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International Immunology Advance Access originally published online on October 31, 2006
International Immunology 2006 18(12):1707-1718; doi:10.1093/intimm/dxl105
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Immunogenic HLA-B7-restricted peptides of hTRT

Xochitl Cortez-Gonzalez1, John Sidney2, Olivier Adotevi3, Alessandro Sette2, Frederick Millard1, Francois Lemonnier3, Pierre Langlade-Demoyen3 and Maurizio Zanetti1

1 Laboratory of Immunology, Department of Medicine and Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0837, USA
2 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, La Jolla, CA 92121, USA
3 Laboratoire d'Immunite Cellulaire Anti-Virale, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris, France

Correspondence to: M. Zanetti; E-mail: mzanetti{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Telomerase reverse transcriptase (TRT) is the first bona fide common tumor antigen. While several 9mer peptides of the human TRT have been identified for HLA-A2, little information exists on peptides for the remaining HLA types. Here, we used a multi-step approach to select and characterize a panel of HLA-B7 9mer peptides as candidate immunogens. In sequence, we used algorithm-based predictions, in vivo immunization of HLA-B7 transgenic (Tg) mice, in vitro immunization of human blood lymphocytes from two normal donors and two cancer patients, in vivo processing in HLA-B7 Tg mice and HLA-B7 supertype binding. We found a correlation between the in vivo immunogenicity and the actual HLA-B7 binding avidity of the seven predicted peptides. Furthermore, endogenous processing correlated with in vitro immunogenicity in human PBMC and HLA-B7 supertype binding. Peptide 1123LPSDFKTIL1131 (p1123) with the wider spectrum of supertype binding displayed the highest immunogenicity overall and was endogenously processed in several human lymphoblastoid cells. Since no single step of the screening/selection process could substitute for the whole approach, we conclude that the identification of MHC class I-restricted peptides for potential vaccination of cancer patients remains, by and large, an empirical process.

Keywords: cancer, supertype, telomerase


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Telomerase is a ribonucleoprotein that mediates RNA-dependent synthesis of telomeric DNA (1). Maintenance of a constant telomere length ensures chromosomal stability, prevents cells from aging and confers immortality (2–4). In vitro studies show that the long-term ectopic expression of human telomerase reverse transcriptase (hTRT) in normal fibroblasts is sufficient for immortalization (5), and the expression of hTRT in combination with two oncogenes (SV40 T antigen and Ras) promotes tumor transformation in normal human epithelial and fibroblast cell lines (6). Thus, although telomerase per se is not tumorigenic, it plays a direct role in oncogenesis by allowing pre-cancerous cells to proliferate continuously and become immortal.

Studies of human cancer cells have shown a striking high expression (>85%) of telomerase activity in tumors of different histological origin and type (7, 8). In contrast, normal tissues display little or no telomerase activity (8, 9). For these reasons, hTRT is considered the prototype common tumor antigen (10). To date, numerous in vitro studies have been published demonstrating that hTRT peptides can be used to expand CD8 T cell precursors and generate CTL in human PBMCs (11–15). Furthermore, several phase 1 trials have also been completed proving that specific CD8 T cell responses can be induced in vivo (16–19) in cancer patients.

T lymphocytes recognize antigens through the intermediary of molecules of the MHC or HLA, a polymorphic system composed of several hundred molecules (MHC restriction). CD8 T cells recognize antigen presented through MHC class I molecules expressed at the surface of every cell after antigen peptides have been processed inside the cell and exported to the cell surface through the endogenous pathway (20). Under normal circumstances, MHC class I molecules present a broad variety of peptides, mainly the product of processing of endogenous proteins. Upon infection by microbial pathogens or tumor transformation, peptides are generated that once complexed with the MHC molecules of an antigen-presenting cell (APC) can activate CD8 T cells and induce CTL responses. However, since the MHC system is highly polymorphic among the human population, it requires that the immunogenicity of antigen peptides be studied in relation to each HLA molecule. An alternative and simpler approach is to test antigen peptides in relation to HLA alleles grouped into large supertype families (21). An HLA supertype is defined by the ability of a peptide to bind multiple HLA molecules (supermotif). The HLA allelic variants that bind peptides possessing a particular HLA supermotif are referred to as HLA supertype. The HLA-B7 supertype includes the B*0702, B*3501-03, B*51, B*5301, B*5401, B*0703-05, B*1508, B*5501-02, B*5601-02, B*6701 and B*7801 alleles. These HLA molecules share a peptide-binding specificity for P in position 2 and a hydrophobic aliphatic (A, L, I, M or V) or aromatic (F, W or Y) residue at the C-terminal position (22).

To date, specific information on the immunogenicity hTRT peptides is limited to one MHC allele (HLA-A*0201) with only initial reports on the HLA-A3 (13) and HLA-A24 (23) types, respectively. Although HLA-A*0201 is the most frequent in the human population [95% of HLA-A2 type which is itself expressed in ~50% of the Caucasian population (24–26)], immunogenic peptides for an equally large segment of the human population need to be identified. The goal of the work presented here was to identify immunogenic hTRT peptides restricted by HLA-B*0702 molecule which is the most prevalent allele within the HLA-B7 type which accounts for ~8.6% of the Caucasian population (27).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
HLA-B7 transgenic (Tg) mice express a chimeric HLA-B7/H2-Db MHC class I molecule, are on a C57BL/6 background and have been previously described (28). Mice were originally produced at the Institut Pasteur (Paris, France). A colony was bred and maintained under specific pathogen-free conditions in the vivarium of the University of California, San Diego (La Jolla, CA, USA). All experimental procedures were performed according to an approved protocol and the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Cell lines
The human T2-B7 transfectants and murine RMA-B7 transfectants lines have been transfected with the HLA-B*0702 allele as described previously (28, 29). Human erythroleukemia K562 cells were purchased from American Type Tissue Culture Collection (ATCC). The EBV-transformed B lymphoblastoid (HLA-A2/B7) JY cells were obtained from Dr Antonella Vitiello (PRI Johnson & Johnson, La Jolla, CA, USA). BC1-B7 and T1-B7 are EBV-transformed lymphoblastoid human cells.

Human blood cells
Buffy coats from HLA-B7+ normal donors were purchased from the San Diego Blood Bank (San Diego, CA, USA). Prostate cancer patients were recruited through the Division of Hematology/Oncology and blood was obtained by venipuncture. HLA-B7 positivity was assessed by flow cytometry. Experiments were performed in accordance with approved Institutional Review Board protocols.

Peptides and mAbs
All synthetic peptides were purchased from the Peptide Synthesis Core Facility of Ohio State University (Columbus, OH, USA). The mAb against HLA-B7, BB7.1, was purchased from American Type Tissue Culture Collection (Manassas, VA, USA). Other antibodies used were FITC-conjugated mouse IgG anti-human CD8 beta (mAb 53-6.7) and PE-conjugated mouse IgG anti-human CD3 (BD PharMingen, San Diego, CA, USA), and FITC-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch, West Grove, PA, USA).

Predictive algorithms
The following predictive algorithms were used: (i) BIMAS which is based on highly favorable and unfavorable dominant anchor residues, as well as auxiliary anchor residues, and scores peptides according to a coefficient (30) (access via: http://thr.cit.nih.gov/molbio/hla_bind/). (ii) SYFPEITHI which is based on known T cell epitopes and MHC ligands (31, 32) (access via: http://www.uni-tuebingen.de/uni/kxi/) and takes into consideration the amino acids in the anchor and auxiliary anchor positions, and scores peptides according to the cumulative (positive or negative) effects of contributing amino acids with ideal anchor residues accounting for 10 points and amino acids regarded as having a negative effect on binding accounting for –1 and 3 points. (iii) PAProC, Prediction Database for Proteasomal Cleavages, a computer-based theoretical model for the cleavage of substrate proteins by yeast and human 20S proteasomes. PAProC predicts cleavability of amino acids sequence (cuts per amino acids) and individual cleavages (positions and estimated strength). Specifically, we used the Type III model, based on human erythrocyte proteasome cleavage of enolase and ovalbumin (33, 34) (access via: http://www.paproc.de/).

MHC binding assays
Relative avidity measurements The relative avidity of hTRT peptides for HLA-B7 was measured using an MHC stabilization assay on T2-B7 cells in comparison with a reference peptide as described previously (14). Results are expressed as values of relative avidity, which is the ratio of the concentration of test peptide necessary to reach 20% of the maximal binding by the reference peptide, so that the lower the value the stronger the binding.

Supertype analysis
Quantitative assays to measure the binding affinity of peptides to purified HLA-B7 supertype molecules (B*0702, B*3501, B*5101, B*5301 and B*5401) and B*0801 were based on the inhibition of binding of a radiolabeled standard peptide, and were performed as previously described (22, 35). Briefly, 1–10 nM of radiolabeled peptide was co-incubated at room temperature with 1 µM–1 nM of purified MHC in the presence of 1–3 µM human beta2-microglobulin (Scripps Laboratories, San Diego, CA, USA) and a cocktail of protease inhibitors. After a 2-day incubation, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC–peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-one, Longwood, FL, USA) coated with the W6/32 antibody, and measuring bound counts per minute (c.p.m.) using the TopCount microscintillation counter (Packard Instrument Co.). Results are expressed as the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled reference peptide. Peptides were typically tested at six different concentrations covering a 100 000-fold dose range, and in three or more independent assays. Under the conditions utilized, where [label] < [MHC] and IC50 ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd values.

In vitro immunization procedures
In experiments shown in Table 3 and Fig. 3, immunizations were performed in 96-well plates. Briefly, 2 x 105 irradiated (6000 rads) human PBMCs were plated in 96 (flat)-well plate in 100 µl of complete human medium (RPMI-1640 medium containing 10% heat-inactivated human AB serum, 2 mM glutamine, 50 µg ml–1 streptomycin and 50 µg ml–1 penicillin) with 100 µg ml–1 of peptide. Twelve wells per peptide were plated per patient. Then, 2 x 105 PBMCs in 100 µl of complete human medium were added into each well. Four days later, 100 µl of medium was replaced with 100 µl of fresh complete human medium containing 80 IU ml–1 of IL-2. At days 6–7, 100 IU ml–1 of IL-2 were added and wells were split into two. On days 10–11, micro-cytotoxicity assay was performed. In experiments shown in Figs 4 and 5, human PBMCs were stimulated in vitro in 24-well plate with autologous, irradiated, peptide-pulsed adherent cells in the presence of IL-2 and IL-7 as previously described (12). On days 4–5 after re-stimulation, effector CTLs were tested in a standard 51Cr-release assay.


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  		Table 3 CTL response in vitro following immunization of normal donors PBMC with HLA-B7-restricted hTRT peptides

 

Figure 3
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  		Fig. 3 Examples of CTL induction in a small-scale in vitro immunization assay using normal donor PBMC. HLA-B7+ human PBMCs were immunized in vitro in a 96-well plate assay, and tested for specific lysis of T2-B7 pulsed with peptide on days 10–11. The micro-CTL assay was performed as described in Methods. All cultures but those with p444 were set with PBMC from the same donor.

 

Figure 4
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  		Fig. 4 Characterization of human CTL generated by in vitro immunization. An example of one of three HLA-B7 + normal donor PBMC and one of two prostate cancer patients. Immunization in vitro was performed using a conventional method (12). (A) Specific lysis of T2-B7 cells pulsed with p1123 by CTL generated in normal donor PBMC. CTLs were tested after five cycles of in vitro re-stimulation with homologous peptide. (B) Surface phenotype analysis using anti-CD3 and anti-CD8 mAbs of the CTL shown in panel (A). The percentage of double-positive cells is indicated. (C) Specific lysis of T2-B7 cells pulsed with p1123 by CTL generated in prostate cancer patient PBMC. CTLs were tested after six cycles of in vitro re-stimulation with homologous peptide. (D) Surface phenotype analysis using anti-CD3 and anti-CD8 mAbs of the CTL from the same patient shown on panel (C) after five cycles of in vitro re-stimulation with homologous peptide. Experiments shown in (A) and (B) are representative of set of similar data from two normal donors examined at different times.

 

Figure 5
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  		Fig. 5 p1123 is endogenously processed in JY lymphoblastoid cells. CTLs from an HLA-B7+ human normal donor PBMC were tested in a 4-h 51Cr-release assay of T2-B7 cells pulsed with p1123 (A), or JY cells (B). Tests were run in duplicates at the indicated E:T ratios. CTLs were used after four cycles of in vitro re-stimulation with homologous peptide. Tests were done in duplicate.

 
In vivo immunization procedures
Peptide immunization HLA-B7 Tg mice (28) were injected subcutaneously at the base of the tail with 100 µg of hTRT peptides along with 120 µg of I-Ab MHC class II helper peptide 128–140 of the hepatitis B virus core protein in incomplete Freunds' adjuvant as described previously (12). Long-term CTL lines were maintained in culture by weekly re-stimulation with irradiated, peptide-pulsed syngeneic spleen cells in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 5 x 10–5 M 2-mercaptoethanol, 50 µg ml–1 streptomycin and 50 µg ml–1 penicillin (complete medium) and supplemented with 40 IU ml–1 of recombinant human IL-2.

DNA immunization
A DNA vector coding for the hTRT expressed under the control of CMV promoter was purified on plasmid Giga Kit columns under endotoxin-free conditions (Qiagen, Hilden, Germany). Anesthetized HLA-B*0702 Tg mice were injected with 50 µl of cardiotoxin into each tibialis anterior muscle 5–6 days prior DNA injection. For vaccination, 50 µl of DNA (1 µg µl–1 in PBS) was injected into each pre-treated muscle at day 0 and day 14. Ten days later, spleen cells of individual mice were separately re-stimulated in vitro with each relevant peptide (10 µg ml–1) for 6 days. Effector CTL cells were tested in a standard 4-h 51Cr-release assay, using RMA-B7 cells (HLA-B*0702 transfected RMA cells) pulsed with test peptide or control peptide (CMV p65-derived R10TV restricted to HLA-B7). Specific percent lysis as indicated below. In vivo immunization procedures were preformed in accordance with approved animal protocols at the University of California, San Diego or the Pasteur Institute, respectively.

CTL assays
Both murine and human CTLs were detected by a 51Cr-release assay performed as previously described (14). Briefly, HLA-B7+ APCs (RMA-B7 or T2-B7 cells) were labeled for 1 h with 100 µ Ci of Na251CrO4 (Perkin Elmer). Washed cells (5 x 103 per well) were mixed in 96-well plates in 100 µl per well with each peptide (at 10 µg ml–1 or lower concentration) and 100 µl of the CTL effector cells (at various E:T ratio) in RPMI medium. In experiments shown in Figs 4 and 5, the CTL assays were performed in excess (10 x 104) of cold K562 cells. Plates were incubated for 4–5 h at 37°C (5% CO2). The supernatants were harvested and counted on a Wallac 1470 Wizard Gamma counter. The percent lysis was calculated as 100 (c.p.m.exp – c.p.m.spont) / (c.p.m.max – c.p.m.spont).

FACS analysis
The phenotypic characteristics of in vitro expanded CTL were determined by FACS analysis. Briefly, on day 6 or 7 after stimulation, cells (0.5 x 106) were incubated with FITC-conjugated mouse anti-human CD8 mAb and PE-conjugated mouse anti-human CD3 mAb (2 µg ml–1) in Hank's Balanced Solution containing 0.1% BSA and 0.05% sodium azide for 30 min at 4°C. For human PBMC typing, cells were incubated with 10 µl of BB7.1 mouse B cell hybridoma supernatant for 20 min at 4°C, followed by 30 min incubation with FITC-conjugated rabbit anti-mouse IgG antibody. For IFN-{gamma} intracellular staining, murine (m) CTL harvested after five rounds of weekly in vitro re-stimulations with peptide were incubated overnight in the presence of Golgi Plug (BD Biosciences) at a 20:1 E:T ratio using the following targets: T1-B7, BC1-B7, T2-B7 cells pulsed with p1123 (positive control) and T2-B7 cells pulsed with p464 (negative control). Cells were washed and then stained with an APC-conjugated rat antibody anti-mouse CD8a (BD PharMingen) and a PerCP-conjugated hamster antibody anti-mouse CD3e (BD PharMingen) for 20 min at room temperature. Cells were fixed with cytoperm/cytofix (BD Biosciences) and incubated at 4°C for 20 min. Lastly, cells were incubated with FITC-conjugated rat antibody anti-mouse IFN-{gamma} (BD PharMingen) for 20 min at room temperature. Samples were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA, USA). One hundred thousand events were collected and analyzed using the CellQuest software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Selection of peptides on predicted algorithms
To limit the number of candidate peptides to a manageable panel, we used two predictive algorithms BIMAS and SYFPEITHI. These were used independently to predict nine amino acid peptides for the HLA-B*0702 allele which accounts for the majority of the members of the HLA-B7 type (27). While BIMAS predicts HLA binding based on overall binding characteristics and the presence of canonic anchor residues, SYFPEITHI predicts peptides whose binding characteristics are extrapolated from naturally occurring MHC ligands as a matrix database. PAProC (Prediction Database for Proteasomal Cleavages), which predicts the proteasomal cleavage of full-length proteins, was used to define cleavage accessibility.

We initially selected ten 9mer peptides with high predicted score in either of the two algorithms or both, and synthesized only seven peptides (Table 1). These peptides were selected based on a consensus prediction by both BIMAS and SYFPEITHI. Among the seven peptides only three had a score >180 using BIMAS, and all but two had a score of 23 using SYFPEITHI. Interestingly, the two peptides that could not be predicted using SYPEITHI scored among the best using BIMAS.


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  		Table 1 Prediction of HLA-B7 binding for hTRT peptides

 
Next, we assessed the actual binding avidity and affinity for HLA-B7 (HLA-B*0702). Two independent assays were used: binding stabilization assay on T2-B7 cells by flow cytometry (12) and a competitive solid-phase radioimmunoassay on immobilized purified HLA-B7 molecule (35). As shown in Table 2, five out of seven peptides (p277, p342, p464, p1107 and p1123) displayed high-avidity binding. The two peptides with weak binding (p444 and p966) were among the top three peptides predicted by BIMAS. There was excellent concordance between the two types of binding assays utilized.


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  		Table 2 RA and affinity of predicted hTRT peptides for HLA-B7

 
In vivo immunization of HLA-B7 Tg mice
In order to assign immunogenicity to each of the peptides and correlate this property with the binding characteristics and the scores of the predictive algorithms, we immunized HLA-B7 Tg mice (28). Ten to eleven days after immunization, mice were sacrificed, the spleen harvested and splenocytes put in culture with LPS/Dextran-activated APC, and re-stimulated 7 days after. Cultures were tested on days 4–5 after re-stimulation in a 4-h 51Cr-release assay. As shown, only five out of seven peptides yielded a meaningful, specific CTL response even after a third cycle of in vitro re-stimulation (Fig. 1). All immunogenic peptides induced a response from the first in vitro re-stimulation and this response increased upon subsequent rounds of antigen re-stimulation. An example of CTL for two of the immunogenic peptides is shown in Fig. 2. As noted, the lysis of peptide-pulsed RMA-B7 target cells increased at each round of in vitro re-stimulation. No lysis occurred on RMA-B7 cells not pulsed with peptide. Thus, the in vivo results together with the actual measure of the avidity of HLA-B7 binding avidity distinguished two groups of 9mer hTRT peptides. One group (p277, p342, p464, p1107 and p1123) displayed both high binding in vitro and good immunogenicity in vivo. The other group (p444 and p966) showed poor binding and poor immunogenicity.


Figure 1
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  		Fig. 1 In vivo CTL responses against p277, p342, p444, p464, p966, p1107 and p1123 in HLA-B7 Tg mice. HLA-B7 Tg mice were vaccinated with 100 µg of individual hTRT peptide together with 120 µg of HBV helper peptide in IFA. Ten days after immunization, spleen lymphocytes were re-stimulated in vitro with peptide and fresh, irradiated syngeneic APC. Re-stimulations were performed on a weekly basis. A standard 4-h 51Cr-release assay was performed on day 5 of the in vitro re-stimulation, using RMA-B7 cells pulsed with the homologous hTRT peptide as targets at an E:T ratio of 25:1. Results are expressed as the mean specific lysis ± SD of responder mice only, whose number is indicated in each panel. Tests were run in duplicate.

 

Figure 2
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  		Fig. 2 Examples of CTL responses induced in vivo by immunization with p277 and p1123. Spleen lymphocytes of HLA-B7 Tg immunized mice were re-stimulated in vitro with the homologous hTRT peptide on a weekly basis. A standard 4-h 51Cr-release assay was performed, using RMA-B7 cells pulsed or not pulsed with peptide as targets, at the indicated E:T ratios. CTL assay was performed after one (a and b), two (c and d) and three (e and f) rounds of in vitro re-stimulation.

 
In vitro immunization of human PBMC from normal donors
To further assess the immunogenicity of the selected peptide candidates as well as their ability to expand precursor CD8 T cell in human PBMC, the following experiment was performed. PBMCs from eight HLA-B7+ normal donors were screened in a small-scale in vitro immunization assay (96-well plate assay) to determine the level of the CTL response against each individual peptide. The cumulative data of this screening step are shown in Table 3. Although, this screening test is mainly qualitative, the response to these peptides varied among the eight donors. Overall, two peptides (p277 and p1123) yielded strong responses in the majority of subjects. Two additional peptides (p342 and p1107) while inducing strong responses in fewer instances also induced low-magnitude CTL responses in other instances. CTL responses against p464 and p444 occurred in few instances only. The response against p966 was not tested because of repeated negative results in HLA-B7 Tg mice. Thus, the results of this in vitro assay narrowed the spectrum of immunogenic peptides beyond those identified in vivo in HLA-B7 Tg mice. A typical result of this type of analysis is shown in Fig. 3, which depicts the induction of CTL and their specificity in each of the 12 wells. As shown, there is considerable variability in the number of positive wells per peptide, and in the percentage lysis that varied from peptide to peptide. In some instances, non-specific lysis, presumably by NK cells, was also encountered (e.g. p464). This variation in the response to each peptide may be related to either an intrinsic characteristic of the peptide (e.g. its avidity) or a variation in the frequency of CD8 T cell precursors for that peptide among donors particularly in view of the format of the assay used.

In vivo processing
Next, we established which among the various candidate peptides was processed and presented from full-length hTRT. To this end, we immunized HLA-B7 Tg mice with hTRT plasmid DNA. Mice were sacrificed on day 24, and splenocytes were re-stimulated in vitro with each of the following peptides: p277, p342, p444, p464, p1107 and p1123. As shown in Table 4, some but not all the peptides were processed and presented in vivo. Three peptides (p277, p1107 and p1123) yielded greater CTL responses than the other peptides, implying either preferential processing and/or better immunogenicity once displayed at the surface of the APC. The remaining three peptides (p342, p444 and p464) were marginally immunogenic if any. Based on this analysis, it appears that only three of the original seven peptides were processed and presented efficiently in vivo. Interestingly, we found that among the three most immunogenic peptides only one (p277) was predicted by PAProC, whereas the other two (p1107 and p1123) were not (Table 1). Thus, selection using PAProC algorithm was per se unable to predict hTRT peptides that would be cleaved and become immunogenic in vivo.


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  		Table 4 In vivo processing and immunogenicity of hTRT peptides in HLA-B7 Tg mice

 
Supertype analysis
HLA molecules are highly polymorphic posing problems to the identification of peptides which could be used to cover the totality of the human population. However, HLA alleles can be clustered into a relatively small number of groups termed supertypes (21). The HLA-B7 supertype includes several serologically distinct alleles sharing a specificity for proline (P) in position 2 and hydrophobic residues at the C-terminus of their peptide ligands. HLA-B7 supertype alleles are predicted to be present in excess of 40% of individuals in the general population, regardless of ethnicity (22). Herein, we decided to test the selected hTRT peptides for their ability to bind the five most common HLA-B7 supertype alleles: B*0702, B*3501, B*5101, B*5301 and B*5401. In addition, the B*0801 allele shares binding features with B*0702, although is not formally part of the HLA-B7 supertype, while is fairly cross-reactive with HLA-B*0702, it reacts only marginally with the other HLA-B7 supertype alleles. This analysis had the purpose to further narrow the selection of putative HLA-B7 immunogens based on supertype binding (Table 5). Only one peptide (p1123) had measurable affinity for all alleles examined. Another peptide (p1107) bound with high affinity three out of five alleles. Two additional peptides (p277 and p342) bound four alleles with intermediate avidity. The remaining peptides (p444, p464 and p966) displayed little HLA-B7 supertype binding. Thus, it appears as if the peptides retained through the in vitro and in vivo screening processes described above for immunogenicity and processing in vivo, ranked best as HLA-B7 supertype binders. This demonstrates that the supertype analysis is a pivotal step in refining the selection process.


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  		Table 5 HLA-B7 supertype binding assay

 
Characterization of human CTL against p1123
To better characterize the response against the peptide with the best characteristics for immunogenicity overall (p1123), new in vitro immunization experiments were performed, using PBMC from three HLA-B7+ normal blood donors and two cancer patients. These new experiments were performed using a conventional in vitro immunization assay (12). After repeated rounds of in vitro re-stimulation, high efficiency CTLs were induced that specifically killed T2-B7 target cells pulsed with p1123 (Fig. 4A). The phenotype of this CTL line was CD8+CD3+ (80%), CD8–CD3+ (10%) and CD56 (8%) (Fig. 4B). Thus, p1123 expanded CD8 T cell precursors which developed into CTL. A similar approach was used with PBMC from two prostate cancer patients. Again, after repeated re-stimulations, we were able to expand CTL that lysed T2-B7 target cells pulsed with p1123 (Fig. 4C). Compared with the CTL responses observed in the three normal blood donors, the CTL response in the cancer patient studied was weaker implying either a different precursors frequency or TCR avidity. Given the low frequency of HLA-B7+ in the population (~1:10) and hence difficulty to find patients suitable to study, it is premature to conclude that in cancer patients CD8 T cell responses against p1123 are of a lower magnitude or strength than in normal individuals. The phenotype of this CTL line was CD8+CD3+ (90%), CD8–CD3+ (8%) and CD56 (3%). The activity was seemingly mediated by CD3/CD8+ lymphocytes double-positive T cells (90%) as indicated by FACS analysis (Fig. 4D). Lysis by NK cells is unlikely since non-pulsed targets were not lysed and in view of the fact that each assay was routinely performed in excess of K562 cells, a target for NK cells. Collectively, these data confirm that CD8 T cell precursors for p1123 exist in the normal CD8 T cell repertoire, and apparently persist after cancer development.

Finally, it was important to demonstrate that CTLs against p1123 were able to lyse transporter antigen protein-competent/hTRT-positive target cells. To this end, we used the JY (a HLA-A2+/B7+ EBV-transformed B lymphoblastoid human cell line), which is highly positive for hTRT. For instance, we have documented by FACS and deconvolution microscopy that JY cells express large number of HLA-A2–p540 complexes, indicating that in these cells hTRT processing and presentation occurs efficiently (our unpublished data). CTL from normal donors that efficiently killed T2-B7 target cells pulsed with p1123 also killed JY cells in the absence of any peptide pulsing (Fig. 5), suggesting that p1123 is naturally processed from endogenous hTRT, and that HLA-B7–p1123 complexes are presented at the cell surface in a way that is recognized by CTL induced by peptide immunization.

An mCTL line recognizes human cells
To further characterize the endogenous processing and presentation of p1123 in human cells, we used an mCTL line specific for p1123 with high lytic activity for human target cells (T2-B7) pulsed with peptide (p1123) (Fig. 6A). Two HLA-B7+ human lymphoblastoid cells were used, T1-B7 and BC1-B7. Although TAP-deficient T2-B7 cells pulsed with p1123 are highly susceptible to lysis by mCTL, non-pulsed TAP-competent hTRT+ HLA-B7+ EBV-transformed B lymphoblastoid human cell lines, T1-B7 and BC1-B7, were not (data not shown). This suggests that the low-affinity interaction between the murine CD8 co-receptor molecule and the human MHC may be compensated by the abundance of MHC–peptide complexes on T2-B7 cells pulsed with peptide.


Figure 6
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  		Fig. 6 mCTL specific for p1123 specifically recognizes hTRT+ human target cells (T1-B7 and BC1-B7). An mCTL line was expanded from p1123-immunized HLA-B7 Tg mice and re-stimulated five times in vitro. (A) Four-hour 51Cr-release assay was performed with mCTL using human T2-B7 as target cells, with or without p1123 pulsing. (B) Intracellular IFN-{gamma} staining of mCTL upon overnight incubation with T1-B7, BC1-B7 lymphoblastoid cells and T2-B7 pulsed with p1123 (positive control) and p464 (negative control). Tests were repeated twice with similar results.

 
As an alternative approach, we tested intracellular synthesis IFN-{gamma} in an mCTL line specific for p1123 in the presence of T1-B7 and BC1-B7 cells reasoning that specific recognition of p1123 would engender IFN-{gamma} synthesis. This was assessed by intracellular staining. As shown in Fig. 6(B), overnight contact with T1-B7 and BC1-B7 lymphoblastoid cells produced an increase in CD8/IFN-{gamma} double-positive cells. This was only slightly at variance with the percentage CD8/IFN-{gamma} double-positive CTL incubated with control T2-B7 cells pulsed with p1123 (positive control). This confirms, therefore, endogenous processing and presentation of hTRT p1123 in human cells.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Defining the immunogenic components of hTRT for each HLA type is a formidable task but a necessary step to develop immunotherapies to target hTRT on tumor cells in the widest assortment of the human population. Previously, this (12, 14) and other (11) laboratories identified immunogenic peptides for the most frequent HLA type, HLA-A2. The outcome of these studies was that humans possess a residual CD8 T cell repertoire for both high- and low-affinity hTRT peptides that can be expanded by immunization in vitro (12, 14, 16). hTRT-specific CD8 T cell precursors have been reported to persist in patients with advanced cancer (12, 14, 15). Here, we expanded our systematic effort to the identification and characterization of immunogenic hTRT peptides restricted to HLA-B7. The results of the present study lead to a series of general considerations.

The conventional algorithms used here proved to be overall useful predictors of immunogenic hTRT peptides for the HLA-B7 type. Previously, we successfully used BIMAS as a way to predict and select HLA-A2-restricted hTRT peptides that fulfill desired criteria for immunogenicity similar to those studied here. In contrast, BIMAS that was effective in predicting binding in some instances could not predict HLA-B7 immunogenic peptides overall. For instance, p444, the top peptide according to BIMAS, was not immunogenic in vivo in HLA-B7 Tg mice, was poorly immunogenic in vitro for human PBMC and was apparently not processed in HLA-B7 Tg mice immunized with full-length hTRT pDNA. Not surprisingly, p444 actual binding avidity for the HLA-B7 molecule was also poor, hence pointing to a discrepancy between predicted affinity, actual avidity and immunogenic function. SYFPEITHI did not predict two peptides (p966 and p464) that were poorly immunogenic, but at the same time did not distinguish between immunogenic and non-immunogenic peptides among the remaining five peptides studied. Finally, predictions based on proteasome cleavage were found not to be useful. For instance, the two peptides with the highest predicted probability for processing and immunogenicity turned out to be non-immunogenic in one case (p966) and poorly immunogenic in the other case (p342). This algorithm did, however, predict p277. Collectively, none of the three algorithms used to guide the initial selection of peptides was per se able to discriminate peptides that fulfill prerequisites for immunogenicity.

In vitro immunization studies support the conclusion that there exists a residual CD8 T cell repertoire for the majority (five out of seven) peptide specificities investigated. Since these peptides also possess good binding avidity for the HLA-B7 molecule, the present findings implies that thymic negative selection (central tolerance) of hTRT CD8 T cell clonotypes restricted to HLA-B7 did not occur or occurred to a limited extent only. This conclusion is in agreement with studies of similar nature regarding HLA-A2-restricted hTRT peptides (10). One can speculate on the reasons why high-affinity (0.9–11 nM) peptides such as p1123 and p277 did not cause negative selection. One simple explanation would be that deletion occurred only for T lymphocytes with high-affinity TCR leaving intact a residual repertoire of low-affinity T cells, a view in agreement with studies in p53 Tg mice (36). A more speculative possibility would be that although telomerase is expressed and seemingly active in fetal thymus (37), translocation of the reverse transcriptase subunit from the nucleus to the cytoplasm and its degradation by the proteasome may be limited or inhibited in the early ontogeny, hence minimizing the toll of negative selection. This view considers that it could be in the developing organisms' advantage not to eliminate hTRT-reactive T cell clonotypes which may be needed during adult life for tumor-immune surveillance (38). Future experiments will need to address this issue.

The response of HLA-B7 Tg mice to in vivo immunization with peptide in immunological adjuvant was immediate and stronger than that of HLA-A2 Tg mice similarly immunized (12, 14, 39). It appears as if, at least with respect of hTRT, HLA-B7–peptide complexes are highly immunogenic. Similarly, high immunogenicity was documented in studies where HLA-B7 Tg mice were immunized with influenza virus peptides (28). A high immunogenicity of HLA-B7–peptide complexes finds partial support in reports showing that the HLA-B7 type is dominant in terms of peptide selection and presentation (40). Whether or not there is a structural substrate to the high immunogenicity observed in vivo remains to be established. Interestingly, based on our previous experience with HLA-A*0201 Tg mice, HLA-B7 Tg CTL recognize human target cells pulsed with peptide in a 51Cr-release assay. Here we show that TAP-competent HLA-B7/hTRT-positive human cell lines activate mCTL and promote intracellular IFN-{gamma} synthesis, but not lysis. One explanation for this discrepancy could be that while IFN-{gamma} synthesis may only need a short contact of the TCR with the MHC–peptide complex, cytotoxicity may require a more stable interaction enabled by the CD8 co-receptor molecule. In all likelihood, the murine CD8 interacts only weakly with the complex formed with a Tg HLA-B7 molecule.

The supertype binding studies proved to be an excellent final checkpoint in the selection of immmunogenic peptides. For instance, p1123 and to a lesser degree p277 and p1107 bound to various alleles of the HLA-B7 supertype. Taken together, the results of our study indicate that candidate immunogenic peptides need to satisfy at least two general criteria, good avidity interaction with the HLA-B7 molecule and good supertype binding. As to the first characteristic, work from others (41–43) and our own laboratory (14, 39) showed that in the case of HLA-A2 peptides there exists a direct correlation between avidity for the MHC molecule and immunogenicity. Moreover, unlike antigenicity, immunogenicity is based on sufficient avidity for the MHC molecule and the presence of canonical anchor residues (39). If a comparison with previous work on HLA-A2-restricted peptides is warranted, one may also need to consider the quality of the interaction between the MHC–peptide complex with the TCR as an additional factor in immunogenicity. As to the second characteristic, our data are consistent with the suggestion that supertype binding peptides are preferentially processed and possess a selective advantage for interaction with molecules of the TAP complex (14, 44, 45).

In conclusion, we presented the successful identification of several immunogenic hTRT peptides restricted to HLA-B7. We show that this identification required a multi-step approach and involved an ensemble of in vitro and in vivo steps using both mice and human PBMCs. This implies that the selection of immunogenic peptides for potential clinical use rests on a series of checkpoints and an element of empiricism overall. To date, such systematic approach has enabled the identification of HLA-A2 (10), and now HLA-B7 peptides with characteristics of immunogenicity that could justify their use in immunotherapy of cancer patients. Together, HLA-A2 and HLA-B7 account for ~60% of the Caucasian population. If one takes into account supertype binding of some of the peptides identified in this study, one may achieve >70% coverage irrespective of ethnicity. Thus, for a complete coverage of the human population, immunogenic peptides for the alleles accounting for the remaining 30–40% of the population still need to be identified systematically using a strategy similar to the one followed herein. However, the challenges may be greater than what encountered to date due to the more limited availability of immunological tools for these HLA types.


   Acknowledgements
 
We are grateful to A. Franco for guidance in the 96-well CTL assay. M.Z. acknowledges the support of NIH grant RO1 CA84062. X.C.-G. is grateful to the University of California, San Diego, Alliance for Graduate Education and the Professoriate (NSF-9978892), and the Training Program in Basic Genetics (NIH/NIGMS 5 T32 GM08666-07).


   Abbreviations
 
APC, antigen-presenting cell
c.p.m., counts per minute
hTRT, human telomerase reverse transcriptase
m, murine
NIH, National Institute of Health
Tg, transgenic

   Notes
 
Transmitting editor: S. Hedric

Received 22 December 2005, accepted 14 September 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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