Valencia, CA) was used for HLA typing.
High-resolution Luminex-based typing for HLA class I and class II was
used, according to the manufacturer’s instructions (Sequence-Specific Oli-
gonucleotides typing; One Lambda, Canoga Park, CA). Where needed, PCR-
based methods were used to provide high-resolution subtyping. (Sequence-
Specific Primer typing; One Lambda).
Ex vivo IFN-g ELISPOT assay
PBMC were incubated at a density of 2 3 105cells/well and were stim-
ulated with either peptide pools (1 mg/ml) or individual peptides (10 mg/
ml), PHA (10 mg/ml), or medium containing 0.25% DMSO (corresponding
to percent DMSO in the pools/peptides) as a control in 96-well flat-bottom
plates (Immobilon-P; Millipore, Bedford, MA) coated with 10 mg/ml anti–
IFN-g mAb (clone AN18; Mabtech, Stockholm, Sweden). Each peptide
was tested in triplicates. Following a 20-h incubation at 37˚C, the wells
were washed with PBS/0.05% Tween 20 and then incubated with 2 mg/ml
biotinylated IFN-g mAb (clone R4-6A2; Mabtech) for 2 h. The spots were
developed using Vectastain avidin/biotin complex peroxidase (Vector
Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole (Sigma-
Aldrich, St. Louis, MO) and counted by computer-assisted image analysis
(KS-ELISPOT reader; Zeiss, Munich, Germany). The level of statistical
significance was determined with a Student t test using the mean of trip-
licate values of the response against relevant pools or individual peptides
versus the response against the DMSO control. Responses against peptides
were considered positive if the net spot-forming cells (SFC) per 106were
$20, the stimulation index $ 2, and p , 0.05.
Magnetic bead separation
For experiments that use depletion of CD4 or CD8 T cells, these cells were
isolated by magnetic bead positive selection (Miltenyi Biotec, Bergisch
Gladbach, Germany), according to the manufacturer’s instructions, and
effluent cells (depleted cells) were used for experiments.
For all experiments that use purified CD4+T cells, the cells were isolated
by magnetic bead positive selection (Miltenyi Biotec), according to the
To determine the HLA locus restriction of identified epitopes, Ab inhibition
assays were performed. CD4+T cells purified by positive selection together
with effluent cells (at a 2:1 ratio) were incubated with 10 mg/ml Abs
(Strategic Biosolutions, Windham, ME) against HLA-DR (LB3.1), -DP
(B7/21), or -DQ (SVPL3) 30 min prior to peptide addition. IFN-g cytokine
production against positive peptides was then measured in an ELISPOT
assay as described above. The pan-MHC class I Ab (W6/32) was used as
MHC class II tetramers conjugated using PE-labeled streptavidin were
provided by the Tetramer Core Laboratory at the Benaroya Research In-
stitute at Virginia Mason (Seattle, WA). CD4+T cells were purified using
the Miltenyi T cell isolation kit II, according to the manufacturer’s
instructions. Purified cells (∼10 3 106) were incubated in 0.5 ml PBS
containing 0.5% BSA and 2 mM EDTA (pH 8) (MACS buffer) with a
The Journal of Immunology5021
1:50 dilution of class II tetramer for 2 h at room temperature. Cells were
then stained for cell surface Ags using anti–CD4-FITC, anti–CCR7-
APCEFluor780, anti–CD45RA-EFluor450, and anti–CD8, -CD14, -CD19-
PerCPCy5.5 (all from eBioscience) to exclude non-T cell containing pop-
ulations of cells that bound tetramer. Tetramer-specific T cell populations
were positively enriched by incubating cells with 50 ml anti-PE microbeads
(Miltenyi Biotec) for 20 min at 4˚C. After washing, cells were resuspended
in 5 ml MACS buffer and passed through a magnetized LS column (Mil-
tenyi Biotec). The column was washed three times with 3 ml MACS buffer,
and after removal from the magnetic field, cells were collected with 5 ml
MACS buffer. Before the acquisition of samples, 7-aminoactinomycin D
(BD Biosciences) was added to exclude dead cells. Samples were acquired
on an LSR II flow cytometer (BD Immunocytometry Systems) and analyzed
using FlowJo software.
Ex vivo T cell responses to common TB Ags vary dramatically
in frequency and magnitude
In a first series of experiments, we analyzed T cell reactivity
against the CFP10 (Rv3874), ESAT-6 (Rv3875), Rv2031c (hspX),
Rv2654c (TB7.7), and Rv1038c (EsxJ) Ags. For this purpose, we
measured ex vivo production of IFN-g by PBMCs from 18 LTBI
donors, using ELISPOT assays and 15-mer peptides overlapping
by 10 aa spanning the entire length of the proteins. The peptides
were arranged in 11 Ag-specific pools (two per Ag, except
Rv2031c which had three) of ∼10 peptides (9 6 0.7) each. The
most frequently recognized proteins were CFP10 and ESAT-6,
which elicited responses in 67 and 56% of the LTBI donors, re-
spectively. Less frequent responses were detected for Rv2031c
(28%) and Rv1038c (11%). None of the LTBI donors studied
responded to Rv2654c, which was somewhat surprising because
this Ag is included in the QuantiFERON-TB Gold In-Tube test.
When the data were scrutinized from the standpoint of magnitude
of responses, a superimposable hierarchy of responses was ob-
served (Fig. 1). As expected, no responses were observed in
PBMCs from 18 TB uninfected control donors (Fig. 1). As a cri-
terion of positivity, consistent positive responses in three out of
three or four independent experiments was required, which, in our
experience, is important for defining the most robust, and thereby
most relevant, responses. Accordingly, in the case of one pool,
where positive responses were noted in only two of four inde-
pendent experiments, the Ag/donor combination was conserva-
tively deemed negative. For all other cases, the criterion of
positivity was met. In conclusion, these results highlighted that,
when responses are evaluated side-by-side using ex vivo analysis
to avoid potential biases introduced by in vitro restimulation, there
are large variations in terms of the frequency and magnitude of
responses against common TB Ags.
The number of epitopes contained within each Ag influences
but does not fully explain immunodominance
Although the data shown above clearly establishes an immuno-
dominance hierarchy within the set of Ags analyzed, it was unclear
whether this dominance might be reflective of a large(r) number of
different epitopes being recognized in the more dominant Ags or,
alternatively, whether dominance at the Ag level might reflect the
presence of a limited number of more dominant epitopes. To ad-
dress this issue, each positive peptide pool was next deconvoluted,
and individual reactive peptides were identified. The patterns of
reactivity derived on the basis of these experiments are shown
in Figs. 2A, 3A, 4A, and 4B. The recognition of overlapping
peptides defines several antigenic regions (one or adjacent over-
lapping peptides recognized, based on response frequency and/or
magnitude). The number of donors recognizing each of the re-
gions of the various Ags is indicated as well as the magnitude of
responses measured in the ELISPOT assay (SFC sum).
Two different patterns became apparent. For Rv1038c (Fig. 4B),
only one antigenic region could be discerned. In the case of the
three remaining Ags, CFP10, ESAT-6, and Rv2031c, four distinct
regions were apparent in each. For ESAT-6, some reactivity over
the entire protein sequence was observed, but discrete dominant
regions could be discerned. The weakest Ag of the four recognized
Ags had the fewest antigenic regions; however, the two immu-
nodominant Ags (ESAT-6 and CFP10) had the same number of
antigenic regions as the less antigenic protein Rv2031c, indicating
that the number of antigenic regions is not the sole determinant of
immunogenicity for these Ags.
Epitope paucity in Rv2654c and Rv1038c is not due to lack of
peptides binding to HLA class II molecules
One potential explanation for the paucity of epitopes/antigenic
regions observed in the case of Rv2654c and Rv1038c is that
these Ags might contain fewer or no HLA class II binding pep-
tides as compared with the more dominant CFP10, ESAT-6, and
Rv2031c Ags, which contain at least four different epitopes each.
To examine this issue, a panel of 27 HLA class II binding assays
was assembled, representative of common HLA class II DR, DP,
and DQ molecules expressed in the general population. The panel
of assays, together with theirgenotypic frequencies and phenotypic
frequencies, is shown in Supplemental Table I. Next, the LTBI
donors analyzed were HLA typed by a high-resolution Luminex-
based method (Supplemental Table II), and the coverage of this
specific donor population afforded by the panel of 27 HLA binding
assays was calculated. Each individual usually expresses a total of
eight HLA class II genes (one copy of HLA DRB1, B3/4/5, DP, and
DQ genes on each chromosome). All (100%) of the individuals in
PBMC from either LTBI donors (black dots, positive responses; white
dots, negative responses) or control donors (gray dots) were incubated with
1 mg/ml pools of peptides from indicated proteins, after which the number
of IFN-g–producing cells were enumerated in an ELISPOT assay. Top
panel shows response frequency in LTBI and control donors to Rv3874
(CFP10), Rv3875 (ESAT-6), Rv2031c (hspX), Rv1038c (EsxJ), and
Rv2654c (TB7.7). Graph shows sums of pool responses (SFC/106PBMC)
per Ag for LTBI versus control donors for indicated Ags. Pool responses
were averaged from at least three independent experiments per donor and
then summed to calculate response per Ag. For each individual experi-
ment, pools were tested in triplicate. ***p , 0.0001, **p , 0.01, *p ,
0.05 (one-tailed Mann–Whitney test).
Differential ex vivo responses to commonly used TB Ags.
5022 EX VIVO T CELL RESPONSES TO COMMONLY USED TB Ags
the donor cohort expressed at least one of the 27 HLA molecules in
our assay panel, and for 83%, the assay panel provided coverage of
six or more donor HLA class II types (Supplemental Fig. 1).
Closely related alleles that have an identical peptide binding region
were considered a match when HLA typing was compared with the
27 HLA molecules in the assay panel. These results demonstrate
that the selected panel of HLA molecules affords high coverage of
the specific donor population investigated.
from ESAT-6, and the number of IFN-g–producing cells was enumerated in an ELISPOT assay. ESAT-6 has four antigenic regions, indicated by capped
lines. The numbers of donors tested for each region were n = 7 for Regions 1 and 2 and n = 5 for Regions 3 and 4. Shown are total SFC (black line and
closed circles) and number of donors responding to each peptide (dashed line and open circles) versus peptides tested (15-mer overlapping by 10) (A).
Minimal epitope core sequence within each antigenic region for ESAT-6. Shown are the average net SFC/106PBMC for each donor in response to the tested
peptides (15-mer overlapping by 14) from at least two independent experiments. Boxes indicate selected epitopes containing the minimal epitope core
sequence. For each individual peptide, samples were tested in triplicate. Error bars indicate SD. The dotted line indicates the 20 net SFC/106cells threshold
used to define positivity (B, C). Selected epitopes identified from Regions 1, 2, and 4, showing donors responding, position in protein, and sequence.
Minimal epitope core sequence is highlighted in bold (D, E).
Determination and characterization of antigenic regions from ESAT-6. PBMC from LTBI donors were incubated with 10 mg/ml peptides
The Journal of Immunology 5023
Each of the overlapping peptides previously tested for recog-
nition by the donor PBMCs was tested for binding to each of 27
purified HLA molecules (IEDB Submission number 1000492). On
average, each peptide bound 6.7 (6 5.6) molecules at the 1000 nM
threshold previously reported to be associated with immunoge-
nicity for HLA class II responses (33–35) and that each Ag con-
tained a minimum of nine peptides binding at least 20% of the
alleles tested (Table I). Importantly, the binding distribution did
not differ appreciably when more dominant (CFP10, ESAT-6, and
Rv2031c) and less dominant (Rv1038c and Rv2654c) Ags were
compared, thus indicating that the incidence of HLA binding
peptides did not account for the immunodominance hierarchy
observed. In conclusion, the subdominance and the paucity of
epitopes observed in the case of Rv2654c and Rv1038c cannot be
CFP10 has four antigenic regions, indicated by capped lines (n = 5 for Regions 1 and 2, n = 9 for Regions 3 and 4) (A). Minimal epitope core sequence
within each antigenic region for CFP10 (B, C). Selected epitopes identified from Regions 2, 3, and 4, showing donors responding, position in protein, and
sequence. Minimal epitope core sequence is highlighted in bold (D, E).
Determination and characterization of antigenic regions from CFP10. For a description of experimental conditions, see the legend for Fig. 2.
5024EX VIVO T CELL RESPONSES TO COMMONLY USED TB Ags
ascribed to these Ags simply containing no or fewer HLA class II
binding peptides as compared with the more dominant Ags,
CFP10, ESAT-6, and Rv2031c.
Further characterization of antigenic regions
We next sought to determine whether the immunodominant
epitopes/antigenic regions corresponded to a single epitope sim-
ilarly recognized by different donors or rather the presence of
overlapping but distinct epitopes. For ESAT-6 and CFP10 three
antigenic regionseach andforRv2031candRv1038cone antigenic
region each were analyzed in more detail.
To determine the epitope core sequence from each antigenic
region, recognition of every possible 15-mer spanning the iden-
tified antigenic region (i.e., 15-mer overlapping by 14 aa) was
analyzed. In general, the same minimal epitope core sequence was
recognized by the different donors responding to a given antigenic
region (Figs. 2B, 2C, 3B, 3C, 4C, 4D) and defined by the pattern
of reactivity to the peptides overlapping by 14 aa. The minimal
epitope sequence was defined by the epitope core region necessary
for optimal reactivity. For example, there is a loss of reactivity
when ESAT-614–28is compared with ESAT-615–29, which suggests
that residue 29 is the C-terminal boundary of the core region.
Similarly, there is loss of reactivity when ESAT-623–37is com-
pared with ESAT-622–36, suggesting that residue 22 is the N-
terminal of the crucial core region. Thus, the minimal epitope
core region needed for optimal reactivity corresponds to ESAT-
622–29 (VTSIHSLL). Accordingly, for each antigenic region,
a representative 15-mer epitope containing the minimal epitope
core sequence and additional flanking residues was chosen for
further investigation. A summary of selected epitopes for each
donor tested is shown in Figs. 2D, E, 3D, 3E, 4E, and 4F.
Taken together, these results define the minimal epitope core se-
quences contained within ESAT-6, CFP10, Rv2031c, and Rv1038c.
Most importantly, the data presented indicate that the immunodo-
minant antigenic regions corresponded to the same core sequence,
similarly recognized by different donors, rather than to multiple
overlapping but distinct regions.
Rv2031c has four antigenic regions (n = 1 for Region 1, n = 3 for Regions 2 and 3, and n = 5 for Region 4), and Rv1038c has one antigenic region (n = 2),
indicated by the capped lines labeled by region numbers (A, B). Minimal epitope core sequence within each antigenic region for Rv2031c and Rv1038c (C,
D). Selected epitopes identified from Region 4 Rv2031c and Region 1 Rv1038c, showing donors responding, position in protein, and sequence. Minimal
epitope core sequence is highlighted in bold (E, F).
Determination of minimal epitopes from Rv2031c and Rv1038c. For a description of experimental conditions, see the legend for Fig. 2.
overlapping by 10 aa covering ESAT-6, CFP10, Rv2031c, Rv2654c, and
Summary of number of HLA molecules bound by 15-mers
AgTotal No. of Peptides
No. of Peptides Binding
$20% of Alleles
The Journal of Immunology 5025
CD4 and HLA class II restriction of selected epitopes
Having defined the minimal core sequences recognized allowed us
to further characterize the phenotype of epitope-reactive cells and
either CD4+or CD8+T cells to establish the major T cell subset
responsible for the responses. As shown in Fig. 5A, a total loss of
peptide reactivity was observed in all of the CD4+T cell-depleted
PBMC fractions, whereas CD8+depletion had no effect. These
results demonstrate that the epitopes identified are CD4, and thus
also likely HLA class II, restricted.
Next, to verify this assumption and we determined the particular
HLA class II loci used. For this purpose, the capacity of Abs
specific for HLA-DR, -DP, or -DQ to inhibit the epitope-specific
response was tested (representative data; Fig. 5B). Locus re-
striction could be determined for 30 of the 35 (86%) recognition
events analyzed (donor/epitope combinations) (Table II). For the
remainder, either lack of a sufficiently strong response or an in-
conclusive inhibition pattern precluded a definitive locus assign-
ment. DR restriction was noted for 18 responses and DP and DQ
restriction for 11 each; 8 responses were restricted by multiple loci
in the same donor (Table II). These data confirmed the CD4/CD8
depletion data and formally demonstrate class II restriction and
also illustrate how a diverse set of alleles and loci are involved in
restricting these responses. Interestingly, several antigenic regions/
epitopes were restricted by multiple loci, as discussed in more
Selective versus promiscuous restriction of selected epitopes
The HLA typing data for each donor responding to a given epitope
was compared with the locus determination data presented above
and the in vitro HLA binding data (Table II). Each of the epitopes
was tested for binding to purified HLA molecules (IEDB Sub-
mission number 1000492). Furthermore, for each donor/epitope
combination for which a restricting locus could be assigned, we
noted the corresponding alleles expressed by the donor for which
the epitope was shown to bind the epitope with either strong
(,100 nM) or intermediate (,1000 nM) affinities. These allelic
molecules are highlighted by green and yellow shading, respec-
tively, in Table II. In approximately half of the cases, the epitope
was restricted by multiple HLA class II loci (ESAT-63–17,
CFP1040–54, CFP1072–86, Rv2031c106–120, and Rv1038c28–42) or
was indeed restricted by a single HLA class II locus, but the
CD4+(N) T cells and were incubated with 10 mg/ml of indicated peptides, and the number of IFN-g–producing cells was enumerated in an ELISPOTassay.
Shown are the net SFC/106cells for representative donors for at least two independent experiments. Each individual peptide was tested in triplicate. The
dotted line indicates the 20 net SFC/106cells threshold used to define positivity (A). Determination of restricting locus. Purified CD4+T cells and remaining
PBMC cells (2:1 ratio) were incubated with Abs against HLA-DR, -DP, -DQ, or pan-MHC class I and stimulated with peptide at indicated concentrations.
The number of IFN-g–producing cells was determined by ELISPOT. Shown are percentage inhibitions by Abs at different peptide concentrations for
representative donors (B).
Characterization of epitopes. CD4 T cells are the dominant cell type responding to tested epitopes. PBMCs were depleted of CD8+(n) or
5026EX VIVO T CELL RESPONSES TO COMMONLY USED TB Ags
individuals responding to the epitope did not share a common
HLA binding molecule that could be considered as a candidate
for a single restriction element (ESAT-670–84and Rv2031c111–125).
These data suggest that promiscuous recognition in the context of
multiple HLA class II molecules may be a mechanism significantly
contributing to epitope immunodominance.
Conversely, in the case of three other epitopes (ESAT-618–32,
ESAT-673–87, and CFP1052–66), all responses were restricted by
a single locus, and all responders shared an allelic variant shown
to bind the epitope with high affinity. In these cases, we were able
to infer specific restriction, as listed in the “Inferred Restricting
HLA Allele” column (Table II). A large majority of the donors
expressing that particular HLA class II allele also responded to
the given epitope. Specifically, four of five donors expressing
DRB5*01:01 responded to CFP1052–66, and three of five donors
expressing DQB1*03:01 responded to ESAT-672–87, as listed in
the “No. of Donors Responding” column (Table II), thus further
strengthening the proposed restrictions.
On average each of these three single locus restricted peptides
bound 6.3 (6 1.2) alleles. By contrast, the promiscuously re-
stricted peptides bound 12.3 (6 5.3) alleles with high binding
affinity (100 nM). These results suggest that certain epitopes are
immunodominant on the basis of an alternative mechanism,
namely that although they are selectively restricted by one HLA
Table II. Summary of epitope characteristics
Bold, restricting HLA locus; green, strong binding (,100 nM); yellow, intermediate binding (100–999 nM); gray, not available.
aMean SFC/106PBMC, average of two independent experiments.
bVariant alleles that have an identical peptide binding region to an allele in the assay panel and the inferred restricting allele.
The Journal of Immunology5027
class II molecule, they are frequently and strongly recognized in
the context of that particular HLA molecule.
Phenotypic characterization of selected epitopes
To further characterize some of the identified epitopes, we selected
one representative selectively restricted and one representative
promiscuously restricted epitope for further experiments. In the
case of CFP1052–66, the experiments above indicated DRB5*01:01
as the restriction element. In the case of the promiscuous epitope
CFP1040–54, DRB1*01:01 was indicated as one of the likely
restricting elements, because three of four donors that responded
express DRB1*01:01, and CFP1040–54binds this allele with high
affinity. Accordingly, respective MHC–peptide tetramer reagents
were prepared for these CFP10 epitopes. To increase sensitivity,
tetramer staining of CD4+purified cells followed by a magnetic
bead enrichment technique was used (36).
The tetramer staining experiments confirmed the HLA re-
striction of the two CD4+T cell responses (Fig. 6). Epitope-
specific T cell responses were detected in three donors at fre-
quencies 0.1–0.5% (mean of 0.33 6 0.23 SD) above background
for DRB1*01:01 CFP1040–54and in six donors at frequencies
0.1–10% (mean of 2.54 6 3.97 SD) above background for
DRB5*01:01 CFP1052–66. Only a small number of background
tetramer+cells were detected with the epitope-specific tetramers
in the HLA-matched control donors and in LTBI donors with an
HLA mismatch for DRB5*01:01 CFP1052–66(Fig. 6A), which
confirmed that tetramer specificity was derived from the epitope
and HLA molecule combination.
The memory subset phenotype was addressed using Abs to
CD45RA and CCR7 (37–39). As shown in Fig. 6B and 6C,
DRB1*01:01 CFP1040–54epitope-specific tetramer+CD4+T cells
predominantly consisted of CD45RA2CCR72effector memory
cells in all three donors analyzed, followed by central memory
T cells (CCR7+CD45RA2). Percentages ranged between 68.3 and
77.5% (SD 6 4.6) for effector memory T cells and 12.6–23.2%
(SD 6 5.5) for central memory T cells. Only a minor fraction of
the tetramer+CD4+T cells appeared to be naive (CCR7+
CD45RA+) or effector T cells (CCR72CD45RA+). Similarly, for
the DRB5*01:01-CFP1052–66response, epitope-specific tetramer+
CD4+T cells predominantly consisted of CCR72CD45RA2ef-
fector memory cells in all six donors analyzed, followed by
central memory T cells (CCR7+CD45RA2), percentages ranging
between 60.2 and 91.6% (SD 6 12.9) for effector memory T cells
and 1.5–20.4% (SD 6 13.0) for central memory T cells. Again,
only a minor fraction of the tetramer+CD4+T cells appeared to be
naive (CCR7+CD45RA+) or effector T cells (CCR72CD45RA+)
Although the QuantiFERON-TB Gold In-Tube and T-SPOT.TB
IGRAs are highly successful as TB diagnostics, they are also
not without problems. Both tests are an empirical mixture of
peptides, and the epitopes have not been characterized ex vivo,
even though the assays are performed in ex vivo settings. This
results in a fundamental lack of understanding of the immunolog-
ical mechanisms and specificity underlying the diagnostic utility.
Several fundamental questions remain to be answered. Namely, 1)
are all Ags equally recognized with similar prevalence (response
possible toidentifyadditional Agsthatmight enhance the antigenic
composition of the IGRAs? 3) What are the molecular features of
Ag recognition at the population level (i.e., what determines which
donors, HLA-matched control donors, or HLA-mismatched LTBI donors (for DRB5*01:01) were stained with MHC class II tetramers, and tetramer+cells
were isolated following magnetic bead enrichment. Plots are gated on CD4+T cells, and the numbers indicate the percentages of tetramer+cells isolated
from each donor’s CD4+population. DRB1*01:01 CFP1040–54, n = 3 LTBI donors and DRB5*01:01 CFP1052–66, n = 6 LTBI donors (A). Memory
phenotype of tetramer+cells for two representative donors per tetramer. Plots are gated on total CD4+T cells (black background) or epitope-specific CD4+
T cells (red dots). The numbers represent the percentages of tetramer+CD4+T cells in the gate (B). Pie chart representation of the proportion of CCR72
CD45RA2, CCR72CD45RA+, CCR7+CD45RA+, and CCR7+CD45RA2CD4+T cells for each tetramer, DRB1*01:01 CFP1040–54 (n = 3) and
DRB5*01:01 CFP1052–66(n = 6). Effector memory T cells are CCR72and CD45RA2, central memory T cells are CCR7+and CD45RA2, naive T cells are
CCR7+and CD45RA+, and effector T cells are CCR72and CD45RA+(C).
HLA restriction and memory phenotype of CFP10-specific CD4+T cells using MHC class II tetramers. CD4-purified cells from LTBI
5028 EX VIVO T CELL RESPONSES TO COMMONLY USED TB Ags
Ags and epitopes are most dominant)? 4) Are a myriad of epitopes
recognized, differing from donor to donor, or are a limited number
of epitopes recognized in the context of multiple donors (preva-
lence) or even across different HLAs (promiscuity)? 5) If dominant
epitopes can be identified, could tetrameric reagents be used to
more thoroughly characterize immune responses, either diagnos-
tically or prognostically?
This study represents, to the best of our knowledge, the first in-
commonly used TB Ags CFP10, ESAT-6, Rv2031c, Rv1038c, and
Rv2654c, as well as an in-depth investigation of the mechanisms
influencing their recognition by human T cells. The direct ex vivo
approach avoids introducing potential bias as a result of in vitro
restimulation and expansion of T cells, and thereby delineates
a more physiologically relevant immune response. Although direct
ex vivo responses to ESAT-6 and/or CFP10 have been investigated
previously (9, 11, 12, 40, 41), most studies used less physiological
techniques such as short-term T cell lines (13–16, 20, 21, 42).
In our side-by-side comparison, we found striking levels of
variation in terms of prevalence of Ag recognition and magnitude
of responses. Interestingly, we could not identify any responses to
Rv2654c, which has been shown to be recognized by T cells during
TB infection and is included in the QuantiFERON In-Tube Gold
diagnostic test for TB (13, 14). Previous studies used short-term
T cell cultures (13, 14), which could explain the discrepancy be-
tween this study and earlier work and highlights the necessity of
revisiting Ag reactivity with approaches more closely reflective of
the techniques used in the assays (i.e., ex vivo detection of IFN-g).
Regarding whether it is possible to identify additional Ags that
might enhance the antigenic composition of IGRAs, we observed
high reactivity to Rv2031c, with 28% of the LTBI donors re-
sponding. This demonstrates that other prevalently recognized
Ags can be identified and potentially included in diagnostic tools
for TB, although this study was not aimed at identifying new Ags
that will address this issue in more detail and may identify many
additional Ags (manuscript in preparation).
This study provides, to the best of our knowledge, the first actual
molecular HLA binding affinity data for these commonly used TB
Ags using a panel of HLA class II molecules representative of the
most common alleles worldwide. Previous studies have reported
bioinformatics predictions of ESAT-6 epitopes (43, 44).
The low frequency or lack of recognition of Rv1038c and
peptides. It has been shown for infectious diseases such as malaria
and HIV, and more recently also for allergy, that peptide binding to
a HLA molecule is vital, but not by itself sufficient, for recognition
by epitope-specific T cells (45–47). Other factors that might play
a role in Ag recognition are the function of the protein, expression
levels, stage of expression, Ag presentation, and so on, factors that
could also be influenced by the disease state. These factors are
also interesting but beyond the scope of the current investigation
and best suited for a genome-wide analysis.
The identified antigenic regions from CFP10, ESAT-6, Rv2031c,
and Rv1038c correlated well with data previously available in the
literature (7, 9, 10, 12, 13, 41, 42). However, the side-by-side
comparison provided additional insights into the frequency and
magnitude of responses directed against these Ags. Most impor-
tantly, the recognition of dominant antigenic regions/epitopes
in multiple donors was mapped to the recognition of the same
minimal epitope core sequence jointly recognized by multiple
donors. This could be explained by either restriction by a single
specific HLA molecule or recognition of the same peptide in the
context of multiple HLA types recognizing largely overlapping
epitopes. Our results indicated that both selective and promiscuous
restriction contribute to immunodominance. The molecular mech-
anisms underlying promiscuous recognition are not clear, and
one hypothesis is that the processing of Ags preferentially gen-
erates certain peptide fragments. Furthermore, it is known that
HLA class II molecules share peptide binding repertoires (33–35,
48), which could also contribute to promiscuous restriction of
a particular epitope. Regardless of the mechanism, these results
indicate that it is possible to identify epitopes prevalently recog-
nized by LTBI subjects and therefore suggest that further studies
identifying the specific epitopes recognized in TB infection could
lead to better molecularly defined diagnostic assays. The reduction
in complexity afforded by the definition of the dominant epitopes
could in turn allow elimination of poorly or unrecognized Ags and
epitopes to make room for highly prevalent epitopes derived from
additional Ags. A substantial number of LTBI patients are nega-
tive for the IGRA Ags. This is consistent with the fact that IGRA
is by necessity a mixture of different Ags. Our results further
emphasize that an approach based on inclusion of only one of the
IGRA Ags is not feasible and rather suggest that inclusion of
additional epitopes and Ags might be beneficial. Future studies
should include a larger study population from different ethnicities
and geographic locations as well as include patients with different
disease states. This would provide answers for different HLA
phenotypes as well as whether patients with different disease
states show a different recognition pattern.
The question of whether dominant epitopes can be used in
conjunction with tetrameric reagents or ICS assays to more thor-
oughly characterize immune responses, either diagnostically or
prognostically, was addressed. We found that all ex vivo-detected
epitopes are CD4 restricted. This demonstrates that CD8-restricted
epitopes for these Ags are a minor component of the ex vivo IFN-g
response measured by IGRAs and is consistent with CD8-
restricted epitopes for ESAT-6 and CFP10 having been defined
using T cell lines (5, 11, 49). Ag-specific T cell phenotypes have
been well described in human viral infections, as well as in some
bacterial infections (40, 50, 51), and these analyses provide im-
portant information regarding effector function. We found the
phenotype of CFP10 epitope-specific CD4+cells to be predomi-
nantly effector memory cells, CD45RA2CCR72(37–39), which
is consistent with previous studies in HIV+LTBI individuals and
infected mice (40, 52, 53). This observation was true for both
promiscuously and selectively restricted epitopes. It is not known
whether there is some degree of bacterial replication ongoing in
the individuals tested in this study, even though they have no
symptoms of active TB (chest X-ray negative), because there is no
accurate test for Ag levels associated with MTB infection in
humans. Some bacterial replication could potentially drive the
cells to maintain an effector memory phenotype, but further
studies are needed to determine whether it is due to persistence of
Ags or whole bacteria or if it represents the longevity of the im-
mune response even in the absence of antigenic stimuli. Further-
more, it has previously been shown that the majority of cells
responding to Rv2031c are effector memory T cells and that ef-
fector memory cells dominate the immune responses in TB (54,
55). Moreover, long-term persistence of TB-reactive cells has
been described previously (54). It would perhaps be unlikely to
detect central memory cells in PBMC samples, because they
mostly reside in lymphoid organs and few circulate. In addition, it
should be noted that there might be a weakness in testing blood
samples for LTBI in that the T cells that are cognizant of infection
in LTBI are probably localized in the lymphoid organs and are not
accessible through peripheral blood. These cells would be ex-
pected to be recruited to the lungs after exposure or after reac-
The Journal of Immunology5029
tivation. In conclusion, these data demonstrate how the epitopes
identified following the approaches described in this paper can be
used to develop tetrameric reagents and suggest that definition and
production of mixtures of highly defined tetrameric reagents could
represent the next generation of diagnostic reagents, allowing for
quantitation and characterization of responses to an unprecedented
level of detail.
Finally, a side-by-side comparison of commonly used TB Ags
shows that different Ags are recognized with drastically different
prevalence (response frequency) and magnitude of responses. The
recognition of Rv2031c also suggests that it is possible to identify
additional Ags that might enhance the antigenic composition of
IGRAs. Furthermore, a limited number of epitopes are recognized in
the context of multiple donors (prevalent) or even across different
HLAs (promiscuous), suggesting that eliminating poorly or not rec-
derived from additional Ags might represent a powerful avenue to
generate more molecularly defined diagnostic reagents. Likewise,
tetrameric reagents could be used to more thoroughly characterize
immune responses, either diagnostically or prognostically.
We thank Amiyah Steen, Sandy Ngo, and Carrie Moore for performing the
HLA binding assays and Scott Southwood for performing the HLA typing
assays. We also thank Julie Hoffman and Hilda Grey for assistance with the
The authors have no financial conflicts of interest.
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