Generation of specific antitumor reactivity by the stimulation of spleen cells from gastric cancer patients with MAGE-3 synthetic peptide

The induction of cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using MAGE peptide has been investigated in order to use MAGE antigens immunotherapeutically. We therefore developed a simplified method for inducing peptide-specific CTL that kill tumor cells expressing MAGE from the PBMC of either healthy donors or even cancer patients. Since the spleen is a major lymphoid organ, we used a simple method to examine the capacity of spleen cells to generate MAGE-specific CTL by in vitro stimulation with MAGE peptide in gastric cancer patients. The CTL responses could thus be induced from unseparated spleen cells in HLA-A2 patients with gastric carcinoma expressing MAGE-3 by stimulating these cells with autologous spleen cells pulsed with HLA-A2-restricted MAGE-3 peptide as antigen-presenting cells and by using keyhole limpet hemocyanin and interleukin-7 for the primary culture. The induced CTL were thus able to lyse HLA-A2-positive carcinoma cells transfected with MAGE-3 and expressing MAGE-3, as well as the target cells pulsed with the peptide, in an HLA-class-I or -A2-restricted manner. Since MAGE-specific CTL could be induced from the spleen cells of gastric cancer patients, the spleen appears to play an important role in either clinical tumor vaccination or the treatment of cancer patients by adoptive immunotherapeutic approaches using the MAGE peptide. Received: 3 December 1998 / Accepted: 30 March 1999     

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     

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.     

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.     

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     

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.     

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8⁺ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201⁺ KLK4 ⁺ cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

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.     

CD8+ T cells specific for the androgen receptor are common in patients with prostate cancer and are able to lyse prostate tumor cells

The androgen receptor (AR) is a hormone receptor that plays a critical role in prostate cancer, and depletion of its ligand has long been the cornerstone of treatment for metastatic disease. Here, we evaluate the AR ligand-binding domain (LBD) as an immunological target, seeking to identify HLA-A2-restricted epitopes recognized by T cells in prostate cancer patients. Ten AR LBD-derived, HLA-A2-binding peptides were identified and ranked with respect to HLA-A2 affinity and were used to culture peptide-specific T cells from HLA-A2+ prostate cancer patients. These T-cell cultures identified peptide-specific T cells specific for all ten peptides in at least one patient, and T cells specific for peptides AR805 and AR811 were detected in over half of patients. Peptide-specific CD8+ T-cell clones were then isolated and characterized for prostate cancer cytotoxicity and cytokine expression, identifying that AR805 and AR811 CD8+ T-cell clones could lyse prostate cancer cells in an HLA-A2-restricted fashion, but only AR811 CTL had polyfunctional cytokine expression. Epitopes were confirmed using immunization studies in HLA-A2 transgenic mice, in which the AR LBD is an autologous antigen with an identical protein sequence, which showed that mice immunized with AR811 developed peptide-specific CTL that lyse HLA-A2+ prostate cancer cells. These data show that AR805 and AR811 are HLA-A2-restricted epitopes for which CTL can be commonly detected in prostate cancer patients. Moreover, CTL responses specific for AR811 can be elicited by direct immunization of A2/DR1 mice. These findings suggest that it may be possible to elicit an anti-prostate tumor immune response by augmenting CTL populations using AR LBD-based vaccines.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs.

We have studied Ags recognized by HLA class I-restricted CTLs established from tumor site to better understand the molecular basis of tumor immunology. HLA-A24-restricted and tumor-specific CTLs established from T cells infiltrating into lung adenocarcinoma recognized the two antigenic peptides encoded by a cyclophilin B gene, a family of genes for cyclophilins involved in T cell activation. These two cyclophilin B peptides at positions 84-92 and 91-99 induced HLA-A24-restricted CTL activity against tumor cells in PBMCs of leukemia patients, but not in epithelial cancer patients or in healthy donors. In contrast, the modified peptides at position 2 from phenylalanine to tyrosine, which had more than 10 times higher binding affinities to HLA-A24 molecules, could induce HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, or healthy donors. PHA-activated normal T cells were resistant to lysis by the CTL line or by these peptide-induced CTLs. These results indicate that a cyclophilin B gene encodes antigenic epitopes recognized by CTLs at the tumor site, although T cells in peripheral blood (except for those from leukemia patients) are immunologically tolerant to the cyclophilin B. These peptides might be applicable for use in specific immunotherapy of leukemia patients or that of epithelial cancer patients.     

Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A.

Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell-mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross-reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy.     

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.     

HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions

 The DNA from human papillomavirus (HPV) can be detected in 90% of cervical carcinomas. To address whether patients infected with HPV can mount efficient T cell responses to this pathogen we examined the cytotoxic T lymphocyte (CTL) response of peripheral blood mononuclear cells (PBMC) from patients with abnormal genital epithelial cells. PBMC from 11 HLA-A2⁺ patients were stimulated with CaSki, a cervical carcinoma cell line that is HPV 16⁺ and HLA-A2⁺. The CTL were screened for reactivity to the cervical carcinoma cell line C33A (HPV^–, HLA-A2⁺) transfected with the HPV 16 E6 or E7 genes or the plasmid without insert. The CTL of 1 patient showed particularly strong CaSki and HPV E6 or E7 protein-specific cytotoxicity in a HLA-A2-restricted fashion. In contrast, these CTL lysed neither a vector-only transfectant, the natural killer cell (NK) target, K562 nor the lymphokine-activated killer cell (LAK) target, Daudi. HLA-A2 restriction was demonstrated by the lack of recognition of a HLA-A2^– CaSki cell line developed in our laboratory. The CTL line was cloned and 99 clones were harvested and screened; 51 clones lysed CaSki, of which 17 did not lyse the A2^– CaSki. Of these HLA-A2^– restricted clones, 8 did not lyse C33A transfectants, 6 lysed all C33A transfectants, 3 lysed C33A-E7 only and none lysed C33A-E6 only. These data imply that, within the bulk CTL line, HLA-A2-restricted recognition of antigens was restricted to CaSki antigens, antigens common to cervical carcinoma (CaSki plus C33A), or HPV-16-E7-derived antigen on the clonal level. The E7-restricted clones were negative for recognition of known HLA-A2-binding peptides from E7. Received: 16 November 1995 / Accepted: 15 January 1996     

HLA-A0201 positive pancreatic cell lines: new findings and discrepancies

Pancreatic cancer is being pursued as an immunotherapy target using antigen-specific vaccine approaches activating CD8(+) CTL and CD4(+) T-helper cells. CD8(+) CTL exert their anti-tumor effects in an HLA-restricted manner and only tumor cells carrying a matched HLA class I sub-type are targets for antigen-specific CTL. In the process of characterizing CD8(+) T cell responses against pancreatic cancer, we screened a number of human pancreatic tumor cell lines for HLA-A0201 positive (HLA-A2(+)) cell lines to be used in the evaluation of CTL function. This analysis revealed some new findings and discrepancies in the literature on the HLA sub-type of some commonly used pancreatic cell lines. We found that Capan-1 cells, originally reported to be HLA-A0201(+), actually only express HLA-A010101 and HLA-A300101 and were targets for HLA-A0201-restricted CTL only after transduction with an HLA-A0201-expressing lentivirus. Panc-1 cells were found to be HLA-A0201 positive, in agreement with published reports, while CF-Pac-1 cells were found to express both HLA-A020101 and HLA-A030101. We also found a normal human pancreatic ductal epithelial cell line, HPDE, to be HLA-A0201 positive. Our findings were verified with two different sequence-based typing methods, antibody staining followed by flow cytometry analysis, and functional analysis using an HLA-A0201-restricted peptide-specific T cell response.     

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.     

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

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.