Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived from Mycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

Mice.HLA-A*0201 transgenic mice (HHD mice) (25) were kindly donated by F. A. Lemonnier (Pasteur Institute, France). HHD mice express a monochain in which the C terminus of human β2-microglobulin is covalently linked to the N terminus of the HLA-A2.1 heavy chain in a chimeric configuration (α3 domain of mouse origin) (25). In HHD mice, the HLA-A*0201 monochain is the only type of major histocompatibility complex (MHC) class I molecule expressed. The mice were kept under specific-pathogen-free conditions and fed autoclaved food and water ad libitum at the Institute for Experimental Animals of the Hamamatsu University School of Medicine. Two- to 3-month-old female mice were used in all experiments. Animal experiments were performed according to the Guidelines for Animal Experimentation, Hamamatsu University School of Medicine. We confirmed that HLA-A*0201 was expressed on spleen cells of the HHD mice that we used (data not shown).

Human subjects.HLA-A*0201⁺ healthy donors who had previously been vaccinated with M. bovis BCG were recruited from the Hamamatsu University School of Medicine. Blood samples were taken after written permission was obtained from the individuals participating in this study.

Peptides.Peptides spanning the entire mature MPT5l amino acid sequence of M. tuberculosis (266 amino acid residues) were synthesized as 20-mers overlapping by 10 residues; the only exception was the carboxyl-terminal 12-mer from amino acid 255 to amino acid 266, which was described previously (34). Briefly, lyophilized peptides were purchased from Invitrogen Corporation (Carlsbad, CA), and the purity was confirmed by mass spectrometry. To identify the potential HLA-A*0201-restricted CD8⁺ T-cell epitopes in a 20-mer peptide, computer-based T-cell epitope prediction algorithm programs were used, which were accessed through the websites of the National Institutes of Health BioInformatics and Molecular Analysis Section (BIMAS) HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (24) and SYFPEITHI Epitope Prediction (http://www.syfpeithi.de/) (27). All peptides were dissolved in distilled water to obtain a concentration of 1 mM and stored at −80°C until use.

Immunization of mice.Mice were immunized with pCI-MPT51, a plasmid DNA vaccine encoding the mature MPT51 molecule (34), employing a gene gun bombardment system. For DNA immunization with the Helios gene gun system (Bio-Rad Laboratories, Hercules, CA), cartridges of DNA-coated gold particles were prepared according to the manufacturer's instructions. To immunize mice, the shaved abdominal skin was wiped with 70% ethanol. Mice were inoculated with 2 μg of the plasmid DNA four times at 1-week intervals.

Cell lines.The human transporter associated with peptide loading (TAP)-deficient T2 cell line (29) was kindly donated by Peter Creswell (Yale University School of Medicine). The cells were cultured in RPMI 1640 medium (Sigma-Aldrich, Inc., St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Thermo Electron, Melbourne, Australia) (RPMI/10FCS) in an incubator with a humidified atmosphere containing 5% CO₂.

Preparation of splenocyte culture supernatants and measurement of IFN-γ amounts.Spleen cells were harvested from MPT51 DNA-immune mice. Recovered cells were plated in 96-well plates at a concentration of 1 × 10⁶ cells per well in the presence or absence of 5 μM of each MPT51 peptide at 37°C with an atmosphere containing 5% CO₂. Supernatants were harvested 24 h later and stored at −20°C until they were assayed. The concentration of IFN-γ in the culture supernatants was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) The ELISA was carried out as described previously (34), with some modifications. The following method was used. The 96-well ELISA plates (EIA/RIA A/2; Costar, Cambridge, MA) were coated with 2 μg ml⁻¹ of capture antibody (anti-murine IFN-γ monoclonal antibody [MAb] R4-6A2; BD Biosciences, San Jose, CA) at 4°C overnight, washed with phosphate-buffered saline supplemented with 0.05% Tween 20 (PBS-Tween), and blocked with Block One blocking solution (Nakalai Tesque, Kyoto, Japan) at room temperature for 45 min. After washing with PBS-Tween, the culture supernatants were added to the plates and the plates were incubated at 4°C overnight. After washing with PBS-Tween, 0.5 μg ml⁻¹ of biotin-labeled anti-murine IFN-γ MAb XMG1.2 (BD Biosciences) was added to the plates, and the plates were incubated for 1 h at room temperature. After washing with PBS-Tween, horseradish peroxidase-conjugated streptavidin (eBioscience, San Diego, CA) was added, and the preparations were incubated for 30 min at room temperature. After washing, TMB (3,3′,5,5′-tetramethylbenzidine) one-component horseradish peroxidase amino hydrogen peroxide microwell substrate (BioFX Laboratories, Owings Mills, MD) was added to the plates to detect bound horseradish peroxidase-conjugated streptavidin. After 5 min, the enzyme reaction was stopped by adding 2 M H₂SO₄, and then the absorbance at 450 nm was measured using an EZS-ABS microplate reader (Asahi Techno Glass, Tokyo, Japan).

MHC stabilization assay.The abilities of peptides to bind to HLA-A*0201 were measured by determining the stabilization of class I molecules on the surface of T2 cells (33). T2 cells (1 × l0⁶ cells ml⁻¹) were cultured at 26°C overnight and then incubated for 1 h in the presence or absence of peptides (50 or 250 μM). Cells were then incubated at 37°C for 2 h and washed with FACS buffer [phosphate-buffered saline supplemented with 1% fetal calf serum], and the cell surface expression of HLA-A*0201 molecules was detected by flow cytometry (EPICS XL; Beckman Coulter, Fullerton, CA) using a mouse MAb specific for HLA class I molecules (34-1-25; Cedarlane, Ontario, Canada), followed by treatment with fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin antibodies (Rockland, Gilbertsville, PA). The results were expressed as the mean fluorescence intensity (MFI) ratio, determined as follows: [(MFI observed in the presence of peptide at 37°C/MFI observed in the absence of peptide at 26°C) − (MFI observed in the absence of peptide at 37°C/MFI observed in the absence of peptide at 26°C)] × 100.

Intracellular IFN-γ staining.An antigen-specific T-cell subset was also identified by simultaneous flow cytometric assessment of the T-cell phenotype and intracellular IFN-γ synthesis.

The methods used for cell surface staining of CD4 and CD8 and intracellular IFN-γ staining have been described previously (34). Intracellular IFN-γ staining was performed using a Cytofix/Cytoperm Plus (with GolgiStop) kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions.

Cytotoxicity assay.One week after the last immunization, immune spleen cells (2 × 10⁷ cells) were cocultured for 5 days with 2 × 10⁷ syngeneic splenocytes treated with 100 μg ml⁻¹ of mitomycin C (Kyowa Hakko, Tokyo, Japan) and pulsed with peptide for 2 h at 37°C. Each well also received 10 U ml⁻¹ of human recombinant interleukin-2 (Hoffmann-La Roche, Nutley, NJ). Cell-mediated cytotoxicity was measured by using a conventional ⁵¹Cr release assay as described previously (34). Briefly, the target cells used in this study were T2 cells pulsed with peptide at a concentration of 1 μM for 15 h at 37°C. Target cells (1 × 10⁴ cells/well) were incubated for 5 h in triplicate at 37°C with serial dilutions of effector cells, and the level of specific lysis of the target cells was determined by using the following equation: percentage of specific lysis = [(experimental counts per minute − spontaneous counts per minute)/(total counts per minute − spontaneous counts per minute)] × 100.

Tetramer staining.A phycoerythrin (PE)-labeled HLA-A*0201/MPT51 p53-62 tetramer complex was kindly supplied by the NIH Tetramer Facility. After 10 days of in vitro stimulation with the MPT51 p53-62 peptide, spleen cells of immune HHD mice were treated with ammonium chloride and potassium chloride lysis buffer for 5 min at room temperature to remove erythrocytes, washed twice with RPMI 1640 medium, and resuspended in RPMI/10FCS. For some experiments, peripheral blood mononuclear cells (PBMCs) from purified protein derivative (PPD)-reactive HLA-A*0201⁺ human healthy subjects were prepared by LeucoSep (Greiner Bio-One, Frickenhausen, Germany) according to the manufacturer's instructions. These cells (1 × 10⁶ cells) were washed twice with FACS buffer and stained with the PE-labeled HLA-A*0201/MPT51 p53-62 tetramer and FITC-labeled anti-mouse or -human CD8 MAb for 30 min at 4°C. The cells were then washed with FACS buffer twice and analyzed with a digital flow cytometer (EPICS XL; Beckman Coulter).

IFN-γ production in response to overlapping synthetic peptides from MPT51 in HHD mice.Splenocytes from HHD mice immunized with a DNA vaccine encoding mature MPT5l (pCI-MPT51) were stimulated with the overlapping MPT51 peptides for 24 h, and the IFN-γ concentrations in culture supernatants were determined by ELISA. As shown in Fig. 1, robust IFN-γ production was observed in splenocytes from MPT51 DNA-vaccinated HHD mice after stimulation with peptide 5l (p51) (amino acid residues 5l to 70) and peptide 171 (p171) (amino acid residues 171 to 190). In addition, weak IFN-γ production was observed in the splenocytes in the presence of peptide 191 (p191) (amino acid residues 191 to 210). Since the HHD mice that we used in this study had a C57BL/6 background (25) and we observed that only CD4⁺ T cells produced IFN-γ in response to p171 and p191, we concluded that CD4⁺ T cells responded to these peptides presented on H2-A^b molecules and produced IFN-γ (34). As expected, spleen cells from naïve HHD mice showed no significant IFN-γ production in response to any MPT51 peptide.

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FIG. 1.

IFN-γ production by spleen cells from HHD mice immunized with pCI-MPT51. The IFN-γ production by splenocytes from HHD mice immunized with the pCI-MPT51 plasmid in response to 1 of 26 overlapping peptides (5 μM) covering the MPT51 molecule or medium alone (−) was evaluated. Splenocytes from naïve HHD mice were also examined as a control. The data are representative of the results of three independent experiments.

Identification of a 10-mer CD8⁺ T-cell epitope in peptide p51-70 of MPT51.Since CD8⁺ T-cell epitopes presented by MHC class I molecules comprise 8 to 10 amino acids and are generally 9 amino acids long, we pursued a line of inquiry to identify the fine HLA-A*0201-restricted CD8⁺ T-cell epitope. We predicted candidate peptides in the 20-mer peptide by using the computer-based programs BIMAS HLA Peptide Binding Predictions and SYFPEITHI Epitope Prediction. Using the BIMAS program, we found that a 9-mer peptide, p53-61 (TLAGKGISV), and a 10-mer peptide, p53-62 (TLAGKGISVV), showed high scores for binding to the HLA-A*0201 molecule in the region containing amino acid residues 51 to 70 (the binding scores were 69.552 for p53-61 and 65.588 for p53-62) (Table 1). In addition, the SYFPEITHI program also produced high scores for these peptides (27 for p53-61 and 28 for p53-62) (Table 1). Therefore, we synthesized p53-61 (TLAGKGISV) and p53-62 (TLAGKGISVV). In addition, we synthesized the p21-29 peptide (FLAGGPHAV) since this peptide had the highest HLA-A*0201 binding scores with the BIMAS and SYFPEITHI programs (319.939 and 29, respectively). Three-color flow cytometric analysis showed that the number of IFN-γ-producing CD8⁺ T cells increased in the presence of p53-62 (TLAGKGISVV) but not in the presence of p53-61 (TLAGKGISV) or p21-29 (FLAGGPHAV) (Fig. 2). The MPT51 p53-62 peptide was confirmed to stimulate splenocytes derived from MPT51 DNA-immune HHD mice in a dose-dependent manner. The minimum concentration of this peptide for inducing IFN-γ production by the splenocytes was approximately 5 × 10⁻⁸ M (50 nM) (Fig. 3).

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FIG. 2.

Identification of a T-cell epitope in the MPT51 p53-62 peptide and the T-cell subset recognizing the epitope in HHD mice. (A) Levels of IFN-γ-producing T-cell subsets in spleens of HHD mice immunized with the pCI-MPT51 plasmid. Three-color flow cytometric analysis was performed for detection of intracellular IFN-γ and cell surface CD4 and CD8 molecules after immune splenocytes were cultured in the presence of the MPT51-derived peptides p51-70 (20-mer peptide), p21-29 (FLAGGPHAV), p53-61 (TLAGKGISV), and p53-62 (TLAGKGISVV). The data are the percentages of IFN-γ-producing CD4⁺ or CD8⁺ cells in the total CD4⁺ or CD8⁺ cells after 4 h of stimulation with peptides. The results of a representative experiment are shown. (B) Representative flow cytometry data for intracellular IFN-γ and cell surface CD8 staining of spleen cells of HHD mice immunized with the pCI-MPT51 plasmid after 4 h of stimulation with the MPT51 p51-70 peptide. The percentages of IFN-γ-producing cells in the total CD8⁺ cells are shown.

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FIG. 3.

MPT51 p53-62 is a dominant T-cell epitope in HHD mice. The IFN-γ production by splenocytes from HHD mice immunized with the pCI-MPT51 plasmid in response to twofold serially diluted doses of candidate peptides MPT51 p53-62 (TLAGKGISVV), p53-61 (TLAGKGISV), and p21-29 (FLAGGPHAV) was evaluated. The data are representative of the results of three independent experiments.

Binding affinity of the p53-62 peptide to the HLA-A*0201 molecule.We then examined the binding affinity of the MPT51 p53-62 peptide to the HLA-A*0201 molecule by measuring the binding stability with T2 cells, and we compared this peptide with several other M. tuberculosis-derived epitopes in terms of binding stability. T2 cells are defective for endogenous class I presentation due to the TAP deficiency, but peptide loading on MHC molecules stabilizes the expression of MHC on the cell surface (33). The MHC molecules stabilized with the appropriate peptides could be detected by flow cytometry with an MAb to the HLA-A*0201 molecule. As shown in Fig. 4A, MPT51 p21-29 (FLAGGPHAV) and MPT51 p53-62 (TLAGKGISVV) were strongly bound to the HLA-A*0201 molecule on T2 cells, whereas MPT51 p53-61 (TLAGKGISV), a known M. tuberculosis Ag85A-derived HLA-A*0201-binding peptide (KLIANNTRV) (30), and an M. tuberculosis ESAT6-derived HLA-A*0201-binding peptide (LLDEGKQSL) (19) were relatively weakly bound to the HLA-A*0201 molecule.

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FIG. 4.

MPT51 p53-62 peptide binds to cell surface HLA-A*0201 molecules and can be recognized by immune T cells in the context of HLA-A*0201. (A) HLA binding assay with T2 cells showing that MPT51 p21-29 (FLAGGPHAV) and MPT51 p53-62 (TLAGKGISVV) bound to HLA-A*0201 strongly, whereas MPT51 p53-61 (TLAGKGISV), the Ag85A-derived peptide KLIANNTRV, and the ESAT6-derived peptide LLDEGKQSL bound to HLA-A*0201 relatively weakly. The MFI ratios in the presence of the indicated peptides at a concentration of 100 μM are shown. The listeriolysin O (LLO)-derived peptide GYKDGNEYI was used as a negative control. The expression of HLA-A*0201 on T2 cells cultured in the absence of any peptide at 37 or 26°C is also shown. Representative data from three independent experiments are shown. (B) Lysis of MPT51 p53-62 peptide-pulsed T2 cells by splenocytes from MPT51 DNA-immune HHD mice. Immune splenocytes (effectors) were incubated with target cells using the effector/target cell ratios (E/T ratio) indicated on the x axis. Representative data from three independent experiments are shown.

To obtain insight into T-cell recognition of the MPT51 p53-62/HLA-A*0201 complex on T2 cells, we examined the cytotoxic T-cell response of immune mice to the peptide-MHC complex. As shown in Fig. 4B, immune splenocytes of MPT51 DNA-immune HHD mice after in vitro stimulation with MPT51 p53-62 peptide-pulsed autologous splenocytes lysed the peptide-pulsed T2 cells substantially. However, the immune splenocytes did not lyse MPT51 p21-29 peptide-pulsed T2 cells after in vitro stimulation with the peptide-pulsed autologous splenocytes (Fig. 4A), although the peptide bound relatively strongly to HLA-A*0201 on T2 cells (data not shown).

Detection of MPT51 p53-62-specific CD8⁺ T cells in PBMCs of HLA-A*0201⁺ PPD-reactive healthy subjects.Finally, we examined whether HLA-A*0201⁺ PPD-reactive healthy subjects do have MPT51 p53-62-specific memory T cells. We screened PBMCs of HLA-A*0201⁺ individuals for the presence of the memory T cells. HLA-A*0201⁺ PPD-reactive PBMCs were subjected to MPT51 p53-62/HLA-A*0201 tetramer staining after in vitro stimulation with mitomycin C-treated, MPT51 p53-62-pulsed autologous PBMCs for 10 days. As shown in Fig. 5A, PBMCs from some HLA-A*0201-positive PPD-reactive individuals showed larger amounts of MPT51 p53-62/HLA-A*0201 tetramer-positive CD8⁺ T cells by flow cytometric analysis than PBMCs from HLA-A*0201-negative individuals. The PBMCs of two of five HLA-A*0201-positive individuals were tetramer positive. In parallel, the tetramer-positive PBMCs produced large amounts of IFN-γ after in vitro stimulation (Fig. 5B).

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FIG. 5.

Detection of MPT51 p53-62-specific memory T cells in PBMCs of HLA-A*0201⁺ PPD-reactive healthy subjects. (A) Flow cytometric analyses to detect MPT51 p53-62-specific memory T cells in PBMCs of HLA-A*0201⁺ PPD-reactive healthy subjects using MPT51 p53-62/HLA-A*0201 tetramer. PBMCs of the healthy subjects were prepared and cultured for 10 days together with mitomycin C-treated, MPT51 p53-62-pulsed autologous PBMCs and then subjected to flow cytometric analysis after treatment with PE-conjugated MPT51 p53-62/HLA-A*0201 tetramer and FITC-conjugated anti-human CD8 MAb staining (graphs A and B). HLA-unmatched PBMCs were used as a negative control (graph C). Representative flow cytometry patterns are shown. The percentages of tetramer-positive cells in the total CD8⁺ cells are indicated. (B) IFN-γ production by PBMCs of HLA-A*0201⁺ PPD-reactive healthy subjects stimulated with MPT51 p53-62 (TLAGKGISVV)- or p53-61 (TLAGKGISV)-pulsed autologous PBMCs for 10 days as evaluated by an IFN-γ ELISA. Samples A and C correspond to graphs A and C in panel A.