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The Journal of Immunology, 2004, 172: 6913-6921.
Copyright © 2004 by The American Association of Immunologists

Induction of In Vivo Functional Db-Restricted Cytolytic T Cell Activity against a Putative Phosphate Transport Receptor of Mycobacterium tuberculosis1

Marta Romano*, Olivier Denis{dagger}, Sushila D’Souza*, Xiao-Ming Wang{ddagger}, Tom H. M. Ottenhoff§, Jean-Marc Brulet and Kris Huygen2,*

Laboratories of * Mycobacterial Immunology, {dagger} Allergology, and {ddagger} Mycobacterial Antigens, Pasteur Institute of Brussels, Brussels, Belgium; § Department of Immunohematology & Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands; and Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4+ T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-{gamma} production. Furthermore, a potent IFN-{gamma}-inducing, Db-restricted CD8+ epitope was identified using MHC class I mutant B6.C-H-2bm13 mice and intracellular IFN-{gamma} and whole blood CD8+ T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this Db-restricted PstS-3 epitope. IFN-{gamma} ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4+ and CD8+ T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2b, p, and f haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2d, k, r, s, and q haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2bm12 mice could be overcome by DNA vaccination. IFN-{gamma}-producing CD8+ T cells could also be detected against the Db-restricted epitope in H-2p haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4+ and particularly CD8+ T cell responses against mycobacterial Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. The only TB vaccine currently available is an attenuated strain of Mycobacterium bovis, termed bacille Calmette-Guérin (BCG). The efficacy of BCG remains controversial, particularly against pulmonary TB in young adults, and the development of an improved vaccine is urgently needed to counter the global threat of this disease (1). The definition of the protective Ags from M. tuberculosis is essential for ultimately achieving this goal (2).

Experimental vaccination studies with mycobacterial culture filtrate (CF) have indicated that secreted and surface-exposed proteins, present abundantly in CF, must be key Ags recognized by the protective immune response against human and bovine tuberculosis (3, 4, 5, 6). However, two-dimensional polyacrylamide gel electrophoresis of M. tuberculosis CF has revealed >200 different protein spots (7), and only a small number of these have been studied in more detail for their vaccine potential.

A promising protein family from mycobacterial CF consists of three putative phosphate transport receptors homologous to the periplasmic ABC phosphate-binding receptor PstS from Escherichia coli (8, 9, 10). The genes encoding these proteins were identified using specific mAbs derived from BCG-vaccinated mice (11) and the proteins were called PstS-1 (also known as a 38-kDa protein (12, 13, 14)), PstS-2, and PstS-3. The genes encoding PstS-1, PstS-2, and PstS-3 are very similar (~75% similarity between pstS-1 and pstS-2 or pstS-3 and 94% similarity between pstS-2 and pstS-3) and are found on a continuous stretch of M. tuberculosis genome. At least pstS-1 and pstS-3 seem to be organized in two distinct operons encoding their proper transmembrane PstC and PstA transporter molecules (9). The three PstS proteins all have a lipoprotein consensus signal and are exposed on the cell surface of the bacillus, as demonstrated by flow cytometric analysis using PstS-specific mAbs (10). These lipoproteins are powerful B cell Ags, and detection of Abs against PstS-1 has been reported to be a valuable tool in the serodiagnosis of tuberculosis (15). We have previously reported on a comparative analysis of immunogenicity and protective efficacy of the three PstS proteins in C57BL/6 (B6) mice. Sustained protection against an i.v. M. tuberculosis challenge was only induced by vaccination with plasmid DNA encoding PstS-3, but not with plasmid DNA encoding PstS-1 or PstS-2 (16).

Whereas a DNA vaccine encoding the mycolyl transferase Ag85A confers its protection against TB in B6 mice exclusively through the stimulation of CD4+ T cells (17, 18), the relative role of CD4+ and CD8+ T cells in protection conferred by the PstS-3 DNA vaccine is unknown at present. In this study, we report on the use of DNA vaccination for the mapping of CD4+ and CD8+ T cell epitopes from PstS-3 in C57BL/6 mice. Besides strong CD4+ T cell epitopes, a potent IFN-{gamma}-inducing, Db-restricted CTL epitope was identified and by adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA-vaccinated or M. tuberculosis-infected recipients, we could demonstrate a functional in vivo CTL activity against this epitope. Specificity of the response was also analyzed in mice vaccinated with DNA encoding PstS-1 and PstS-2 and in B10 congenic mice with seven different H-2 haplotypes and in B6.C-H-2bm mutant mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/10 (B10) and C57BL/6 (B6; H-2b), B10.D2 (H-2d), B10.BR (H-2k), B10.P (H-2p), B10.M (H-2f), B10.RIII (H-2r), B10.S (H-2s), and B10.Q (H-2q) mice with eight different haplotypes and B6.C-H-2bm1 (mutated in Kb allele), B6.C-H-2bm12 (mutated in the {beta}-chain of I-Ab), and B6.C-H-2bm13 (mutated in Db allele) mice were bred in the Animal Facilities of the Pasteur Institute of Brussels from breeding pairs originally obtained from Dr. R. ten Berg (Netherlands Cancer Institute, Amsterdam, The Netherlands) as described before (11). C57BL/6 mice genetically deleted in the {beta}2-microglobulin gene ({beta}2-microglobulin ({beta}2M) knockout) were a kind gift from Dr. O. Leo (Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium). Only female mice, 6–10 wk old at the start of vaccination, were used.

Infection

C57BL/6 mice were infected i.v. in the tail vein with 106 CFU of luminescent M. tuberculosis H37Rv, grown as a surface pellicle on synthetic Sauton medium for 14 days, and stored in 20% glycerol in frozen aliquots at –70°C (19).

Plasmid construction

Plasmid DNA encoding the mature Ag85A, PstS-1, PstS-2, and PstS-3 protein from M. tuberculosis was prepared as described before (16). In these V1J.ns-tPA vector-based plasmids, the genes are expressed under the control of the promoter of IE1 Ag from CMV, including intron A, and they are preceded by the signal sequence of human tissue plasminogen activator.

Immunization

Mice were anesthetized with ketamine/xylazine and injected i.m. in both tibia anteriors with 2 x 25 µg of plasmid DNA encoding PstS-3 formulated in vaxfectin in a 2:1 plasmid DNA:cationic lipid molar ratio (20, 21). All mice received three immunizations at 3-wk intervals. For the ELISPOT and the in vivo CTL assessment and for the analysis of cross-reactivity of the immune response with the two other phosphate-binding proteins, B6 mice were vaccinated with 100 µg of DNA in saline, three times at 3-wk intervals, as described before (16).

Antigens

The gene fragment coding for the mature PstS-3 protein was cloned into pQE60 (Qiagen, Valencia, CA) in frame with a His6 encoding headpiece (IPO-10 plasmid obtained from Dr. B. Gicquel, Institut Pasteur, Paris, France). The construction was transformed into the competent E. coli M15 (pRER4; Qiagen). For protein expression, cells were grown in Luria-Bertani medium with 100 µg of ampicillin/ml and 50 µg of kanamycin/ml to an OD at 600 nm of 0.6–0.8 and then induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside for 2 h at 37°C. The harvested bacterial cells were washed with 20 mM Tris-HCl (pH 8.0) buffer and lysed by sonication or with French press. The overexpressed recombinant protein formed inclusion bodies and was recovered in the insoluble fraction. It was purified by metal affinity chromatography under denaturing conditions on a HiTrap Chelating Sepharose HP column (1 ml; Amersham Pharmacia Biotech, Piscataway, NJ) preloaded with nickel sulfate. Thirty-six 20-mer peptides (overlapping by 10 aa residues) covering the entire 369 aa long PstS-3 sequence (including the 22-aa signal sequence), were synthesized as described before (22). The alignment of the amino acid sequence of the three PstS proteins from M. tuberculosis and PstS from E. coli is given in Table I. Recombinant GST fusion proteins of PstS-1, PstS-2, and PstS-3 were prepared as described before (16).


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  		Table I. Amino acid sequences of PstS-1, PstS-2, and PstS-3 from M. tuberculosis and the PstS homologue of E. colia

 
Ab ELISA

Sera from PstS-3 DNA-immunized mice were collected by retro-orbital bleeding 3 wk after the third injection. Levels of total anti-PstS-3 Ig{kappa} Abs were determined by ELISA in sera from individual mice (three per group) against recombinant His-tagged PstS-3 coated at 400 ng/well in borate buffer. Serum titers are expressed in units per milliliter by comparison to a mix of two PstS-3-specific mAbs (2F8-3 and 2C1-5 (11)) attributed a theoretical activity of 1000 U/ml. For statistical comparison, all data were converted to log10 units per milliliter.

Cytokine production

Vaccinated mice were sacrificed 3 wk after the third immunization and spleens were removed aseptically. Spleen cells (4 x 106 white blood cells/ml) from three mice per group were tested individually for cytokine response to purified recombinant His-tagged PstS-3 (5 µg/ml) and CF from M. tuberculosis (25 µg/ml) and as a pool for peptide mapping (10 µg/ml) (22). Supernatants were harvested after 24 h (IL-2) and 72 h (IFN-{gamma}), when peak values of the respective cytokines can be measured. Supernatants from at least three separate wells were pooled and stored frozen at –20°C until assayed. Experiments were performed at least three times and data from one experiment are reported.

IL-2 assay

IL-2 activity was measured using a bioassay as described before (23). Each sample was tested in duplicate. IL-2 levels are expressed as mean cpm. SD was below 10% when not indicated. A typical international standard curve of this assay was published recently (19).

IFN-{gamma} assay

IFN-{gamma} activity was quantified by sandwich ELISA using coating Ab R4-6A2 and biotinylated detection Ab XMG1.2 (both from BD PharMingen, San Diego, CA). Sensitivity of ELISA was 5 pg/ml.

Intracellular IFN-{gamma} measurement using flow cytometry

Intracellular IFN-{gamma} measurement using flow cytometry was performed as described before (24, 25). In short, spleen cells (2.5 x 106 cells/ml) were cultured in 48-well tissue culture plates in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with penicillin and streptomycin, 2 mM L-glutamine, 5 x 10–5 M 2-ME, 10% FCS, and 10 µg/ml PstS-3 peptide spanning aa 285–293. Cells were maintained in 5% CO2 at 37°C. After 1 or 6 days of culture, living cells were harvested on a Ficoll gradient and IFN-{gamma} production was analyzed by intracellular staining and FACS analysis directly or after a second restimulation of 24 h in the presence of RMA-S cells or RMA-S cells pulsed with the PstS-3 peptide. Brefeldin A (Sigma-Aldrich, St. Louis, MO) was added to the cells at a concentration of 10 µg/ml during the last 4 h of culture, and cells were harvested, washed once in staining buffer, and surface stained. One million cells in 100 µl of staining buffer (PBS plus 0.1% NaN3 plus 5% FCS) were incubated with FITC-conjugated monoclonal rat anti-mouse CD8 Abs (clone 53-6.7; BD PharMingen) for 30 min at 4°C. Cells were washed twice in staining buffer and fixed in PBS containing 3% (w/v) paraformaldehyde for 30 min. Paraformaldehyde-fixed cells were washed once with staining buffer and then permeabilized in staining buffer supplemented with 0.1% (w/v) saponin. Cells were incubated for 20 min. at 4°C with PE-conjugated rat anti-mouse IFN-{gamma} Abs (clone XMG1.2; BD PharMingen) diluted in staining buffer supplemented with 0.1% saponin. A PE-conjugated control rat IgG1 (BD PharMingen) was used as control to evaluate the nonspecific staining. Cells were washed twice in staining buffer containing saponin and finally resuspended in PBS containing paraformaldehyde. Samples were acquired on a flow cytometer (FACSCalibur; BD Biosciences, Mountain View, CA) calibrated with the CaliBRITE beads (BD Biosciences) and fluorescence was analyzed using CellQuest software. For each sample, 6 x 104 lymphocytes were gated on the basis of their characteristic forward and side scatter profile.

IFN-{gamma} ELISPOT assay

Specific spleen cell IFN-{gamma} secretion was assayed by ELISPOT. Briefly, 96-well flat-bottom nitrocellulose plates (MAHA S4510; Millipore, Billerica, MA) were incubated overnight at 4°C with 50 µl of capture-purified anti-mouse IFN-{gamma} in PBS (15 µg/ml) (BD PharMingen) and then saturated with RPMI 1640 medium (Life Technologies) supplemented with penicillin, streptomycin, 5 x 10–5 M 2-ME, and 10% FCS for 2 h at 37°C. Spleen lymphocytes (pool of three mice per group) were added at a known concentration in the same medium in the presence or absence of peptides (10 µg/ml) and plates were incubated for 20–24 h at 37°C in 5% CO2. Plates were washed with PBS-0.05% Tween 20 and PBS, incubated overnight at 4°C with 50 µl of biotinylated rat anti-mouse IFN-{gamma} (2 µg/ml; BD PharMingen), washed as before, and incubated for 45 min at 37°C in 5% CO2 with 0.76 U/ml alkaline phosphatase-labeled streptavidin (Sigma-Aldrich). After washing, spots were revealed with a Bio-Rad (Hercules, CA) alkaline phosphatase conjugate substrate kit according to the manufacturer’s instructions, and plates were analyzed on a Bioreader 3000 LC (BioSys, Karben, Germany). Data are reported as spot-forming cells (SFC) per million lymphocytes.

Whole blood CD8+ staining with Db-PstS-3285–293 tetramers

Blood from Ag85A DNA- and PstS-3 DNA-vaccinated B6 female mice was collected by tail bleeding in anticoagulant EDTA 6 wk after the third vaccination and analyzed for staining of CD8+ T cells by Db-PstS-3285–293 tetramers as described by Whelan et al. (26). Briefly, RBCs were lysed in ammonium chloride lysis buffer (150 mM NH4Cl/10 mM NaHCO3/0.4% EDTA (pH 7–7.5)), and cells were washed and incubated at room temperature with soluble Db-SGVGNDLVL MHC-peptide tetramer streptavidin-PerCP conjugate (1.2 x 10–2 µg/ml; ProImmune, Oxford, U.K.) and FITC-anti-mouse CD8a (clone 53-6.7, 1 x 10–2 µg/ml; BD PharMingen). Cells were washed, fixed in 4% paraformaldehyde, and analyzed on a FACSCalibur cytofluorometer.

In vivo CTL activity assessment by adoptive transfer of CFSE-labeled target cells

Spleens from naive B6 female mice were removed aseptically and homogenized by gentle disruption in a loosely fitting Dounce homogenizer. Erythrocytes were depleted from spleen cell suspension by lysis in ammonium chloride solution, washed in RPMI 1640, resuspended at 20 x 106 cells/ml in RPMI 1640–10% FCS, and incubated for 1 h at 37°C/5% CO2 either alone or with 10 µg/ml Db-restricted peptide SGVGNDLVL. After incubation, cells were washed, resuspended in RPMI 1640 at 20 x 106 cells/ml, and labeled for 10 min at 37°C in the dark with CFSE (Molecular Probes Europe, Breda, The Netherlands) either at 1 µM (unpulsed cells; CFSElow) or 10 µM (peptide-pulsed cells; CFSEhigh). Cells were washed and resuspended in PBS at 100 x 106 cells/ml. Twenty million cells of a 1:1 mixture of CFSElow:CFSEhigh was adoptively transferred into female PstS-3 DNA-vaccinated or M. tuberculosis-infected B6 mice (adapted from Refs.27 and 28). Eighteen hours later, adoptively transferred mice were sacrificed, spleens were removed and homogenized, erythrocytes were depleted, and cells were washed and resuspended in PBS for acquisition on a FACSCalibur cytofluorometer. To evaluate the percentage of specific lysis, the ratio of CFSEhigh:CFSElow in vaccinated or infected mice was compared with the ratio in transferred naive control mice. For each experimental group, at least three animals were tested. Results from one representative experiment are shown.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of IL-2- and IFN-{gamma}-inducing epitopes of PstS-3 protein in C57BL/10 mice vaccinated with DNA encoding PstS-3

Vaccination of C57BL/10 mice with DNA encoding PstS-3 enabled the rapid identification of immunodominant IL-2- and IFN-{gamma}-inducing epitopes. As shown in Fig. 1B, spleen cells from C57BL/10 mice vaccinated with DNA encoding PstS-3 produced high IFN-{gamma} levels (between 5,000 and 15.000 pg/ml) in response to four synthetic 20-mer peptides from PstS-3: aa 151–170, 161–180, 191–210, and 281–300. Lower, albeit significant, titers were found following stimulation with three peptides spanning aa 61–80, 131–150, and 261–280. IL-2 was detected in response to the same peptides, except for the peptide spanning aa 281–300, which stimulated exclusively IFN-{gamma} production (Fig. 1A).



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  		FIGURE 1. Th1 T cell epitope mapping in PstS-3 DNA-vaccinated C57BL/10 mice. IL-2 (A) and IFN-{gamma} (B) production in 24- and 72-h culture supernatants of spleen cells from C57BL/10 mice vaccinated with plasmid DNA encoding PstS-3 and restimulated in vitro with 20-mer synthetic peptides (10 µg/ml), overlapping by 10 aa, spanning the entire 369 aa PstS-3 protein.

 
Identification of a Db-restricted CTL epitope on PstS-3 protein using MHC class I and class II mutant B6 mice

Analyzing the sequence of the PstS-3 protein, two sequences with MHC class I consensus motifs for Db binding according to Falk and Rötzchke (29), i.e., 1234N678(M,I,L,V), were found: aa 285–293 SGVGNDLVL and aa 159–167 ITQWNNPAI. Anchor residues are underlined. Using a HLA peptide motif (http://bimas.dcrt.nih.gov/molbio/hla_bind/), a half-time dissociation score of 1408 was calculated for the aa 285–293 peptide, reflecting a strong theoretical affinity of this PstS-3 sequence for the Db allele. The aa 159–167 peptide has a predictive score of only 68. To prove the Db restriction of the IFN-{gamma} response to the former peptide, we analyzed the immune response of two MHC class I mutant B6 mice with mutations in the Kb and Db alleles (Table II). The peptide spanning aa 281–300 was not recognized at all by B6.C-H-2bm13 mice, which have a mutation in the Db allele. In contrast B6.C-H-2bm1 mice with a mutation in the Kb allele recognized the peptide (and all other dominant B6 peptides for that matter) to the same extent as wild-type B6 mice. MHC class II B6.C-H-2bm12 mutant mice displayed impaired IFN-{gamma} and IL-2 production in response to aa 131–150 and 191–210, but responses to aa 61–80 and 151–170 were comparable to parental B6 responses. Interestingly, the IFN-{gamma} response to aa 281–300 was also impaired to some extent in this MHC class II mutant mouse strain, which may have resulted from the overall decreased CD4+ T cell help. IFN-{gamma} responses to recombinant PstS-3 protein were about 5-fold lower in B6.C-H-2bm12 and B6.C-H-2bm13 mutant mice than in B6.C-H-2bm1 mutant or B6 wild-type mice, the latter suggesting that the response to the Db-restricted epitope contributes significantly to the total response to the PstS-3 Ag (data not shown).


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  		Table II. Spleen Th1 cytokine responses in PstS-3 DNA-vaccinated B6 mice, mutated in the Kb, I-Ab, or Db allele

 
In vitro cytolytic T cell activity of spleen cells from B6 mice vaccinated with PstS-3 DNA

As expected from the results obtained in the B6.C-H-2bm13 mice and from the predicted binding scores, in vitro cytolytic T cell activity against 51Cr-labeled RMA-S target cells could be measured in spleen cell cultures from B6 mice vaccinated with PstS-3 DNA only in response to peptide SGVGNDLVL (21) but not to peptide ITQWNNPAI (data not shown).

Intracellular IFN-{gamma} secretion by CD8+ T cells in B6 vaccinated with PstS-3 DNA

To quantify the magnitude of the IFN-{gamma} response to the Db-restricted peptide, intracellular IFN-{gamma} staining was performed on spleen cells from PstS-3 DNA-vaccinated mice. After 1 day of in vitro stimulation with PstS-3285–293, ~1% of the total lymphocyte population in the spleen was IFN-{gamma}-producing CD8+ T cells (Fig. 2Ab). A 6-day stimulation with PstS-3 peptide spanning aa 285–293 and restimulation of these cells with peptide-pulsed RMA-S cells increased the total number of IFN-{gamma}-producing CD8+ T cells to >4% of total lymphocytes (Fig. 2Bc).



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  		FIGURE 2. Intracellular IFN-{gamma} secretion by CD8+ T cells from PstS-3 DNA-vaccinated C57BL/6 mice. A, Spleen cells from PstS-3 DNA-vaccinated B6 mice were stimulated for 1 day in the absence (a) or presence (b) of PstS-3 peptide 285–293 and analyzed by flow cytometry for intracellular IFN-{gamma} staining of CD8+ T cells. B, Spleen cells from PstS-3 DNA-vaccinated B6 mice were stimulated for 6 days in the presence of PstS-3 peptide 285–293, Ficoll purified, and analyzed by flow cytometry for intracellular IFN-{gamma} staining of CD8+ T cells (a). After Ficoll purification, cells were restimulated for 1 day with RMA-S cells (b) or RMA-S cells pulsed with the PstS-3 peptide 285–293 (c). The percent represents the number of IFN-{gamma}-producing cells in the total lymphocyte population.

 
Enumeration by ELISPOT of Db-PstS-3285–293-specific IFN-{gamma}- secreting cells in C57BL/6 mice vaccinated with DNA encoding PstS-3 or infected with M. tuberculosis

Specific IFN-{gamma} secretion by spleen cells of C57BL/6 mice vaccinated with PstS-3 DNA or infected with M. tuberculosis for 2 mo was quantified by ELISPOT assay following a 20- to 24-h incubation with Db-PstS-3285–293 peptide (Fig. 3A). In PstS-3 DNA-vaccinated B6 mice, 246 SFC/million spleen cells were detected. In TB-infected animals, 177 SFC/106 spleen cells were specific for the PstS-3285–293 peptide, clearly indicating that a genuine MHC class I-restricted IFN-{gamma} response is generated to the PstS-3 protein during TB infection. The magnitude of this response was about half the magnitude of the MHC class II-restricted response to the immunodominant I-Ab-restricted epitope from the mycolyl transferase Ag85B240–260 (19). IFN-{gamma} SFC to the PstS-3285–293 peptide were undetectable in spleen from PstS-3 DNA-vaccinated C57BL/6 {beta}2M–/– mice lacking functional CD8+ T cells, but clearly positive in response to the I-Ab-restricted PstS-3 191–210 peptide (Fig. 3B).



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  		FIGURE 3. Specific IFN-{gamma} secretion in C57BL/6 mice by ELISPOT assay. A, Spleen cells of naive B6 mice (a), B6 mice vaccinated with DNA encoding PstS-3 (b), or B6 mice infected with M. tuberculosis for 2 mo (c) were incubated for 24 h with RPMI 1640 ({square}), Db-PstS-3285–293 peptide ({blacksquare}), or Ag85B240–260 peptide (), and the number of IFN-{gamma}-producing cells was determined by ELISPOT. B, {beta}2M–/– mice were vaccinated with DNA encoding PstS-3 DNA and the number of SFC/106 spleen lymphocytes was determined in unstimulated cells ({square}) and in cells stimulated with Db-PstS-3285–293 peptide ({blacksquare}) or I-Ab PstS-3191–210 peptide ().

 
Whole blood tetramer staining of Db-restricted CD8+ T cells in B6 vaccinated with PstS-3 DNA

As shown in Fig. 4, PstS-3-specific Db-restricted CD8+ T cells could be visualized in whole blood from B6 mice by Db-PstS-3285–293 tetramer staining 6 wk after the third PstS-3 DNA immunization (Fig. 4B), but not in blood from B6 mice vaccinated with Ag85A DNA (Fig. 4A). Approximately 7.3% of circulating CD8+ T cells (which made up ~9% of total blood lymphocytes) were stained by the Db-PstS-3285–293 tetramers. In contrast to the spleen ELISPOT results, blood from TB-infected B6 mice scored negative in this Db-PstS-3285–293 tetramer assay, probably because of sequestration of the Db-PstS-3285–293-specific CD8+ cells to the infected spleen and lungs. The CD8-negative population stained by the Db-PstS-3285–293 tetramers are probably B cells interacting nonspecifically with the {beta}2M of the tetramer.



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  		FIGURE 4. Db-restricted peptide tetramer staining of CD8+ T cells from PstS-3 DNA-vaccinated C57BL/6 mice. Whole blood Db-restricted peptide tetramer staining of CD8+ T cells from C57BL/6 mice vaccinated with Ag85A DNA (A) or DNA encoding PstS-3 (B) 6 wk after the third immunization. The percentage indicates the number of Db-restricted peptide tetramer-positive cells in the CD8+ population.

 
In vivo detection of a Db-restricted CTL response using CFSE-labeled target cells

Using a more recently described technique of adoptive transfer of peptide-pulsed, CFSE-labeled spleen cells from naive mice as targets into H-2 syngeneic mice (27), we could demonstrate that the Db-restricted CTLs induced in B6 mice by vaccination with DNA encoding PstS-3 were able to exert an in vivo cytolytic activity. Specific lysis after 18 h was estimated around 41% (40.8 ± 1.9%) in adoptively transferred PstS-3 DNA-vaccinated mice as compared with the values observed in naive animals (Fig. 5, A and C). Confirming the ELISPOT results, we found that the PstS-3285–293 peptide is indeed processed and presented to CD8+ T cells during M. tuberculosis infection and a specific lysis of ~25% (25.3 ± 6.1%) was found in adoptively transferred B6 mice that had been infected with M. tuberculosis 2 mo before (Fig. 5D) and of 31% (31.0 ± 5%) in infected mice, injected once with PstS-3 DNA 1 wk before the assay (Fig. 5E). In naive mice injected once with PstS-3 DNA 1 wk before assay, a specific lysis of 23% (28.8 ± 11.6%) was observed (Fig. 5B). As an additional control for the specificity of the CFSE assay, we performed the test in PstS-3 DNA-vaccinated C57BL/6 {beta}2M–/– mice. Confirming the ELISPOT results, no PstS-3285–293-specific lysis could be detected in these CD8-deficient animals (data not shown). Time course experiments in TB-infected mice showed that in vivo cytotoxic activity was not detectable in the early stage and that it appeared only after 8–10 wk of infection (data not shown). This is, to our knowledge, the first time that an in vivo CD8+-mediated cytolytic T cell activity has been demonstrated toward a mycobacterial Ag.



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  		FIGURE 5. In vivo CTL activity against the Db-restricted PstS-3 epitope. Flow cytometric analysis of naive B6 mice (A), B6 mice vaccinated once with PstS-3 DNA (B), B6 mice vaccinated three times with PstS-3 DNA (C), B6 mice infected for 9 wk with M. tuberculosis (D), and B6 mice infected for 9 wk with M. tuberculosis and vaccinated once with PstS-3 DNA in week 8 (E) were adoptively transferred with a 1:1 mixture of unpulsed CFSElow and peptide-pulsed CFSEhigh naive B6 spleen cells 18 h previously. Percentage of lysis is reported for each experimental animal.

 
Lack of cross-reactive Th1 responses to PstS-3 peptides in B6 mice vaccinated with DNA encoding the two other phosphate-binding proteins PstS-1 and PstS-2

To analyze whether the immunodominant CD4+ and CD8+ epitopes of PstS-3 were shared with the two other putative phosphate transport receptors PstS-1 and PstS-2 from M. tuberculosis, we examined the cytokine production in spleen cell cultures from B6 mice vaccinated with DNA encoding these two proteins. Confirming previous findings (16), IL-2 responses to purified recombinant GST fusion proteins of PstS-1, PstS-2, and PstS-3 were completely specific for each Ag. IL-2 responses of PstS-1 and PstS-2 DNA-vaccinated B6 mice were also completely negative following stimulation with the three IL-2-inducing peptides of PstS-3. Similarly, the IFN-{gamma} response to each of the four IFN-{gamma}-inducing peptides of PstS-3 (or any other PstS-3 peptide for that matter) was below detection level in B6 mice vaccinated with DNA encoding PstS-1 or PstS-2 (data not shown).

Influence of MHC on PstS-3-specific Ab production in mice vaccinated with DNA encoding PstS-3

MHC-linked genes are known to exert a profound influence on the Ab response against secreted and surface-exposed culture filtrate proteins in mice vaccinated with live M. bovis BCG (11). Strong Ab production against the PstS-3 Ag was detected in sera from BCG-vaccinated B10 (H-2b), B10.M (H-2f), and B10.P (H-2p) mice, but not in B10.D2 (H-2d), B10.BR (H-2k), B10.Q (H-2q), B10.S (H-2s), or B10.RIII (H-2r) mice (30). Analysis of the serum from BCG-vaccinated B6.C-H-2bm12 mutant mice demonstrated that the I-Ab allele is essential for the production of a high Ab response to this Ag. To analyze whether a similar MHC-linked influence exists following PstS-3 DNA vaccination, we analyzed the Ab response in seven B10 congenic strains with different H-2 haplotypes and in the three B6.C-H-2bm mutant strains by Ab ELISA against recombinant His-tagged PstS-3 protein. As shown in Fig. 6, MHC-linked genes influenced the magnitude of the response of PstS-3 DNA-vaccinated mice, H-2p, f and b haplotype mice producing strong, and H-2d, k, s, r, and q haplotype mice producing only weak Ab responses. These genetic differences were identical to the ones observed in mice vaccinated with live M. bovis BCG. In contrast, analysis of the MHC class II mutant B6.C-H-2bm12 mouse strain showed that DNA vaccination could help to overcome its low responsiveness observed following live BCG vaccination (11). Indeed, Ab responses were only slightly lower in B6.C-H-2bm12 mice as compared with responses in parental B6 mice, but they were significantly higher than in the nonresponder B10 congenic strains. Ab titers in the MHC class I mutant B6.C-H-2bm1 and B6.C-H-2bm13 mice were not significantly different from titers in B10 mice.



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  		FIGURE 6. Influence of H-2 haplotype on PstS-3-specific IgG production. PstS-3-specific total IgG production in serum from B6.C-H-2 mutant, B10, and B10 congenic mice vaccinated with DNA encoding PstS-3 and analyzed 3 wk after the third DNA immunization. Titers are expressed in log10 arbitrary units (AU) per milliliter as compared with a mixture of two PstS-3-specific mAbs, with a theoretical activity of 3 log10 arbitrary units per milliliter. The H-2 haplotype is indicated at the top.

 
Influence of MHC on Th1-type cytokine secretion in mice vaccinated with DNA encoding PstS-3

The various B10 congenic mouse strains were also analyzed for their spleen cell Th1-type cytokine secretion following PstS-3 DNA vaccination. A clear correlation was observed between the Ab and the Th1-type cytokine responses, and only the two B10 congenic mouse strains with good Ab responses, i.e., B10.P (H-2p) and B10.M (H-2f), also produced significant IFN-{gamma} levels in response to some peptides from PstS-3 (Fig. 7). B10.P mice recognized three peptide regions: aa 61–80, 161–180, and 281–300. These peptides were the same as some of the peptides recognized by B10 mice, but aa 61–60 elicited a stronger IFN-{gamma} response in B10.P than in B10 mice, as compared with the other peptides. IFN-{gamma} responses in B10.M mice were of a lower magnitude, but a significant response was observed against one peptide spanning aa 261–280, a region that was also recognized by B10 mice. Spleen cells from B10.BR mice showed a weak IFN-{gamma} response (~300 pg/ml) to one peptide spanning aa 151–170 (data not shown). IFN-{gamma} responses in spleen cells from B10.S, B10.Q, and B10.RIII mice finally were undetectable or below 100 pg/ml (data not shown). All IFN-{gamma}-inducing peptides, except aa 281–300, also induced significant IL-2 production in spleen cell cultures from B10.P and B10.M mice vaccinated with PstS-3 DNA (data not shown). Intracellular IFN-{gamma} staining of CD8+ T cells following stimulation with peptide 281–300 was positive in B10.P but not in B10.M mice (data not shown), suggesting that the Dp allele has a similar restriction specificity as the Db allele.



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  		FIGURE 7. IFN-{gamma}-inducing epitopes of PstS-3 in H-2p (B10.P) and H-2f (B10.M) mice. H-2p (A) and H-2f (B) mice were vaccinated with plasmid DNA encoding PstS-3 and spleen cells were restimulated in vitro with 20-mer synthetic peptides (10 µg/ml), overlapping by 10 aa, spanning the entire 369 aa PstS-3 protein 3 wk after the third immunization. IFN-{gamma} content was determined in 72-h culture supernatant by ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The attenuated strain of M. bovis, BCG, continues to be widely administered to children worldwide, although its efficacy is disappointingly low against the pulmonary form of tuberculosis in young adults (2). A number of factors are thought to lie at the basis of this low efficacy, including exposure to environmental mycobacteria and genetic variations of both host and pathogen. It has also been postulated that the current BCG vaccines have been weakened, particularly with respect to their induction of CD8+-mediated immune responses. These CD8+ responses are known to be important for protection, particularly during the persistent phase of infection (31). A number of experimental vaccine approaches have focused precisely on increasing the CD8+ T cell-mediated responses of BCG by boosting with recombinant poxviruses (32, 33) or by genetically modifying the BCG for increased escape toward the host cell cytoplasm (1). So far, only a small number of CTL epitopes have been identified for M. tuberculosis Ags, and this seriously hampers detailed analysis of the relative role of the CD8+ T cell compartment in protection against the Koch bacillus.

DNA vaccination is a powerful method for the induction of CD4+ and particularly CD8+-mediated immune responses and it offers an easy and rapid tool for the screening of protective Ags of M. tuberculosis (34). We have previously shown that DNA vaccination of mice with plasmids encoding the three mycolyl transferases Ag85A, Ag85B, and Ag85C can induce responses to dominant CD4+ epitopes, also presented during M. tuberculosis infection or M. bovis BCG vaccination. Furthermore, DNA vaccination could also induce responses against subdominant CD4+ epitopes and more importantly against MHC class I-restricted CD8+ epitopes, responses that are below detection level for Ag85 in mycobacterially infected mice (19, 35) but that have been defined in infected and BCG-vaccinated humans (36, 37, 38). Induction of responses to dominant and subdominant epitopes has also been described in two separate studies with DNA vaccines encoding an influenza and a Sendai virus nucleoprotein (39, 40). To what extent this broader DNA vaccine-induced response to multiple epitopes also results in a broader central and/or effector memory T cell repertoire remains an open question (41, 42). DNA vaccination was also shown to be effective in HLA-transgenic mice for the identification of HLA-A*0201-restricted epitopes on the Ag85B protein (36) and of a dominant HLA-DR3-restricted epitope on the PstS-3 protein (A. Geluk, unpublished data). The definition of CD4+ and CD8+ epitopes on a particular Ag offers an easy working tool for subsequent analysis of immune responses by bypassing the need for purified Ag. Moreover, peptides can be used in experimental vaccination strategies such as peptide-pulsed dendritic cells (43), and finally definition of new epitopes for known MHC haplotypes can help to increase the accuracy of existing models predicting MHC class I- and class II-restricted epitopes in general.

In this study, we have identified the immunodominant epitopes on the phosphate-binding protein PstS-3 protein recognized by CD4+ T cells of C57BL/6 mice. Furthermore, DNA vaccination enabled the identification of an IFN-{gamma}-inducing Db-restricted CTL epitope. This response could be visualized by ELISPOT, whole blood CD8+ Db tetramer staining, and in vivo cytolytic activity could be measured using adoptive transfer of CFSE-labeled syngeneic target cells, pulsed with this Db epitope, into PstS-3 DNA-vaccinated mice. More importantly, this Db-restricted CTL epitope was found to be genuinely processed and presented during mycobacterial infection, as demonstrated by the lysis of CFSE-labeled, peptide-pulsed targets in M. tuberculosis-infected mice and by the presence in spleen from M. tuberculosis-infected mice of a significant number of cells staining positive in an IFN-{gamma} ELISPOT assay using the Db-restricted peptide. This is to our knowledge the first time that in vivo CD8+-mediated cytolytic T cell activity has been demonstrated toward a mycobacterial Ag. Interestingly, the cytolytic activity as well as the specific IFN-{gamma} production in M.tuberculosis-infected mice were observed only at late time points, i.e., 2 mo but not at the onset of infection. There is evidence that CD8+ T cells take over a substantial portion of IFN-{gamma} production during the persistent phase of infection in the mouse model, although CD4+ T cells continue to play an important role in protection (44) and that these CD8+ T cells are essential for controlling latency (31). It is possible that during the persistent phase, lung macrophages become overloaded with mycobacteria and that this would result in leakage of the PstS-3 protein from the phagosome into the cytoplasm. For the 19-kDa lipoprotein, this alternate class I MHC Ag processing has been reported before (45). A slow generation of PstS-3-specific MHC class I-restricted epitopes could explain why the best protective effects with the PstS-3 DNA vaccine are found at late time points after infection, in contrast to the early protection observed for the Ag85A DNA vaccine (16). In contrast, it has been described that alternate MHC class I Ag processing, but not MHC class I expression, can be inhibited in macrophages by M. tuberculosis (46), and this could explain the lower magnitude of the PstS-3-specific in vivo CTL response in TB-infected mice as compared with PstS-3 DNA-vaccinated mice and the fact that we have been unable so far to detect a PstS-3-specific CTL response against macrophages infected in vitro with TB (D. Canaday, unpublished data).

Despite the high similarity between the three PstS genes, particularly the ones encoding PstS-2 and PstS-3, T cell and B cell immune responses against the PstS-3 protein were found to be completely component specific, and none of the immunodominant T cell epitopes defined on the PstS-3 molecule was shared with the PstS-1 or PstS-2 protein. The strongest I-Ab-restricted epitope for B6 mice on PstS-3 was present in the region spanning aa 191–210. Comparison to the corresponding sequences revealed that 4 of 20 and 12 of 20 residues differed from the PstS-2 and PstS-1 molecules, respectively. The Db-restricted epitope SGVGNDLVL on PstS-3 shares its two anchor residues asparagine and leucine with the corresponding sequence in PstS-2, but apparently the shifts at position 285 (serine substituted by methionine) and position 287 (valine substituted by glutamine) impair the interaction with the Db molecule. The corresponding sequence in the PstS-1 molecule differs in nine of nine residues. For the PstS-1 protein, a Kb-restricted CTL epitope has been identified using MHC class I prediction motifs, MHC class I binding experiments, and in vivo immunization with peptide in IFA (47, 48, 49). Comparison of this Kb-restricted sequence INEYAIV of PstS-1 (aa 326–333) to the corresponding, common sequence LATYEIV in the PstS-2 and PstS-3 proteins shows that four of seven of the amino acids are different but that the two anchor residues are conserved. Nevertheless, this region did not induce any IFN-{gamma} response in PstS-3 DNA-vaccinated B6 mice, confirming the notion that predictions on the sole basis of anchor motifs are not accurate enough to identify MHC class I-restricted epitopes and as such identification of entire new epitopes is of great value for the construction of more reliable algorithms. Also, for the three mycolyl transferases of the Ag85 complex, we have found highly component-specific immunodominant Th1 helper T cell epitopes, despite substantial sequence homology (19).

With the entire M. tuberculosis genome sequence now available, it has become clear that gene duplication and triplication has been a frequent event in mycobacterial evolution and a number of proteins are actually members of protein families (50). This is the case among others for the mycolyl transferases of the Ag85 complex and for the phosphate transport receptors discussed here. The components of these protein families may be differentially expressed in time, depending on changes in the environment, such as changes in pH, oxygen, or nutritional factors. In 1991, Content et al. (51) suggested that the expression of the different members of the Ag85 complex was regulated independently at the transcriptional level, and Betts et al. (52) have demonstrated, using microarray and proteome analysis, that in an in vitro nutrient starvation model of M. tuberculosis the genes of the Ag 85 complex are indeed differentially expressed, fbpA and fbpB being down-regulated, but fbpC2 being significantly up-regulated. Also in vivo in the lungs of M. tuberculosis-infected mice, it was found that mRNA synthesis of the Ag85B protein decreased at the moment of transition from growth to persistence associated with the onset of Th1-mediated immunity (53). We hypothesize that differential antigenic expression of family members (with shared enzymatic function) but with highly specific immunodominant epitopes could act as source of antigenic variation and eventually of immune evasion for mycobacteria.

MHC polymorphisms have been associated with susceptibility or resistance to infectious diseases such as malaria, AIDS, hepatitis B, leprosy, and TB (54). In this study, we have shown that the H-2-linked variations in B cell and T cell response against the PstS-3 protein are the same in B10 congenic mice vaccinated with plasmid DNA or vaccinated with live M. bovis BCG. The only discrepancy between the DNA- and BCG-induced response was found in B6.C-H-2bm12 mutant mice, which produced significant Ab and Th1 cytokine responses following PstS-3 DNA but not following BCG vaccination. It has been speculated that the immune response defects in B6.C-H-2bm12 mutant mice are caused by a suboptimal epitope presentation rather than by a lack of MHC class II molecules to associate with the epitopes (55). These mutant mice have indeed two to three times less surface Ia than wild-type B6 mice, probably because their mutant {beta}-chain fails to associate as effectively with its {alpha} partner as the wild-type {beta}-chain does (55). In contrast, it has also been reported that the nonresponsiveness of B6.C-H-2bm12 mice (to the male H-Y Ag) results from the active generation of suppressor T cells (56). It is not clear at the moment whether the improved immune response to PstS-3 in DNA-vaccinated B6.C-H-2bm12 mice is caused by an increased Ag-presenting activity or by the inhibition of suppressor T cells. In contrast, it must be underlined that immunological nonresponsiveness, caused by complete lack of MHC-peptide interaction and subsequent presentation, apparently cannot be overcome by the plasmid DNA vaccination approach.

In conclusion, DNA vaccination is a powerful means for the induction and characterization of CD4+ and particularly CD8+ T cell responses against mycobacterial Ags and it has enabled for the first time the demonstration of an in vivo functional, Db-restricted PstS-3-specific CTL activity in M. tuberculosis-infected mice. These results also warrant further studies on the protective efficacy of the PstS-3 protein as part of a multisubunit protein or DNA vaccine.


   Acknowledgments
 
We are very grateful to Dr. B. Gicquel (Pasteur Institute of Paris, Paris, France) for giving us the IP0–10 clone. We also thank Dr. Carl Wheeler (Vical Inc., San Diego, CA) for the generous gift of vaxfectin and Dr. Oberdan Leo (Université Libre de Bruxelles, Gosselies, Belgium) for the {beta}2M knockout mice.


   Footnotes
 
1 This work was partially supported by Grant G.0266.00 from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen, by the European Union (Tuberculosis Vaccine Cluster QLK2-CT-1999-01093), by the Brussels Hoofdstedelijk Gewest, and by the Damiaanaktie Belgium. Back

2 Address correspondence and reprint requests to Dr. Kris Huygen, Mycobacterial Immunology, Pasteur Institute of Brussels, 642 Engelandstraat, B1180 Brussels, Belgium. E-mail address: khuygen{at}pasteur.be Back

3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacille Calmette-Guérin; CF, culture filtrate; {beta}2M, {beta}2-microglobulin; SFC, spot-forming cell. Back

Received for publication May 2, 2003. Accepted for publication March 22, 2004.


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 Materials and Methods
 Results
 Discussion
 References
 

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