Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996     

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.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Cytotoxic T cell lines recognize autologous and allogeneic melanomas with shared or cross-reactive HLA-A

Summary Cytotoxic T lymphocytes (CTL), CD3⁺, / T-cell-receptor-positive, are important effector cells with specific immunity in melanoma patients. The establishment and expansion in vitro of CTL of a specific phenotype to tumor cells strongly depends on the method of activation and sensitization with tumor cells. We generated CD3⁺ CTL lines to melanoma by co-culturing peripheral blood lymphocytes with autologous irradiated melanoma cells and repetitive stimulation with high-dose interleukin-4 in a cocktail culture medium. CTL lines were investigated for their specificity to kill autologous and allogeneic melanoma. Histocompatibility locus antigen (HLA) class I (A, B) molecules are important restrictive recognition antigens for CTL. Although these antigens are highly polymorphic, they can share a similar immunogenic molecular epitope(s) and can be immunologically cross-reactive. The CTL lines generated were found to kill not only autologous melanoma, but also allogeneic melanomas having class I HLA-A antigens shared or cross-reactive with autologous HLA-A. These CTL lines were poor killers of melanomas bearing non-shared or non-cross-reactive HLA-A. Cold-target inhibition assays demonstrated this CTL cross-reactivity to allogeneic melanoma specificity. Epstein-Barr-virus-transformed autologous and allogeneic B lymphoblastoid cell lines failed to block autologous melanoma killing, indicating that CTL were not recognizing major histocompatibility complex antigens, serum proteins or culture medium products as the primary target antigen. HLA-A2 was the major shared HLA-A antigen recognized by CTL lines on the melanoma lines studied. CTL lines also recognized shared HLA-A11 and A24 on allogeneic melanoma. There were no CTL lines showing restriction to HLA-B. These results suggest that common tumor-associated antigens are present on melanomas and are recognized in association with distinct HLA-A epitopes by CTL.This study was supported by grant CA12 582 awarded by the National Cancer Institute, USA     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

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     

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

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.     

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

Identification of novel MAGE-A6- and MAGE-A12-derived HLA-A24-restricted cytotoxic T lymphocyte epitopes using an in silico peptide-docking assay

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called ??-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24⁺ melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.     

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.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.     

Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects

The spontaneous cytotoxic T cell responses to melanoma differentiation antigens and influenza matrix peptide were compared in 20 HLA-A2⁺ melanoma patients and 17 healthy A2⁺ individuals. Cytotoxic T lymphocyte (CTL) responses were determined by mixed lymphocyte peptide culture (MLPC) involving two stimulations of unfractionated peripheral blood lymphocytes (PBLs) with peptide in vitro. CTL responses to Melan-A 9-mer (amino acids 27–35, AAGIGILTV) peptide were detected in 4 out of 16 normal individuals, but in none of the melanoma patients. CTL specific for influenza matrix peptide were frequently found in both normal individuals and melanoma patients, suggesting that generalized immuno-suppression was not responsible for this difference. No significant responses were observed in either normal individuals or melanoma patients to Melan-A 10-mer (26–35, EAAGIGILTV), two gp100 epitopes (280–288, YLEPGPVTA; 457–466, LLDGTATLRL) and two tyrosinase epitopes (1–9, MLLAVLYCL; 368–376, YMDGMSQV). Melan-A (27–35)-specific CTL cells generated by normal individuals and melanoma patients recognized both synthetic peptide-pulsed T2 cells and two HLA-A2⁺, Melan-A⁺ melanoma cell lines (ME272, LAR1) in an antigen-specific, MHC class I restricted manner. T cells generated against Melan-A 9-mer were also able to recognize Melan-A 10-mer-pulsed target cells. Spontaneous CTL responses to Melan-A 9-mer from three known responder normal individuals were further evaluated over a prolonged time course (6–11 months). All 3 subjects demonstrated specific Melan-A 9-mer responses throughout the study period, although lytic activity fluctuated over time for a given individual. We found the MLPC assay to be reliable and easy to perform for monitoring T cell responses, although it may still not be sufficiently sensitive to detect low numbers of precursor T cells. Received: 21 May 1998 / Accepted: 23 July 1998     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

Cancer/testis antigens can be immunological targets in clonogenic CD133<Superscript>+</Superscript> melanoma cells

“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133⁺ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133⁺ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8⁺ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.