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-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment1
*
Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and
Wellcome Centre for Clinical Tropical Medicine, Imperial College School of Medicine, Northwick Park Hospital, London, United Kingdom
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting CD4 T cells specific for ESAT-6, a secreted Ag that
is highly specific for M. tuberculosis, and a target of
protective immune responses in animal models. We found that frequencies
of circulating ESAT-6 peptide-specific IFN--secreting CD4 T cells
were higher in latently infected healthy contacts and subjects with
minimal disease and low bacterial burdens than in patients with
culture-positive active pulmonary tuberculosis (p =
0.009 and p = 0.002, respectively). Importantly,
the frequency of these Ag-specific CD4 T cells fell progressively in
all groups with treatment (p = 0.005), suggesting
that the lower responses in patients with more extensive disease were
not due to tuberculosis-induced immune suppression. This population of
M. tuberculosis Ag-specific Th1-type CD4 T cells appears
to correlate with clinical phenotype and declines during successful
therapy; these features are consistent with a role for these T cells in
the containment of M. tuberculosis in vivo. Such
findings may assist in the design and evaluation of novel tuberculosis
vaccine candidates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(4), and TNF-
(5) are
essential for protection. In humans, various subsets of CD8 T cells
have been identified (6, 7, 8, 9, 10), but their role in
containment of Mycobacterium tuberculosis infection remains
uncertain (11). In contrast, the greatly increased risk of
active tuberculosis caused by HIV infection (12), and
other observations (11, 13, 14), point to a crucial role
for Th1-type CD4 T cells.
Selection of Ags for inclusion in novel subunit vaccines depends upon
identifying the targets of protective immunity in vivo, yet little is
known about the Ags targeted by protective immune responses in humans.
In rodent models, secreted Ags are the predominant targets of
protective immunity (15), and vaccination with secreted
Ags can confer protection (16, 17). ESAT-6, a secreted Ag
that is highly specific for the M. tuberculosis complex, is
a major target of IFN--secreting CD4 T cells in the memory immune
mouse model (15), and immunization with ESAT-6 induces
protective immunity against tuberculosis (17, 18), which
is mediated by ESAT-6 peptide-specific IFN--secreting CD4 T cells
(18). In humans, the recent observation that ESAT-6 is
recognized by a large proportion of patients with active tuberculosis
(19, 20, 21, 22), together with the absence of ESAT-6 from
Mycobacterium bovis bacillus Calmette-Guérin
(BCG)3 (23, 24), has generated much interest in this Ag as a diagnostic
reagent. However, it is not yet known whether, as in murine models,
ESAT-6 is a target of T cells with a protective role in vivo. Given the
important role of CD4 T cells and IFN- in tuberculosis, we asked
whether the levels of ESAT-6-specific IFN--secreting CD4 T cells
correlate with clinical phenotype in humans. Importantly, the highly
restricted species specificity of ESAT-6 means that T cell responses to
this Ag are M. tuberculosis specific and not a result of
cross-reactivity with BCG vaccination (20, 25).
The result of infection with M. tuberculosis is determined largely by the host immune response. The clinical outcome ranges from asymptomatic latent infection associated with long-term immunological control of the bacillus (healthy contacts), through mild forms of disease with minimal symptoms, frequent spontaneous resolution, and low bacterial burdens (e.g., tuberculous lymphadenitis), to sputum smear-positive pulmonary tuberculosis with severe symptoms, extensive disease, and high bacillary loads. This wide spectrum of clinical phenotypes offers the opportunity to investigate the relationship between immune responses and the degree of containment of the bacillus in vivo.
Using conventional T cell assays that rely on Ag-driven proliferation
in vitro, weaker responses have sometimes been found in patients with
more extensive disease (26, 27, 28). This probably reflects
nonspecific suppression of cellular immune responses during active
tuberculosis, which recover with therapy (28, 29, 30). We
reasoned that direct enumeration of Ag-specific T cells from peripheral
blood, without an in vitro proliferation step, would delineate a more
physiologically relevant immune response and might be less susceptible
to the nonspecific immune suppression resulting from tuberculosis
itself. This direct ex vivo approach has recently provided important
insights into the protective role of Ag-specific CD8 and CD4 T cells in
chronic viral infections, including HIV, hepatitis B virus, and EBV
(31, 32, 33, 34, 35, 36). We therefore used the sensitive ex vivo ELISPOT
assay for IFN- (33, 37, 38) to directly quantitate
IFN--secreting CD4 T cells specific for a panel of overlapping
peptides spanning the ESAT-6 molecule. We studied five precisely
clinically defined groups of individuals: tuberculin skin test
(TST)-positive healthy household contacts (HHCs) of sputum
smear-positive tuberculosis cases, patients with tuberculous
lymphadenitis (TBLN), patients with self-healed culture-negative
pulmonary tuberculosis (C-PTB), patients with
culture-positive pulmonary tuberculosis (C+PTB),
and healthy unexposed controls (UCs). The quantitative readout of the
ex vivo ELISPOT assay permitted meaningful comparisons of
ESAT-6-specific T cell frequencies between these groups and, in some
individuals, we longitudinally tracked these Ag-specific T cells during
treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult patients and contacts were recruited prospectively at
Northwick Park Hospital (London, U.K.) and the hospitals of the Oxford
Radcliffe National Health Service Trust (Oxford, U.K.). Ethical
approval for the study was granted by the Harrow and Central Oxford
Research Ethics Committees. A heparinized blood sample was drawn after
obtaining informed consent. No subjects had features of HIV infection,
and patients known to be HIV infected were excluded. Demographic
characteristics are shown in Table I
.
|
Thirty-two healthy UCs with no history of tuberculosis and no known tuberculosis contact were recruited prospectively. Most were laboratory staff, and 28 had been BCG vaccinated.
Twenty-five patients with C+PTB had clinical and radiographic findings consistent with tuberculosis and positive cultures for M. tuberculosis from one or more respiratory specimens. Eighteen of twenty-five (72%) PTB patients had received either no therapy, or less than 4 wk of therapy at the time of venepuncture.
Eight patients with self-healed C-PTB had a clinical diagnosis made on the basis of highly suggestive appearances on chest radiography and positive tuberculin skin tests. All were asymptomatic and had no previous history of treatment for tuberculosis. All respiratory samples, which consisted of three sputum specimens (or three gastric washings in the absence of productive cough) and a bronchoalveolar lavage for each patient, were negative for M. tuberculosis culture. Six of eight (75%) of this group had received either no therapy, or less than 4 wk of therapy.
Eleven patients with TBLN had consistent clinical findings, positive TSTs, and a good response to antituberculous chemotherapy. Seven had lymph node biopsies, of which five grew M. tuberculosis on culture. Five of eleven (45%) TBLN patients had received either no therapy, or less than 4 wk of therapy at the time of venepuncture.
Seventeen patients with nonlymph node extrapulmonary tuberculosis (EPTB) were recruited on the basis of clinical findings and positive cultures for M. tuberculosis from one or more clinical specimens. Four patients had tuberculous osteomyelitis, two had musculoskeletal tuberculosis (psoas abscess), four had pleural disease, three had miliary disease, three had abdominal disease, and one had meningitis. The small number of patients precluded statistical comparisons of T cell frequencies between the subgroups of EPTB.
Peptides
Seventeen peptides spanning the length of the ESAT-6 molecule were synthesized by solid-phase F-moc chemistry (Research Genetics, Huntsville, AL). Each peptide was 15 aa in length and overlapped its adjacent peptide by 10 residues. Identity was confirmed by mass spectrometry, and purity by HPLC. Sequence homology searches of the SwissProt and translated GenBank protein databases confirmed that these peptides are uniquely restricted to the ESAT-6 protein of M. tuberculosis complex.
Ex vivo ELISPOT assay for single cell IFN- release: enumeration
of circulating ESAT-6 peptide-specific T cells from peripheral blood
PBMC were separated from 20 ml blood by Ficoll gradient
centrifugation and suspended in RPMI supplemented with 2 mM
L-glutamine, 100 µg/ml ampicillin, 50 µg/ml gentamicin,
1 mM sodium pyruvate, and 10% heat-inactivated FCS (Sigma, St. Louis,
MO) (R10). As previously described (7, 37), 96-well
polyvinylidene difluoride-backed plates (MAIPS45; Millipore, Bedford,
MA), precoated with 15 µg/ml anti-IFN- mAb 1-D1K (Mabtech,
Stockholm, Sweden), were blocked with R10 for 2 h. A total of
3 x 105 PBMC was added in 100 µl
R10/well, and peptides were added individually to single wells at 10
µg/ml. PPD (batch RT49; Statens Seruminstitut, Copenhagen, Denmark)
was also tested at 20 µg/ml. PHA (ICN Biomedicals, Aurora, OH) at 5
µg/ml was added to duplicate positive control wells, and no peptide
to duplicate negative control wells. rESAT-6 (a kind gift of A. Whalen
and M. Vordermeier, VLA, Addlestone, U.K.) was added to single wells at
10 µg/ml for 35 subjects.
After 14-h incubation at 37°C, 5% CO2 plates
were washed with PBS 0.05% Tween 20 (Sigma). Fifty microliters of 1
µg/ml biotinylated anti-IFN- mAb, 7-B6-1 biotin (Mabtech),
were added. After 2 h, plates were washed, and
streptavidin-alkaline phosphatase conjugate (Mabtech) was added to
1:1000. After 1 h and further washing, 50 µl diluted chromogenic
alkaline phosphatase substrate (Bio-Rad, Hercules, CA) was added. After
20 min, plates were washed and allowed to dry.
ELISPOT assay for single cell IL-4 release
These assays were conducted as described above using high affinity IL-4-specific Abs (IL-4-I catcher and biotinylated IL-4-II detector; Mabtech) and a 60-h incubation period.
Enumeration of IFN- spot-forming cells (SFCs)
IFN- SFCs were counted using a magnifying glass, and
responses were scored as positive if test wells contained at least five
IFN- SFCs more than negative control wells, and this number was at
least twice that in negative control wells. This cutoff (five IFN-
SFCs per 3 x 105 PBMC) translates into a
detection threshold of 1/60,000 PBMC. The person performing and reading
the assays was blinded to the clinical status of the different groups
of patients and HHCs, but not to the UCs. Background numbers of SFCs in
negative control wells were below five. After subtraction of background
values, the number of IFN- SFCs specific for each peptide was
summated, and this total number of ESAT-6 peptide-specific SFCs for a
given individual was used for comparisons between groups of subjects
and between different time points during treatment of individual
patients.
In vitro generation of peptide-specific T cell lines
T cell lines were generated as previously described
(7). Briefly, PBMC were suspended at 5 x
105 cells/ml in AB medium (RPMI supplemented with
2 mM L-glutamine, 100 µg/ml ampicillin, 5 µg/ml
gentamicin, and 10% heat-inactivated pooled human AB serum; National
Blood Transfusion Service, Bristol, U.K.), and 200 µl was added per
well in a round-bottom 96-well plate. Peptide was added at 10 µg/ml.
A total of 10 U/ml IL-2 in the form of Lymphocult-T (Biotest, Dreiech,
Germany) was added every 3 days. After 12 days, T cell lines were
immunomagnetically depleted and tested against the stimulating peptide
in duplicate wells in ELISPOT assays for IFN- using 1–5 x
104 cells/well.
Immunomagnetic cell depletions
CD4 and CD8 T cells were depleted from T cell lines by 30-min incubation with anti-CD4 or anti-CD8 mAbs conjugated to ferrous beads at a ratio of 10 beads to 1 cell using Dynabeads M-450 (Dynal, Oslo, Norway) in 200 µl R10 on ice. Following dilution in R10, the conjugate-coated cells were removed by a magnet (Dynal). These Dynabeads reliably deplete >99% of the target cell population.
[3H]Thymidine incorporation lymphoproliferation assays
A total of 2 x 105 PBMC was seeded per well in 200 µl AB medium in 96-well round-bottom plates. Peptides were added at 10 µg/ml, negative control wells had medium only, and positive control wells contained PPD at 20 µg/ml. All conditions were set up in triplicate wells. Following incubation at 37°C, 5% CO2 for 5 days, 1 µCi [3H]thymidine was added to each well. After 18 h, wells were harvested, and [3H]thymidine incorporation was measured in a beta counter (Wallac; PerkinElmer Life Sciences, Cambridge, U.K.). Results were scored as positive if the stimulation index was 3 or more.
Inhibition of T cell responses in ELISPOT assays with anti-MHC class II Abs
HLA class II restriction of the CD4 T cell response to two immunodominant peptides, ESAT-61–15 and ESAT-671–85, was investigated using T cell lines as well as ex vivo PBMC. The murine mAbs L243, L2, and B7.21, which block peptide presentation to CD4 T cells by HLA-DR, HLA-DQ, and HLA-DP, respectively, were added at 10 µg/ml to three separate pairs of duplicate wells in ELISPOT assays, followed by 10 µg/ml peptide 90 min later. Control duplicate wells received peptide only, or neither peptide nor Ab.
Statistical methods
Summated frequencies of ESAT-6 peptide-specific IFN- SFCs for
responders were compared between patient groups using the nonparametric
Mann-Whitney test (two tailed), and between the first and last time
points sampled in the treatment course of individual patients using the
nonparametric Wilcoxon signed rank test (two tailed). The proportional
decrease in Ag-specific T cell frequencies during therapy was
calculated using the Student’s t test on log
transformed data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting T cells circulate in
M. tuberculosis-infected individuals at frequencies that
correlate with clinical disease state
ESAT-6 peptide-specific IFN--secreting T cells were detected in
almost all tuberculosis patients and the majority of contacts.
Responses were observed in 10 of 11 TBLN patients, 7 of 8
C-PTB patients, 23 of 25
C+PTB patients, and 23 of 27 HHCs (Table I
). It
is possible that the four HHCs who did not respond to ESAT-6 were not
infected with M. tuberculosis; their positive TSTs may have
resulted from prior BCG vaccination. By contrast, none of the 32 UCs
(of whom 28 were BCG vaccinated) responded to ESAT-6 peptides in the ex
vivo ELISPOT assay for IFN- (Table I), consistent with
ESAT-6-specific responses being M. tuberculosis specific.
Frequencies of ESAT-6 peptide-specific IFN--secreting T cells for
all responders from each group are shown in Fig. 1 and summarized in Table I. For the HHCs
that responded to ESAT-6 (n = 23), frequencies of
ESAT-6-specific T cells were significantly higher
(p = 0.009) than among the
C+PTB patients who responded (n =
23) and higher (p = 0.044) than the PTB
(C+ and C-) group as a
whole (n = 30). Both groups of ESAT-6-responsive
patients with minimal, paucibacillary disease, TBLN (n
= 10) and C-PTB (n = 7), also
had significantly higher frequencies of ESAT-6 peptide-specific T cells
than the C+PTB patients
(p = 0.002 and p = 0.029,
respectively). The range of ESAT-6-specific T cell frequencies within
each patient group is broad and probably reflects the natural range of
interindividual variability for such responses.
|
-secreting T
cells in tuberculosis patients decline progressively with treatment
If lower frequencies of ESAT-6-specific IFN--secreting T cells
in patients with active C+PTB were a result of
nonspecific disease-associated immunosuppression, T cell frequencies
would be expected to rise during effective treatment. We therefore
longitudinally tracked 12 patients (4 C+PTB, 3
TBLN, and 5 EPTB) on antituberculous chemotherapy and observed a
decline in the overall frequency of ESAT-6 peptide-specific T cells
(p = 0.005) (Fig. 2). The average decrease over the mean
follow-up period of 18.6 wk was by a factor of 0.62 95%
confidence interval (CI), 0.37–0.76), that is, to 38% of the initial
values, and the rate of decay was 5.5% (95% CI, 2.4–8.4) per week.
The frequencies of T cells specific for each of the peptides declined
in parallel, but only the summated responses to all ESAT-6-derived
peptides are shown for clarity (Fig. 2).
|
Using the ex vivo ELISPOT assay, each of the overlapping
ESAT-6-derived 15-mer peptides was recognized by IFN--secreting T
cells from one or more of 88 tuberculosis patients and contacts tested
(Fig. 3). In this population, peptides
ESAT-61–15, ESAT-66–20,
and ESAT-671–85 were particularly widely
recognized by 59%, 45%, and 37% of subjects, respectively.
|
-secreting T cells are mostly CD4
positive
Fifty-nine T cell lines were generated from several patients and
HHCs against 15 ESAT-6-derived peptides. After 12 days of culture with
IL-2 supplementation, ELISPOT assays were performed before and after
immunomagnetic depletion of CD4 or CD8 T cells. For 55 of 59 T cell
lines, peptide-specific responses were abrogated by CD4
depletion (Table II); 4 T cell lines
specific for certain peptides were CD8 positive (7, 39).
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-secreting T cells are capable of
rapid effector function
A response in the ex vivo ELISPOT assay for IFN- indicates T
cell-mediated IFN--secretion within 14 h of exposure to
peptide. To establish how quickly these T cells can release IFN-
upon Ag encounter, we conducted 6-h ex vivo ELISPOT assays in three
subjects (two HHCs and one C-PTB) with 11
peptides. In each case, IFN- SFCs were readily detected at 6 h
to all the peptides that gave a response at 14 h (data not shown).
The frequency of peptide-specific IFN--secreting T cells enumerated
at 6 h was 80–90% of that at 14 h, indicating that ESAT-6
peptide-specific T cells can secrete IFN- within 6 h of Ag
contact, and are thus capable of rapid effector function
(37).
T cells specific for certain ESAT-6 epitopes are not detected by assays dependent on in vitro proliferation
Using PBMC from 15 subjects, lymphoproliferation assays were
performed in parallel to ex vivo IFN- ELISPOT assays using 16
different peptides. Surprisingly, we did not detect proliferation to
peptides ESAT-61–15 and
ESAT-671–85 in several subjects with
IFN--secreting T cells specific for these peptides in the ex vivo
ELISPOT (Table II). Twelve subjects responded to
ESAT-61–15 by ex vivo ELISPOT (mean
peptide-specific IFN- SFCs for responders,
77/106 PBMC (IQ range, 38–100)) compared with
only three by lymphoproliferation. For
ESAT-671–85, 10 subjects responded by ex vivo
ELISPOT (mean peptide-specific IFN- SFCs for responders,
121/106 PBMC (IQ range, 65–171)) compared with 5
by lymphoproliferation. However, lymphoproliferative responses to the
positive control, PPD, were strong: mean stimulation index, 82 (IQ
range, 35–98). Nonetheless, peptide-specific T cell lines were readily
generated by peptide stimulation in vitro with IL-2 supplementation;
thus, these ESAT-6 peptide-specific T cells, while displaying rapid
effector function upon Ag contact, do not proliferate in vitro in the
absence of exogenous IL-2. For several other peptides, however, there
was a broad concordance between the two assays (Table II).
Immunodominant peptides ESAT-61–15 and ESAT-671–85 are HLA-DQ restricted
We were interested to determine the MHC restriction of the two
peptides that were immunodominant by ex vivo ELISPOT but gave
discrepant results in lymphoproliferation assays. We used mAbs to block
presentation of peptide to T cell lines and ex vivo PBMC in ELISPOT
assays. For ESAT-61–15, in all three individuals
tested, IFN- SFCs were markedly diminished by anti-HLA-DQ Ab,
but not by Abs to HLA-DR or HLA-DP (Fig. 4A–C). For
ESAT-671–85, two individuals were tested, and in
both cases, the response was blocked only by the anti-HLA-DQ Ab
(Fig. 4, D and E). In contrast, ex vivo ELISPOT
responses to the other peptides tested
(ESAT-66–20, ESAT-651–65,
ESAT-666–80, and
ESAT-676–90) were shown to be HLA-DR restricted
(data not shown).
|
In 35 subjects (28 patients and 7 HHCs), rESAT-6 Ag was tested in
the ex vivo ELISPOT assays in parallel with ESAT-6 peptides, and 30 of
35 (86%) responded to rESAT-6 (Fig. 3). The 5 subjects who did not
respond to the protein were subjects who also failed to respond to any
of the 17 peptides. Thus, of 30 patients who responded to one or more
peptides, all responded to rESAT-6 Ag, indicating that rESAT-6 is
processed and presented to T cells in the ex vivo ELISPOT assay.
ESAT-6 peptide-specific CD4 T cells are Th1 polarized
Freshly isolated PBMC from eight tuberculosis patients
(four C+PTB and four EPTB) and eight HHCs were
tested ex vivo against all 17 ESAT-6-derived peptides in an ELISPOT
assay for IL-4 in parallel with the ELISPOT assay for IFN-. The
numbers of SFCs were summated for each peptide. Among the tuberculosis
patients, eight of eight had ESAT-6 peptide-specific IFN--secreting
T cells (mean, 425 IFN- SFCs/million PBMC), while zero of eight had
ESAT-6 peptide-specific IL-4-secreting T cells. For the HHCs, six of
eight responded in the IFN- ELISPOT assay (mean frequency in
responders, 414 ESAT-6 peptide-specific IFN- SFCs/million PBMC)
compared with two of eight responders in the IL-4 ELISPOT assay (mean
frequency in responders, 93 ESAT-6 peptide-specific IFN-
SFCs/million PBMC). Both the contacts who responded in the IL-4 ELISPOT
assay also responded in the IFN- ELISPOT assay. Positive controls
for IFN- and IL-4 ELISPOT assays with PHA stimulation were always
over 500 IFN- or IL-4 SFCs/106 PBMC,
respectively (data not shown).
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Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-secreting CD4 T cells that circulate in the blood
of almost all M. tuberculosis-infected individuals, but are
absent in unexposed, BCG-vaccinated controls. The presence of
circulating IFN--secreting ESAT-6-specific CD4 T cells in
PPD-positive, latently infected HHCs, who were clinically and
radiographically free of disease, indicates that these T cells are not
necessarily associated with tissue pathology per se. Rather, their
presence in these individuals may be consistent with a role in the
containment of M. tuberculosis in vivo. This hypothesis is
supported by the observation that ESAT-6-specific CD4 T cells circulate
at higher frequencies in HHCs than in C+PTB
patients (Fig. 1). Among subgroups of tuberculosis patients, the
frequency of these T cells was higher in those groups who manifest
relatively more successful containment of M. tuberculosis in
vivo. Thus, TBLN patients (with highly localized disease, minimal
symptoms, and frequent spontaneous resolution) and self-healed
C-PTB patients (with no symptoms and no evidence
of ongoing bacterial replication in multiple clinical specimens) both
had higher frequencies of ESAT-6-specific CD4 T cells than
C+PTB patients with M. tuberculosis
actively growing in respiratory secretions (Table I and Fig. 1).
However, active tuberculosis causes an ill-defined nonspecific
immunosuppression. It is therefore possible that immune responses that
are weaker in patients with more extensive active disease than in
patients with minimal disease are merely secondarily suppressed as a
consequence of active tuberculosis itself. If this were the case,
immune responses should rise with treatment and, in general,
Ag-stimulated or PPD-stimulated IFN- secretion from cultured PBMC
and lymphoproliferative responses do increase during antituberculous
therapy (28, 29, 30, 40, 41). However, longitudinal follow-up
of tuberculosis patients with the ex vivo ELISPOT assay showed that the
frequency of ESAT-6-specific IFN--secreting CD4 T cells actually
fell during therapy (p = 0.005) (Fig. 2). This
indicates that the lower frequency of ESAT-6-specific T cells in
patients with more extensive disease is not a secondary effect of
disease-related immunosuppression nor a result of sequestration of
M. tuberculosis-specific T cells at sites of active disease.
Rather, the decline in ESAT-6-specific T cells with therapy, which
reduces bacterial load by several orders of magnitude, suggests that,
within a given individual, the frequency of ESAT-6-specific T cells is
related to Ag load. This observation was made possible by using an
assay that directly quantitates Ag-specific T cells without involving
in vitro proliferation (33, 37, 38). Our finding is
somewhat analogous to the decay in virus-specific CD8 and CD4 T cell
frequencies, directly enumerated from peripheral blood, in HIV-infected
patients during combination antiretroviral therapy (31, 32, 34) and, in general for intracellular pathogens, frequencies of
Ag-specific T cells, when directly quantitated ex vivo, appear to be
driven by Ag load (33, 42, 43).
Although there is no quantitative absolute measure of bacterial or Ag
load in tuberculosis, they must be directly and closely interrelated.
Ag load is almost certainly much lower in HHCs than in patients and,
among patients, will obviously be lower in TBLN patients and
C-PTB patients than in
C+PTB patients. Given that Ag load would be
expected to drive ESAT-6-specific CD4 T cell frequencies, it is
remarkable that the M. tuberculosis-infected subjects with
lower Ag loads have higher levels of ESAT-6-specific CD4 T cells. This
inverse correlation is similar to that observed for virus-specific CD8
T cell frequencies and plasma viral load in HIV-infected patients
(31), a finding that provided important support for the
protective role of CD8 T cells in control of HIV and that depended
crucially upon a means to quantify Ag-specific T cells directly ex vivo
(33, 38). Thus, consideration of the frequencies of
ESAT-6-specific T cells in the context of the differing bacterial load
in the different groups of M. tuberculosis-infected subjects
lends further support to the hypothesis that these T cells mediate
containment of M. tuberculosis in vivo. All groups of
patients had similar proportions of individuals who had undergone
treatment except for the HHCs, none of whom had received
chemoprophylaxis. Moreover, the effect of treatment on ESAT-6
peptide-specific T cell frequencies appears to be similar for all
groups of patients followed up (Fig. 2). Thus, the differences in
ESAT-6-specific CD4 T cell frequencies between the three groups of
patients are unlikely to result from differences in duration of
treatment, although this may not apply to the HHCs.
Our interpretation of these findings is that, at the time of initial
infection with M. tuberculosis, HHCs mount a strong, high
frequency Th1-type CD4 T cell response to M. tuberculosis
and, in particular, to ESAT-6, and so help to limit bacterial
replication. Individuals who go on to develop active disease, in
contrast, make a weak CD4 T cell response, and the bacteria are allowed
to reach a higher equilibrium bacterial load, resulting in disease.
Thus, HHCs maintain a high frequency of ESAT-6-specific CD4 T cells
with limited antigenic stimulation from a low bacterial load, while in
C+PTB patients, a high bacterial load stimulates
weaker, less efficient proliferation of Ag-specific CD4 T cells in
vivo. This model is analogous to that proposed for certain chronic
viral infections, in which virus-specific CD8 T cells are believed to
mediate protective immunity (44). The difference between
HHCs and PTB patients may lie in an individual’s T cell
responsiveness, which has previously been defined as the rate at which
pathogen-specific T cells proliferate in vivo after encountering an
infected host cell (44). Indeed, the early emergence and
efficient proliferation of IFN--secreting CD4 T cells in vivo have
recently been identified as crucial factors in the early containment of
mycobacterial infection in murine models (1, 45, 46).
ESAT-6 contains multiple CD4 T cell epitopes, as previously described
(19, 47). Fig. 3 shows that several ESAT-6-derived
peptides are widely recognized by T cells from an ethnically and
genetically diverse range of patients and HHCs, suggesting that these
peptides may be permissively restricted by a wide range of HLA class II
haplotypes. The hierarchy of immunodominance using the ex vivo ELISPOT
assay (Fig. 3) is quite different to that reported by others using more
conventional assays that depend on in vitro proliferation
(19). In particular, the striking immunodominance of
peptides ESAT-61–15 and
ESAT-671–85 has not been previously recognized
with other assays, and might reflect the fact that the ex vivo ELISPOT
assay can detect T cells that do not proliferate well in vitro
(33, 37). We therefore tested these, and the other
ESAT-6-derived peptides, for their ability to stimulate T cells in
lymphoproliferation assays. Although for most peptides there was a
broad concordance between T cell responses in these assays and in the
ex vivo ELISPOT, for peptides ESAT-61–15 and
ESAT-671–85 there was a marked discrepancy
(Table II). The impaired ability of
ESAT-61–15-specific and
ESAT-671–85-specific T cells to proliferate in
vitro probably explains why the immunodominance of these peptides was
not hitherto fully appreciated. Interestingly, however, these T cells
did proliferate in vitro if stimulated with IL-2 as well as
peptide.
Because peptides ESAT-61–15 and
ESAT-671–85 are recognized ex vivo by T cells
from a very high proportion of M. tuberculosis-infected
subjects, their HLA restriction is of special interest. Immunodominant,
permissively restricted CD4 epitopes are usually HLA-DR restricted, and
this is the case in tuberculosis (48). Surprisingly,
peptides ESAT-61–15 and
ESAT-671–85 were shown to be HLA-DQ restricted
in ELISPOT assays (Fig. 4). These are, to our knowledge, the first
immunodominant HLA-DQ-restricted mycobacterial epitopes to be
identified.
Having found that most M. tuberculosis-infected individuals
have high frequencies of ESAT-6-specific IFN--secreting CD4 T cells,
we asked whether ESAT-6-specific Th2-type CD4 T cells are also induced
to a similar extent. Ex vivo ELISPOT assays for IL-4 in a subset of
patients and HHCs showed that, in contrast to the almost universal
presence of ESAT-6-specific IFN--secreting T cells in M.
tuberculosis-infected subjects, IL-4-secreting T cells specific
for ESAT-6 are rare. ESAT-6-specific CD4 T cells induced by natural
M. tuberculosis infection thus have a highly Th1-polarized
pattern of cytokine secretion.
We have identified a population of circulating IFN--secreting
M. tuberculosis Ag-specific CD4 T cells that circulate at
high frequencies in asymptomatic individuals with latent M.
tuberculosis infection, as well as tuberculosis patients. Our
direct quantitative ex vivo approach led to certain key findings that
have not been recognized using previous assays. Unlike other cellular
immune responses in tuberculosis, frequencies of ESAT-6-specific CD4 T
cells decay progressively with treatment, suggesting that these T cell
frequencies are driven, at least in part, by Ag load. Therefore,
patients with more extensive disease and higher bacterial loads would
be expected to have the highest Ag-specific T cell frequencies.
However, we observed the opposite: between groups of tuberculosis
patients, the frequencies of these T cells appear to correlate
inversely with inferred bacterial load. These findings are consistent
with a role for this population of Th1-type Ag-specific CD4 T cells in
the containment of M. tuberculosis in vivo. These results,
together with the fact that ESAT-6 contains multiple CD8 T cell
epitopes (7, 39), suggest that this Ag may be a target of
protective immune responses in M. tuberculosis-infected
humans; its absence from M. bovis BCG (23, 24)
might thus account, in part, for the limited efficacy of BCG
vaccination. Our findings thus support the development of an
ESAT-6-based tuberculosis subunit vaccine that aims to induce Th1-type
CD4 and CD8 T cells. Such a vaccine would, however, limit the clinical
utility of ESAT-6-specific T cell responses as a diagnostic marker for
M. tuberculosis infection; these potentially contrasting
roles for ESAT-6 will need to be reconciled over the next few years.
Finally, the decay of ESAT-6-specific CD4 T cells in the ex vivo
ELISPOT during effective antituberculous chemotherapy may suggest a
novel approach for monitoring the efficacy of new pharmacological or
immunological interventions for tuberculosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ajit Lalvani, Nuffield Department of Clinical Medicine, University of Oxford, Level 7, John Radcliffe Hospital, Oxford OX3 9DU, U.K. E-mail address: ajit.lalvani{at}ndm.ox.ac.uk
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; C+PTB, culture-positive pulmonary tuberculosis; C-PTB, culture-negative pulmonary tuberculosis; CI, confidence interval; EPTB, extrapulmonary tuberculosis; HHC, healthy household contact; PPD, purified protein derivative; SFC, spot-forming cell; TBLN, tuberculous lymphadenitis; TST, tuberculin skin test; UC, unexposed control.
Received for publication April 10, 2001. Accepted for publication August 22, 2001.
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