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Originally published online as doi:10.1189/jlb.0403138 on September 12, 2003

Published online before print September 12, 2003
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(Journal of Leukocyte Biology. 2003;74:1008-1014.)
© 2003 by Society for Leukocyte Biology

Human CD8+ T cells recognize epitopes of the 28-kDa hemolysin and the 38-kDa antigen of Mycobacterium tuberculosis

Homayoun Shams*,{dagger},1, Peter F. Barnes*,{dagger},{ddagger}, Stephen E. Weis§, Peter Klucar*,{dagger} and Benjamin Wizel*,{dagger}

* Center for Pulmonary and Infectious Disease Control, Departments of
{dagger} Microbiology and Immunology and
{ddagger} Medicine, University of Texas Health Center, Tyler; and
§ Department of Internal Medicine, University of North Texas Health Sciences Center, Fort Worth

1Correspondence: CPIDC, UT Health Center, 11937 U.S. Hwy. 271, Tyler, TX 75708-3154. E-mail: amir.shams{at}uthct.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis antigens that are recognized by human CD8+ T cells are potentially important vaccine target molecules. We used a motif-based strategy to screen selected proteins of M. tuberculosis for peptides predicted to bind to human leukocyte antigen (HLA)-A*0201. We identified two 10 amino acid peptides that elicited cytolytic T lymphocyte activity and interferon-{gamma} production by CD8+ T cells from HLA-A*0201+ healthy tuberculin reactors. These peptides were derived from the 38-kDa antigen and the 28-kDa hemolysin, the latter being a novel target for CD8+ T cells. We speculate that hemolysins may alter the phagosomal membrane surrounding intracellular M. tuberculosis, allowing themselves and other antigens to gain access to the major histocompatibility complex class I processing pathway.

Key Words: cytolytic T lymphocyte • HLA-A*0201 • Bacillus Calmette-Guerin • Interferon gamma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis is one of the world’s most successful human pathogens, causing approximately 8 million new cases and 2 million deaths worldwide annually [1 ]. Development of a vaccine is likely to be the most cost-effective means to reduce worldwide morbidity from tuberculosis, and identification of mycobacterial antigens that elicit protective immunity against M. tuberculosis is an area of intensive investigation. CD4+ T cells play a pivotal role in protection against M. tuberculosis, as evidenced by the severe manifestations of tuberculosis in persons with human immunodeficiency virus infection and defective CD4+ T cell function [2 ]. CD8+ T cells also contribute to immunity against M. tuberculosis. CD8+ T cell-deficient mice show enhanced susceptibility to tuberculosis [3 ], and in humans infected with M. tuberculosis, CD8+ T cells recognize mycobacterial antigens, lyse M. tuberculosis-infected monocyte-derived macrophages and alveolar macrophages [4 5 6 7 8 9 10 11 ], and directly kill M. tuberculosis by secreting the antimicrobial peptide granulysin [12 ].

CD8+ T cell epitopes are recognized in the context of major histocompatibility complex (MHC) class I molecules, and human leukocyte antigen (HLA)-A*0201 is a class I allele that is expressed at high frequency in individuals of a variety of ethnic backgrounds [13 ]. Therefore, identification of M. tuberculosis peptides that are recognized by HLA-A*0201+ persons is important for vaccine development. We used a motif-based strategy to first screen secreted proteins of M. tuberculosis for peptides predicted to bind to HLA-A*0201. These peptides were then empirically tested for binding to HLA-A*0201 and for their capacity to elicit cytolytic T lymphocyte (CTL) activity and interferon-{gamma} (IFN-{gamma}) production by CD8+ T cells from healthy tuberculin reactors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic peptides
Sequences of eight M. tuberculosis antigens were inspected for the presence of nine or 10 amino acid sequences conforming to the expanded HLA-A*0201-binding motif [14 ]. The algorithm analyzes peptide sequences for the presence of residues at nonanchor positions that are favorable or detrimental for binding to HLA-A2.1 molecules. Of 1387 motif-bearing segments, 298 were predicted to bind HLA-A*0201 molecules with intermediate-to-high affinity, using an algorithm that takes into account the role of secondary anchors in peptide binding to class I molecules [14 , 15 ]. Of these, 51 sequences from different proteins that had the highest scores were selected for peptide synthesis. We also synthesized control peptides that are known to bind HLA-A*0201 and/or to elicit CTL activity: the M. tuberculosis Ag85B143-152 (FIAGSLSAL), the M. tuberculosis Ag85B199-207 (KLVANNTRL), the M. tuberculosis Ag85A242-250 (KLIANNTRV), the hepatitis B virus core HBc18-27 F6 Y peptide (FLPSDFFPSV), influenza A virus matrix FluM58-66 (GILGFVFTL), and melanoma Melan-A27-35 (AAGIGILTV) [4 , 10 , 16 17 18 ]. Peptides were synthesized by the Molecular Genetics Instrumentation Facility at the University of Georgia (Athens) and by ResGen-Invitrogen Corp. (Carlsbad, CA), using Fmoc chemistry. The purity of the peptides was ~70%, as assayed by high-pressure liquid chromatography, and their composition was verified by mass spectrometry. Lyophilized peptides were dissolved at 20 mg/ml in Dulbecco’s modified Eagle’s medium, aliquoted, and stored at -70°C.

Peptide binding–HLA-A*0201 stabilization assay
The binding of synthetic peptides to HLA-A*0201 molecules was measured by their ability to stabilize these molecules on the surface of T2 cells [19 , 20 ]. Briefly, T2 cells that had been incubated in complete Iscove’s medium (Invitrogen-Life Technologies, Gaithersburg, MD) at room temperature the previous night to increase cell-surface MHC class I molecule expression were then incubated in incomplete medium overnight at 37°C with 10 µM each peptide. Stability of HLA-A*0201 was assayed by staining the cells with anti-HLA-A*02 monoclonal antibody (mAb) BB7.2 [American Type Culture Collection (ATCC), Manassas, VA] and fluorescein isothiocyanate–F(ab')2 goat anti-mouse immunoglobulin G (Southern Biotechnology, Birmingham, AL), followed by analysis with an EPICS C flow cytometer (Beckman Coulter, Hialeah, FL). The HBc18-27 peptide was used as a positive control.

Study subjects
This study was approved by the Institutional Review Boards of the University of North Texas Health Science Center at Fort Worth and the University of Texas Health Center at Tyler. Subjects were healthy adult tuberculin reactors who had received no more than 4 weeks of isoniazid at the time of their enrollment. Of 56 subjects, 19 were HLA-A*02+, as determined by flow cytometric analysis of peripheral blood mononuclear cells (PBMC) stained with mAb BB7.2. Six volunteers were HLA-A*0201+, based on a sequence-specific primer polymerase chain reaction, conducted at the Riley Hospital Histocompatibility Laboratory (Indianapolis, IN). The eight healthy tuberculin reactors who were HLA-A*02+ had a skin test in duration of 12–30 mm. Six of these eight were born in the United States, and none had received Bacillus Calmette-Guerin (BCG) vaccination. Four of the eight donors had known extensive exposure to M. tuberculosis, and PBMC from three of three donors tested produced IFN-{gamma} in response to the M. tuberculosis-specific antigen ESAT-6. Three tuberculin-negative HLA-A*02+ donors and a tuberculin-positive HLA-A*02- donor were recruited as controls.

Preparation of peptide-specific CTL effectors
Heparinized blood was obtained from each subject, and PBMC were isolated by density gradient centrifugation over Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden). PBMC were washed, resuspended in RPMI 1640 (Invitrogen-Life Technologies), supplemented with 10% heat-inactivated human AB serum (Atlanta Biologicals, Norcross, GA), 20 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (all from Invitrogen-Life Technologies), and 50 U penicillin (Sigma Chemical Co., St. Louis, MO), and seeded in 24-well plates (Becton Dickinson, Franklin Lakes, NJ) at 3 x 106 cells/well. Individual peptides (10 µg/ml) were included in each 2 ml culture. At day 3 of culture, 100 U/ml recombinant human interleukin (IL)-2 (Proleukin, Chiron Corporation, Emeryville, CA) was added to each well. After 1 week of incubation at 37°C and 6% CO2, cells in each well were restimulated by adding 3 x 106 peptide-pulsed, irradiated (3300 rad), autologous PBMC and 100 U/ml IL-2. Six days after restimulation, effector cells were tested for CTL activity in a standard 5-h 51Cr release assay [21 ]. Short-term cell lines were generated by restimulation of effector cells with PBMC for 6–8 weeks.

In some cases, CD8-enriched and CD8-depleted effectors were isolated to test for CTL activity. For enrichment, CD8+ T cells were purified by positive selection with magnetic beads coated with anti-CD8 antibodies (Miltenyi Biothech, Auburn, CA). The negative fraction was further depleted of residual CD8+ T cells by panning on Petri dishes coated with anti-CD8 (OKT8, Beckman Coulter) following standard procedures [22 ]. Enriched and depleted effector cells contained >95% and <2% CD8+ T cells, respectively.

Preparation of target cells and 51Cr release assay
Target cells were HLA-A*0201+ JY cells (ATCC), which had been incubated overnight at 37°C in the presence of 100 µCi Na2 51CrO4 (Amersham Life Science Corp., Arlington Heights, IL) and 10 µg/ml peptide. After extensive washing, target cells were suspended in complete medium, and 104 cells per well were added in triplicate to round-bottom 96-well plates, each well containing 6 x 105 stimulated effector cells, giving an effector-to-target (E:T) cell ratio of 60:1. In some cases, CD8-enriched and CD8-depleted effector cells were also used at E:T ratios of 60:1. Plates were centrifuged at 500 g for 2 min and then incubated for 5 h at 37°C. Supernatants were collected (Skatron, Inc., Sterling, VA), and 51Cr release was expressed as the mean percent-specific lysis of triplicate wells, calculated as follows: 100 x [(experimental release–spontaneous release)/(maximum release–spontaneous release)]. Maximum and spontaneous release was determined in wells containing target cells only, in the presence or absence of 2% Triton X-100, respectively. Spontaneous release was <25% of maximum release in all experiments. Net percent-specific lysis was calculated by subtracting the lysis of unpulsed target cells from the lysis of peptide-pulsed target cells.

Enzyme-linked immunospot (ELISPOT) assay
CD14+ and CD8+ cells were separated from PBMC by positive selection with immunomagnetic beads conjugated to anti-CD14 and anti-CD8 (Miltenyi Biotech) using standard methods. CD40 ligand trimer (5 µg/ml; Amgen Corporation, Seattle, WA) and IL-2 (100 U/ml; PharMingen, San Diego, CA), with or without peptides (10 µg/ml), were added to CD8+ T cells alone, CD14+ cells alone, or CD8+ T cells mixed with CD14+ cells at a ratio of 5:1. Seventy-two hours later, supernatants were collected, and cells were washed, transferred in triplicate to 96-well nitrocellulose plates (Millipore Corp., Bedford, MA), coated with 15 µg/ml anti-human IFN-{gamma} mAb (1-DlK, Mabtech, Nacka, Sweden), and incubated at 37°C with 5% CO2. The detection antibody was biotinylated anti-human IFN-{gamma} mAb 7-B6-1 (Mabtech). The wells were developed, according to the manufacturer’s instructions, and the spots in the air-dried plates were counted with a stereomicroscope.

Enzyme-linked immunosorbent assay (ELISA)
Supernatants were collected, and ELISA measured IFN-{gamma} concentrations, according to the manufacturer’s protocol (PharMingen). The lower limit of sensitivity was 5 pg/ml.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Prediction and identification of peptides that bind with high affinity to HLA-A*0201
To identify M. tuberculosis peptides that elicit CTL activity of MHC class I-restricted CD8+ T lymphocytes, we first selected eight M. tuberculosis proteins that have been shown to elicit responses by CD8+ T cells [8 , 23 , 24 ] or are known to be secreted [25 , 26 ]. These proteins are therefore likely to reach the cytoplasm of host cells and become available to the endogenous MHC class I antigen-processing pathway. The proteins selected were ESAT-6, the 28-kDa hemolysin, pep A, antigens 85A and 85B, and antigens of 19, 38, and 45/47 kDa. Using an algorithm that takes into account the role of secondary anchors in peptide binding to HLA-A*0201 molecules [14 , 15 ] to screen these proteins, we selected 51 sequences of nine to 10 amino acids for peptide synthesis (Table 1 ).


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  		Table 1. Peptides of M. tuberculosis Proteins That Are Predicted to Bind HLA-A*0201

 
To empirically determine the capacity of these 51 peptides to bind to a MHC class I molecule, we measured their capacity to stabilize a HLA-A*0201 class I molecule on T2 cells. This method has been shown to identify peptides that bind to HLA molecules with high affinity [19 , 20 ]. Results were expressed as relative mean fluorescence intensity (MFI), calculated as the MFI of peptide-treated cells/MFI of cells treated with the HBc18-27 peptide, which is known to bind HLA-A*0201. Of the 51 peptides, 11 showed the greatest stabilization of HLA-A*0201, with relative MFIs of 0.6 or higher. These peptides were derived from hemolysin, pep A, and antigens of 19, 38, and 45/47 kDa (Table 2 ). The MFIs of the other 40 peptides were 0.42–0.58.


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  		Table 2. Binding of Predicted Peptides from M. tuberculosis Proteins to HLA-A*0201

 
Capacity of peptides to elicit CTL activity in HLA-A*02+ donors
PBMC from two to four HLA-A*02+ healthy tuberculin reactors were cultured with each of the 11 peptides, as outlined in Materials and Methods, and after one round of restimulation, CTL activity was measured against unpulsed and peptide-pulsed targets. CTL activity was considered to be positive if net-specific lysis was >=10%. Five peptides (#2, #23, #28, #42, and #47) did not elicit CTL activity in the first two to four donors and were not evaluated further (Table 3 ). Four peptides (#15, #35, #39, and #43) induced CTL activity in only one donor. Peptide 21 stimulated CTL activity in six of eight donors. Peptide 36 elicited CTL activity in two of seven donors, and net-specific lysis was 5–8% in an additional three volunteers (% lysis of unpulsed cells was 0–1% in these three persons). Therefore, these two peptides were selected for further evaluation. PBMC, from three HLA*0201+ donors, were stimulated with three M. tuberculosis peptides that previously have been reported to be HLA*0201-restricted [4 , 10 ] (Table 3) . However, significant CTL activity was not elicited. We cultured PBMC from four HLA*0201+ donors with melanoma peptide and observed no cytotoxicity (Table 3) . These findings suggest that our culture conditions did not artifactually induce development of CTL in vitro but expanded pre-existing, peptide-specific CTL (Table 3) . In addition, CTL activity was not elicited from PBMC of three HLA-A*0201+, tuberculin-negative donors or from PBMC of a HLA-A*02-negative, tuberculin-positive donor.


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  		Table 3. CTL Activity of PBMC from HLA-A*02+ Donors in Response to Peptides from M. tuberculosis and Other Sources*

 
The contribution of CD8+ T cells to CTL activity
To determine if the CTL activity elicited by peptides 21 and 36 was mediated by CD8+ T cells, PBMC from three donors were cultured with each of these peptides and restimulated with peptide-pulsed, irradiated PBMC after 1 week, and 6 days later, CD8+ T cells were depleted from an aliquot of PBMC. The CTL activity of PBMC and CD8-depleted PBMC was measured against target cells pulsed with peptide and medium alone. Depletion of CD8+ T cells reduced net-specific lysis by 65–100% (Fig. 1 ), indicating that CD8+ T cells predominantly mediated the CTL activity.



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  		Figure 1. Effect of depletion of CD8+ T cells on CTL activity against targets pulsed with peptide 21 and peptide 36. PBMC from three healthy tuberculin reactors were stimulated with peptide 21 or peptide 36 and restimulated with peptide-pulsed, irradiated PBMC after 1 week, and 6 days later, CD8+ T cells were depleted from an aliquot of PBMC. PBMC and CD8-depleted PBMC were used as effectors against HLA-A*0201+ JY cells, pulsed with peptide or medium alone, at an E:T ratio of 60:1. Mean values and SEM of triplicates are shown.

 
As an additional means to confirm that CD8+ CTL recognized peptides 21 and 36, we generated short-term lines with these peptides. PBMC from two HLA-A*0201+ donors were stimulated weekly with peptide and autologous, irradiated PBMC for five to eight cycles. CTL lines (PBMC) from both donors lysed targets pulsed with relevant peptide (Fig. 2A ). All lines also contained 775–4296 pg/ml IFN-{gamma} in their supernatants. CD8+ T cells purified from the peptide 36-stimulated lines showed modestly higher cytolytic activity than PBMC (Fig. 2B) . The increase in CTL activity with purified CD8+ cells may have been small, as the original lines were already 55–75% CD8+, as determined by flow cytometry.



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  		Figure 2. CTL activity and IFN-{gamma} production by short-term T cell lines generated against peptide 21 and peptide 36. PBMC from two healthy tuberculin reactors were stimulated with peptide 21 or peptide 36 for five to eight cycles, as outlined in Materials and Methods. Mean values and SE of triplicates are shown in each panel. (A) CTL activity of short-term T cell lines against HLA-A*0201+ JY cells. (B) CTL activity of CD8+-enriched T cells against HLA-*0201+ JY cells pulsed with peptide #36. (C) Production of IFN-{gamma} by short-term T cell lines. Concentrations of IFN-{gamma} in supernatants of short-term T cell lines are shown.

 
Peptides 21 and 36 elicit production of IFN-{gamma} by CD8+ T cells in PBMC
To determine if peptides 21 and 36 elicited IFN-{gamma} production by CD8+ T cells in blood of healthy tuberculin reactors, we used the ELISPOT assay. When purified CD8+ T cells from PBMC of healthy tuberculin reactors were cultured with autologous monocytes at a ratio of 5:1 in the presence of peptide for periods ranging from 1 to 5 days, no IFN-{gamma}+ cells were observed. We previously found that the capacity of CD8+ T cells to produce IFN-{gamma} in response to M. tuberculosis antigens depends on CD4+ T cell help [27 ] and that CD4+ T cell help can be replaced by the addition of CD40 ligand trimer and IL-2 [28 ]. When freshly isolated, purified CD8+ T cells were stimulated with CD40 ligand trimer, IL-2, and peptide 21, peptide 36, or influenza peptide, the number of IFN-{gamma}+ cells was four- to fivefold higher than those present in the absence of peptide (Table 4 ). The number of IFN-{gamma}-producing CD8+ cells paralleled IFN-{gamma} production, as measured by ELISA, as well as the magnitude of CTL activity of peptide-stimulated PBMC.


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  		Table 4. CTL Activity and IFN-{gamma} Production Elicited by Peptides 21 and 36

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a HLA*0201-binding motif-based strategy to screen secreted antigens of M. tuberculosis, we identified two 10 amino acid peptides that are recognized by CD8+ T cells from HLA-A*0201+ persons with latent tuberculosis infection. These peptides were derived from the 28-kDa hemolysin and the 38-kDa antigen and elicited CTL activity and IFN-{gamma} production by CD8+ T cells from healthy tuberculin reactors. The peptides did not elicit CTL activity by PBMC from tuberculin-negative donors or from a tuberculin-positive person who was HLA-A*0201–, confirming that recognition of these peptides requires prior infection with M. tuberculosis. It is important to note that these two peptides induced CTL activity after PBMC had been cultured for only 13 days, including one round of restimulation with peptide (Table 3) . Prior studies that identified M. tuberculosis epitopes for human CD8+ cells demonstrated CTL activity only after multiple rounds of restimulation with the relevant peptides [4 ]. These previously identified peptides did not elicit CTL activity during the brief periods of culture used in this study (Table 3) , suggesting that the precursor frequencies of peptide-specific CTL may be lower than those of the peptides described in the current work.

The identification of M. tuberculosis antigens that are recognized by human T cells is a high priority for development of an effective subunit vaccine against tuberculosis. As CD8+ T cells are essential for effective immunity against M. tuberculosis in animals [3 ] and are likely to contribute to immunity in humans, antigens recognized by CD8+ T cells are potentially important vaccine-target molecules. HLA-A*02 is expressed by 50% of Caucasians, 35% of African-Americans, and 36% of Asian/Pacific Islanders [13 ], so epitopes recognized in the context of this haplotype are particularly relevant. Epitopes recognized by HLA-A*0201-restricted CD8+ T cells have previously been identified on antigens 85A and 85B, the 19-kDa lipoprotein, the 16-kDa {alpha}-crystallin, a predicted 44-kDa protein product of the M. tuberculosis gene Rv0341, thymidylate synthase, RNA polymerase ß-subunit, and permease protein A-1 [4 , 8 , 10 , 29 30 31 ]. Microbial antigens for CD8+ T cells are generally those that are secreted into the host cytosol, permitting processing and presentation via the MHC class I pathway. However, several of the above antigens recognized by CD8+ T cells are somatic, indicating that these proteins enter the host cytoplasm, perhaps through a pore in the phagosome where M. tuberculosis resides [32 , 33 ].

Identification of multiple peptides recognized by CD8+ T cells is important for two reasons. First, the epitopes can be linked in a multiepitope vaccine that may increase the strength and depth of the CD8+ response, preventing immune evasion by epitope mutation. Second, the proteins containing these epitopes can be more fully evaluated for additional CD8+ T cell epitopes restricted by other HLA class I alleles and for recognition by CD4+ cells. This can be done by motif-based screening or by using overlapping peptides that span the entire protein to empirically identify immunogenic peptides [6 ]. Proteins that contain epitopes for CD4+ and CD8+ T cells may be particularly valuable for eliciting a durable, protective immune response against M. tuberculosis.

We found that CD8+ T cells from HLA-A*0201+ persons recognized peptides from the 38-kDa antigen and the 28-kDa hemolysin of M. tuberculosis. The 38-kDa antigen is a phosphate-binding protein that previously has been found to elicit responses by murine CD4+ and CD8+ T cells, as well as human CD8+ T cells [25 , 34 , 35 ], although the CTL epitopes recognized by human CD8+ T cells have not been characterized. Several M. tuberculosis molecules with hemolytic properties have been described [36 37 38 39 ], but they have not been previously shown to contain epitopes for CD8+ T cells. It is intriguing to speculate that hemolysins may alter the phagosomal membrane surrounding intracellular M. tuberculosis, allowing molecules to pass through more readily. In this event, hemolysin and other antigens may gain access to the MHC class I processing pathway and be recognized by CD8+ T cells. This has been previously observed in the case of listeriolysin, which is a major target of CD8+ CTL in Listeria infection [40 ]. In addtion, recombinant BCG organisms that express listeriolysin induce potent CD8+ CTL responses [41 ].


   ACKNOWLEDGEMENTS
 
The Potts Memorial Foundation, the National Institutes of Health (A127285), the Center for Pulmonary and Infectious Disease Control, and the Cain Foundation for Infectious Disease Research supported this study. P. F. B. holds the Margaret E. Byers Cain Chair for Tuberculosis Research. We thank Amgen Corporation for the gift of recombinant CD40 ligand trimer.

Received April 4, 2003; revised July 3, 2003; accepted August 4, 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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