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,
* Center for Pulmonary and Infectious Disease Control, and Departments of
Microbiology, Immunology, and
Medicine, University of Texas Health Center, Tyler, TX 75708;
Department of Internal Medicine, University of North Texas Health Science Center, Fort Worth, TX 76107;
¶ Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom;
|| Benaroya Research Institute, Seattle, WA 98101;
# Division of Pulmonary and Critical Care Medicine, Oregon Health and Science University/Portland Veterans Affairs Medical Center, Portland, OR 97207; and
** Statens Seruminstitut, Copenhagen, Denmark
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Abstract |
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production and CTL activity by both CD4+ and CD8+ T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP1071–85 contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4+ cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8+ cells of A2+ donors. T6 elicited responses by CD4+ cells in the context of DRB1*04 and DQB1*03, and by CD8+ cells of B35+ donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN- production, suggesting that they are minimal epitopes for both CD4+ and CD8+ cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP1071–85 to stimulate IFN- production and CTL activity by CD4+ and CD8+ cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The only available antituberculosis vaccine is bacillus Calmette-Guérin (BCG),3 a live attenuated Mycobacterium bovis that was created in 1921. Vaccination with M. bovis BCG reduces the severity of tuberculosis in children, but does not protect against development of tuberculosis. Furthermore, vaccination can cause life-threatening disease in immunocompromised patients, such as those with HIV infection (4).
T cells play a pivotal role in protection against tuberculosis, and many studies have shown that CD4+ T cells are essential for immunity (5). A growing body of evidence in animals and in humans suggests that CD8+ cells also contribute significantly to immune defenses against tuberculosis through lysis of infected cells, production of IFN-, and direct microbicidal activity (6, 7, 8, 9, 10, 11, 12). Therefore, the most effective vaccine is likely to be one that elicits responses by both CD4+ and CD8+ T cells (12).
Most published evidence indicates that secreted M. tuberculosis Ags stimulate protective immunity (13). Two important secreted proteins are 6-kDa early secretory antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP10), which form a tightly bound 1:1 heterodimeric complex (14). The encoding genes are cotranscribed (15) and are part of the RD1 region of the M. tuberculosis genome, which is deleted from M. bovis BCG. Restoration of RD1 enhanced the capacity of BCG vaccination to protect mice against subsequent infection with M. tuberculosis (16).
ESAT-6 and CFP10 stimulate T cells to produce IFN- and exhibit CTL activity in animal models and in humans infected with M. tuberculosis, making them excellent candidates for inclusion in an antituberculosis subunit vaccine (17, 18, 19, 20). T cells from a high percentage of persons with latent tuberculosis infection recognize ESAT-6 and CFP10 (20, 21), suggesting that they either contain multiple epitopes that are restricted by different MHC molecules, or epitopes that are promiscuously recognized in the context of multiple MHC molecules. Several epitopes for CD4+ and CD8+ T cells, restricted by different MHC molecules, have been identified in ESAT-6, providing an explanation for its widespread recognition (18, 22, 23). In contrast, only two CD8 epitopes for CFP10 have been identified (24). In this study, we wished to determine the molecular basis for the recognition of CFP10 by most individuals with latent tuberculosis infection. We identified and characterized a 15-mer peptide of CFP10 that elicited IFN- production and CTL activity by both CD4+ and CD8+ T cells from the majority of persons with latent tuberculosis infection, including those expressing several different MHC class I and class II molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was approved by the Institutional Review Boards of the University of North Texas Health Science Center (Fort Worth, TX) and the University of Texas Health Center (Tyler, TX). Blood was obtained from 10 healthy tuberculin-negative donors without prior contact with tuberculosis patients, and from 132 donors who were recent contacts of patients with pulmonary tuberculosis. Seventy-six (58%) donors were Hispanic, 29 (22%) were white non-Hispanic, 21 (16%) were African American, and 6 (5%) were Asian. All donors had no symptoms of tuberculosis with normal chest radiographs. Donors were classified as having latent tuberculosis infection if they had a tuberculin skin test showing at least a 5-mm diameter of induration and their PBMC produced IFN- in response to CFP10 or ESAT-6, based on the ELISPOT assay.
Peptides
We selected 15-mer peptides that overlapped by 10 aa and spanned the CFP10 protein. Truncated peptides were also synthesized, as outlined in the results. Peptides were synthesized by the Molecular Genetics Instrumentation Facility at the University of Georgia (Athens, GA) and by Invitrogen Life Technologies (Carlsbad, CA), using Fmoc chemistry. Peptide purity was >70%, as assayed by HPLC, and their composition was verified by mass spectrometry. Lyophilized peptides were dissolved at 25 mg/ml in DMSO, aliquoted, and stored at 4°C.
Antibodies
We used Abs to framework MHC class I (ATCC clone W6/32; American Type Culture Collection (ATCC), Manassas, VA) and MHC class II (ATCC clone 9.3F10; ATCC).
Isolation of PBMC and cell subpopulations
PBMC were obtained by centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden) and cultured in RPMI 1640 (Invitrogen Life Technologies, Gaithersburg, MD), supplemented with 10% heat-inactivated human AB serum (Atlanta Biologicals, Norcross, GA). In some experiments, CD4+, CD8+, or CD14+ cells were isolated from PBMC by positive selection with magnetic beads conjugated to the appropriate Abs (Miltenyi Biotech, Auburn, CA). Positively selected cells were >95% pure, as determined by flow cytometry.
Measurement of the frequency of IFN--producing cells
To measure the frequency of cells in PBMC that produced IFN- in response to mycobacterial Ags or peptides, 2 x 105 cells per well were cultured in RPMI 1640 and 10% heat-inactivated human AB serum, with purified protein derivative (1 µg/ml; Statens Seruminstitut, Copenhagen, Denmark), CFP10 (10 µg/ml; Lionex, Braunschweig, Germany), ESAT-6 (10 µg/ml; Statens Seruminstitut) or CFP peptides (10 µg/ml) for 16–20 h in 96-well plates that were precoated with 15 µg/ml anti-human IFN- mAb (1-DlK; Mabtech, Nacka, Sweden).
To measure the frequency of IFN--producing CD4+ or CD8+ cells, PBMC were cultured in T-25 flasks at 1.5 x 106 cells/ml, in medium alone, or with peptide (10 µg/ml), or purified protein derivative (1 µg/ml) for 48–72 h. Preliminary studies showed that this period of stimulation yielded the maximum number of IFN-+ cells. After 48–72 h, cells were washed three times, and one aliquot was placed on an anti-IFN--precoated ELISPOT plate for 16–20 h. From two other aliquots, CD4+ and CD8+ cells were positively selected and placed on an ELISPOT plate for 16–20 h.
ELISPOT plates were washed with PBS plus 0.05% Tween 20, and anti-human IFN- mAb 7-B6-1 conjugated to alkaline phosphatase (Mabtech) was added as the detection Ab. After 90 min, the plates were washed and 5-bromo-4-chloro-3-indolyl phosphate/NBT substrate (Moss, Pasadena, MD) was added for 2–5 min or until spots appeared. The spots in air-dried plates were counted using a stereomicroscope. Responses were considered positive if the Ag-stimulated well contained a mean of at least five more spot-forming cells than the mean of the negative control wells, and the Ag-stimulated value was at least twice the mean of the negative control value (20).
In some experiments, freshly isolated CD4+ cells (25,000 cells/well) or CD4+ clones (100 cells/well) were cultured with transfected bare lymphocyte syndrome (BLS) cells (25,000 cells/well), expressing a single HLA molecule (25) as APCs on an ELISPOT plate for 16–20 h. The number of IFN--producing cells was determined, as outlined above.
Expansion of peptide-specific CTLs
PBMC were washed, resuspended in RPMI 1640 containing 10% human AB serum, 20 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (all from Invitrogen Life Technologies), 50 U penicillin (Sigma-Aldrich, St. Louis, MO), and 10 µg/ml peptide, and seeded in 24-well plates (BD Biosciences, San Jose, CA) at 3 x 106 cells/well. After 3 days, 100 U/ml recombinant human IL-2 (Proleukin; Chiron, Emeryville, CA) was added to each well. After 7 days, 3 x 106 peptide-pulsed irradiated (3300 rad) autologous PBMC and 100 U/ml IL-2 were added to each well. Six days later, effector cells were tested for CTL activity in a 51Cr release assay.
In some cases, positively selected CD4+ and CD8+ effectors were isolated from peptide-expanded short-term lines, using immunomagnetic beads. Purity of these cells was 95–99%, as assessed by flow cytometry.
Generation of peptide-specific T cell clones
Clones were generated by previously described methods (26, 27). Briefly, autologous dendritic cells were generated as described below (preparation of target cells), pulsed with peptide T1, irradiated, and cultured at 104 cells per well in 96-well round-bottom plates with 300 CD4+ T cells in each well. Wells that showed visible growth were tested for reactivity with peptide T1 by ELISPOT. IFN-+ T cell clones were expanded with anti-CD3 mAb (OKT3; Ortho Biotech, Bridgewater, NJ), irradiated allogeneic PBMC, and an EBV-transformed B cell line lymphoblastoid cell lines (LCL).
Assessment of CTL activity
Target cells were autologous dendritic cells, generated by incubating positively selected CD14+ macrophages with IL-4 (10 ng/ml; R&D Systems, Minneapolis, MN) and GM-CSF (10 ng/ml; R&D Systems) for 5 day. Dendritic cells were either unstimulated, infected with M. tuberculosis H37Rv for 48 h, or pulsed with a peptide overnight.
Targets were labeled overnight with 100 µCi of Na251CrO4 (Amersham Life Science, Arlington Heights, IL) at 37°C. After extensive washing, they were suspended in complete medium containing 10% FBS, and 104 cells/well were added in triplicate to round-bottom 96-well plates, each well containing 6 x 105 effector cells, an E:T ratio of 60:1. Plates were centrifuged at 500 x g for 2 min, then incubated for 5 h at 37°C. Supernatants were collected (Skatron, Sterling, VA), and 51Cr release was expressed as the mean percent specific lysis, calculated as: 100 x ([experimental release – spontaneous release]/[maximum release – spontaneous release]). Net specific lysis was calculated by subtracting the percent specific lysis of unpulsed target cells from the percent specific lysis of peptide-pulsed or M. tuberculosis-infected target cells. Maximum and spontaneous release were determined in wells containing target cells only, with or without 2% Triton X-100, respectively. Spontaneous release was always <15% of maximum release.
MHC typing
DNA was extracted from PBMC, using Wizard Genomic (Promega, Madison, WI). Low resolution HLA typing was performed, using PCR with sequence-specific primers (Combi Tray; GenoVision, West Chester, PA). Briefly, DNA samples (30 ng/µl) were mixed with a master mix containing Taq polymerase (GenoVision), added into the plates containing sequence-specific primers and amplified by PCR. PCR products (10 µl) were electrophoresed on a 2% agarose gel containing ethidium bromide. MHC alleles were identified with the GenoVision version of HELMBERG-SCORE Virtual Sequencing software.
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Results |
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production by T cells from most persons with latent tuberculosis infection
PBMC from 132 close contacts of patients with pulmonary tuberculosis were stimulated with the M. tuberculosis-specific proteins, CFP10 and ESAT-6, which have previously been used to identify persons with latent tuberculosis infection (20, 21, 23). The ELISPOT assay was used to identify IFN--producing cells. Fifty-three subjects were classified as having latent tuberculosis infection, based on having positive tuberculin skin tests and IFN--producing PBMC in response to either ESAT-6 or CFP10. Of these 53, 49 responded to CFP10.
To identify the regions of CFP10 that induced IFN- production, we tested overlapping 15-aa peptides that spanned CFP10 (20). CFP1071–85 and CFP1076–90 were the most potent, and were recognized by 83 and 91% of responders, respectively (Fig. 1). CFP1071–85 and CFP1076–90 did not elicit IFN- production by PBMC from 10 tuberculin-negative persons who had no history of contact with tuberculosis patients.
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production and CTL activity by CD4+ and CD8+ T cells
We stimulated PBMC from eight donors with CFP1071–85 and CFP1076–90. After 48 h, we obtained CD4+ and CD8+ cells by positive immunomagnetic selection, and cultured them on ELISPOT plates overnight. CFP1071–85 induced IFN- production by PBMC from eight donors (mean 300 ± 124 (SE) IFN-+ cells per 2.5 x 105 cells; Fig. 2A), by CD4+ cells from seven donors (mean 205 ± 86 IFN-+ cells per 2.5 x 105 cells; Fig. 2B) and by CD8+ cells from eight donors (mean 173 ± 85 IFN-+ cells per 2.5 x 105 cells; Fig. 2C). CFP1076–90 was recognized by PBMC from six donors (mean 320 ± 176 IFN-+ cells per 2.5 x 105 cells; Fig. 2A), by CD4+ cells from seven donors (mean 146 ± 64 IFN-+ cells per 2.5 x 105 cells; Fig. 2B), and by CD8+ cells from five donors (mean 121 ± 83 IFN-+ cells per 2.5 x 105 cells; Fig. 2C).
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CFP1071–85 elicited IFN- production by both CD8+ and CD4+ cells from most donors with latent tuberculosis infection, suggesting that it is recognized in the context of multiple MHC alleles. To evaluate these possibilities, we performed MHC typing of seven donors whose CD4+ and CD8+ cells produced IFN- in response to CFP1071–85 (Table I). No single MHC class I or class II allele was shared between all donors. However, of these seven individuals, six expressed DQB1*03 and four expressed DRB1*04. This suggests that CFP1071–85 contains a single epitope that is recognized promiscuously in the context of multiple MHC molecules, or that it contains two or more epitopes, each restricted by different MHC molecules.
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To identify the epitopes in CFP1071–85, we first truncated 1–5 aa from the N terminus (T1-T5, Table II). We also used a motif-based algorithm (29) to identify additional sequences in CFP1071–85 that were predicted to bind with high affinity (score, >9) to 4–8 MHC class I alleles (T6-T8, Table II). Finally, we identified a peptide that was predicted to bind with high affinity to three MHC class I alleles (T9, Table II).
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+ cells was measured by ELISPOT (Table III). The 9-mer T6 and CFP1071–85 elicited comparable numbers of IFN-+ cells for all donors. The 10-mer T1 yielded similar numbers of IFN-+ cells as CFP1071–85 for four donors, and 20–60% fewer IFN-+ cells for two donors.
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production by PBMC (Table IV).
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To identify the restriction elements for peptides T1 and T6, we treated PBMC with anti-MHC class I, anti-MHC class II, or control Ab for 4 h before addition of peptide on the IFN- ELISPOT plate. Neutralization of MHC class I reduced the mean number of T1- and T6-responsive IFN-+ cells by 50% (T1, mean 119 ± 18 vs 51 ± 15 cells per 2.5 x 105 cells, p = 0.02; T6, mean 139 ± 26 vs 65 ± 14 cells per 2.5 x 105 cells, p = 0.04; Table V). Anti-MHC class II reduced the number of IFN-+ cells by >90% (T1, mean 119 ± 18 vs 9 ± 5 cells per 2.5 x 105 cells, p = 0.0001; T6, mean 139 ± 26 vs 8 ± 2 cells per 2.5 x 105 cells, p = 0.0005; Table V). Isotype control Abs had no effect on the number of IFN-+ cells (data not shown).
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production and CTL activity by CD4+ and CD8+ T cells
PBMC from four donors were stimulated with peptides T1 and T6. Forty-eight to 72 h later, positively selected CD4+ and CD8+ cells were placed on an IFN- ELISPOT plate. The frequency of peptide-responsive IFN-+ cells was similar in CD4+ cells and PBMC (Fig. 4, A and B). The number of IFN-+ CD8+ cells was lower than that of CD4+ cells, but higher than corresponding values for unstimulated cells in three donors for peptide T1 and four donors for peptide T6 (Fig. 4C).
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Dendritic cells infected with M. tuberculosis express peptides T1 and T6
To determine whether peptides T1 and T6 are expressed by APCs during M. tuberculosis infection in vivo, we cultured PBMC from three donors with T1 and T6, and tested their capacity to lyse autologous dendritic cells infected with H37Rv. T1-primed effector PBMC and CD4+ cells showed modest lytic activity (net specific lysis 7–13%; Fig. 5, A and C). T6-primed effector PBMC and CD4+ cells lysed infected cells from two of three donors (net specific lysis 0–32%; Fig. 5, B and D). CD8+ effector cells pulsed with T1 or T6 also lysed infected dendritic cells, but nonspecific lysis was higher than for CD4+ cells (net specific lysis 2–32%; Fig. 5, E and F).
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All nine donors whose T1- or T6-primed CD4+ T cells exhibited CTL activity or produced IFN- expressed DQB1*03, and seven of nine also expressed DRB1*04 (Table VI). All five donors whose T1-primed CD8+ T cells showed CTL activity or IFN- production were HLA A*02+, whereas all five donors whose T6-primed CD8+ T cells showed CTL activity or IFN- production were HLA B*35+. Three donors whose CD8+ T cells responded to T1 and T6 expressed both HLA A*02 and B*35 alleles.
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specifically in response to DRB1*0401+ or DQB1*0302+ targets pulsed with peptide T1 or T6, but not to unpulsed targets. In contrast, CD4+ cells from CFP10-negative donors did not produce IFN- in response to peptide-pulsed targets (Table VII).
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+ cells were only observed in the presence of peptide, and BLS cells alone elicited IFN- production by 
10% of the clones (Fig. 6). The frequency of IFN-+ cells in both clones was 3- to 6-fold higher when BLS cells expressing DRB5*0101 or DRB1*0401 were used, and BLS cells expressing DQB1*0602 yielded a 2-fold higher response by clone B9. Cells expressing DRB1*0401 induced the strongest response in both clones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and CTL activity by both CD4+ and CD8+ T cells from a high proportion of persons with latent tuberculosis infection. CFP1071–85 was recognized by CD4+ and CD8+ T cells from persons expressing multiple MHC class II and class I molecules, respectively (Table I), and contains at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4+ cells in the context of at least DRB1*0401, DRB5*0101, and DQB1*0302, and by CD8+ cells of A2+ donors (Tables VI and VII, and Fig. 6). T6 elicited responses by CD4+ cells in the context of DRB1*0401 and DQB1*0302, and by CD8+ cells of B35+ donors (Tables VI and VII). Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN- production, suggesting that they are minimal epitopes for both CD4+ and CD8+ cells. As far as we are aware, these are the shortest microbial peptides that are known to stimulate responses by both T cell subpopulations. The capacity of CFP1071–85 to stimulate IFN- production and CTL activity by CD4+ and CD8+ cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in an antituberculosis vaccine.
CD4+ and CD8+ T cells play complementary roles in protective immunity to many intracellular pathogens, including M. tuberculosis. CD4+ cells are the major source of the macrophage-activating factor IFN-, whereas CD8+ cells predominate in lysing infected cells (28). CD4+ cells also enhance the CD8+ cell response to Ag through interactions between CD40L on the surface of CD4+ cells and CD40 on APCs and on CD8+ cells (30, 31, 32), and we have recently shown that the CD40/CD40L pathway contributes significantly to the human CD8+ T cell response to M. tuberculosis (33). To maximize the protective immune response, it is theoretically appealing to vaccinate with peptides that contain epitopes for both CD4+ and CD8+ T cells. Such peptides can be presented by the same APC to both T cell subpopulations, and their close physical proximity may favor CD40/CD40L interactions and cytokine effects that enhance CD8+ cell effector function. Administration of a peptide containing a CTL epitope of HIV fused to a Th epitope yielded increased CTL responses (34), and vaccination with a 35-mer peptide containing both a CTL and a Th epitope of human papillomavirus completely eradicated papilloma virus-expressing tumors in a murine model (35).
Although epitopes for CD4+ cells and those for CD8+ cells can be fused to create chimeric peptides, naturally occurring peptides recognized by CD4+ and CD8+ cells may elicit more effective immunity because they are more likely to undergo appropriate Ag processing. Fused peptides can also create junctional epitopes that inhibit the immune response to the desired epitopes (36). An epitope comprising 15 aa capable of binding to both MHC class I and class II molecules in a murine model has been identified for HIV (37) and CD8 epitopes within CD4 epitopes are present in Plasmodium falciparum (38). CD8 epitopes of P. falciparum that were nested within CD4 epitopes were more antigenic for humans than other CD8 epitopes, supporting the enhanced immunogenicity of peptides that stimulate both classes of T cells.
Peptides within the M. tuberculosis proteins Ag 85B, ESAT-6, mce2, and the 16-kDa proteins MPB70 and 
-crystallin are recognized by T cells from persons expressing more than one MHC class II haplotype (39, 40, 41, 42, 43, 44). In most of these studies, peptides of 16–25 aa were studied, minimal epitopes were not delineated by peptide truncation, or responses of purified CD4+ cells were not tested (39, 40, 41, 42, 43). Therefore, these peptides may contain more than one CD4 epitope, or a CD4 and CD8 epitope, rather than a single promiscuous CD4 epitope. Valle and colleagues identified a 12-aa peptide of Ag85 that elicited proliferation by PBMC from 89% of healthy tuberculin reactors (44). However, because a proliferative response was defined as only 2-fold that of background levels, and MHC typing of the donors was not performed, it is uncertain whether this peptide is truly promiscuous. The current study provides the most definitive evidence to date that M. tuberculosis peptides of only 9–10 aa can be recognized by persons expressing multiple MHC class II alleles. These peptides are shorter than the 13–16 aa peptides that have generally been found to bind MHC class II molecules.
Anti-MHC class I reduced the number of peptide T1- and T6-responsive IFN-+ cells by 50%, whereas anti-MHC class II almost completely abrogated the response (Table V). These results suggest that MHC class I-restricted CD8+ T cells contribute significantly to IFN- production induced by M. tuberculosis peptides. However, this response depends on the presence of CD4+ cells. These findings extend the results of prior studies indicating that the capacity of CD8+ T cells to produce IFN- in response to heat-killed M. tuberculosis requires CD4+ cells, probably through CD40/CD40L interactions (33, 45).
CFP10 is recognized by T cells from the majority of persons with latent tuberculosis infection and by persons with active tuberculosis, including patients with HIV infection (20, 46, 47). The carboxy end of the molecule is highly immunogenic, and peptides 71–90 elicit responses by 30–50% of PBMC from CFP10-responsive persons in Zambia and India (20, 46). The current results confirm and extend these findings, demonstrating that CFP1071–85 contains at least two epitopes for CD4+ T cells, and is recognized in the context of DRB1*0401, DRB5*0101, and DQB1*0302 (Tables VI and VII, and Fig. 6). The responsiveness of CD4+ T cells from subject T225 to the CFP10 peptides in the absence of DRB1*04 or DQB1*03 (Table I) may be due to the expression of DRB5*0101, which is linked to the DRB1*1501 allele in HLA-DR2+ subjects. Peptide T1 contains isoleucine at position 1, alanine at position 4, and valine at position 6, conforming to the motif predicting strong binding to HLAB1*0401, which is the most common subtype of HLAB1*04 in the United States (48). In contrast, peptide T6 shows no features of this motif. Because some donors whose CD4+ T cells produced IFN- in response to CFP1071–85 expressed other MHC class II alleles (Table I, and data not shown), peptides T1 and T6, or other epitopes on CFP1071–85, are likely to be presented by additional class II molecules. Our findings are consistent with previous work demonstrating that certain peptides can bind to at least seven common DR types, including DRB1*0401 (49).
Previous work has shown that CFP1085–94 and CFP102–11 are HLA-B14- and HLA-B44-restricted epitopes, respectively, for human CD8+ T cell clones (24). We found that CFP1071–85 contains at least two epitopes for CD8+ T cells, one recognized by persons expressing HLA-A*02 and the other by persons expressing HLA-B*35 (Table VI). CD8+ T cells from persons expressing other MHC class I alleles may also recognize these epitopes, as formal restriction analysis with cells expressing a single allele was not performed. HLA-A*02 is part of the HLA-A2 supertype, which is expressed by 39–46% of Caucasians, North American Blacks, Hispanics, and Asians (50). HLA-B*35 is part of the HLA-B7 supertype, which is expressed by 43–57% of these ethnic groups. Therefore, CFP1071–85 is likely to be recognized by CD8+ T cells from the majority of people in different populations throughout the world.
The capacity of CFP1071–85 to elicit IFN- production and CTL activity by CD4+ and CD8+ T cells from persons bearing multiple MHC class I and class II alleles makes it an intriguing candidate for inclusion in an antituberculosis vaccine. DNA vaccines encoding short peptides or peptide-based vaccines are attractive because they are substantially easier to produce than vaccines based on whole proteins. In addition, epitopes in proteins that elicit suppressive or immunopathogenic responses can be avoided. Peptides such as CFP1071–85, perhaps in combination with other immunodominant M. tuberculosis peptides, may also be useful to develop a diagnostic test for latent tuberculosis infection, based on an ELISPOT assay that detects IFN--producing cells. However, a vaccine that includes CFP1071–85 would limit the clinical utility of CFP1071–85-based diagnostic tests in the vaccinated population. These potentially contrasting roles will need to be reconciled in the future.
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Acknowledgments |
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ELISPOT plates, and to Ortho Biotech for provision of anti-CD3 mAb. We thank Sharon Kochik for excellent technical assistance in handling BLS cells and transfectants. |
Footnotes |
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1 This work was supported by grants from the National Institutes of Health (AI44935), the Cain Foundation for Infectious Disease Research, the Wellcome Trust, and the Center for Pulmonary and Infectious Disease Control. P.F.B. holds the Margaret E. Byers Cain Chair for Tuberculosis Research. A.L. is a Wellcome Senior Research Fellow in Clinical Science. 
2 Address correspondence and reprint requests Dr. Homayoun Shams, Center for Pulmonary and Infections Disease Control, University of Texas Health Center, 11937 US Highway 271, Tyler, TX 75708. E-mail address: amir.shams{at}uthct.edu
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; BLS, bare lymphocyte syndrome; CFP, culture filtrate protein; ESAT, early secretory antigenic target.
Received for publication December 16, 2003. Accepted for publication May 24, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95:270.-secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease. Eur. J. Immunol. 30:2713.[Medline]
-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J. Immunol. 167:5217. interferon production in human tuberculosis. Infect. Immun. 68:3002. interferon production in human tuberculosis. Infect. Immun. 69:3497.This article has been cited by other articles:
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  T. S. C. Hinks, D. P. S. Dosanjh, J. A. Innes, G. Pasvol, S. Hackforth, H. Varia, K. A. Millington, X.-Q. Liu, M. Bakir, A. Soysal, et al. Frequencies of Region of Difference 1 Antigen-Specific but Not Purified Protein Derivative-Specific Gamma Interferon-Secreting T Cells Correlate with the Presence of Tuberculosis Disease but Do Not Distinguish Recent from Remote Latent Infections Infect. Immun., December 1, 2009; 77(12): 5486 - 5495. [Abstract] [Full Text] [PDF]
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 X. Wang, P. F. Barnes, K. M. Dobos-Elder, J. C. Townsend, Y.-t. Chung, H. Shams, S. E. Weis, and B. Samten ESAT-6 Inhibits Production of IFN-{gamma} by Mycobacterium tuberculosis-Responsive Human T Cells J. Immunol., March 15, 2009; 182(6): 3668 - 3677. [Abstract] [Full Text] [PDF]
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J. S. Woodworth, Y. Wu, and S. M. Behar Mycobacterium tuberculosis-Specific CD8+ T Cells Require Perforin to Kill Target Cells and Provide Protection In Vivo J. Immunol., December 15, 2008; 181(12): 8595 - 8603. [Abstract] [Full Text] [PDF]
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J. S. Woodworth, S. M. Fortune, and S. M. Behar Bacterial Protein Secretion Is Required for Priming of CD8+ T Cells Specific for the Mycobacterium tuberculosis Antigen CFP10 Infect. Immun., September 1, 2008; 76(9): 4199 - 4205. [Abstract] [Full Text] [PDF]
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 D. L. Hava, N. van der Wel, N. Cohen, C. C. Dascher, D. Houben, L. Leon, S. Agarwal, M. Sugita, M. van Zon, S. C. Kent, et al. Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation PNAS, August 12, 2008; 105(32): 11281 - 11286. [Abstract] [Full Text] [PDF]
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B. Samten, J. C. Townsend, S. E. Weis, A. Bhoumik, P. Klucar, H. Shams, and P. F. Barnes CREB, ATF, and AP-1 Transcription Factors Regulate IFN-{gamma} Secretion by Human T Cells in Response to Mycobacterial Antigen J. Immunol., August 1, 2008; 181(3): 2056 - 2064. [Abstract] [Full Text] [PDF]
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Y. Wu, J. S. Woodworth, D. S. Shin, S. Morris, and S. M. Behar Vaccine-Elicited 10-Kilodalton Culture Filtrate Protein-Specific CD8+ T Cells Are Sufficient To Mediate Protection against Mycobacterium tuberculosis Infection Infect. Immun., May 1, 2008; 76(5): 2249 - 2255. [Abstract] [Full Text] [PDF]
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 H. M. Vordermeier, J. Brown, P. J. Cockle, W. P. J. Franken, S. M. Arend, T. H. M. Ottenhoff, K. Jahans, and R. G. Hewinson Assessment of Cross-Reactivity between Mycobacterium bovis and M. kansasii ESAT-6 and CFP-10 at the T-Cell Epitope Level Clin. Vaccine Immunol., September 1, 2007; 14(9): 1203 - 1209. [Abstract] [Full Text] [PDF]
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K. A. Millington, J. A. Innes, S. Hackforth, T. S. C. Hinks, J. J. Deeks, D. P. S. Dosanjh, V. Guyot-Revol, R. Gunatheesan, P. Klenerman, and A. Lalvani Dynamic Relationship between IFN-{gamma} and IL-2 Profile of Mycobacterium tuberculosis-Specific T Cells and Antigen Load J. Immunol., April 15, 2007; 178(8): 5217 - 5226. [Abstract] [Full Text] [PDF]
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 K. Ewer, K. A. Millington, J. J. Deeks, L. Alvarez, G. Bryant, and A. Lalvani Dynamic Antigen-specific T-Cell Responses after Point-Source Exposure to Mycobacterium tuberculosis Am. J. Respir. Crit. Care Med., October 1, 2006; 174(7): 831 - 839. [Abstract] [Full Text] [PDF]
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H. Shams, S. E. Weis, P. Klucar, A. Lalvani, P. K. Moonan, J. M. Pogoda, K. Ewer, and P. F. Barnes Enzyme-linked Immunospot and Tuberculin Skin Testing to Detect Latent Tuberculosis Infection Am. J. Respir. Crit. Care Med., November 1, 2005; 172(9): 1161 - 1168. [Abstract] [Full Text] [PDF]
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