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Evaluation of T-cell responses to peptides with MHC class I-binding motifs derived from PE_PGRS 33 protein of Mycobacterium tuberculosis

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

Correspondence
R. Nayak
nayak{at}mcbl.iisc.ernet.in

Received 5 September 2006
Accepted 12 December 2006

Abbreviations: BCG, bacille Calmette–Guérin; ELISPOT, enzyme-linked immunosorbent spot assay; IFN-, gamma interferon; IL, interleukin; LDH, lactate dehydrogenase; MHC, major histocompatibility complex; PPD, protein-purified derivative; TB, tuberculosis.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Infection with Mycobacterium tuberculosis continues to be a major cause of morbidity and mortality throughout the world, leading to 3 million deaths and over 8 million new cases of tuberculosis (TB) per year (Kochi, 1991). Although bacille Calmette–Guérin (BCG) has been the most widely used vaccine in the world to date, it has been shown to be ineffective against adult pulmonary tuberculosis (Fine, 1995). Thus there is a renewed search for better vaccine candidates.

Protective immunity against TB is dependent on an intact cellular immune response. Several studies have demonstrated that cellular immunity mediated by CD4⁺ T cells plays a primary role in the protective immune response against M. tuberculosis (Flynn & Chan, 2001; Kaufmann, 2001; Xing, 2001). However, major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T lymphocytes (CTLs) also contribute to a protective immune response against M. tuberculosis in both the mouse (Flynn et al., 1992; Ladel et al., 1995) and in humans (Pathan et al., 2000; Lalvani et al., 1998; Lewinsohn et al., 1998; Canaday et al., 1999). ß2-Microglobulin-deficient knockout mice, which lack an effective CD8 response, show increased susceptibility to M. tuberculosis infection (Sousa et al., 2000). Release of the genome sequence of M. tuberculosis has revealed a unique multigene PE family (Dheenadhayalan et al., 2006; Cole et al., 1998), of which 37 genes code for highly homologous proteins in M. tuberculosis H37Rv (PE genes), whilst 63 genes of the PE_PGRS subfamily encode more complex proteins with a PE domain at the N-terminus and a C-terminal glycine-rich domain (Brennan & Delogu, 2002). However, the function of this large family of proteins remains to be elucidated. The Rv1818c gene, which expresses one of the best-studied PE_PGRS proteins, has been detected in lung granuloma, and the pericavity and distant lung of pulmonary tuberculosis patients (Rachman et al., 2006). It has been demonstrated that a Mycobacterium bovis BCG strain carrying a transposon insertion within the Rv1818c gene is defective in infecting and surviving within macrophages, suggesting that Rv1818c may be an important member of this family of genes (Brennan et al., 2001). Recent work has suggested that expression of the Rv1818c protein provides the non-pathogenic Mycobacterium smegmatis with some specific properties more typical of virulent mycobacteria, including increased survival in macrophages and host tissues (Dheenadhayalan et al., 2006). Infection of mice has revealed that the PE region of Rv1818c can elicit an effective cellular immune response and that the Gly-Ala-rich PGRS domain can induce a strong humoral response (Delogu & Brennan, 2001).

Relatively few epitopes in mycobacterial antigens have been identified so far for human CD8⁺ T cells. A recent in silico analysis of the genes for all of the PE_PGRS proteins revealed a large number of potential CTL epitopes (Chaitra et al., 2005). In this work, we describe the identification of CTL epitopes from Rv1818c using synthetic peptides with MHC class I-binding motifs. The peptides were evaluated for their ability to induce a CTL response in vitro and their ability to induce cytokine production from spleen cells primed in vivo with whole protein.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Animals and immunization. BALB/c mice (8–10 weeks old) were obtained from and maintained in the Central Animal Facility, Indian Institute of Science, Bangalore, India. All experiments were performed according to the institutional ethical committee guidelines for animal experimentation.

For DNA vaccination, six mice were immunized intramuscularly with 100 µg plasmid DNA (pFCN7, see later) and boosted with the same amount of DNA on days 21 and 36. For the DNA prime–protein boost, six mice were immunized intramuscularly with pFCN7 DNA and boosted subcutaneously with 100 µg of purified recombinant Rv1818c protein emulsified in Ribi adjuvant (MPL/TDM adjuvant system; Sigma) on days 21 and 36. Sera, splenocytes and lymph node cells were isolated 1 week after the last immunization. Immunization of mice with pFLAG vector alone was used as the control.

Antigens and synthetic peptides. The full-length Rv1818c gene cloned into pET15b fused to a histidine (His) tag was a kind gift from Dr M. J. Brennan (CBER, FDA, Bethesda, USA). The His-tagged recombinant protein was expressed in Escherichia coli (C41 strain) and purified by Ni-NTA chromatography, as described previously (Razeghifard, 2004). The His-tagged Rv1818c protein was purified under denaturing conditions and dialysed against 10 mM Tris-buffered saline at pH 8. The protein sequence of Rv1818c was analysed in silico employing BIMAS (Parker et al., 1994) and SYFPEITHI (Rammensee et al., 1999) algorithms for binding of all overlapping nonameric peptides generated from Rv1818c to class I HLA and class I mouse MHC. Based on this prediction, two peptides from the Rv1818c protein of M. tuberculosis containing MHC class I-binding motifs, ³⁸⁵ALGGGATGV³⁹³ (Peptide_385–393) and ⁶TIPEALAAV¹⁴ (Peptide_6–14) were chosen. The TIPEALAAV peptide has also been predicted to bind to HLA A_0201 with a T_1/2 of 90 and the ALGGGATGV peptide to bind to HLA A_0201 with a T_1/2 of 70 (Chaitra et al., 2005). The peptides were synthesized (Peptron) and dissolved in 500 µl DMSO, diluted in 0.9 % NaCl to a concentration of 10 mM and stored at –70 °C.

Transient expression of Rv1818c in mammalian cells. The full-length Rv1818c gene was PCR amplified from the genomic DNA of M. tuberculosis H37Rv using the sequence-specific primers 5'-TCGATAGATCTGATGTCATTTGTGGTCACGATC-3' (forward) containing a BglII site and 5'-TAGCGAGGATCCCTACGGTAACCCGTTCATCCC-3' (reverse) with a BamHI site, and cloned into the mammalian expression vector pFLAG-CMV4 (Sigma-Aldrich). The chosen clone was termed pFCN7. P815 cells were transfected with pFCN7 (pFLAG-Rv1818 recombinant vector) and the pFLAG vector alone as a control. Expression of Rv1818c protein was confirmed by immunostaining with rabbit anti-1818c polyclonal antibody (raised in rabbit by immunizing with purified recombinant Rv1818c protein). Plasmid DNA was amplified in E. coli DH5 cells, purified using a plasmid purification kit (Qiagen) and redissolved in PBS.

Detection of IgG isotypes by ELISA. Serum from immunized and unimmunized control mice was tested by ELISA. ELISA plates (BD Falcon) were coated overnight with purified recombinant Rv1818c protein (100 ng per well) in PBS at 4 °C. Plates were washed three times with PBS plus 0.1 % Tween 20. Wells were blocked with 3 % gelatin for 1 h at 37 °C. After washing three times, the plates were incubated with the immunized mice sera at different dilutions for 2 h at 37 °C. Plates were washed three times and incubated with goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 antibody conjugated to HRP (Bangalore Genie) for 1 h. Plates were washed three times with PBS/0.1 % Tween 20 and developed using o-phenylenediamine. The A₄₉₀ was recorded in an ELISA reader.

Cell lines. P815 (H-2^d), a mastocytoma cell line, was maintained in RPMI 1640, supplemented with 10 % fetal bovine serum (Life Technology). RMAS-K^d, a peptide transport-deficient mouse lymphoma cell line transfected with H-2K^d (Chen et al., 1999), was generously provided by Dr Jonathan Yewdell (NIH, Bethesda, MD, USA).

In vitro proliferation assay and cytokine assay. Splenocytes (0.5x10⁶ cells) from plasmid DNA-immunized mice were cultured in RPMI 1640 in 96-well plates in the presence of 20 µg protein-purified derivative (PPD) ml^–1 or 20 µg recombinant purified Rv1818c protein ml^–1, or with irradiated P815 cells that had been pre-pulsed with the synthetic peptides (10 µg ml^–1 for 2 h), at 37 °C in 5 % CO₂. For blocking experiments, anti-CD8 mAb (eBioscience) was added to the splenocytes.

Plates were incubated for 96 h at 37 °C. [³H]Thymidine (1 µCi per well; Perkin-Elmer) was added for the last 16 h of incubation. Cells were harvested on glass fibre filters using a semi-automated cell harvester (Nunc). [³H]Thymidine incorporation was measured in a scintillation spectrometer. Supernatants from parallel cultures were harvested after 72 h of the same culture and stored at –70 °C until assayed for specific cytokines. Interleukin (IL)-4 and gamma interferon (IFN-) levels were measured by sandwich-ELISA.

In vitro expansion of antigen-specific CTLs. Spleens were excised by splenectomy under sterile conditions. Each spleen was mashed between glass slides and the cells were suspended in 5 ml RPMI 1640. Cells were spun (room temperature, 1000 g, 10 min) and resuspended in 5 ml of red cell lysis solution containing 155 mM NH₄C1, 10 mM KHCO₃ and 0.11 mM EDTA. After 1 min, the solution was neutralized by the addition of 5 ml RPMI 1640. Cells were centrifuged (room temperature, 1000 g, 10 min) and washed twice with RPMI 1640. Spleen cells (2x10⁶) from in vivo-primed mice were co-cultured with 1x10⁵ cells per well of P815 cells pulsed with peptides in RPMI 1640 for 7 days at 37 °C. Recombinant IL-2 (30 U ml^–1; Roche Diagnostics) was added after 48 h of culture and every third day for 2 weeks. The antigen-specific T cells were used for enzyme-linked immunosorbent spot (ELISPOT) and CTL assays.

Intracellular cytokine assay. After expansion of antigen-specific T cells from immunized mice for 6 days, the cells were restimulated with the peptides (10 µg ml^–1). The expanded T cells were cultured in vitro with irradiated (5000 rads) P815 cells (1x10⁴) pulsed with peptides, in a total volume of 200 µl RPMI 1640 for 6 h. Brefeldin-A (10 µg ml^–1; Sigma) was added to the culture and cells were incubated at 37 °C in 5 % CO₂ for 6 h. The cells were then harvested and stained for surface CD8 and for intracellular IFN- and perforin using conjugated mAbs, as described previously (Koksoy et al., 2004). Cells were stained for CD8 with an FITC-conjugated rat anti-mouse CD8 mAb (clone 53-6.7; eBioscience) by adding 0.6 µg antibody to 1x10⁶ cells, followed by incubation at room temperature for 30 min. Surface-stained cells were washed three times with washing buffer (PBS containing 0.2 % BSA and 0.2 % sodium azide) and then fixed with 4 % paraformaldehyde at 4 °C for 15 min and permeabilized with 0.1 % saponin (Sigma) at 37 °C for 15 min. Permeabilized cells were stained for intracellular IFN- or perforin with a phycoerythrin-conjugated rat anti-mouse IFN- mAb (1 : 40 dilution, clone XMG1.2; eBioscience) or anti-mouse perforin mAb (1 : 40 dilution, clone eBioOMAK-D; eBioscience), respectively, diluted in saponin buffer at 37 °C for 30 min. FITC-conjugated rat IgG2a (eBioscience) and phycoerythrin-conjugated rat IgG1 were used as isotype controls for CD8 and IFN- antibodies, respectively. Finally, cells were washed three times with washing buffer, fixed with 1 % paraformaldehyde and stored at 4 °C until analysis by flow cytometry. The predicted frequency of CD8⁺ IFN-⁺ cells was determined by subtracting the percentage of unsensitized CD8⁺ IFN-⁺ cells from the percentage of antigen-sensitized CD8⁺ IFN-⁺ cells.

ELISPOT assay. Millipore 96-well nitrocellulose plates were coated with 50 µl anti-IFN- mouse mAb (code 1598-00, diluted 1 : 500 in PBS; eBioscience) overnight at room temperature. Wells were washed with PBS/0.1 % Tween 20, incubated with 200 µl RPM1 1640 plus 10 % fetal bovine serum for 2 h at 37 °C and then washed once with PBS. In vitro-expanded antigen-specific T lymphocytes were then seeded at a concentration of 4x10³ cells per well together with 1x10⁵ cells per well of uncultured P815 pulsed with or without peptide as antigen-presenting cells in the wells and treated with 100 IU IL-2 ml^–1. P815 cells used as antigen-presenting cells had been pulsed with 1 pg peptide ml^–1 for 2 h at 37 °C and washed twice to remove free peptide. After incubation for 24 h at 37 °C and 5 % CO₂, the plates were washed three times with PBS/0.05 % Tween 20. PBS/0.05 % Tween 20 was used for all further washing and incubation steps. Wells were incubated with 100 µl polyclonal rabbit anti-mouse IFN- antibody (1 : 250 dilution) at 4 °C overnight, washed and 100 µl polyclonal HRP-conjugated goat anti-rabbit IgG antibody (1 : 500 dilution; eBioscience) was added. After 3 h incubation at 37 °C, wells were washed with PBS and 50 µl substrate [3-amino-9-ethylcarbazol (100 mg ml^–1; Sigma), dissolved in 8 ml dimethylformamide and diluted 1 : 50 in 5 ml sodium acetate buffer at pH 5.0 plus 20 µl 30 % H₂O₂] was added. After 7–15 min, 100 µl PBS was added per well, the supernatants were discarded, the plates were air-dried overnight and coloured spots were enumerated using a microscope.

MHC I peptide-binding stabilization assay. Binding assays were performed, as described elsewhere (Zhou et al., 1992), using TAP-deficient RMAS-K^d cells expressing H-2K^d. Briefly, the RMAS-K^d (H-2^d) cells at exponential phase (1x10⁶ cells ml^–1) were collected, washed with RPMI 1640 and cultured for 16 h at 25 °C in RPMI 1640 to allow ‘empty’ HLA class I molecules to accumulate on the cell surface. The RMAS-K^d cells (4–5x10⁵ cells per well) were then washed with RPMI 1640, incubated with different concentrations of the synthetic peptides at 25 °C in 5 % CO₂ for 2 h and incubated for an additional 2 h at 37 °C. After incubation, cells were washed once and incubated on ice for 30 min with FITC-conjugated anti-MHC class I mAb (clone 34-1-2S; eBioscience), which recognizes mouse H-2K^d. The cells were then washed twice with PBS. The stained cells were fixed with 1 % paraformaldehyde and analysed with a FACScan flow cytometer (Becton Dickinson). Results are expressed as mean fluorescence intensity. In the RMAS stabilization assay (Zhou et al., 1992), cells that had been cultured at 25 °C for 16 h were incubated at 25 °C for 1 h with 100 µM peptide and then at 37 °C for 2 h. The cells were washed to remove unbound peptides and the incubation was continued at 37 °C for the indicated times (0, 1, 2, 4, 6 and 12 h), followed by washes at 4 °C in RPMI 1640. For the zero time point determination, cells were washed immediately at 4 °C after the 37 °C incubation. Each sample was stained for H-2K^d on the cell surface, as described earlier. The stability of peptide–MHC complexes was expressed as the time (h) required for 50 % of the molecules to decay (DT₅₀).

CTL assay. A CTL assay was performed as described previously (Sinnathamby et al., 2001), employing a non-radioactive method based on the release of lactate dehydrogenase (LDH) from target cells. Briefly, antigen-specific splenocytes were collected from in vitro-expanded cultures and washed once with RPMI 1640. Viable cells were purified by Ficoll density gradient separation and resuspended in RPMI 1640. P815 target cells were incubated with the peptides (10 µg ml^–1) for 2 h at 37 °C. After washing, 5x10³ cells per 100 µl were added to 100 µl of various numbers of effector cells that had been plated in 96-well plates to get target : effector ratios of 1 : 20, 1 : 40 and 1 : 100. The medium or the target cells alone were kept as a low control (spontaneous LDH release). For the high control (maximum LDH release), 2 % Triton X-100 was added to the target cells. The cells were incubated for 12 h and then 100 µl of cell-free culture supernatant was collected and assayed for LDH release using a Cytotoxicity Detection kit (LDH) (Roche Applied Science). The percentage of cell-mediated cytotoxicity was determined as: cytotoxicity (%)={[(effector target cell mix–effector cell control)–low control]/(high control–low control)}x100.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
An effective vaccine for tuberculosis should be a good inducer of a cellular immune response, mediated by the induction of IFN--secreting cells and CD8⁺ CTLs. This type of response is crucial for protection against TB in both humans (Ottenhoff & Mutis, 1995; Rees et al., 1988) and animals (Cooper et al., 1993; Flynn et al., 1993). Several studies have provided evidence that MHC class I-restricted CD8⁺ T cells recognize M. tuberculosis-infected cells and can release IFN-, lyse infected target cells and kill intracellular bacteria (Ladel et al., 1995; Caccamo et al., 2002; Sousa et al., 2000). For these reasons, there has been an intense effort to define M. tuberculosis epitopes that can be presented by MHC class I molecules to CD8⁺ T cells (Cho et al., 2000; Klein et al., 2001; Geluk et al., 2000). In a previous study employing an immunoinformatics approach, we analysed the PE_PGRS proteins of M. tuberculosis to identify MHC class I-binding peptides with the capability of activating CD8⁺ T cells using both prediction algorithms and molecular modelling (Chaitra et al., 2005). This analysis revealed the presence of promising MHC class I binders that were predicted to be promiscuous epitopes and also to bind to more than one MHC. In the present work, we used two of these predicted T-cell epitopes (Peptide_6–14 and Peptide_385–393) from the Rv1818c protein and demonstrated that they are recognized by CD8⁺ T cells from Rv1818c DNA-immunized mice.

Rv1818c DNA vaccine elicits a CD8⁺-mediated T-cell response

In order to determine the nature of the effector T-cell population, mouse T lymphocytes were primed by immunization with pFCN7 plasmid DNA and the pFLAG vector DNA as a control. The lymph node cells from immunized mice were restimulated in vitro with purified Rv1818c protein or PPD, or with the synthetic peptides from the Rv1818c protein. In vitro restimulation of splenocytes with full-length Rv1818c protein induced an approximately eight- to ninefold increase in the proliferation of primed T cells compared with control T cells. Blocking experiments with anti-CD8 mAb showed that Rv1818c DNA generated CD8⁺ T cells that were likely to contribute to the immune response against Rv1818c protein expressed in vivo by the plasmid DNA, as shown in Fig. 1(a). Immunization of mice with purified recombinant Rv1818c protein also resulted in a good T-cell response in terms of proliferation and cytokine secretion (unpublished data). The Rv1818c immune T cells were able to proliferate following stimulation with the synthetic peptides (Fig. 1b, c). This showed that the peptides from Rv1818c were being processed and presented in vivo and were able to elicit a T-cell response. To characterize the functionality of the induced T cells, the cytokines (IL-4 and IFN-) elicited by the different plasmid DNAs were analysed. The amounts of IL-4 and IFN- produced by spleen cells from Rv1818c DNA-immunized mice were measured after 72 h of in vitro restimulation. Significant levels of IFN- were secreted by spleen cells from Rv1818c DNA-vaccinated mice after in vitro stimulation with the recombinant Rv1818c protein compared with vector DNA-immunized splenocytes (Fig. 2). IL-4 production was low or undetectable for the in vitro stimulation (data not shown). Peptide_6–14 and Peptide_385–393 could also induce cytokine secretion from the cells of Rv1818c DNA-immunized mice (Fig. 2). Production of IFN- following restimulation in vitro with purified Rv1818c protein or with Peptide_6–14 was significantly higher in spleen cell cultures from mice that had been immunized with a DNA prime–protein boost regimen than in spleen cell cultures from mice that had been vaccinated with Rv1818c DNA only (Table 1). Mouse T cells primed with the Rv1818c DNA recognized these two epitopes (peptides) generated from the full-length Rv1818c protein after natural processing. In addition, the primed splenocytes were also stimulated by culture filtrates of M. tuberculosis (PPD). The epitope specificity of IFN--producing cells generated by DNA plasmid vaccination demonstrated that both of the peptides triggered IFN- secretion from cells derived from immune animals primed with the DNA vaccine. Previous studies in mice have suggested the relevance of these macrophage-activating cytokines for the control of mycobacterial infection (Bonato et al., 1998; Tan et al., 1997).

View larger version (20K): [in this window] [in a new window]   Fig. 1. (a) In vitro T-cell proliferative response specific for Rv1818c. Rv1818c DNA-primed T cells were restimulated in vitro with either the full-length Rv1818c protein (20 µg ml–1) or PPD (20 µg ml–1) for 96 h and tested for T-cell proliferation. For blocking experiments, splenocytes were added with anti-CD8 mAb (5 µg per 106 cells) prior to restimulation. Unpulsed P815 cells alone were used as a control. Filled bars, DNA immunization; open bars, DNA prime–protein boost. (b) In vitro proliferation of RV1818c DNA-primed splenocytes in response to Rv1818 Peptide6–14. Lymphocytes from the Rv1818c DNA immunized mice were restimulated in vitro with Rv1818c-derived Peptide6–14 (10 µg ml–1)-pulsed P815 cells for 96 h and peptide-specific T-cell proliferation was measured in terms of [3H]thymidine incorporation into dividing T cells. Filled bars, DNA immunization; open bars, DNA prime–protein boost. (c) In vitro proliferation of RV1818c DNA-primed splenocytes in response to Rv1818 Peptide385–393. Lymphocytes from Rv1818c DNA-immunized mice were restimulated in vitro with Rv1818c-derived Peptide385–393 (10 µg ml–1)-pulsed P815 cells for 96 h and T-cell proliferation was measured in terms of [3H]thymidine incorporation into dividing T cells. Filled bars, DNA immunization; open bars, DNA prime–protein boost.

View larger version (10K): [in this window] [in a new window]   Fig. 2. IFN- secretion by Rv1818c DNA-primed splenocytes. Splenocytes from Rv1818c DNA-primed or DNA prime–protein boosted mice were incubated with full-length Rv1818 protein, Peptide6–14 (10 µg ml–1) or Peptide385–393 (10 µg ml–1) and in the presence of mAb to CD8. Supernatants were tested for IFN- levels with recombinant IFN- as the standard. Results are presented as means±SD of the pooled response of three mice from three independent experiments. Filled bars, DNA immunization; open bars, DNA prime–protein boost.

View this table: [in this window] [in a new window]   Table 1. Rv1818c peptide-specific IFN- production in spleen cell cultures from mice vaccinated with Rv1818c DNA alone or DNA primed and boosted with Rv1818c protein

Antigen-specific proliferation and IFN- production by Rv1818c-specific T-cell lines

View larger version (30K): [in this window] [in a new window]   Fig. 3. Functional activity of peptide-specific CD8+ T-cell lines. (a) Intracellular IFN- staining of antigen-specific T cells following in vitro stimulation with synthetic peptides. Primed T cells were expanded in vitro in the presence of the synthetic peptides (10 µg ml–1) and the percentage of CD8+ T cells secreting IFN- was determined by intracellular staining for IFN-. (b) Intracellular perforin staining of peptide-specific T cells.

Peptide–MHC class I (H-2K^d) binding and stabilization by the Rv1818c peptide–MHC complex

In order to validate the in silico MHC class I-binding prediction, peptides from Rv1818c with known mouse MHC class I-binding motifs were studied for their ability to upregulate MHC class I molecules on RMAS-K^d cells. RMAS-K^d cells are TAP-deficient cells in which low levels of unstable MHC class I are expressed on the cell surface at 37 °C. However, incubation of cells at low temperature enhances transport, stability and surface expression of these MHC molecules (Ljunggren et al., 1990; Rock et al., 1991). Culturing TAP-deficient cells in the presence of class I-binding synthetic peptides increases class I surface expression. This upregulation is measured by anti-MHC class I mAb, which is structure dependent. Peptide binding was dose dependent with an optimum binding at a peptide concentration of 100 µM. At this concentration, a threefold increase in mean fluorescence was observed for Peptide_6–14 above the background level, similar to the positive control peptide, whereas Peptide_385–393 showed a twofold increase in its mean fluorescence intensity.

It has been demonstrated previously that the capacity of a peptide to bind and stabilize MHC class I molecules is correlated directly with its ability to induce specific CTL responses (Zhou et al., 1992; Sinnathamby et al., 2001). Therefore, the stability of peptide–MHC complexes was measured by following HLA class I surface expression of peptide-pulsed RMAS-K^d cells over a 2–10 h period at 37 °C. Stability was expressed as DT₅₀, the incubation time (h) required for 50 % of the complexes to decay. The binding and stabilization of Rv1818c peptides was tested in comparison with a positive-control peptide, LLFGYPVYV (Ogata et al., 2003). Peptide_6–14 could stabilize the MHC complex on the cell surface for more than 6 h, whereas Peptide_385–393 could stabilize the complex only for about 3 h. As peptide–MHC binding is also an important factor in determining the cytolytic activity of the epitopes, it is of value to look at the strength or stability of peptide–MHC binding. In vitro binding assays revealed that Peptide_6–14 and Peptide_385–393 could both upregulate MHC class I molecules on the cell surface and displayed low dissociation rates of peptide–MHC complexes. This indicated that the peptides were able to stay on the cell surface for a considerable time, increasing the chances of circulating T cells recognizing the MHC-bound peptide.

Induction of a cytotoxic T-cell response for Rv1818c peptides

To establish that Rv1818c peptides are effective in inducing CTLs, splenocytes from Rv1818c DNA-immunized mice were expanded in vitro in the presence of peptides. Cytotoxicity was measured after in vitro restimulation, using peptide-pulsed P815 target cells. Fig. 4 shows that DNA immunization with Rv1818c resulted in strong CTL responses, which were significantly above the background level. Both of the peptides from Rv1818c induced a CTL response comparable to that of the full-length Rv1818c protein, although the N-terminal peptide-specific T-cell line showed better CTL activity compared with that of the C-terminal peptide. CTLs specific for Peptide_6–14 and Peptide_385–393 were analysed further for the presence of intracellular perforin. As shown in Fig. 3(b), after stimulation with Peptide_6–14, more than 60 % of CD8⁺ T cells stained positive for perforin, whilst with Peptide_385–393 more than 30 % of cells stained positive for perforin. These results indicated that CD8⁺ CTL lines have cytolytic potential in addition to their cytokine-producing function.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Cytotoxic T-cell responses after immunization with Rv1818c DNA. Rv1818c DNA-primed splenocytes were expanded in vitro in the presence of the peptides or Rv1818c protein. The ability to induce CTLs was tested by a cytotoxicity assay against different numbers of antigen-stimulated P815 cells. The percentage of specific lysis was measured for different target : effector (T : E) cell ratios. The specific lysis of peptide-pulsed P815 cells was significantly higher compared with unpulsed 815 cells (P <0.05 by Student’s t-test). , Rv1818 full-length protein; , Rv1818 Peptide6–14; , Rv1818 Peptide385–393; x, unpulsed P815 cells.

Conserved PE_PGRS genes of mycobacteria encode cell-surface proteins that may influence virulence and infection of host cells by mycobacteria (Brennan et al., 2001). Although PE_PGRS 33 (Rv1818c) gene expression did not change significantly under the different experimental conditions tested (Delogu et al., 2006), it has been shown to give a survival advantage for the organism in macrophage cultures in vitro, as well as in mice models, which is associated with the production of increased amounts of tumour necrosis factor (De Libero et al., 1988).

In conclusion, the present work has clearly shown that PE_PGRS 33 has access to the MHC class I processing pathway and that short peptide fragments of this protein can be presented to CD8⁺ T cells. It would be of interest to characterize the immune responses against these peptides using peripheral blood mononuclear cells from BCG-vaccinated healthy individuals of known HLA genotype.

	ACKNOWLEDGEMENTS

 
We thank the Department of Biotechnology, Government of India, and Indian Council for Medical Research for providing support for infrastructure facilities. R. N. is an Emeritus Medical Scientist of the Indian Council for Medical Research.

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TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
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