Isolation of broadly reactive, tumor-specific, HLA Class-I restricted CTL from blood lymphocytes of a breast cancer patient

Blood lymphocytes of a HLA-A2 positive breast cancer patient were stimulated with either MCF-7 or MDA-MB-231, i.e., HLA-A2-matched allogeneic breast carcinoma cell lines. Several CD8⁺ CTL clones with reactivity against the stimulator cells but not against K562 were generated. Reactivity could be blocked with monoclonal antibody (mAb) W6/32, MA2.1, and/or BB7.2, indicating that the clones are HLA-class I and HLA-A2 restricted. The CTL clones generated following stimulation with MCF-7, recognized various other allogeneic HLA-A2⁺ tumor cell lines, including breast carcinoma, renal cell carcinoma, and melanoma cell lines, but not HLA-A2⁻ tumor cell lines. The CTL clones did not recognize normal HLA-A2⁺ cells including breast epithelial cells, renal proximal tubular epithelial cells (PTEC), or EBV-transformed B cells including the autologous EBV cell line. In contrast to the CTL clones induced with MCF-7, the reactivity of the clones stimulated with MDA-MB-231, was limited to the stimulator cell MDA-MB-231. Cytotoxicity assays utilizing T2 cells loaded with peptides as target cells indicated that none of the examined CTL-epitopes derived from HER-2/neu, Muc-1, Ep-CAM-1, and p53 were recognized by the CTL clones generated. Our findings underscore that breast cancer is an immunogenic tumor and that HLA-class I-matched allogeneic tumor cells can be used as stimulator cells to generate tumor-specific CTL from peripheral blood of a breast cancer patient with specificity for an antigenic determinant that is broadly expressed on tumor cells from various origins or with specificity limited to the breast cancer stimulator cell.     

An HLA-modified ovarian cancer cell line induced CTL responses specific to an epitope derived from claudin-1 presented by HLA-A*24:02 molecules

In an attempt to induce cytotoxic T lymphocytes (CTLs) that react to ovarian cancer cells, we isolated a CTL clone that specifically recognizes claudin-1 in an HLA-A*24:02-restricted manner. Naïve CD8⁺ T lymphocytes were obtained from a healthy adult donor and stimulated twice in vitro with HLA-modified TOV21G cells that were originally derived from an ovarian clear-cell carcinoma line. The TOV21G modification involved RNAi-mediated gene silencing of intrinsic HLA molecules and lentiviral transduction of a synonymously mutated HLA-A*24:02. Then, cDNA library construction using mRNA extracted from the parental TOV21G cells and subsequent expression cloning were conducted. These experiments revealed that a CTL clone obtained from the bulk culture recognized a minimal epitope peptide RYEFGQALF, which was derived from an autoantigen claudin-1 presented by HLA-A*24:02 molecules. This clone exhibited cytolytic activities against three ovarian cancer cell lines and normal bronchial epithelial cells in an HLA-A*24:02-restricted manner. Our data indicate that HLA-modified cancer cells can be used as an artificial antigen-presenting cell to generate antigen-specific CTLs in a manner restricted by an HLA allele of interest.     

Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors

Although specific immunotherapy is one candidate treatment of brain tumor, the molecular basis of T-cell-mediated recognition of brain tumors has not yet been elucidated. In this study, we tried to identify brain tumor antigens using HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs). As an HLA-A2-restricted OK-CTL line contained CTLs capable of responding to HLA-A2+ malignant glioma cells, this cell line was used for identification of brain tumor antigens. After screening a cDNA library from brain tumor cells, this CTL line was found to produce interferon (IFN)-gamma when cultured with COS-7 cells, which were cotransfected with both a cDNA clone (clone 1) and HLA-A0207 cDNA. Data base searches indicated that the clone 1 was 98% identical to that of the human ADP-ribosylation factor 4-like (ARF4L). Two peptides, ARF4L 15-24 and ARF4L 69-77, possessed the ability to induce HLA-A2-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells of patients with brain tumors. Although ARF4L seemed to be ubiquitously expressed at the mRNA level, ARF4L-reactive CTLs failed to exhibit cytotoxicity against normal lymphoid blasts. These results indicate that these two ARF4L peptides could be targets for immunotherapy of HLA-A2+ patients with brain tumors.     

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

HLA Class-I-Restricted and Colon-Specific Cytotoxic T Cells from Lamina Propria Lymphocytes of Patients with Ulcerative Colitis

We established cytotoxic T-lymphocyte (CTL) lines against colonic epithelial cell line from LPLs of patients with ulcerative colitis by continuous stimulation with human lymphocyte antigen A (HLA-A) matched colonic epithelial cell lines and clones from LPLs using polyclonal stimulation with phytohemagglutinin. The established CTL lines and clones showed cytotoxicity against HLA-A-matched colonic epithelial cell line but not against HLA-mismatched colonic epithelial cell lines, and HLA-A-matched lung and esophagus cell lines. The CTL response was HLA class I-restricted and mediated by CD8-positive T-lymphocytes. Moreover, the CTL line showed cytotoxicity against autologous B-LCLs pulsed with peptides extracted from HLA-A-matched colonic epithelial cell line but not against other organ-derived peptides pulsed and unpulsed autologous B-LCLs. CTL lines and clones established from LPLs of patients with ulcerative colitis showed colon-specific and HLA class I-restricted killing of human colonic epithelial cell line, suggesting that these CTLs may play a role in colonic epithelial cell damage in some patients with ulcerative colitis.     

A novel human HER2-derived peptide homologous to the mouse K(d)-restricted tumor rejection antigen can induce HLA-A24-restricted cytotoxic T lymphocytes in ovarian cancer patients and healthy individuals

A mouse HER2-derived peptide, HER2p63 (A) (TYLPANASL), can induce K(d)-restricted mouse cytotoxic T lymphocytes (CTL) and also function as a tumor rejection antigen in an in vivo assay. Since the anchor motif of mouse K(d) for peptide binding has much similarity to that of human HLA-A2402, we asked if human HER2p63 (T) (TYLPTNASL) could induce HER2-specific CTL in HLA-A2402-positive individuals. Peripheral blood mononuclear cells (PBMC) of HLA-A2402-positive individuals were sensitized in vitro with HER2p63-pulsed autologous dendritic cells prepared from PBMC. CTL clone derived from these specifically lysed HER2-expressing cell lines bearing HLA-A2402. Cytotoxic activity of the CTL clone against the HER2-expressing cell line bearing HLA-A2402 was blocked by antibodies against CD3, CD8, HLA-A24 or MHC class I, and was also inhibited by the addition of excess HER2p63-pulsed C1R bearing HLA-A2402. Killer cells were generated from PBMC of seven healthy individuals and five ovarian cancer patients, all of HLA-A2402 type, by in vitro sensitization with HER2p63-pulsed autologous antigen presenting cells. These killer cells selectively lysed HER2-expressing SKOV3 transfected with HLA-A2402 cDNA, indicating high immunogenicity of HER2p63 in all 12 individuals examined.     

Differences in the antigens recognized by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection

We have studied the patterns of antigens recognized by autologous cytolytic T lymphocytes (CTL) on two melanoma cell lines derived from metastases that were removed from patient LB33 at several years distance. Cell line LB33-MEL. A was obtained after surgery in 1988. A large number of CTL clones directed against LB33-MEL. A was obtained with blood lymphocytes collected from the patient in 1990. In vitro selection of melanoma cells that were resistant to these CTL clones indicated that at least five different antigens were recognized on LB33-MEL. A by autologous CTL. Four of these antigens were found to be presented by HLA-A28, B13, B44 and Cw6, respectively. The patient remained disease-free until 1993 when a metastasis was detected and was used to obtain cell line LB33-MEL. B. This cell line proved resistant to lysis by all the CTL clones directed against the LB33-MEL. A cells and showed no expression of HLA class I molecules except for HLA-A24. Using LB33-MEL. B cells to stimulate blood lymphocytes collected from the patient in 1994 we derived CTL clones that lysed these cells. All these CTL clones recognized a new antigen presented by HLA-A24. These results suggest that in patient LB33 the melanoma cells may have lost the expression of several HLA molecules under the selective pressure of an anti-tumor CTL response.     

Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumor-infiltrating lymphocytes

Three melanoma-specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor-infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA-A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA-A2, but by a B or C HLA antigen. HLA-A2-restricted CTL clones M17-1 and M17-2 lysed 5 and 12 out of 15 HLA-A2⁺ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein-Barr virus B cells, HLA-A2.1-transfected P815 cells,13 HLA-A2⁺ non-melanoma tumor cell lines and 10 HLA-A2^? melanomas, these clones appeared specific for melanoma-restricted epitopes presented by the HLA-A2.1 molecule. We then tried to determine why a few HLA-A2⁺ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA-A2 genes, we show that failure to lyse did not result from low expression or polymoprhism of the HLA-A2 molecule, or from deficient expression of the adhesion molecules ICAM-1 and LFA-3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA-A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA-A2⁺ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.     

The HER2/neu-derived peptide p654–662 is a tumor-associated antigen in human pancreatic cancer recognized by cytotoxic T lymphocytes

The protooncogene HER2/neu encodes a 185-kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that cytotoxic T lymphocytes (CTL) derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. To evaluate whether this HLA-A2-binding peptide is a tumor-associated antigen (TAA) in pancreatic cancer, the ability of HER2/neu-reactive CTL to lyse human pancreatic carcinoma cells was tested. CTL were generated from tumor-associated T lymphocytes from HLA-A2⁺ HER2/neu⁺ breast and ovarian cancer patients. All CTL recognized autologous and allogeneic HER2/neu⁺ tumor cells in an HLA-A2-restricted fashion. Furthermore, all CTL recognized p654–662 (GP2) derived from HER2/neu. These CTL also recognized HER2/neu⁺ pancreatic cancer cells in an HLA-A2-restricted fashion. HER2/neu⁺ HLA-A2^? pancreatic cancer cells were not or only poorly lysed. Repeated stimulation of HLA-A2⁺ PBL from pancreatic cancer patients using the HER2/neu-derived peptide resulted in specific recognition of this peptide and, more importantly, HER2/neu⁺ pancreatic tumors in an HLA-A2-restricted fashion. Autologous HLA-A2⁺ fibroblasts or HLA-A2⁺ malignant melanoma cells were not recognized. HLA-A2^? peptide-stimulated T lymphocytes showed no significant cytotoxicity. These results demonstrate that this HER2/neu-derived peptide is a shared TAA among several adenocarcinomas including pancreatic carcinoma, suggesting a common mechanism of recognition of these human tumors by T lymphocytes. The identification of the HER2/neu-derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies.     

Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T-lymphocyte responses

Because the epithelial cell adhesion molecule (Ep-CAM) is expressed in almost all carcinomas and human leucocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, including Japanese, the identification of peptide sequences, which elicit HLA-A*2402-restricted Ep-CAM-specific cytotoxic T-lymphocyte (CTL) responses, would facilitate specific immunotherapy for various histological types of carcinomas. An epitope was identified through the following steps: (i) computer-based epitope prediction from the amino acid sequence of Ep-CAM, (ii) major histocompatibility complex (MHC) stabilization assay to determine the affinity of the predicted peptide with HLA-A*2402 molecules, (iii) stimulation of CD8+ T cells with peptide-pulsed dendritic cells and (iv) testing the CTL specificity by means of enzyme-linked immunospot (ELISPOT) assays, CTL assays and MHC/peptide-tetramer staining. Peripheral CD8+ T cells of four of five healthy donors after three rounds of stimulation with the peptide Ep-CAM173-181 (RYQLDPKFI) secreted interferon-gamma in ELISPOT assays when exposed to the peptide. A CTL clone specific to the peptide efficiently lysed Ep-CAM-expressing cancer cell lines in an HLA-A*2402-restricted fashion. Endogenous processing and presentation of the peptide in a lung cancer cell line were confirmed by means of cold target inhibition assays. The CTL clone was also lytic to normal bronchial epithelial cells but to a lesser extent at low effector: target ratios. All these data suggest that the peptide-specific CTL responses may play some roles both in anti-cancer and autoimmune reactions. The peptide should prove useful to study anti-Ep-CAM CTL responses among population possessing HLA-A*2402.     

Identification of HLA-A24-restricted CD8(+) cytotoxic T-cell epitopes derived from mammaglobin-A, a human breast cancer-associated antigen

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8⁺ cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8⁺ CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8⁺ CTL lines lysed the HLA-A24⁺/Mam-A⁺ stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24^-/Mam-A⁺ and HLA-A24⁺/Mam-A^- breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8⁺ CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.     

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32 % in Caucasian populations, since 49 % of individuals carry the HLA-A2 allele and 65 % of melanomas express MAGE-3.     

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.     

应用肺癌细胞系诱生肿瘤特异性T细胞的初步研究

目的 探讨通过构建表达人白细胞抗原(HLA)HLA-A2的肺癌细胞系以诱生肿瘤特异性细胞毒性T淋巴细胞(CTL).方法 将编码HLA-A2基因的质粒pcDNA3.1D/V5转染人肺腺癌A549细胞,得到能够稳定表达HLA-A2的肺腺癌细胞系.将其用丝裂霉素(MMC)处理后,行混合淋巴细胞肿瘤细胞培养(mixed lymphocyte tumor cell culture,MLTC)以诱导HLA-A2 的健康人外周血淋巴细胞(peripheral blood lymphocyte, PBL)产生肿瘤特异性CTL,并通过特异性杀伤实验、单克隆抗体阻断实验、淋巴细胞增殖实验进行检验.结果 HLA-A·0201基因在A549细胞中的瞬时与稳定表达率分别为0.32、0.91.成功获得稳定表达HLA-A2的A549细胞株.MLTC诱导最终产生4组T淋巴细胞,且符合肿瘤特异性CTL的形态学特征与大量增殖的特点,但CTL杀伤实验和单克隆抗体阻断实验结果示肿瘤细胞未被有效杀伤,可能与肿瘤特异性CTL的数量较少及活性较低有关.结论 本研究通过构建表达HLA-A2的肺癌细胞系,对建立肿瘤特异性CTL模型作了初步探讨,为进一步完善肺癌特异性CTL细胞模型并为研究肿瘤免疫逃避、免疫耐受及反杀伤淋巴细胞等生物学行为打下基础.     

Cellular and humoral immune responses to a human pancreatic cancer antigen, coactosin-like protein, originally defined by the SEREX method

Among a number of human tumor antigens identified using the serological analysis of recombinant cDNA expression libraries (SEREX), only MAGE-1, tyrosinase, and NY-ESO-1 have been reported to be immunogenic tumor antigens that have the potential to elicit both humoral and cellular immunity. In this study, we determined whether our SEREX-defined pancreatic cancer antigens could be recognized by CTL, and report that one SEREX-defined antigen, coactosin-like protein (CLP), encoded cellular epitopes recognized by HLA-A2-restricted and tumor-reactive CTL. Three CLP peptides at positions 15-24, 57-65, and 10-113 possessed the ability to induce HLA-A2-restricted and tumor-reactive CTL from the PBMC of cancer patients. Subsequently, humoral responses to these peptides were investigated. IgG antibodies specific to the CLP 15-24, 57-65, and 104-113 peptides were detected in sera from 12, 0, and 12 of 12 cancer patients tested, and were also found in 5, 0, and 0 of 9 healthy donors, respectively. IgE antibodies specific to these peptides were also detected in sera from certain cancer patients and healthy donors. Since peptide-specific IgE was detected, type-I allergy to these peptides was tested. Unexpectedly the CLP 57-65 peptide, to which IgE was found in only 2 healthy donors, but not the other two peptides, was found to elicit an immediate-type hypersensitivity in all 10 healthy volunteers tested. These results indicate that identical antigenic peptides can be recognized by both cellular and humoral immune systems to a tumor-associated antigen. The CLP 15-24 and 104-113 peptides might be appropriate vaccine candidates for peptide-based immunotherapy of HLA-A2(+) cancer patients.     

Naturally occurring peptides associated with HLA-A2 in ovarian cancer cell lines identified by mass spectrometry are targets of HLA-A2-restricted cytotoxic T cells

Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA-restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer-related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA-A2-bound peptides from HLA-A2(+) ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA-A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8(+) CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA-A2-restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA-A2(+) tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide-pulsed targets. Furthermore, lack of recognition of several HLA-A2(+) control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL-defined antigens that could lead to the development of multi-epitope vaccines for the treatment of ovarian cancer.     

Effect of prolactin on carcinoembryonic antigen-specific cytotoxic T lymphocyte response induced by dendritic cells

The cytokine hormone prolactin (PRL) has been shown previously to modulate native cellular responses and maturation of antigen-presenting cells. Here we have addressed its effect on the antigen-specific response of cytotoxic T lymphocytes (CTL). CTL were generated from HLA-A2 lymphocytes after three rounds of stimulation with autologous dendritic cells loaded with HLA-A2-restricted carcinoembrionic antigen (CEA) Cap-1 (YLSGANLNL) peptide. Selected cultures were expanded on cytokine-supplemented feeder-layers, enriched for CD8+ lymphocytes and analysed for PRL-receptor (PRL-R) expression and PRL responsiveness. Resting CD8+ lymphocytes were negative for PRL-R, whereas antigen-activated CD8+ lymphocytes derived from long-term cultures were highly positive. Results of a 51Cr release assay showed CTL killing of CEA-loaded, but not unloaded, T2 cell line and the CEA-positive gastric carcinoma cell line KATO, but not of the CEA-negative T leukaemia cell line Jurkat. Interferon (IFN)-gamma release, evaluated in an ELISPOT assay against CEA-loaded T2, was enhanced (P < 0.05) by concentrations of PRL (12-25 ng/ml) very close to the physiological levels (6-20 ng/ml), but was decreased (P < 0.05) by high concentrations (200 ng/ml). Pre-incubation of the stimulators with the anti-MHC class I MoAb W6.32 induced a 40-60% decrease of the PRL-boosted IFN-gamma release, thus proving the MHC restriction of the lymphocyte response. Cytotoxicity against CEA-loaded T2 and KATO cell lines was also increased by 12-25 ng (P < 0.05) and decreased (P < 0.05) by 200 ng PRL. Pre-incubation of CTL with an antibody specific for the PRL-R almost completely abrogated this effect.     

Cytotoxic T cells to an epitope in the islet autoantigen IA-2 are not disease-specific

Cytotoxic CD8 T lymphocytes (CTL) are effectors of pancreatic islet beta-cell destruction in type 1 diabetes but, with the exception of a single report, CTL to islet antigen peptides have not been identified. We used autologous blood monocyte-derived dendritic cells to elicit HLA-A2-restricted CTL to a peptide, MVWESGCTV (aa 797-805), that is contiguous with a dominant CD4 T-cell epitope in the islet antigen tyrosine phosphatase IA-2. IA-2 peptide-specific CTL activity measured as 51Cr release from autologous lymphoblasts was detected in 2/6 islet antibody-positive relatives at high risk for type 1 diabetes but also in 2/6 closely HLA-matched controls. All subjects had CTL activity to an HLA-A2-restricted Epstein-Barr virus peptide. CTL to the IA-2 self-peptide were therefore not disease-specific, consistent with other evidence that autoreactive T cells are present in healthy individuals.     

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivities directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC : peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95·8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.