Efficient induction of antitumor cytoxic T lymphocytes from a healthy donor using HLA-A2-restricted MAGE-3 peptide in vitro

 The antigenic peptides encoded by tumor-rejection antigen genes, MAGE-1 and -3, have been identified, and various methods have been utilized for the in vitro induction of MAGE-specific, cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using synthetic peptides. However, all of these methods are technically demanding and thus have a relatively limited usefulness. We herein report a simple and efficient method for the in vitro induction of specific CTL by using the HLA-A2-restricted MAGE-3 peptide from the PBMC of a healthy donor. CTL responses could thus be efficiently induced from unseparated PBMC by stimulation with freshly isolated, peptide-pulsed PBMC as antigen-presenting cells and by using interleukin-7 and keyhole limpet hemocyanin for the primary culture. The induced CTL could thus recognize and lyse not only HLA-A2 target cells pulsed with the peptide but also HLA-A2 tumor cells expressing MAGE-3, in an HLA-class-I-restricted manner. This simple method may, therefore, become a useful tool for investigating the potential peptides for tumor antigens as well as for developing various immunotherapeutic approaches for human malignant tumors. Received: 15 October 1996 / Accepted: 6 December 1996     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.     

Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because they are tumor specific and shared by tumors of different histological types. Several clinical trials are in progress with MAGE peptides, proteins, recombinant poxviruses, and dendritic cells (DC) pulsed with peptides or proteins. The use of gene-modified DC would offer the major advantage of a long-lasting expression of the transgene and a large array of antigenic peptides that fit into the different HLA molecules of the patient. In this study, we tested the ability of gene-modified DC to prime rare Ag-specific T cells, and we identified a new antigenic peptide of clinical interest. CD8(+) T lymphocytes from an individual without cancer were stimulated with monocyte-derived DC, which were infected with a second-generation lentiviral vector encoding MAGE-3. A CTL clone was isolated that recognized peptide EGDCAPEEK presented by HLA-Cw7 molecules, which are expressed by >40% of Caucasians. Interestingly, this new tumor-specific antigenic peptide corresponds to position 212-220 of MAGE-2, -3, -6, and -12. HLA-Cw7 tumor cell lines expressing one of these MAGE genes were lysed by the CTL, indicating that the peptide is efficiently processed in tumor cells and can therefore be used as target for antitumoral vaccination. The risk of tumor escape due to appearance of Ag-loss variants should be reduced by the fact that the peptide is encoded by several MAGE genes.     

Generation of specific anti-melanoma reactivity by stimulation of human tumor-infiltrating lymphocytes with MAGE-1 synthetic peptide

The MAGE-1 gene encodes a tumor-specific antigen, MZ2-E, which is recognized by cloned, specific cytolytic T cells (CTL) derived from the peripheral blood of a patient with melanoma. We have produced a MAGE-1-specific CTL line derived from the tumor-infiltrating lymphocytes (TIL) of a melanoma patient by weekly restimulation with autologous EBV-B cells pulsed with the synthetic HLA-A1-restricted MAGE-1 epitope nonapeptide EADPTGHSY. The 1277. A TIL line grew in long-term culture in low-dose interleukin-2 (IL-2) and IL-4, and exhibited antigen-specific, MHC-class-I-restricted lysis of HLA-A1-bearing MAGE-1⁺ cell lines. Cytolysis of target cells pulsed with the synthetic MAGE-1 decapeptide KEADPTGHSY was superior to that of cells pulsed with the immunodominant nonapeptide. Single amino-acid or even side-chain substitutions in the immunodominant nonamer abrogated cytolysis. 1277. A TIL specifically secreted tumor necrosis factor after co-incubation with HLA-A1-expressing MAGE-1⁺ cell lines or fresh tumor. These data suggest that tumor-antigen-specific, MHC-restricted CTL may be grown from TIL in the presence of synthetic epitope peptides and expanded for adoptive immunotherapy in melanoma patients.     

Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8(+), and CD4(+) T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8(+) T cell response to HLA-A2-restricted peptide MAGE-3 271-279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4(+) T cell response to HLA-DP4-restricted peptide 243-258. One patient simultaneously developed CD8(+) T cells to HLA-A1-restricted peptide 168-176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4(+) T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

Polyclonal CTL responses observed in melanoma patients vaccinated with dendritic cells pulsed with a MAGE-3.A1 peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3(168-176) peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 x 10(-6) to 9 x 10(-4) of CD8(+) blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.     

Generation of T cells specific for the wild-type sequence p53(264-272) peptide in cancer patients: implications for immunoselection of epitope loss variants

Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53(264-272) epitope to generate CTL from circulating precursor T cells of HLA-A2.1(+) healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53(264-272) peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53(264-272) peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53(264-272) epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53(264-272) epitope. These findings suggest that in vivo, CTL specific for the wt p53(264-272) peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.     

Enhanced induction of human WT1-specific cytotoxic T lymphocytes with a 9-mer WT1 peptide modified at HLA-A*2402-binding residues

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

CD8+ T cell-mediated HLA-A*0201-restricted cytotoxicity to transaldolase peptide 168-176 in patients with multiple sclerosis

Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168-176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2- MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-gamma-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vbeta repertoire analysis revealed use of the TCR Vbeta14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vbeta14 on the cell surface. Moreover, Ab to TCR Vbeta14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.     

A MAGE-3 peptide presented by HLA-DR1 to CD4+ T cells that were isolated from a melanoma patient vaccinated with a MAGE-3 protein

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4(+) T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize an HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3(267-282) and the MAGE-12(267-282) protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4(+) clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.     

Cross-reactive cytotoxic T lymphocytes against human immunodeficiency virus type 1 protease and gamma interferon-inducible protein 30

The gamma interferon (IFN-gamma)-inducible protein 30 (IP-30) signal peptide -11 to -3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-gamma, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-gamma. Neither high levels of exogenous IFN-gamma nor incubation of PBMC with other HIV peptides triggering substantial IFN-gamma release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-gamma release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-gamma release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide -11 to -3, which has implications for immune memory and autoimmunity.     

A tumor-infiltrating lymphocyte from a melanoma metastasis with decreased expression of melanoma differentiation antigens recognizes MAGE-12

Twenty separate tumor infiltrating lymphocyte (TIL) bulk cultures and a tumor cell line were originated simultaneously from a fine needle aspiration biopsy of a metastasis in a patient with melanoma (F001) previously immunized with the HLA-A*0201-associated gp100:209-217(210 M) peptide. None of the TIL recognized gp100. However, 12 recognized autologous (F001-MEL) and allogeneic melanoma cells expressing the HLA haplotype A*0201, B*0702, Cw*0702. Further characterization of F001-MEL demonstrated loss of gp100/PMel17, severely decreased expression of other melanoma differentiation Ags and retained expression of tumor-specific Ags. Transfection of HLA class I alleles into B*0702/Cw*0702-negative melanoma cell lines identified HLA-Cw*0702 as the restriction element for F001-TIL. A cDNA library from F001-MEL was used to transfect IFN-alpha-stimulated 293 human embryonal kidney (293-HEK) cells expressing HLA-Cw*0702. A 100-gene pool was identified that induced recognition of 293-HEK cells by F001-TIL. Subsequent cloning of the pool identified a cDNA sequence homologous, except for one amino acid (aa 187 D-->A), to MAGE-12. Among 25 peptide sequences from MAGE-12 with the HLA-Cw*0702 binding motif, MAGE-12:170-178 (VRIGHLYIL) induced IFN-gamma release by F001-TIL when pulsed on F001-EBV-B cells at concentrations as low as 10 pg/ml. Peptide sequences from MAGE-1, 2, 3, 4a, and 6 aligned to MAGE-12:170-178 were not recognized by F001-TIL. In summary a TIL recognizing a MAGE protein was developed from an HLA-A*0201 expressing tumor with strongly reduced expression of melanoma differentiation Ags. Persisting tumor-specific Ag expression maintained tumor immune competence suggesting that tumor-specific Ags/melanoma differentiation Ags may complement each other in the context of melanoma Ag-specific vaccination.     

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.     

Identification of natural antigenic peptides of a human gastric signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.     

Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer

Prostate-specific antigen (PSA) is a potentially useful antigen for targeted T-cell immunotherapy of prostate cancer (CaP). Our laboratory has identified a synthetic nonamer peptide (PSA 146-154) homologue of PSA, which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T-lymphocyte (CTL) responses from normal individuals of the HLA-A2 phenotype. In the present study, we report on the induction of CTL from peripheral blood mononuclear cells (PBMC) of patients with hormone-refractory CaP, which exhibit the same specificity. T-cell lines were established from two patients by stimulation of PBMC with PSA 146-154 peptide in vitro. The T-cell lines exhibited specific cytolytic activity against T2 cells pulsed with PSA 146-154 peptide, but not a control HLA-A2 binding peptide (HIV-RT 476-484) via chromium release assay (CRA). The T-cell lines also showed PSA 146-154 peptide-specific IL-4 responses, but no detectable interferon-gamma (IFN-gamma) responses via enzyme-linked immuno-spot assays. Magnetic immuno-selection studies of one of the T-cell lines demonstrated that both cytolytic and interleukin-4 (IL-4) responses were mediated by CD8(+), but not by CD4(+) T cells. This Tc2 line was further characterized for the ability to recognize endogenously processed PSA epitopes. The line specifically secreted IL-4 in response to HLA-A2(+) target cells transfected to express PSA and specifically lysed the PSA(+) target cells, but not control transfected cells. The results indicate that the PSA 146-154 peptide emulates a naturally processed and presented peptide epitope of PSA that is within the T-cell repertoire of HLA-A2(+)patients with CaP.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.