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*
Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, United Kingdom;
Molecular Immunology Group, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom;
Tuberculosis Research Programme, MRC Laboratories, Fajara, The Gambia; and
§
Glaxo-Wellcome Research and Development, Medicines Research Centre, Stevenage, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release was used to
screen CD8+ T cells with overlapping peptides spanning the
mycobacterial major secreted protein, Ag85A. Three peptides
consistently induced a high frequency of IFN- responsive
CD8+ T cells, and two HLA-A*0201 binding motifs,
P48–56 and P242–250, were revealed within the
core sequences. CD8+ T cells responding to the 9-mer
epitopes were visualized within fresh blood by ELISPOT using free
peptide or by binding of HLA-A*0201 tetrameric complexes. The class
I-restricted CD8+ T cells were potent CTL effector cells
that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with
peptide as well as autologous macrophages infected with
Mycobacterium tuberculosis or recombinant vaccinia virus
expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold
higher frequency of peptide-specific T cells than IFN- ELISPOT
assays, indicating functional heterogeneity within the CD8+
T cell population. These results demonstrate a previously unrecognized,
MHC class I-restricted, CD8+ CTL response to a major
secreted Ag of mycobacteria and supports the use of Ag85A as a
candidate vaccine against tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular arm of the immune response mediated by Ag-specific
IFN--secreting CD4+ T cells has long been
established as an essential component of the protective immune response
against M. tuberculosis (3, 4, 5, 6, 7, 8). However, more
recently, the importance of other T cell populations in protective
immunity against TB has come to light. There is increasing evidence in
both the murine model of TB infection and human studies that a major
role exists for MHC class I-restricted 
ß
TCR+ CD8+ T cells
(9, 10, 11, 12, 13, 14, 15, 16).
Ag-specific CD8+ T cells can act as
cytokine-secreting cells, producing the type 1 cytokines IFN- and
TNF-, which activate macrophage mycobactericidal mechanisms
(17); in addition, CD8+ T cells can
perform a CTL function, lysing infected cells and releasing bacteria
for either extracellular killing, or for uptake by activated
macrophages (17). Human CD8+ T cells
have been shown to perform both cytokine production and cytolytic
effector functions when stimulated with mycobacteria (9, 12, 13, 14, 15). In the murine model, perforin, granzyme, and Fas
pathway knockout mice that lack the cytolytic functions of
CD8+ T cells appear to be no more susceptible to
M. tuberculosis infection than wild-type mice, at least
during the early acute infection (18, 19). In addition,
the transfer of immunity with CD8+ T cells has
been shown to be dependent upon the ability of the
CD8+ T cells to secrete IFN-
(20), and an increase in IFN--secreting
CD8+ T cells has been observed in the lungs of
M. tuberculosis-infected mice (21, 22).
Clearly, CD8+ T cells play an important role in
immunity against TB; however, their exact function remains unclear.
The specific Ags to which CD8+ T cells respond to are poorly characterized. At present, only three human CD8+ T cell reactive epitopes have been identified, two in ESAT-6 and one in the 19-kDa Ag (23, 24). Strong CD8+ T cell reactivity has also been observed against the 38-kDa protein of M. tuberculosis (15, 25). Murine vaccination experiments have also shown CD8+ T cell reactivity against Ag85A (26) and heat shock protein-65 (27, 28). Previously, we have found human M. bovis BCG-reactive CD8+ T cells to give strong CTL activity against Ag85A (15). In the present study, we have investigated the human CD8+ T cell response to Ag85A to define the epitopes recognized.
Ag85A is one of three distinct but highly conserved proteins (85A, -B,
and -C) that together make up the Ag 85 complex, a highly
cross-reactive Ag found in all mycobacterial species tested so far.
Ag85A is a secreted Ag that constitutes a major portion of the secreted
proteins present in the culture filtrate of both M.
tuberculosis and M. bovis BCG (29).
CD8+ CTL responses have been observed against
culture filtrate proteins of both M. bovis BCG and M.
tuberculosis (30, 31). Furthermore, the Ag85 complex
has been shown to induce good proliferative, IFN-, and cytolytic
responses in BCG-vaccinated, or M. tuberculosis- and
Mycobacterium leprae-infected individuals (15, 32, 33, 34). Mouse immunization experiments have shown plasmid DNA
vaccination encoding Ag85A to generate a strong Th1
CD4+ T cell and CD8+ T
cell-mediated CTL response in mouse immunization studies (26, 35, 36).
In this paper, we describe the CD8+ T cell epitope mapping of Ag85A in healthy BCG-vaccinated donors. M. bovis BCG-stimulated CD8+ T cells were used to screen 33 overlapping 20-mer peptides covering the entire Ag85A protein from M. tuberculosis. Once peptides had been identified as possessing CD8+ T cell reactivity, peptide-specific cell lines were generated and used in cytotoxicity assays to identify HLA restriction of the epitopes. In addition, reactivity of these peptide-specific lines to whole protein expressed by recombinant vaccinia virus (rVV), or live M. bovis BCG and M. tuberculosis infection, was demonstrated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten healthy U.K. laboratory donors who had previously received M. bovis BCG vaccination were recruited from the London School of Hygiene and Tropical Medicine. Blood samples were taken after gaining written permission from those individuals participating in the study. Ethical permission was obtained from the Ethics Committee at the London School of Hygiene and Tropical Medicine.
Four healthy BCG-vaccinated HLA-A*0201 Gambian donors were recruited from the Banjul Blood Bank, Royal Victoria Hospital, after informed consent was obtained. Ethical permission was given by the Medical Research Council (MRC), The Gambia, Scientific Coordinating Committee, and The Gambia Government/MRC Ethics Committee.
Tissue typing
Subjects were HLA typed by amplification mutation system (ARMS)-PCR using sequence-specific oligonucleotide primers.
Synthetic peptides
The HLA-A*0201-restricted influenza matrix epitope FMP M158–66 (GILGFVFTL) and a total of 33 sequential peptides of 20 amino acids, overlapping by 10 and spanning the complete Ag 85A protein sequence for M. tuberculosis, H37Rv (Sanger Center database: http://www.sanger.ac.uk) were commercially synthesized by Research Genetics (Huntsville, AL). Peptide synthesis was confirmed by HPLC and mass spectrometry profiles and were >80% purity.
M. bovis BCG and M. tuberculosis culture
M. bovis BCG (Glaxo Evans strain; Evans Medical, Leatherhead, U.K.) and M. tuberculosis (H37Rv) were grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, MI) supplemented with Middlebrook ADC enrichment (Difco) and 0.05% Bacto-Glycerol (Difco). Bacteria were harvested and frozen at -70°C. Bacterial counts were determined by counting CFUs either grown on Middlebrook 7H10 agar supplemented with OADC enrichment (Difco) or in liquid 7H9 agar supplemented with ADC enrichment (Difco). Vials were thawed and washed once with RPMI 1640 (Life Technologies, Paisley, U.K.) and sonicated for 10 s in a water bath sonicator (Grant XB2; Fisons, Leicester, U.K.) to disrupt clumps before use.
rVV construction
rVV expressing the mycobacterial proteins Ag85A were constructed by Dr. A. Malin as previously described (15). rVV were cultivated by growth in the thymidine kinase-disrupted osteosarcoma cell line 143 (TK-143). Viruses were harvested and purified by sucrose cushion density centrifugation before aliquoting at -70°C. On the day of use, aliquots were thawed and sonicated before use in assays.
THP-1 cell culture
The human monocyte cell line THP-1 (American Type Culture Collection, Manassas, VA; TIB-202) was maintained in T75 tissue culture flasks (Nunclon, Roskilde, Denmark). The growth medium used was RPMI 1640 supplemented with 2 mM L-glutamine (Life Technologies) and 10% heat-inactivated FCS (Life Technologies).
PBMC separation
PBMC were isolated from heparanized venous blood by density gradient centrifugation over Ficoll-Histopaque (Sigma, Poole, Dorset, U.K.). Nonadherent cells (NAC) and monocytes were separated by adherence to plastic. Briefly, PBMC were resuspended at 1 x 106 cells/ml in RPMI 1640 and plated at 200 µl/well of a 96-well round-bottom tissue culture plate. Cells were incubated for 1 h at 37°C before NAC were removed by pipetting off the supernatant. Adherent monocytes were then resuspended in culture medium (CM) and incubated at 37°C, 5% CO2 for use as target cells in CTL assays. CM consisted of RPMI 1640 supplemented with 2 mM L-glutamine (Life Technologies), 50 µg/ml ampicillin (Sigma), and 10% autologous plasma. NAC were used for the generation of peptide specific short-term cell lines (STCL).
Generation of CD8+ T cell lines
NAC alone were prepulsed with 50 µM peptide for 1 h in a cell pellet and then diluted up to 1 x 106 cells/ml in CM supplemented with 25 ng/ml rhIL-7 (R&D Systems, Abingdon, U.K.) and seeded at 100 µl/well in a 96-well round-bottom tissue culture plate. CM containing 10% Lymphocult-T (Biotest, Solilhull, U.K.) was added to each well at 3- to 4-day intervals. For the generation of M. bovis BCG cell lines, PBMC were aliquoted at 100 µl/well and infected with 2 x 104 bacilli per well. Cells were cultured as for peptide-specific cell lines.
CD8+ T cell separation
Ag-specific CD8+ T cells were prepared by
positive enrichment using the MACS system (Miltenyi Biotech,
Bergisch-Gladbach, Germany). In brief, PBMC were labeled with CD8
microbeads (Miltenyi Biotech; 20 µl/107 cells)
in incubation buffer (PBS/0.5% BSA/2 mM EDTA; 80
µl/107 cells) for 15 min at 4°C. After one
washing, step cells were resuspended in incubation buffer (1 ml
buffer/107 cells), and enrichment was performed
using LS+ columns and the MidiMACS magnet
according to manufacturer’s instructions. The resulting
CD8+ T cell population was >95% pure as
determined by flow cytometric analysis for the surface markers ß
TCR, CD3, CD8, and contamination with CD4 and CD56.
Enzyme-linked immunospot (ELISPOT) assay for IFN-
Ninety-six-well polyvinylidene difluoride-backed plates (MAIP S
45; Millipore, Bedford, U.K.) were precoated with 15 µg/ml
anti-IFN- mAb (1-D1K; MABTECH, Stockholm, Sweden) overnight at
4°C. Plates were then washed six times with RPMI 1640 and blocked
with RPMI 1640 supplemented with 10% heat-inactivated FCS for 1 h
at 37°C. Purified CD8+ T cells from PBMC or
STCL were washed twice with RPMI 1640, resuspended in RPMI 1640
supplemented with L-glutamine and 10% autologous plasma,
and dispensed at known input cell numbers per well in duplicate wells
without additional APCs. In this sensitive ELISPOT assay, efficient
presentation of peptide occurs without additional APCs, because
CD8+ T cells can present peptide through HLA
class I to each other (23, 37). Peptide was added directly
to the supernatant at a final concentration of 2 µM, and the plates
were incubated at 37°C for 18 h. Cells were then shaken off and
the plate washed six times with PBS 0.05% Tween 20 (Sigma). Plates
were then incubated for 3 h at 37°C with the second biotinylated
anti-IFN- mAb (7-B6-1-biotin at 1 µg/ml; MABTECH). A further
wash with PBS 0.05% Tween 20 was performed before the addition of a
1:1000 dilution of streptavidin-alkaline phosphatase conjugate
(MABTECH) for 2 h. Wells were again washed six times, and 100 µl
of chromogenic alkaline phosphatase substrate (Bio-Rad, Hemel
Hempstead, Hertfordshire, U.K.) was added to each well. After 30 min,
the colormetric reaction was terminated by washing with tap water and
the plates were allowed to air dry. After drying, spots were counted
by eye.
51Cr release cytotoxicity assays
Autologous monocyte-derived-macrophages or the cell line THP-1 were seeded at 10,000 cells/well of a 96-well round-bottom plate and used as target cells in cytotoxicity assays. Target cells were incubated overnight at 37°C in CM (RPMI supplemented with L-glutamine plus 10% autologous plasma for macrophages or 10% heat inactivated pooled human AB serum for THP-1 cell line), loaded with Ag (10 µg/ml peptide; 10:1 (CFU/macrophage) M. tuberculosis; 25:1 (PFU/macrophage) rVV expressing Ag85A; or no Ag), and pulsed with 2 µCi 51Cr. Target cells were washed three times with RPMI and either treated with 5 µg/ml w6/32 mAb (Sigma) or resuspended in CM alone before being used in the cytotoxicity assay. Purified CD8+ T cells were added as effector cells to the 96-well plate at various E:T ratios (50:1, 5:1, 0:1) in a final volume of 100 µl/well and incubated at 37°C for 6 h. Supernatants (50 µl) were harvested, and 51Cr release was measured using a gamma counter. The remaining supernatant was removed and replaced with 100 µl/well 5% SDS (Sigma) for 1 h at 37°C to lyse the remaining cells. Spontaneous release was measured in wells containing target cells alone. The percent specific lysis was calculated by the following formulas: % isotope release = [cpm supernatant/(cpm supernatant + cpm pellet)] x 100; and % specific lysis = [(% isotope release test wells) - (% isotope release control wells)].
Tetramers
Tetrameric complexes were synthesized as previously described (38, 39). Briefly, purified HLA heavy chain and ß2-microglobulin were synthesized using a prokaryotic expression system (pET; R&D Systems). The heavy chain was modified by deletion of the transmembrane cytosolic tail and COOH-terminal addition of a sequence containing the biotin-protein ligase BirA (Avidity, LLC, Denver, CO) enzymatic biotinylation site. Heavy chain, ß2-microglobulin, and peptide were refolded by dilution. The 45-kDa refolded product was isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma), adenosine 5'-triphosphate (Sigma), and Mg2+ (Sigma). Streptavidin-PE conjugate (Sigma) was added in a 1:4 molar ratio, and the tetramic product was concentrated to 1 mg/ml.
Flow cytometric analysis
Fresh PBMC or peptide-specific STCL were incubated for 30 min at 37°C in staining buffer (PBS with 0.1% BSA and 0.1% sodium azide) containing 3 µl of PE-labeled tetrameric complex. Cells were given one wash in warm staining buffer. Cells were then incubated at 4°C in staining buffer containing saturating amounts of anti-CD8 mAb conjugated to FITC (Becton Dickinson, Mountain View, CA) or TriColor (Caltag, Buckingham, U.K.). In addition, saturating amounts of anti-CD45Ra or CD45Ro Abs (Becton Dickinson) conjugated to FITC were added to certain samples for phenotype characterization. The stained cells were fixed in PBS containing 4% paraformaldehyde. Samples were analyzed by FACS using CellQuest software (Becton Dickinson). Lymphocytes were gated by forward and side angle light scatter; >105 events were acquired.
Intracellular cytokine staining
Intracellular cytokine production by PBMC was assessed by
three-color flow cytometry. Briefly, cells were stained with tetramer
in 100 µl RPMI. Cells were then resuspended into 1 ml CM containing
10% autologous plasma. Cells were stimulated with peptide at 10
µg/ml for 6 h at 37°C. Cytokine secretion was blocked by
incubation with 10 µg/ml brefeldin A (Sigma) for the duration of the
stimulation. Cells were washed twice in cold PBS and stained for
surface CD8 with TriColor-conjugated Ab (Caltag) at 4°C for 30 min in
incubation buffer (PBS/0.5% BSA/0.1% Na azide). Cells were washed
twice in cold incubation buffer before fixation and permeabilization by
incubation for 30 min in FACS Cytofix/Cytoperm solution (PharMingen,
San Diego). Cells were washed in 1x PermWash solution (PharMingen).
Staining for intracellular cytokine was performed in 1x PermWash with
FITC-conjugated mAb against IFN-, or IL-4 (PharMingen) for 30 min at
4°C. Cells were then washed in PBS and analyzed using CellQuest
software on a FACScan or FACScalibur (Becton Dickinson);
>105 events were acquired for each sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that CD8+
CTL are elicited by exposure to M. bovis BCG and that these
cells recognize Ag85A (15). In this study, we employed the
same approach using PBMC from BCG-vaccinated individuals to generate
M. bovis BCG-specific cell lines and to isolate the
CD8+ T cells. PBMC from 10 healthy BCG-vaccinated
donors were stimulated for 14 days with live M. bovis BCG in
the presence of IL-2 and IL-7 to produce BCG-reactive STCL.
CD8+ T cells were purified from the lines by
MACS-positive selection and used in a sensitive IFN- ELISPOT assay
to detect single cell cytokine production. The
CD8+ T cells were stimulated overnight with one
of the 33 20-mer overlapping peptides from Ag85A to determine which
peptides contained CD8+ T cell-reactive epitopes
(Fig. 1
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production was observed in response to peptides 5, 24, and 25
(Fig. 1
). Closer sequence analysis of these three peptides revealed
HLA-A*0201 binding motifs within the core region of the peptides (Table I). Thus, the HLA-A*0201 motif was
identified as the active region within the peptide 5, because no
reactivity was observed against the flanking peptides 4 and 6.
Similarly, equivalent responses to both peptide 24/25 and not the
flanking peptides indicated reactivity to the overlapping 10 amino
acids congruent with the HLA-A*0201 motif. T cell lines reactive to
peptide 5, or peptides 24/25, were then tested for reactivity with
9-mer peptides containing the HLA-A*0201 binding motif, and both 9-mer
and 20-mer peptides induced equivalent responses. A molecular tissue
type of the five donors who responded to the peptides revealed the
donors all to be HLA-A*0201 (Table II).
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To determine the frequency of circulating peptide-specific
CD8+ T cells, fresh CD8+ T
cells from five HLA-A*0201 donors were assayed in an ex vivo ELISPOT
assay. The mean frequency of circulating
P48–56-reactive CD8+ T
cells was 1/20,000 (range 1/18,519–1/22,727) compared with 1/23,810
(range 1/20,833–1/27,777) P242–250-specific
cells. The frequency of P48–56- and
P242–250-specific IFN- spot-forming cells
(SFCs) was of the same order of magnitude as circulating cells specific
for the HLA-A*0201-restricted influenza matrix epitope FMP
M158–66 (Fig. 2).
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To confirm the HLA restriction of these two epitopes,
CD8+ T cell lines were established by a 14-day
culture of PBMC in the presence of either P48–56
or P242–250. The CTLs elicited were assayed for
their cytotoxic activity against the peptide-pulsed human HLA-A2-,
-A9-, -B5-restricted monocyte cell line THP-1. Peptide-specific
HLA-A2-restricted CTL activity was observed by both
P48–56- and
P242–250-specific CD8+ T
cells (Fig. 3). As seen in Fig. 3A, P48–56-stimulated
CD8+ T cells showed strong CTL activity against
THP-1 cells pulsed with P48–56 (76.4 ±
4.3% specific lysis at 50:1 E:T ratio), but no significant lysis
against either FMP M158–66 or
P242–250 pulsed targets (<10% specific lysis
at 50:1 E:T ratio). Conversely, as shown in Fig. 3B,
P242–250-reactive CD8+ T
cells showed potent CTL activity against
P242–250-pulsed THP-1 cells (76.8 ± 7.0%
specific lysis at 50:1 E:T ratio), but no significant lysis against FMP
M158–66 or P48–56 pulsed
target cells. The cytolytic activity of these Ag-specific
CD8+ CTLs was completely inhibited by the
addition of an anti-MHC class I mAb w6/32. Blocking with this mAb
reduced the levels of lysis to <10%. The addition of an
isotype-matched control mAb (IgG2a) had no significant effect upon
lysis of target cells (data not shown). These results demonstrate that
the peptide-specific CD8+ T cell response is HLA
class I-dependent and, in the case of the donors matched only at HLA-A2
with the target cell line, HLA-A2 restricted.
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To examine the ability of peptide-specific CTLs to recognize
endogenously generated epitopes, autologous monocyte-derived
macrophages were infected overnight with either M.
tuberculosis H37Rv bacilli, or rVV expressing the whole Ag85A
protein. Fig. 4 shows that
P48–56- and
P242–250-specific CTLs are able to recognize and
lyse M. tuberculosis and rVV85A-infected macrophages. The
lysis observed against M. tuberculosis bacilli was shown to
be MHC class I-dependent by blocking with mAb w6/32. These results
demonstrate antimycobacterial activity of peptide-specific CTLs by the
recognition of endogenous generated peptide in a MHC class I-restricted
manner.
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To determine whether the observed recognition of
P48–56 and P242–250 was
unique to U.K. BCG vaccinees, four HLA-A*0201 BCG vaccinated donors
from The Gambia were tested for peptide specific responses. STCL were
generated against both peptides and were tested for CTL activity
against autologous peptide and M. tuberculosis-infected
macrophages. As shown in Fig. 5, peptide
reactivity was observed in all four of the Gambian donors.
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The Ag85A HLA-A*0201 epitopes were used to synthesize tetramer complexes able to stain the peptide-specific CD8+ T cells from PBMC and cell lines. To determine the specificity of each tetramer, fresh blood PBMC from an HLA-A*0201-negative BCG vaccinee were stained with each tetramer; <0.01% of CD8+ T cells stained with either tetramer. In addition, PBMC from three HLA-A*0201 BCG-vaccinated donors were used to generate FMP M158–66-specific T cell lines; tetramer staining was then performed using the Ag85A tetramers: again, <0.01% of CD8+ T cells stained positive for either tetramer.
Fresh blood tetramer staining revealed that 
1/3500 circulating
CD8+ T cells were specific for
P48–56 or P242–250, so
that at least 1/1750 cells are specific for Ag85A as a whole (Table III). The frequency of circulating
peptide-specific CD8+ T cells was 6-fold
higher when detected by tetramer compared with ELISPOT (Table III).
After 10 days of culture with peptide, the frequency of
tetramer-specific T cells increased to 1/20
CD8+ T cells (Fig. 6).
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but not IL-4
To determine the phenotype of the tetramer-specific
CD8+ T cells, fresh blood PBMC from three
HLA-A*0201 BCG-vaccinated donors were stained for surface CD45Ra/CD45Ro
expression in addition to intracellular IFN- or IL-4 production.
As shown in Table IV, the majority
(70–85%) of P48–56-specific
CD8+ T cells expressed the CD45Ro isoform of
CD45, indicative of a memory-like phenotype. In addition, as shown in
Fig. 7 and Table IV, 20% of
tetramer-specific CD8+ T cells produced IFN-;
however, no significant IL-4 was found.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activation of CD4+ T cells has been known for some time to be important for protection against TB; however, there is now mounting evidence from animal models that CD8+ T cells may also be required (10, 11). Nevertheless, the existence of CD8+ T cells specific for M. tuberculosis in human disease has only recently come to light (9, 10, 11, 12, 13, 14, 15). As such, only three human CD8+ T cell epitopes have been identified within M. tuberculosis-specific proteins: two within the secreted protein ESAT-6 (23) and one within the 19-kDa protein (24).
At present, the only available vaccine against TB is the live attenuated M. bovis BCG, which is fully protective in some studies and completely ineffective in others (40). The same vaccine is also the benchmark for protection in animal models of TB where no experimental vaccine has yet proven to be superior. A clearer understanding of how BCG works, or a way to dissect out which of the multiple responses elicited by this vaccine are important, would be of enormous benefit in understanding what protective immunity entails. Unlike the more focused "peptide-based" approach, here, we use the whole live BCG organism to restimulate T cell reactivity from naturally primed individuals. The use of the intact bacillus enables identification of Ags that are preferentially immunogenic as well as those that are processed and presented through a natural route. The data strongly suggest that, of the many proteins secreted during an early infection, Ag85A is at least one that is readily presented through HLA class I. The fact that both of the Ag85A CTL epitopes were presented by macrophages within 24 h of an in vitro infection with M. tuberculosis shows that the microbial secreted Ag is rapidly processed and presented through HLA-class I as well as being immunogenic in M. tuberculosis or M. bovis BCG-infected macrophages.
Of the two epitopes identified, among available sequences, P242–250 is specific for Ag85A of M. tuberculosis and M. bovis, whereas P48–56 is also found in Ag85B, Ag85C proteins of M. tuberculosis, and in Ag85A sequences of a number of other mycobacteria, including M. bovis, M. paratuberculosis, M. avium, and M. leprae (http://www.sanger.ac.uk). It is therefore highly probable that the P48–56 CTL epitope will also be conserved in mycobacteria, where the Ag85 complex proteins are as yet unsequenced. The cross-reactive nature of the P48–56 epitope offers the potential for CTL responses to be primed or boosted through natural exposure to environmental bacilli. Of particular significance was the finding that P48–56- and P242–250-specific CTL were able to recognize and lyse macrophages infected in vitro with M. tuberculosis. The response is therefore likely to have some functional importance in the immune response against infection with the pathogen.
Despite quite diverse environmental and genetic backgrounds, groups of HLA-A*0201 individuals from both Britain and The Gambia responded to the Ag85A CTL epitopes that we describe. Together these data demonstrate that Ag85A CTL epitopes are naturally primed in vivo and that these are likely to be common in different population groups.
Studies by ELISPOT, to investigate the frequency of Ag85A
peptide-specific CD8+ T cells in circulation ex
vivo, revealed a frequency of T cells comparable to that of the
immunodominant HLA-A*0201 epitope of the influenza virus matrix
protein. Because none of the individuals had current influenza virus or
clinical mycobacterial disease, it is likely that a proportion of these
cells possess an immediate effector or memory cell phenotype
(23). The number of peptide-specific
CD8+ T cells is remarkably high considering that
these cells were likely to have been primed by BCG vaccination 10–30
years earlier. When M. bovis BCG-specific lines were
established, the frequency of peptide-specific
CD8+ T cell epitopes increased 10- to 20-fold,
demonstrating the existence of highly effective
CD8+ T cell memory populations.
The identification of CTL epitopes allowed the formation of peptide-MHC
tetrameric complexes that stain epitope-specific CTL directly. These
are the first bacterial-derived peptide tetramers constructed to be
used in the human system; all the previous studies with human cells
have used tetramers containing tumor or viral peptide epitopes
(37, 38, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53). Interestingly, higher frequencies of
peptide-specific CD8+ T cells were seen by
tetramer staining when compared with ELISPOT frequencies. Detection of
peptide-specific CD8+ T cells using such tetramer
complexes is not dependent on T cell function, and these cells do not
all secrete IFN-. Previous studies have also found higher
frequencies of virus- and tumor-specific CD8+ T
cells by tetramer when compared with other methodologies, such as
limiting dilution or ELISPOT (44).
The different estimates of epitope-specific T cell frequencies obtained using ELISPOT and tetramer staining suggest that the "memory pool" of Ag-specific T cells within PBMC is functionally heterogeneous. In the present study, tetramer-specific CD8+ T cells were identified in both the peripheral blood and Ag-specific cell lines, and marked increases in frequency were obtained following stimulation with peptide in vitro. Ex vivo staining of tetramer-specific CD8+ T cells showed the majority of these cells to be CD45Ro-positive, indicative of a memory phenotype. Further studies with the tetrameric complexes will be invaluable to monitor alteration in the frequency of Ag-specific CD8+ T with differing functional phenotypes in studies following M. tuberculosis infection, BCG vaccination, and for testing new vaccines.
Ag85A is a major secreted Ag that is present at a high concentration in
mycobacterial culture filtrate proteins (29) and to which
CD8+ CTL activity has been observed in the mouse
(30, 31). Ag85A is reported to induce proliferative and
IFN- responses by Th1 CD4+ T cells in both
human and murine models (32, 33). It was therefore of
particular interest to assess the proportion of circulating
CD8+ T cells that would recognize peptides from
Ag85A. The data obtained to date suggest that as many as 1 in 1000
circulating CD8+ T cells may respond to this
naturally secreted Ag, which is capable of eliciting classically
restricted CD8+ CTL in humans. Furthermore,
vaccination of mice with plasmid DNA encoding Ag85A will induce
CD8+ T cells specific for MHC class I-restricted
epitopes within Ag85A and confer protection against M.
tuberculosis challenge (26, 35, 36).
Other groups have shown that heat shock fusion proteins and soluble Ags from M. tuberculosis can be presented via the MHC class I pathway (54, 55). However, the means by which Ags, from a bacteria which has been shown to reside within the phagosome (a site inaccessible to the MHC class I presentation pathway) are presented to CD8+ T cells is unclear (56, 57). However, recent evidence showing phagosome permeability of infected macrophages and the identification of a M. tuberculosis hemolysin suggest possible mechanisms for presentation to CD8+ T cells through diffusion of Ags into the cytosol (58, 59).
In conclusion, we found that Ag85A-specific CTL are present at high
frequencies in healthy individuals, both in Britain and in The Gambia,
identifying frequent responses to conserved epitopes. At least one of
the epitopes identified is found in environmental mycobacteria, which
suggests the response could be maintained by such exposure. Only a
minority of the CD8+ T cells-specific for the
epitopes identified using tetramers released IFN- in ELISPOT and
FACS assays suggesting substantial functional heterogeneity within
these CD8+ T cell populations. Finally, we show
that these CTL were able to recognize and lyse macrophages infected in
vitro with a live MTB infection. Cumulatively, these and other murine
vaccination data (26, 35, 36) strongly support a role for
Ag85A in the development of an improved vaccine against TB.
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Acknowledgments |
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Footnotes |
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2 S.M.S. and R.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Steven M. Smith, Immunology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, U.K.
4 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin; rVV, recombinant vaccinia virus; ELISPOT, enzyme-linked immunospot; STCL, short-term cell lines; NAC, nonadherent cells; CM, culture medium; SFC, spot forming cells.
Received for publication March 16, 2000. Accepted for publication September 15, 2000.
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ß TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway. J. Immunol. 162:327.
interferon. Infect. Immun. 66:830. interferon-secreting CD8+, as well as CD4+, T cells in the lungs following aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67:3242.-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95:270.-secreting CD8+ T cells in healthy contacts of tuberculosis. Immunology 95:585.[Medline]
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) and
P242–250 (
)-specific CD8+ T cells were
tested for cytotoxicity against autologous macrophages infected
overnight with either M. tuberculosis H37Rv or rVVAg85A.
M. tuberculosis-infected macrophages were pretreated
with either no mAb or anti-HLA-A, B, C (w6/32) mAb. Results show
mean % specific lysis (±SD) for five donors.
) and
P242–250 (
)-specific CD8+ T cells were
tested for cytotoxicity against autologous macrophages loaded with
either peptide or infected overnight with M.
tuberculosis H37Rv. Results show individual donors and mean
lysis ().
