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Identification of Major Epitopes of Mycobacterium tuberculosis AG85B That Are Recognized by HLA-A*0201-Restricted CD8⁺ T Cells in HLA-Transgenic Mice and Humans¹

Annemieke Geluk^2,*, Krista E. van Meijgaarden^*, Kees L. M. C. Franken^*, Jan W. Drijfhout^*, Sushila D’Souza, Antje Necker, Kris Huygen and Tom H. M. Ottenhoff^*

^* Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands; Department of Mycobacterial Immunology, Pasteur Institute of Brussels, Brussels, Belgium; and Immunotech, Marseille, France

	Abstract

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CD8⁺ T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8⁺ CTLs, epitopes for classical MHC class I-restricted CD8⁺ T cells, which most likely represent a majority among CD8⁺ T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K^b transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4⁺ T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8⁺ T cells against Ag85B of M. tuberculosis in HLA-A2/K^b transgenic mice and HLA-A*0201⁺ humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8⁺ CTLs were identified. These CD8⁺ T cells produced IFN- and TNF- and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8⁺ T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201⁺ individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8⁺ CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8⁺ T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

	Introduction

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Tuberculosis is a reemerging disease that represents a major public health problem in many countries, especially in the developing world (1). Mycobacterium tuberculosis is primarily transmitted via the respiratory route and causes disease in 5–10% of infected individuals, causing 3 million deaths annually. The currently available means for controlling tuberculosis are inadequate: the highly variable protection induced by the commonly used vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG)³ (2), along with the HIV pandemic and the increasing multidrug resistance in M. tuberculosis strains have highlighted the need for new effective vaccines.

Although the mechanisms of protection against tuberculosis are not yet completely understood, effective cell-mediated immunity is essential to control infection with M. tuberculosis. Many studies have indicated a prominently protective role for CD4⁺ T cells (3), and several HLA class II-restricted epitopes have been identified on proteins of M. tuberculosis (4, 5). Since it was reported that ₂-microglobulin (₂m)-deficient mice, which lack CD8⁺ T cells, show increased susceptibility to experimental tuberculosis (6), the role of CD8⁺ T cells has drawn increasing attention. For CD1-restricted, CD8⁺ T cells, several nonprotein ligands have recently been identified. These CD8⁺ T cells were capable of lysing M. tuberculosis-infected target cells and concomitantly kill intracellular pathogens via a granule-exocytosis pathway (7), yet their precise contribution in intracellular infections remains unknown. The recent application of DNA vaccines to tuberculosis has provided evidence for MHC class I-restricted, CD8⁺ T cell-mediated protection in mouse models of tuberculosis (8, 9). In humans, the existence of Mycobacterium-reactive MHC class I-restricted CD8⁺ T cells has been demonstrated (10, 11), but very little is known about which Ags are recognized by such T cells. Only two mycobacterial epitopes that are recognized by HLA class I-restricted CD8⁺ T cells have been reported: an HLA-B52-restricted epitope of M. tuberculosis ESAT-6 (12) and an HLA-A*0201-restricted peptide of the 19-kDa lipoprotein (13).

Secreted extracellular Ags are likely to be important in the induction of protective immunity (14, 15), especially during the early phase of infection. Up to 30% of M. tuberculosis culture filtrate proteins is composed of Ag 85 (Ag85) (16), a family of three highly homologous, 30–32-kDa proteins: 85A, 85B, and 85C. Each of these Ags is associated with mycolyltransferase activity in vitro (17), suggesting their essential involvement in the synthesis of the characteristic cell wall of mycobacteria. Ag85 induces strong T cell proliferation and IFN- secretion in most healthy individuals exposed to M. tuberculosis (18), in BCG-vaccinated mice and humans (19, 20), whereas Ab against Ag85 are more prevalent in active tuberculosis patients with decreased cellular immune response (21). Furthermore, vaccination with plasmid DNA encoding the 85A or 85B component generated strong Th1-type, CD8⁺-mediated immune responses (22) and induced protection against M. tuberculosis challenge in mice (9) and guinea pigs (15). Ag85 thus represents a prominent candidate vaccine Ag.

Since the Ag specificity of the human T cell response is known to be strongly controlled by HLA polymorphism (4), the immunogenic potential of candidate vaccines needs to be defined in the context of major HLA polymorphism. Proper tools to examine the influence of HLA polymorphism in vivo have become available only recently by the generation of HLA-transgenic (tg) mice (23). In this study, we have used DNA vaccination of HLA-A2/K^b tg mice to examine the in vivo induction and specificity of CD8⁺ T cell responses against M. tuberculosis in the context of a major human MHC class I allele. We report the identification of peptide-specific CD8⁺, HLA-A*0201-restricted cytotoxic T cells in HLA-A*0201⁺ mice and humans.

	Materials and Methods

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Peptide selection and synthesis

Candidate HLA-A*0201-binding peptides in Ag85B were selected using MOTIFS software (24). Positive scores were given for each potential anchor residue found in the peptide, and negative scores were given to inhibitory residues. The overall peptide score was the sum of the scores for individual anchor and inhibitor residues. Scores ranged from -9 to 65. All 8-, 9-, 10-, and 11-mer peptides scoring 45 were synthesized, as described previously (4).

HLA-A*0201 peptide-binding assay

Recombinant HLA-A*201 was overexpresssed in Escherichia coli, purified as described (25), and dissolved in 8 M urea. HLA-A2*0201 was titered in the presence of 100 fmol of standard peptide to determine the HLA concentration necessary to bind 20–50% of the total fluorescent signal. All subsequent inhibition assays were performed at this concentration. HLA-A2*201 was incubated in 96-well serocluster plates (Costar, Cambridge, MA) at 20°C for 48 h with 0.5 µl ₂m (15 pmol) and 1 µl (100 fmol) fluorescent labeled peptide in 92.5 µl assay buffer (100 mM sodium phosphate, 75 mM NaCl, and 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, pH 7), 2 µl protease inhibitor mixture (1 µM chymostatin, 5 µM leupeptin, 10 µM pepstatin A, 1 mM EDTA, 200 µM pefabloc), and 2 µl test peptide. As a standard peptide, hepatitis B virus core 47–56 (52C) was used. The HLA-peptide complexes were separated from free peptide by gel filtration on a Synchropak GPC 100 column (250 mm x 4.6 mm; Synchrom, Lafayette, IN) using assay buffer containing 5% CH₃CN. Fluorescent emission was measured at 528 nm on a Jasco FP-920 fluorescence detector (B&L Systems, Maarssen, The Netherlands). The percentage of labeled peptide bound was calculated as the amount of fluorescence bound to MHC divided by total fluorescence. The concentration of peptide inhibitor yielding 50% inhibition was deduced from the dose-response curve.

Ag85B protein

Ag85B protein was obtained by cloning the gene coding for Ag85B in pET19b vector (Novagen, Madison, WI) using PCR. The protein was expressed as a fusion protein, containing 10 histidine residues plus a 13-aa-containing linker sequence attached to its N terminus. For overexpression, E. coli B strain BL21 (DE3) (26) was used, in which the T7 RNA polymerase gene is under control of the lacUV5 promoter. Expression in E. coli was induced at an OD₆₀₀ of 0.6 by addition of 1 mM isopropyl-D-thiogalacto-pyranoside. The cells were harvested after 5-h culture at 37°C and centrifuged at 5000 x g for 15 min. Ag85B protein was purified by Ni-chelate affinity chromatography (Qiagen, Chatsworth, CA) (27).

Plasmid construction

Plasmid DNA encoding Ag85B was prepared as described previously (9). Briefly, the gene from plasmid pAg85B (28) of M. tuberculosis was amplified without its mycobacterial signal sequence by PCR with BglII site-containing primers. Amplified DNA was digested with BglII isolated on a 1% agarose gel, and extracted on Prep A Gene (Bio-Rad, Richmond, CA). Fragments were ligated to the BglII-digested and dephosphorylated V1J-ns-tPA vector, transformed into competent E. coli DH5 (Biological Research Labs, Breda, The Netherlands) cells, and plated on Luria-Bertani agar medium containing kanamycin (50 µg/ml). Recombinant plasmid DNA was amplified in E. coli DH5 and purified on two CsCl₂-ethidium bromide gradients, extracted with 1-butanol and phenol-chloroform, and precipitated with ethanol. In this plasmid, the Ag85B gene is expressed under the CMV promotor of IE1 form preceded by a tPA leader sequence, and followed by a polyadenylation site of the bovine growth hormone.

Mice

HLA-A*0201/K^b (HLA-A2/K^b) tg mice (23) were kindly provided by L. Sherman (Scripps Laboratories, San Diego, CA) and bred under specific pathogen-free conditions at TNO-PG (The Netherlands). Besides the H2-K^b and H2-D^b molecules, these mice express a chimeric HLA-A*0201/K^b gene encoding the murine H-2K^b 3 domain and the HLA-A*0201 1 and 2 domains. This allows the murine CD8 molecule on the murine CD8⁺ T cells to interact with the syngeneic 3 domain of the hybrid MHC class I molecule. Surface expression of the HLA-A*0201/K^b molecule was confirmed by FACS analysis.

Immunizations

Mice were anesthesized by i.p. injection of ketamine/xylazine (100 and 10 mg/kg, respectively) and injected i.m. three times (at 3-wk intervals) in both quadriceps (2 x 50 µl) with Ag85B plasmid (1 mg/ml) or control DNA (empty vector) in PBS. Splenocytes were harvested 3 wk after the last DNA injection. For peptide immunizations, equal volumes of peptide in PBS and IFA (Difco, Detroit, MI) were administered s.c. in the base of the tails, and cells were harvested 7 days postinjection.

Cytotoxicity assays

The human EBV-BLCL JY (HLA-A*0201, -B7, -Cw7) was incubated at 37°C for 1 h with 0.1 mCi Na⁵¹Cr (Amersham, U.K.), washed, and plated with effector cells in triplicates in 96-well round-bottom plates (2500 cells/well) along with medium, peptide (2 µg), or 5% Triton X-100. After 6 h, supernatants were harvested and percent specific lysis was calculated as: (release - spontaneous release)/(maximum release - spontaneous release) x 100%.

CD4 depletion

After peptide immunizations, splenocytes were incubated with anti-mouse CD4 mAb (GK1.5) for 30 min at 4°C, washed, and added to a 10-fold excess of magnetic beads coupled to goat anti-mouse IgG (Dynal, Oslo, Norway). After 30 min at 4°C, beads were removed from the cell suspension and the cells were checked for expression of CD4 and CD8 by FACS analysis. Efficiency of depletion was >95%.

Proliferation tests

Splenocytes or lymphocytes (10⁶ cells/well) in RPMI 1640 (Life Technologies, Rockville, MD)/10% heat-inactivated FCS were added to 96-well flat-bottom plates and in triplicates stimulated with Ag. For Ag85B presentation, APC were treated by hypertonic shock (29) to provide excess to the class I pathway. After 24 h, 1 µCi [³H]thymidine was added. After 18 h, radioactivity incorporated into the DNA was determined by liquid scintillation counting. For blocking experiments, the following Abs were used: BB7.2 (anti-HLA-A), FK18 (anti-CD8), B8.11.2 (anti-HLA-DR).

Determination of anti-Ag85 Abs

Ab levels of serum from immunized mice were determined by ELISA, as described previously (9). Serum titer was converted to Ab concentration by comparison with standard Ag85-specific mAb (17-4). Mean Ab concentration was calculated from three points of the linear part of the titration curve.

Generation of Ag85B-specific human CTL

Human PBMCs, from healthy HLA-A*0201⁺ individuals, were depleted of T lymphocytes by using SRBCs, followed by adherence in six-well plates (Costar) at 37°C in IMDM-10% FCS (Life Technologies) (10⁷ cells/well). After 2 h, GM-CSF (800 U/ml) and IL-4 (500 U/ml) (Genzyme, Cambridge, MA) were added, followed after 4 days by the addition of Staphylococcus aureus culture supernatant medium ((30) to accomplish maturation of dendritic cells (DCs). Mature DCs were then pulsed with Ag85B peptides for 6 h. Autologous T lymphocytes were enriched for CD8⁺ T cells using anti-CD8-labeled MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). DCs were pulsed with peptide (10 µg/ml), washed, and added to CD8-enriched T cells in IMDM-10% human serum (HS) containing IL-12 (100 pg/ml) and IL-7 (10 ng/ml; Genzyme). After 7 days, rIL-2 (25 U/ml; Cetus, Amsterdam, The Netherlands) was added. T cells were checked weekly for CD8 expression by FACS analysis and restimulated (for four to six rounds) using autologous, peptide-pulsed PBMCs.

Generation of BCG-activated short-term cultured T cell line (STCL)

PBMCs (3 x 10⁶ cells/well) were incubated in 24-well plates for 2 h in antibiotic-free culture medium. After removal of nonadherent cells, BCG was added for 16 h at a multiplicity of infection (MOI) of 2:1, mycobacteria to macrophages. Nonadherent cells were enriched for CD8⁺ T cells (MACS magnetic beads) and activated by addition to BCG-infected adherent cells.

Cytokine assays

TNF- production was detected by addition of 24-h supernatants from 2 x 10⁴ target cells BLM (HLA-A*0201⁺ melanoma cell line) and 2500 effector cells to WEHI cells, as described elsewhere (31). IFN- levels were determined after 72 h in supernatants from 3 x 10⁴ target cells (JY) and 2 x 10⁴ effector T cells. Detection capture mAb and polyclonal detection Ab for IFN- were obtained from P. van der Meide (UcyTech, Utrecht, The Netherlands) (sensitivity 3 pg/ml). For Ag85B presentation, APC were first treated by hypertonic shock (29).

BCG infection of monocytes

PBMCs (3 x 10⁵ cells/well) were cultured in 96-well flat-bottom plates for 2 h in antibiotic-free culture medium. After removal of nonadherent cells, BCG (MOI 2:1; estimating 3 x 10⁴ macrophages/well) was added for 16 h. Adherent cells were washed with medium (37°C) before addition of Ag85B-specific CD8⁺ T cells (8 x 10³ cells/well). An aliquot of infected monocytes was analyzed to verify infection by acid-fast staining.

Tetramers

MHC/peptide tetramers were produced as described previously (32), but to abrogate CD8 binding, a mutated HLA-A*0201 heavy chain (denoted A2 m) was used, containing the mutation A₂₄₅V. Briefly, rA2 m heavy chain and r₂m were produced as inclusion bodies in E. coli XA90F'LacQ1, washed extensively, dissolved in 8 M urea, and refolded as HLA monomeric proteins in the presence of 12.5 mmol Ag85B p199–207 in 100 mM Tris, 0.4 M arginine, 2 mM EDTA (pH 8), 0.5 mM glutathione disulfide, 5 mM reduced glutathoine, and protease inhibitors. Monomers were concentrated, dialyzed (Tris 10 mM, pH 8), and biotinylated with 6 µg/ml of BirA enzyme for 4 h at 30°C. Biotinylated complexes are dialyzed and purified by ion exchange chromatography (monoQ; Pharmacia France, St. Quentin en Yvelines, France) to remove free biotin. Tetramerization was achieved by addition of PE-conjugated streptavidin (Immunotech, Marseille, France) in a ratio of 4:1 and controlled by gel filtration on a Superdex 200 (Pharmacia France).

Tetramer staining

CD8-enriched STCLs (10⁵/well) were incubated in V-bottom 96-well plates, washed twice (PBS, 1% FCS), and stained for 1 h at 4°C with PE-labeled tetramers (1 µg) and peridinin chlorophyl protein (PerCP)-labeled anti-human CD8 (Becton Dickinson, Mountain View, CA), washed, and analyzed by flow cytometry on a FACScalibur analyzer (Becton Dickinson).

	Results

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Identification of high-affinity HLA-A*0201-binding peptides in the sequence of M. tuberculosis Ag85

Immunodominant T cell epitopes often display high binding affinity for MHC molecules (33). Since HLA-A*0201 is one of the most prominent HLA class I alleles, we selected 30 candidate peptide epitopes (8, 9, 10, and 11 mers) from the M. tuberculosis Ag85B sequence based on the presence of a described HLA-A*0201 peptide-binding motif (24). Peptides were synthesized and tested for binding to HLA*0201 molecules. Six of the predicted peptides bound to HLA-A*0201 with high affinity (IC₅₀ <1 µM), 10 with intermediate affinity (IC₅₀, 1–10 µM), and 14 peptides bound weakly or not at all (IC₅₀ >10 µM) (Table I). The 16 highest affinity HLA-A*0201-binding peptides were used in the subsequent study.

HLA-A*0201 tg mice represent a powerful model for the induction and examination of HLA-A*0201-restricted CD8⁺ CTL responses in vivo (23). To analyze CD8⁺ T cell responses against the major M. tuberculosis Ag, Ag85B, HLA-A2/K^b tg mice were immunized three times at 3-wk intervals with Ag85B-encoding or control plasmid DNA. Two weeks after the last immunization, splenocytes were harvested, restimulated with a mix (5 µg/ml per peptide) of the 16 best binding Ag85B peptides (Table I), and analyzed 1 wk later for their ability to lyse the human target cell JY, which expresses HLA-A*0201, but not H2-K^b or H2-D^b. Target cells were pulsed separately with each of the 16 Ag85B peptides. Four of the 16 peptides (p143–152, p126–135, p199–207, and p158–166) were strongly recognized by CTL in a dose-dependent fashion (Fig. 1A). The fact that recognition was induced against only 4 of 16 peptides, and the fact that many of the best binding peptides failed to be recognized by CD8⁺ T cells argues strongly against possible in vitro sensitization by the peptide mix used for restimulation. Similarly, no responses were found against the unrelated, HLA-A*0201-binding matrix protein epitope of influenza virus (p58–66) (Fig. 1). As an additional control, to rule out such in vitro peptide sensitization, splenocytes from mice immunized with control vector DNA and stimulated with the same peptide mix were tested as well. Cells from these animals failed to recognize any of the Ag85B peptides tested (Fig. 1B).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Cytotoxic activity of splenocytes derived from HLA-A2/Kb mice, immunized with Ag85B plasmid DNA (A) or with control vector DNA (B). Two weeks after the last immunization, splenocytes were harvested, restimulated with a mix of the 16 best binding Ag85B peptides (final concentration 5 µg/ml per peptide), and analyzed 1 wk later for their ability to lyse human JY target cells. Ag85B-derived peptides used to pulse HLA-A*0201+ target cells are indicated on the x-axis, in order of decreasing HLA-A*0201-binding affinity. The HLA-A*0201-binding peptide of influenza A matrix 58–66 (F58–66) (GILGFVFTL) was used as a control.

In addition to determining CTL activity, serum Abs against Ag85 were measured in Ag85-DNA-immunized HLA-A2/K^b mice (Fig. 2). All four immunized mice produced significant levels of anti-Ag85 Ab, whereas control DNA-immunized animals did not produce any anti-Ag85 Ab, demonstrating efficient induction of both cellular and humoral immunity in HLA-A2/K^b tg animals following Ag85 DNA immunization (Fig. 2).

View larger version (10K): [in this window] [in a new window]   FIGURE 2. Quantification of serum Abs to Ag85 in HLA-A2/Kb mice (four mice per group) following immunization with Ag85-encoding DNA or with control vector DNA. The anti-Ag85 Ab titer is given on the y-axis (ng/ml).

To determine whether the above-defined Ag85B epitopes are naturally processed and may represent candidate subunit components for antimycobacterial vaccination, HLA-A2/K^b mice were immunized separately with each of the four above-identified Ag85B epitopes: p126–135, p143–152, p158–166, and p199–207. After 10 days, splenocytes were depleted for CD4⁺ T cells and the remaining cells stimulated in vitro with various peptides, Ag85B protein, or BCG (Fig. 3). CD8⁺ T cells from Ag85B p143–152- or p199–207-immunized mice proliferated (Fig. 3, A and B) and produced IFN- (data not shown) in response to the immunizing peptide, but not to control peptide or any of the other Ag85B peptides. In addition, p143–152- or p199–207-immunized mice also responded strongly against the whole Ag85B protein as well as M. bovis BCG, indicating that natural processing of these CTL epitopes not only occurs following DNA immunization (Fig. 1), but also from BCG-infected splenocytes. Unexpectedly, the two other Ag85B peptides identified by DNA immunization, p126–135 and p158–166, failed to induce any CD8⁺ T cell proliferation against Ag85B or BCG, while inducing proliferation against the immunizing peptides (data not shown). This indicates that DNA immunization could also induce T cells directed against peptides that are only processed following DNA plasmid immunization, but not following whole Ag pulsing or BCG infection. Alternatively, some peptides may be tolerogenic rather than immunogenic, depending on the mode of delivery.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Proliferative responses of (A and C) and specific lysis induced by (B and D) CD8+ splenocytes from Ag85B p143–152 (A and B)- or Ag85B p199–207 (C and D)-immunized HLA-A2/Kb mice. Peptides used for in vitro challenge are indicated on the x-axis.

We next investigated whether CD8⁺ T cells that recognize Ag85B p143–152 or p199–207 can be detected in the human repertoire in the context of HLA-A*0201. Stable CD8⁺ T cell lines were generated against either p143–152 or p199–207, using CD4 depletion and peptide-pulsed autologous DCs derived from HLA-A*0201⁺, BCG-responsive individuals. These T cell lines were able to lyse HLA-A*0201⁺ peptide-pulsed targets (Fig. 4, A and D) and produced the proinflammatory cytokines IFN- (Fig. 4, B and E) and TNF- (Fig. 4, C and F) in response to specific peptide only, whereas no responses were detected against other Ag85B peptides or the control HLA-A*0201-binding influenza A matrix peptide. Importantly, both CD8⁺ CTL lines were able to recognize whole Ag85B protein, demonstrating natural processing of both p132–152 and p199–207 by human cells (Fig. 4, A–C). Furthermore, T cell responses were CD8^- and HLA-A*0201 dependent, since CD8⁺ T cell activation was inhibited by anti-CD8 mAb or anti-HLA-A*0201 mAb, but not anti-HLA-DR mAb.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. CTL activity (A and D), IFN- production (B and E), and TNF- production (C and F) of human HLA-A*0201-restricted CD8+ T cells specific for Ag85B p143–152 (A–C) or Ag85B p199–207 (D–F). Peptides used for in vitro challenge are indicated on the x-axis. Influenza A matrix 58–66 (F58–66) was used as a negative control.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Human Ag85B 199–207-specific CTL efficiently recognize BCG-infected, HLA-matched (), but not HLA-mismatched () monocytes. Ag85B p199–207-specific human CD8+ T cells were cocultured with BCG-infected (MOI 2:1) monocytes from HLA-A*0201+ or HLA-A*0201- individuals, and IFN- levels were determined after 72 h in supernatants.

Detection of M. tuberculosis Ag85-specific, HLA-A*0201-restricted human T cells using Ag85B peptide/HLA-A*0201 tetramers

To visualize and enumerate human CD8⁺ T cells against M. tuberculosis, tetrameric complexes of HLA-A*0201 and Ag85B p199–207 were constructed using previously described procedures (32). Of the two Ag85B epitopes, p199–207 was chosen in view of the slightly more efficient recognition of this peptide compared with p143–152 by murine CD8⁺ T cells after DNA immunization (Fig. 1A). As negative controls, HLA-A*0201 tetramers were used containing influenza A matrix peptide, HPV16 E7 peptide, or no peptide at all. PE-labeled tetramers were used to identify and determine the frequency of p199–207-specific T cells in BCG-responsive donors. Virtually no specific staining could be detected in unstimulated, freshly isolated, or frozen PBMC directly ex vivo probably due to the relative low precursor frequency of these cells among circulating PBMC in these healthy individuals (data not shown). However, in short-term cultured, BCG-stimulated PBMCs (STCL) from these donors, CD8⁺ T cells were detected that bound to HLA-A*0201/Ag85B p199–207 tetramers, ranging from 6 to 23% of the CD8⁺ T cell population (Fig. 6).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Specific staining of BCG-activated CD8+ T cells from BCG-responsive human donors using PE-labeled HLA-A*0201/Ag85B p199–207 tetramers. STCL from healthy, BCG-responsive, HLA-A*0201+ individuals (donors 1–3), from a HLA-A*0201-, BCG-responsive individual (donor 4) or from a HLA-A*0201+, BCG-nonresponsive individual (donor 5) were incubated with PerCP-labeled anti-CD8 mAb and PE-labeled tetrameric complexes of HLA-A*0201/Ag85B p199–207. PerCP-CD8 vs PE-HLA-A*0201/Ag85B p199–207 plots are shown for the gated CD8+ population in the upper panel. Histograms of the events that fall in the boxed portions of the dot plots are shown in the lower panel. No specific staining was observed after incubation of BCG-induced STCLs with tetramers of HLA-A*0201/HPV16 peptide, nor did HLA-A*0201/Ag85B p199–207 tetramers stain HLA-A*0201-restricted T cells that recognize influenza A matrix p58–66 (data not shown).

	Discussion

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Although it is well known that CD4⁺ T cells are crucial in the protection against infectious disease caused by M. tuberculosis, the contribution of CD8⁺ T cells has been less well established in this respect. A protective role for CD8⁺ T cells has been suggested earlier in experimental tuberculosis in mice, since depletion of CD8⁺ T cells (34) or adoptive transfer thereof (35) was accompanied by a reduction of the number of live M. tuberculosis bacteria detected in infected organs. In addition, ₂m^-/- mice, which lack CD8⁺ T cells, were shown to be highly susceptible to tuberculosis (6). Since ₂m molecules can pair not only with classical MHC class I molecules, but also with the class I-like molecules CD1, H2-M3, TL, Qa-1, and Qa-2, the increased susceptibility of ₂m^-/- mice could result from either the absence of class I-restricted CD8⁺ T cells, or the lack of CD1-restricted or nonclassical MHC class I-restricted CD8⁺ T cells. This topic was addressed in a recent study that demonstrated increased susceptibility to experimental tuberculosis in the absence of TAP, but not CD1d (36). Since Ag recognition by MHC class I- or Qa-1-restricted (but not CD1d) CD8⁺ T cells is TAP dependent, these results demonstrated an important contribution of such cells in protection against tuberculosis in mice. More recently, Sousa et al. (37) elegantly compared a series of knockout mice. Their results confirm that, besides classical MHC class I-restricted CD8⁺ T cells, other ₂m-dependent T cell populations can contribute to protection against experimental tuberculosis and that protective responses are predominantly TAP dependent. Furthermore, in addition to what was reported earlier (38), their results suggested that the perforin molecule does not play a role in early protective responses against M. tuberculosis, but only in the late phases on infection.

Despite the fact that MHC class I-restricted CD8⁺ T cells are likely to represent an important component of the protective immune response to human tuberculosis, the identification of human CD8⁺ T cells specific to mycobacteria has been hard to achieve (10, 12, 13). Moreover, only very few Ags and corresponding epitopes have been described that are recognized by HLA class I-restricted CD8⁺ T cells, whereas a number of ligands have been identified for CD1-restricted CD8⁺ T cells (7). Recently, cytolytic and IFN--secreting human CD8⁺ T cells could for the first time be identified: these were directed against M. tuberculosis ESAT-6 (p69–76), in the context of HLA-B52 (12), or to the 19-kDa lipoprotein in the context of HLA-A*0201 (13). Also, CD8⁺ T cells have been reported in BCG-responsive donors (11) and leprosy patients (10), but the antigenic epitopes recognized remained unknown.

The M. tuberculosis Ag85 protein is an immunodominant Ag in the CD4⁺ T cell and B cell response against M. tuberculosis in mice and humans (18, 19, 20). In mice, vaccination with Ag85-encoding plasmid DNA generated strong type 1-like CD4⁺ and CD8⁺ T cell-mediated immune responses in mice (22), affording protection against M. tuberculosis challenge (9).

Human CD8⁺ T cells were recently shown to be activated by Ag85 protein (39), but no Ag85 epitopes for CD8⁺ could be identified to date. Since the epitope and Ag specificity of human T cells is tightly controlled by HLA polymorphism, preclinical screening of candidate vaccine subunits needs to be conducted in the context of HLA. We have therefore used HLA-A2/K^b tg mice to examine induction of CD8⁺ T cell responses to the immunodominant Ag85B of M. tuberculosis in the context of HLA class I in vivo.

CD8⁺ T cells recognizing Ag85 p143–152 and p199–207 were detected in HLA-A2/K^b tg mice and HLA-A*0201⁺, BCG-responsive individuals, but not in HLA-A*0201^- donors. Importantly, these Ag85B-reactive CD8⁺ T cells not only lysed peptide- or Ag85B-pulsed target cells, but also BCG-infected HLA-A*0201⁺ human macrophages. This shows that Ag85B p143–152 and p199–207 are naturally processed from mycobacteria after infection. Furthermore, Ag85B-reactive CD8⁺ T cells could be detected in HLA-A*0201⁺, but not in A*0201^- individuals, nor in HLA-A*0201⁺, BCG-nonresponsive donors using p199–207/HLA-A*0201 tetramers. Restimulation of PBMCs from patients or healthy donors with M. bovis BCG was necessary to visualize Ag85B p199–207-reactive, CD8⁺ T cells, indicating that the precursor frequency of these cells in the periphery directly ex vivo is too low for efficient detection. This may be due to the absence of recent infection in our donors as opposed to newly diagnosed tuberculosis patients, such that the cells required reactivation before they could be detected. Using tetramer technology, however, T cells reactive to Ag85B peptides can indeed be detected in several HLA-A*0201⁺, BCG-responsive individuals.

Following DNA immunization of HLA-A2/K^b tg mice, four HLA-A*0201-restricted Ag85 epitopes for CD8⁺ T cells were identified (Fig. 1). Unexpectedly, only against two epitopes, p143–152 and p199–207, HLA-A*0201-restricted CD8⁺ T cells could be identified in humans, whereas no such responses were observed to p126–135 and p158–166. This could be due to the mode of Ag delivery: DNA immunization may induce T cells directed against epitopes that are processed following i.m. DNA plasmid injection, but not following protein/peptide immunization or BCG vaccination. In this respect, DNA immunization has indeed been shown to stimulate a broader repertoire of T cell epitopes compared with stimulation with M. tuberculosis infection (40). Alternatively, such differences in peptide recognition may be due to the tolerance of some peptides in mice, as has been shown in several tumor model systems (41).

It remains unresolved which effector function of CD8⁺ T cells is critical to protection against tuberculosis in vivo. It has been suggested that the role of M. tuberculosis-specific CD8⁺ T cells is not limited to perforin-dependent cytotoxicity since CD8-deficient mice show increased susceptibility to experimental tuberculosis compared with perforin-deficient animals (37). It is thus likely that CD8⁺ T cells, in addition to CD4⁺ T cells, contribute to protection against tuberculosis by perforin-independent mechanisms. One of these may well be the production of type 1 cytokines such as IFN- and TNF-, which are crucial for the elimination of intracellular pathogens by macrophages. Mice with disrupted genes for IFN--R, IFN-, or TNF--R (42), or humans with IFN-R or IL-12R deficiency (43) are highly susceptible to mycobacterial diseases. Furthermore, in M. tuberculosis-infected, MHC class II-deficient mice, transfer of CD8⁺ T cells from control mice, but not from IFN--deficient animals, is protective (44). The CD8⁺ CTL we describe produced both IFN- and TNF-, in addition to being able to kill infected macrophages, and thus are clearly type 1 CD8⁺ T cells.

Ag85 may therefore represent a promising antigenic subunit for antimycobacterial vaccination since it is widely expressed by mycobacteria, including the pathogens M. tuberculosis, Mycobacterium leprae, M. bovis, Mycobacterium ulcerans, and Mycobacterium avium, whereas no human homologue exists for Ag85, thus avoiding the risk of autoreactivity in vivo. The above-described Ag85B epitopes are likely to represent promising components for future vaccine design against human tuberculosis.

	Acknowledgments

 
We thank Dr. R. R. P. de Vries and Dr. T. Mutis for critically reading this manuscript, and G. Schijff and R. Brandt for providing tg mice.

	Footnotes

 
¹ These studies were supported by the Royal Netherlands Academy of Arts and Sciences, the Science and Technology for Development Program of the European Community (Commission of the European Communities), The Netherlands Leprosy Foundation (NSL), The Netherlands Organization for Scientific Research, the Fonds voor Wetenschappelÿk Onderzoek-Vlaanderen (G.0355.97), and the World Health Organization.

² Address correspondence and reprint requests to Dr. A. Geluk, Department of Immunohematology & Blood Transfusion, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

³ Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; ₂m, ₂-microglobulin; DC, dendritic cell; MOI, multiplicity of infection; PerCP, peridinin chlorophyl protein; STCL, short-term cultured T cell line; tg, transgenic.

Received for publication March 28, 2000. Accepted for publication August 31, 2000.

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