Characterization of a Mycobacterium tuberculosis Peptide That Is Recognized by Human CD4 + and CD8 + T Cells in the Context of Multiple HLA

Alleles
This information is current as of July 7, 2011 Homayoun Shams, Peter Klucar, Steven E. Weis, Ajit Lalvani, Patrick K. Moonan, Hassan Safi, Benjamin Wizel, Katie Ewer, Gerald T. Nepom, David M. Lewinsohn, Peter Andersen and Peter F. Barnes J Immunol 2004;173;1966-1977
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References

This article cites 47 articles, 33 of which can be accessed free at: http://www.jimmunol.org/content/173/3/1966.full.html#ref-list-1 Article cited in: http://www.jimmunol.org/content/173/3/1966.full.html#related-urls

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright 2004 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

The Journal of Immunology

Characterization of a Mycobacterium tuberculosis Peptide That Is Recognized by Human CD4 and CD8 T Cells in the Context of Multiple HLA Alleles1
Homayoun Shams,2* Peter Klucar,* Steven E. Weis, Ajit Lalvani, Patrick K. Moonan, Hassan Sa,* Benjamin Wizel,* Katie Ewer, Gerald T. Nepom, David M. Lewinsohn,** Peter Andersen, and Peter F. Barnes*
The secreted Mycobacterium tuberculosis 10-kDa culture ltrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP1071 85, that elicited IFN- 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. The Journal of Immunology, 2004, 173: 1966 1977. ight million new cases and 1.8 million deaths annually worldwide are attributed to Mycobacterium tuberculosis, one of the leading causes of death from a single infectious agent (1). The burgeoning epidemic of HIV infection in regions where tuberculosis is common has created a growing population of persons that are highly susceptible to M. tuberculosis. In addition, the continued spread of multidrug-resistant tuberculosis threatens to overwhelm the public health capacity of many jurisdictions (2, 3). These unfavorable factors will cause tuberculosis to remain a major health problem in the coming decades, and increase the urgency for development of an effective vaccine. The only available antituberculosis vaccine is bacillus CalmetteGuerin (BCG),3 a live attenuated Mycobacterium bovis that was
*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; Nufeld 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 Received for publication December 16, 2003. Accepted for publication May 24, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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@uthct.edu 3 Abbreviations used in this paper: BCG, bacillus Calmette-Guerin; BLS, bare lym phocyte syndrome; CFP, culture ltrate protein; ESAT, early secretory antigenic target.

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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 signicantly to immune defenses against tuberculosis through lysis of infected cells, production of IFN- , and direct microbicidal activity (6 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 ltrate 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 (1720). 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 identied
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Copyright 2004 by The American Association of Immunologists, Inc.

The Journal of Immunology in ESAT-6, providing an explanation for its widespread recognition (18, 22, 23). In contrast, only two CD8 epitopes for CFP10 have been identied (24). In this study, we wished to determine the molecular basis for the recognition of CFP10 by most individuals with latent tuberculosis infection. We identied 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.

1967

Materials and Methods

Study subjects
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 classied 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.

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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 veried by mass spectrometry. Lyophilized peptides were dissolved at 25 mg/ml in DMSO, aliquoted, and stored at 4C.

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).

FIGURE 2. CFP1071 85 and CFP1076 90 stimulate production of IFNby PBMC. PBMC from eight CFP10-responsive individuals were stimulated with peptides CFP1071 85 and CFP1076 90 for 48 h. PBMC and positively selected CD4 or CD8 cells were then cultured overnight on IFN- ELISPOT plates. Values shown are the means and SEM of triplicate wells. Unstimulated PBMC showed 0 2 IFN- cells per 2.5 105 cells. A, B, and C show results for PBMC, CD4 cells, and CD8 cells, respectively. FIGURE 1. Capacity of CFP10 peptides to stimulate IFN- production by PBMC from persons with latent tuberculosis infection. PBMC from 49 CFP10-responsive persons were cultured overnight on IFN- ELISPOT plates with 15-mer overlapping peptides spanning the CFP10 protein. The values shown are the percent of CFP10 responders that produced 7 IFN- cells per 2.5 105 cells to each individual peptide. Unstimulated PBMC showed 0 2 IFNcells per 2.5 105 cells.

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,

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A MYCOBACTERIAL PEPTIDE RECOGNIZED BY CD4 and CD8 T cells

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FIGURE 3. CTL activity of short-term T cell lines stimulated with CFP1071 85 and CFP1076 90. PBMC from four donors were stimulated with peptides CFP1071 85 and CFP1076 90 for 1314 days. These short-term lines were used as effectors. Positively selected CD4 or CD8 cells from these lines were also used as effectors. Targets were autologous dendritic cells, either unpulsed or pulsed with relevant peptides. A, B, and C show results, using peptide-stimulated PBMC, CD4 cells, and CD8 cells as effectors, respectively. Values shown are the means and SEM of triplicate wells.

The Journal of Immunology

Table I. HLA typing of donors whose T cells recognized CFP1071 85a
Donor CD8 IFNCells HLA-A HLA-B HLA-C CD4 IFNCells HLA-DQ HLA-DR

1969

T225 T262 T221 T193 T040 T198 T021

15 32 314 700 70 223 15

2 4 26 52 12 4 3

A*01, *24 A*24, *31 A*02, *02 A*02, *68 A*26, *68 A*11, *11 A*02, *03

B*07, *53 B*40, *51 B*39, *40 B*35, *51 B*40, *44 B*15, *3525 B*40, 58

Cw*04, *07 Cw*03, *15 Cw*03, *07 Cw*04, *08 Cw*03, *05 Cw*07, *08 Cw*03, *06

78 85 535 633 223 39 78

7 2 58 56 25 3 2

B1*05, *06 B1*03, *04 B1*03, *03 B1*03, *03 B1*03, *03 B1*03, *03 B1*03, *05

B1*1001, *15 B1*04, *08 B1*04, *04 B1*04, *04 B1*04, *04 B1*11, *12 B1*1001, *11

a PBMC were cultured with or without CFP1071 85 for 48 h, and positively selected CD4 and CD8 cells were placed on an ELISPOT plate for 16 20 h to detect IFNcells. The values shown (mean SEM of triplicate wells) are the number of peptide-stimulated IFNcells per 2.5 105 cells. Unstimulated wells contained 0 2 positive cells per 2.5 105 cells.

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 ow cytometry.

peptide T1 by ELISPOT. IFNT cell clones were expanded with antiCD3 mAb (OKT3; Ortho Biotech, Bridgewater, NJ), irradiated allogeneic PBMC, and an EBV-transformed B cell line lymphoblastoid cell lines (LCL).

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, 2 105 cells per well were cultured in RPMI 1640 and 10% heat-inactivated human AB serum, with puried protein derivative (1 g/ml; Statens Seruminstitut, Copenhagen, Denmark), CFP10 (10 g/ml; Lionex, Braunschweig, Germany), ESAT-6 (10 g/ml; Statens Seruminstitut) or CFP peptides (10 g/ml) for 16 20 h in 96-well plates that were precoated with 15 g/ml anti-human IFNmAb (1-DlK; Mabtech, Nacka, Sweden). To measure the frequency of IFN- -producing CD4 or CD8 cells, PBMC were cultured in T-25 asks at 1.5 106 cells/ml, in medium alone, or with peptide (10 g/ml), or puried protein derivative (1 g/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 25 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 ve 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.

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 Ci of Na251CrO4 (Amersham Life Science, Arlington Heights, IL) at 37C. 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 6 105 effector cells, an E:T ratio of 60:1. Plates were centrifuged at 500 g for 2 min, then incubated for 5 h at 37C. Supernatants were collected (Skatron, Sterling, VA), and 51Cr release was expressed as the mean percent specic lysis, calculated as: 100 ([experimental release spontaneous release]/[maximum release spontaneous release]). Net specic lysis was calculated by subtracting the percent specic lysis of unpulsed target cells from the percent specic 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.

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MHC typing
DNA was extracted from PBMC, using Wizard Genomic (Promega, Madison, WI). Low resolution HLA typing was performed, using PCR with sequence-specic primers (Combi Tray; GenoVision, West Chester, PA). Briey, DNA samples (30 ng/ l) were mixed with a master mix containing Taq polymerase (GenoVision), added into the plates containing sequencespecic primers and amplied by PCR. PCR products (10 l) were electrophoresed on a 2% agarose gel containing ethidium bromide. MHC alleles were identied with the GenoVision version of HELMBERG-SCORE Virtual Sequencing software.

Expansion of peptide-specic 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 g/ml peptide, and seeded in 24-well plates (BD Biosciences, San Jose, CA) at 3 106 cells/ well. After 3 days, 100 U/ml recombinant human IL-2 (Proleukin; Chiron, Emeryville, CA) was added to each well. After 7 days, 3 106 peptidepulsed 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 9599%, as assessed by ow cytometry.

Table II. Truncated peptides derived from CFP1071 85a CFP107185 CFP107690 T110 aa T211 aa T312 aa T413 aa T514 aa T69 aa T79 aa T810 aa T910 aa EISTNIRQAGVQYSR IRQAGVQYSRADEEQ IRQAGVQYSR NIRQAGVQYSR TNIRQAGVQYSR STNIRQAGVQYSR ISTNIRQAGVQYSR EISTNIRQA NIRQAGVQY TNIRQAGVQY RQAGVQYSRA

Generation of peptide-specic T cell clones

Clones were generated by previously described methods (26, 27). Briey, 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

a A ladder of 10 14 aa peptides was made from CFP1071 85 (T1-T5). Four other peptides (T6-T9) were predicted to bind to 3 8 MHC class I alleles with high afnity, using a scoring system that allocates values to every amino acid in a peptide, based on the frequency of the respective amino acid in natural ligands, T cell epitopes, or binding peptides (28).

1970

A MYCOBACTERIAL PEPTIDE RECOGNIZED BY CD4 and CD8 T cells T cells showed lower levels of lysis (127%; Fig. 3C). Recent studies show that M. tuberculosis-responsive CD8 T cell clones are more potent CTL than CD4 T cell clones, using a low E:T ratio and suboptimal peptide concentrations (28). The different results in our experimental system may be due to our use of shortterm T cell lines as effectors, a high E:T ratio and high peptide concentrations. The precursor frequency of CD4 CTL may also be higher than that of CD8 CTL in peptide-stimulated PBMC. CFP1071 85 is recognized by donors expressing different HLA alleles 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. Identication of minimal T cell epitopes within CFP1071 85 To identify the epitopes in CFP1071 85, we rst truncated 15 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 afnity (score, 9) to 4 8 MHC class I alleles (T6-T8, Table II). Finally, we identied a peptide that was predicted to bind with high afnity to three MHC class I alleles (T9, Table II). PBMC from six CFP1071 85-responsive donors were stimulated with peptides T1-T9, and the frequency of IFN- cells was measured by ELISPOT (Table III). The 9-mer T6 and CFP1071 85 elicited comparable numbers of IFNcells 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. To determine whether peptides T1 and T6 contain minimal epitopes, we deleted 12 amino acids from the N or C terminus of these peptides. Removal of a single amino acid from either end of both peptides markedly reduced IFN- production by PBMC (Table IV).

Results
Two CFP10 peptides elicit IFN- 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-specic 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 classied 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. CFP1071 85 and CFP1076 90 elicit IFN- 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 2.5 105 cells; Fig. 2A), by CD4 cells from seven donors (mean 205 86 IFN- cells per 2.5 105 cells; Fig. 2B) and by CD8 cells from eight donors (mean 173 85 IFNcells per 2.5 105 cells; Fig. 2C). CFP1076 90 was recognized by PBMC from six donors (mean 320 176 IFN- cells per 2.5 105 cells; Fig. 2A), by CD4 cells from seven donors (mean 146 64 IFNcells per 2.5 105 cells; Fig. 2B), and by CD8 cells from ve donors (mean 121 83 IFN- cells per 2.5 105 cells; Fig. 2C). To determine whether CFP1071 85 and CFP1076 90 elicited CTL activity, PBMC were cultured with either peptide for 7 days, then restimulated for another 6 7 days. This brief period reduced the likelihood of artifactual induction of peptide-responsive CTL. For four donors, PBMC showed net specic lysis ranging from 6 to 45% against peptide-pulsed autologous dendritic cells (Fig. 3A). CD4 T cells isolated from restimulated PBMC showed similar levels of net specic lysis as PBMC (6 46%; Fig. 3B) and CD8

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Table III. Capacity of truncated peptides to stimulate IFN- production by PBMCa

Donor CFP10 Response T1 (10aa) T2 (11 aa) T3 (12 aa) T4 (13 aa) T5 (14 aa) T6 (9 aa) T7 (9 aa) T8 (10 aa) T9 (10 aa) CFP1071 85 (15 aa)

T221 T225 T262 T267 T283 T294 T296 T298 T299 T300 T301 T303 T302 T297

571 103 313 121 255 178 0 2 0 0 0 0 1 7

671 190 325 119 266 141 0 1 0 0 0 0 6 3

603 284 378 121 263 111 0 0 0 0 0 0 1 6

606 284 315 138 265 149 0 1 0 0 0 0 5 5

590 240 381 146 305 179 3 0 0 0 0 0 7 4

578 213 394 165 296 156 0 2 1 0 0 0 0 6

346 4 163 69 130 29 0 2 0 0 0 0 0 3

384 6 228 82 171 62 0 3 1 0 0 0 0 2

19 4 16 6 23 1 0 1 0 0 0 0 1 2

569 246 309 153 293 189 0 1 0 0 0 0 1 2

a Freshly isolated PBMC from donors who responded to CFP1071 85 were stimulated overnight on IFN- ELISPOT plates with peptide (10 g/ml). For each donor, 23 experiments were performed. Values shown are the mean number of IFNcells per 2.5 105 cells, based on duplicate wells in a representative experiment. Unstimulated wells contained 0 5 positive cells per 2.5 105 cells.

The Journal of Immunology

Table IV. Effect of truncation on the capacity of peptides T1 and T6 to elicit IFN- production by PBMCa
T1 (IRQAGVQYSR) T11 (RQAGVQYSR) T12 (IRQAGVQYS) T13 (IRQAGVQY) T6 (EISTNIRQA) T6 1 (ISTNIRQA) T6 2 (EISTNIRQ)

1971

CFP1071 85 (EISTNIRQAGVQYSR)

T225 T262 T267 T283 T294

149 163 153 191 228

4 4 6 0 0

3 18 18 63 60

0 18 9 1 14

151 185 149 178 147

0 0 4 1 1

3 1 3 0 1

216 204 152 252 208

a Peptides T1 and T6 were truncated from the amino or carboxy terminus, and the effect of truncation was assessed in the ELISPOT assay. Freshly isolated PBMC from CFP1071 85-responsive donors were stimulated overnight with 10 g/ml peptide on IFN- ELISPOT plates. Values shown are the mean number of IFN- cells per 2.5 105 cells, based on duplicate wells. Unstimulated PBMC showed 0 4 IFNcells per 2.5 105 cells.

Peptides T1 and T6 are presented by MHC class I and II molecules 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 2.5 105 cells, p 0.02; T6, mean 139 26 vs 65 14 cells per 2.5 105 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 2.5 105 cells, p 0.0001; T6, mean 139 26 vs 8 2 cells per 2.5 105 cells, p 0.0005; Table V). Isotype control Abs had no effect on the number of IFNcells (data not shown). Peptides T1 and T6 elicit IFN- 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 IFNcells 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). We next evaluated the ability of peptides T1 and T6 to elicit CTL activity. PBMC from ve donors were cultured with peptides T1 and T6 for 7 days and restimulated with peptide for 6 7 more days. PBMC effectors lysed autologous peptide-pulsed target cells (T1, net specic lysis 8 22%; T6, net specic lysis 6 17%; Fig. 4D). Positively selected CD4 T cells showed similar results (T1 and T6, net specic lysis 8 39% and 8 20%, respectively; Fig. 4E). CD8 T cells also lysed comparable numbers of peptidepulsed autologous target cells. However, because CD8 cells lysed a high percentage of unpulsed targets, net specic lysis was relatively low (T1, 214%; T6, 4 10%; Fig. 4F).

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 specic lysis 713%; Fig. 5, A and C). T6-primed effector PBMC and CD4 cells lysed infected cells from two of three donors (net specic lysis 0 32%; Fig. 5, B and D). CD8 effector cells pulsed with T1 or T6 also lysed infected dendritic cells, but nonspecic lysis was higher than for CD4 cells (net specic lysis 232%; Fig. 5, E and F). MHC restriction of T cells that recognize peptides T1 and T6 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 ve donors whose T1-primed CD8 T cells showed CTL activity or IFN- production were HLA A*02 , whereas all ve 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. To more denitively demonstrate that peptides T1 and T6 are restricted by DRB1*04 and DQB1*03, we used transfected BLS cells expressing a single HLA allele as target cells. Freshly isolated CD4 T cells were obtained from ve persons who responded to CFP10 and were infected with M. tuberculosis, and from three uninfected persons who did not respond to CFP10. CD4 cells from the CFP10-responsive donors produced IFN- specically 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). The results above suggest that peptides T1 and T6 are recognized in the context of more than one MHC class II allele. To

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Table V. Effect of Abs to MHC class I and MHC class II on peptide-induced IFN- production by PBMCa
Peptide T1 Anti-MHC I Peptide T1 Anti-MHC II Peptide T6 Anti-MHC I Peptide T6 Anti-MHC II

Peptide T1

Peptide T6

T221 T225 T262 T267 T343 T375

100 87 170 123 65 168

48 18 108 ND 35 45

0 4 10 31 0 8

139 94 179 189 38 195

63 81 94 ND 15 73

4 5 8 18 10 5

a Freshly isolated PBMC from donors responsive to peptides T1 and T6 were incubated with Abs for 4 h, then incubated overnight with peptide on IFN- ELISPOT plates. Values shown are the mean number of IFN- cells per 2.5 105 cells, based on duplicate wells. Unstimulated PBMC showed 0 4 IFNcells per 2.5 105 cells.

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FIGURE 4. Peptides T1 and T6 stimulate production of IFN- and CTL activity. PBMC from four donors were stimulated with peptides T1 and T6 for 48 h, and then PBMC (A) and puried CD4 (B) and CD8 T cells (C) were cultured overnight on IFN- ELISPOT plates. Values shown are the means and SEM of triplicate wells. Unstimulated cells showed 0 2 cells per 2.5 105 PBMC. PBMC from ve donors were cultured with peptides T1 or T6 for 1314 days. These short-term lines (D), or positively selected CD4 (E) or CD8 cells (F) from these lines, were used as effectors. Targets were autologous dendritic cells, either unpulsed or pulsed with relevant peptides. Values shown are the means and SEM of triplicate wells.

conrm these ndings at the level of the single cell, we used two T1-specic CD4 T cell clones as effector cells. BLS cells lacking endogenous MHC class II expression (BLS-1) and six BLS cell lines, each expressing a single transfected HLA class II allele, were used as APCs. Signicant numbers of IFNcells 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 IFNcells 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

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FIGURE 5. CTL activity of effector cells stimulated with peptides T1 and T6 against M. tuberculosis-infected target cells. PBMC from three donors were cultured with peptides T1 (A, C, and E) or T6 (B, D, and F) for 14 days. These short-term lines, and positively selected CD4 or CD8 cells from these lines, were used as effectors. Targets were autologous dendritic cells, either uninfected or infected with H37Rv. Means and SEM of triplicate wells are shown. Effectors used in A and D are peptide-stimulated PBMC, effectors in B and E are CD4 cells, and those in C and F are CD8 cells.

response by clone B9. Cells expressing DRB1*0401 induced the strongest response in both clones.

Discussion
Based on data using puried CD4 and CD8 primary T cells, T cell lines, and T cell clones, we demonstrated that a 15-mer peptide in the secreted mycobacterial protein CFP10 elicits IFN- 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

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A MYCOBACTERIAL PEPTIDE RECOGNIZED BY CD4 and CD8 T cells

Table VI. HLA typing results and responses of CD4 and CD8 T cells to peptide T1 and peptide T6a
Donor CD4 CTL/IFNDRB1 DQB1 CD8 CTL/IFNHLA-A HLA-B HLA-C

Peptide T1 T294 T283 T295 T013 T215 T221 T193 T234 T235 Peptide T6 T294 T283 T295 T013 T215 T221 T193 T234 T235
a

/ND /ND /ND / / / ND/ ND/ / /ND /ND /ND / / / ND/ ND/ /

DRB1 *15 *04, DRB1 *08 *04, DRB1 *04, *04 DRB1 *08 *04, DRB1 *04, *04 *04, *04 DRB1 DRB1 *04, *04 DRB1*03, *13 DRB1*03, *13 DRB1 *15 *04, DRB1 *08 *04, DRB1 *04, *04 DRB1 *08 *04, DRB1 *04, *04 DRB1 *04, *04 DRB1 *04, *04 DRB1*03, *13 DRB1*03, *13

DQB1 *06 *03, DQB1 *04 *03, DQB1 *03, *03 DQB1 *04 *03, DQB1 *03, *03 DQB1 *03, *03 DQB1 *03, *03 DQB1*02, *03 DQB1*02, *03 DQB1 *06 *03, DQB1 *04 *03, DQB1 *03, *03 DQB1 *04 *03, DQB1 *03, *03 DQB1 *03, *03 DQB1 *03, *03 DQB1*02, *03 DQB1*02, *03

/ND /ND /ND / /ND / ND/ ND/ / /ND /ND /ND / /ND / ND/ ND/ /

A*24, *3108 A*24, *68 A*01, *02 A *24 *02, A *11 *02, A *02 *02, A *68 *02, A*24, *74 A*24, *74 A*24, *3108 A*24, *68 A*01, *02 A*02, *24 A*02, *11 A*02, *02 A*02, *68 A*24, *74 A*24, *74

B*15, *4406 B*15, *40 B*40, *57 B*15, *35 B*35, *40 B*39, *40 B*35, *51 B*35, *41 B*35, *41 B*15, *4406 B*15, *40 B*40, *57 B*15, *35 B *40 *35, B*39, *40 B *51 *35, B *41 *35, B *41 *35,

Cw*01, *05 Cw*01, *03 Cw*03, *06 Cw*01, *04 Cw*03, *04 Cw*03, *07 Cw*04, *08 Cw*04, *17 Cw*04, *17 Cw*01, *05 Cw*01, *03 Cw*03, *06 Cw*01, *04 Cw*03, *04 Cw*03, *07 Cw*04, *08 Cw*04, *17 Cw*04, *17

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Boxes show HLA class I and class II alleles that appeared to be associated with either CTL activity or IFN- production by CD8 and CD4 cells, respectively.

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 32), and we have recently shown that the CD40/CD40L pathway con tributes signicantly 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 identied 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 44). In most of these studies, peptides of 16 25 aa were studied, minimal epitopes were not delineated by peptide truncation, or responses of puried CD4 cells were not tested (39 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 identied a 12-aa peptide of Ag85 that elicited proliferation by PBMC from 89% of healthy tuberculin reactors (44). However, because a proliferative response was dened 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 denitive 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 1316 aa peptides that have generally been found to bind MHC class II molecules. Anti-MHC class I reduced the number of peptide T1- and T6responsive IFNcells 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 signicantly to IFN- production induced by M. tuberculosis peptides. However, this response depends on the presence of CD4 cells. These ndings 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 7190 elicit responses by 30 50% of PBMC from CFP10-responsive persons in Zambia and India (20, 46). The current results conrm and extend these ndings, demonstrating that CFP1071 85

The Journal of Immunology

Table VII. Presentation of peptides T1 and T6 by APC expressing a single HLA allelea
Donors HLA Type

1975

Peptide T1

Peptide T6

CFP10 Donors response

DR B1*04 B1*04 B1*04 B1*08 B1*04 B1*08 B1*04 B1*15 B1*04 B1*13 B1*01 B1*13 B1*03 B1*04 B1*01 B1*13

DQ B1*03 B1*03 B1*03 B1*04 B1*03 B1*04 B1*03 B1*06 B1*03 B1*06 B1*05 B1*06 B1*02 B1*03 B1*05 B1*05

DQB1*0302 DRB1*0401 DQB1*0302 DRB1*0401 peptide DQB1*0302 DQB1*0302 peptide DRB1*0401 DRB1*0401 peptide DQB1*0302 DQB1*0302 peptide DRB1*0401 DRB1*0401 CD4 CD4 peptide CD4 CD4 peptide CD4 CD4 peptide CD4 CD4 peptide

T040 T262 T283 T294 T030 T062 T185 T281

80 78 58 68 7 19 3 12

8 5 18 10 8 14 5 18

3 3 0 3 0 0 0 0

45 70 40 65 2 4 2 2

5 8 0 10 3 7 7 4

0 0 0 0 0 0 0 0

113 85 70 120 5 22 4 18

20 23 13 10 8 16 1 12

3 3 0 3 0 0 0 0

110 80 58 55 8 5 5 2

8 5 8 5 5 5 6 4

0 0 0 0 0 0 0 0

a BLS cells expressing DRB1*0401 or DQB1*0302 were used as target cells, either unpulsed or pulsed with peptides T1 or T6. Puried CD4 T cells from freshly isolated PBMC from four donors were added to target cells, and the number of IFN- cells was measured by ELISPOT. Values shown are the mean number of IFN- cells per 2.5 105 cells, based on duplicate wells.

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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 HLADR2 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 ndings 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 CFP108594 and CFP10211 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 HLAB*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 4357% 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.

FIGURE 6. Peptide T1 is recognized by T cell clones in the context of different HLA class II alleles. T1-responsive clones B9 and F10 were generated from a donor whose MHC class II alleles were DQB1*03, DQB1*02, DRB1*04, and DRB1*07. Thirty-ve percent and 58% of B9 and F10 cells produced IFN- in response to T1-pulsed autologous macrophages, respectively. Clones were incubated with BLS cells lacking endogenous MHC class II alleles, and with BLS cells expressing single HLA class II alleles, in the presence or absence of peptide at concentrations of 10, 1, and 0.1 g/ml. Clones without BLS cells were also used as controls. A total of 25,000 BLS cells and 100 clones were placed into each well in duplicate, and the number of IFN- -producing cells was enumerated after 16 h. Values shown are the mean of duplicate results obtained with a peptide concentration of 1 g/ml. A total of 10 g/ml peptide yielded essentially identical results. The number of IFNcells was reduced by 90% when the peptide concentration was 0.1 g/ml.

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Acknowledgments
We are grateful to Mabtech and Staffan Paulie for providing us with precoated IFN- ELISPOT plates, and to Ortho Biotech for provision of antiCD3 mAb. We thank Sharon Kochik for excellent technical assistance in handling BLS cells and transfectants.

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