A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients

Melanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315–324 (SADVEFCLSL). We derived two distinct additional specific CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this specific repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316–324 nonapeptide induced the growth of specific cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-specific T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma 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.     

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.     

Immune therapy of melanoma: Overview of therapeutic vaccines

Melanoma is the most serious type of skin cancer which develops from the occurrence of genetic mutations in the melanocytes. Based on the features of melanoma tumors such as location, genetic profile and stage, there are several therapeutic strategies including surgery, chemotherapy, and radiotherapy. However, because of the appearance resistance mechanisms, the efficiency of these treatments strategies may be reduced. It has been demonstrated that therapeutic monoclonal antibodies can improve the efficiency of melanoma therapies. Recently, several mAbs, such as nivolumab, pembrolizumab, and ipilimumab, were approved for the immunotherapy of melanoma. The antibodies inhibit immune checkpoint receptors such as CTL4 and pd-1. Another therapeutic strategy for the treatment of melanoma is cancer vaccines, which improve clinical outcomes in patients. The combination therapy using antibodies and gene vaccine give us a new perspective in the treatment of melanoma patients. Herein, we present the recent progressions in the melanoma immunotherapy, especially dendritic cells mRNA vaccines by reviewing recent literature.     

Non-fastidious,melanoma-specific CD8+ cytotoxic T lymphocytes from choroidal melanoma patients

To characterize the anti-melanoma reactivity of CD8⁺ cytotoxic T lymphocytes (CTL) from choroidal melanoma patients, CTL clones were isolated from the peripheral blood of three patients after mixed lymphocyte/tumor cell culture (MLTC). Clones were derived from lymphocytes stimulated by allogeneic (OCM-1, A24, A28) or autologous (OCM-3, Al, A30) melanoma cells. Their reactivity against a panel of HLA-typed melanoma and nonmelanoma cells was assessed, to determine whether a single CTL clone could recognize and lyse a variety of allogeneic melanoma cell lines. While proportionately more clones derived from autologous MLTC were melanoma-specific than allogeneic MLTC (42% versus 14%), melanoma-specific CTL were recovered from both. Notably, a novel melanoma specificity was identified. These CTL clones were termed non-fastidious because they were capable of lysing melanoma cells with which they had no HLA class I alleles in common. Nonetheless, lysis was mediated by the HLA class I molecule. Since lysis was specific for melanoma cells, these CTL appeared to recognize a shared melanoma peptide(s). Because of their prevalence, we propose that non-fastidious CTL are integral to human anti-melanoma T cell immunity. This reinforces clinical findings that allogeneic melanomas can substitute for autologous tumors in active specific immunotherapy. By circumventing the need for autologous melanoma, it is possible to treat patients after removal of the primary choroidal melanoma in an attempt to prevent metastasis.Supported by USPHS grants EY-09031 and EY-09427, and the Lucy Adams Choroidal Melanoma Research Fund to J. K.-M.     

A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.     

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.     

Shared human melanoma antigens. Recognition by tumor-infiltrating lymphocytes in HLA-A2.1-transfected melanomas.

Three predominantly CD8+ CTL lines, TIL 501, TIL 620, and TIL 660, were generated from three HLA-A2+ melanoma patients by culturing tumor-infiltrating lymphocytes in 1000 U/ml IL-2. These tumor-infiltrating lymphocytes lysed 12 of 18 HLA-A2+ autologous and allogeneic melanomas, but none of 20 HLA-A2-negative melanomas. They also did not lyse the MHC class I negative lymphoma-leukemia cell lines, Daudi, K562, or HLA-A2+ non-melanoma cell lines including PHA or Con A-induced lymphoblast, fibroblast, EBV-transformed B cell, Burkitt's B cell lymphoma, and colon cancer cell lines. Autologous and allogeneic melanoma lysis was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag among melanoma cell lines in a TCR-dependent, HLA-A2-restricted manner. Six HLA-A2-negative melanoma cell lines obtained from five HLA-A2-negative patients were co-transfected with the HLA-A2.1 gene and pSV2neo. All 17 cloned transfectants expressing cell surface HLA-A2 molecules, but none of 12 transfectants lacking HLA-A2 expression, were lysed by these three HLA-A2-restricted, melanoma-specific CTL. Lysis of the HLA-A2+ transfectants was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag on transfectants in a TCR-dependent, HLA-A2-restricted manner. These results identify the HLA-A2.1 molecule as an Ag-presenting molecule for melanoma Ag. They also suggest that common melanoma Ag are expressed among melanoma patients regardless of HLA type. These findings have implications for the development of melanoma vaccines that would induce antitumor T cell responses.     

Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-alpha. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.     

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.     

Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines

Melanoma is a cancer where the immune system is believed to play an important role in the control of malignant cell growth. To study the variability of the immune response in melanoma patients, we derived melanoma cell lines from several HLA-A2⁺ and HLA-A2^– patients. The melanoma cell lines studied were designated FM3, FM6, FM9, FM28, FM37, FM45, FM55_P, FM55_M1 and FM55_M2 and were established from eight metastatic tumors as well as from one primary tumor from a total of seven different patients. On the basis of the ability of tumor cells to induce specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes (PBL) in mixed lymphocyte/tumor culture with HLA-A2⁺ melanoma cells, the FM3 cell line was characterized as highly immunogenic. To investigate the expression of different melanoma-associated antigens recognized by CTL on different melanoma cell lines, we selected the cell line FM3 for restimulation and further T cell cloning experiments. The lytic activity of CTL clones with good proliferative activity was examined using a panel of HLA-A2⁺ and HLA-A2^– melanoma cell lines. None of the tested HLA-A2^– melanoma cell lines were susceptible to lysis by the CTL clones, whereas allogeneic HLA-A2⁺ melanoma cell lines were lysed only by a few CTL clones. On the basis of their reactivity with different melanoma cell lines, it was possible to divide the present CTL clones into at least four groups suggesting the recognition of at least four different antigens. Three of these target structures probably are different from already-described HLA-A2-restricted melanoma-associated antigens, because their expression in the different melanoma cell lines do not correlate with the recognition of melanoma cells by these CTL. The results first indicate that poorly immunogenic melanoma cells may express melanoma-associated antigens, and also suggest that, by using CTL clones obtained against different HLA-class-I-matched melanoma cells, it is possible to define such antigens.     

Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma

Previous studies have suggested that immunotherapy with dendritic cell (DC) vaccines may be effective in treatment of patients with AJCC stage IV melanoma. We examined this treatment in phase I/II studies in 33 patients with good performance status and low volume disease. Nineteen patients received DCs plus autologous lysates and 14 patients DCs plus peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1. Keyhole limpet hemocyanin (KLH) was used as a helper protein and influenza peptide was given as a positive control. DCs were produced from adherent cells in blood lymphocytes (monocytic DCs), grown in IL-4 and GM-CSF without a maturation step. The DCs were injected into inguinal lymph nodes at weekly intervals (×4), 2 weeks (×1), and 4-weekly intervals (×2). There were 3 responses (3 partial responses) and 1 mixed response in the 19 patients treated with DCs plus autologous lysates. No responses were seen in the group treated with DCs plus peptides. Stable disease (defined as no progression over a period of 3 months) was seen in 4 patients in group 1 and 5 patients in group 2. Treatment was not associated with significant side effects. We examined whether DTH skin tests or assays of IFN- cytokine production may be useful predictors of clinical responses. Twenty-two of 30 patients had DTH responses to KLH and 12 of 13 patients had DTH responses to the influenza peptide. Five of 15 DTH responses were seen against autologous lysates. This was strongly correlated with clinical responses. Approximately half the patients had responses to MART-1 peptide and a third to the other melanoma peptides. Similarly, cytokine production assays showed responses to influenza in 7 of 13 patients, and approximately one third of patients had responses to the other peptides. No IFN- responses were seen in 5 patients against their autologous lysates. There was no correlation between assays of IFN- production and clinical responses. The present studies suggest that autologous lysates may be more effective than the melanoma peptides used in the study as the source of antigen for DC vaccines. DTH responses to autologous lysates appear useful predictors of clinical responses, but further work is needed to identify other measures associated with clinical responses.Abbreviations DC dendritic cells - DTH delayed hypersensitivity skin tests - KLH keyhole limpet hemocyanin - CTL cytotoxic T lymphocytes     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stability and CTL activity of N-terminal glutamic acid containing peptides.

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.     

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.     

Cloned cytotoxic T lymphocytes as target cells. II. Polarity of lysis revisited

The original polarity of lysis experiments suggested that CTL are themselves sensitive to whatever mechanism it is that CTL use to lyse their targets. This concept has placed certain limitations on possible mechanisms of lysis by CTL. Recently, we found in studies with cloned CTL as targets that cloned CTL are in fact highly resistant to lysis by other CTL, as well as to their cytotoxic granule proteins. We show here that although cloned CTL are extremely resistant to lysis by primary and cloned CTL, they are readily inactivated functionally by all primary CTL and by at least one CTL clone. Moreover, cloned CTL are also functionally inactivated by cytotoxic granule proteins. The activation of CTL, which we call inhibitin, is Ca2+ insensitive and distinct from hemolytic activity, and is, thus, unlikely to be perforin. These experiments suggest a possible alternative interpretation of the original polarity of lysis experiments.     

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.     

High frequency of autologous anti-melanoma CTL directed against an antigen generated by a point mutation in a new helicase gene

We have identified an Ag recognized by autologous CTL on the melanoma cells of a patient who enjoyed an unusually favorable clinical evolution. The antigenic peptide, which is presented by HLA-A28 molecules, is encoded by a mutated sequence in a new gene. This gene, which was named MUM-3, is expressed ubiquitously and shows homology with the RNA helicase gene family. Limiting dilution analysis indicated that at least 0.15% of the blood CD8 T cells were tumor-specific CTL precursors. The MUM-3 Ag was recognized by 90% of these CTL, indicating that it is the dominant target Ag of the tumor-specific CTL response. The high frequency of anti-MUM-3 CTL was confirmed with tetramers of soluble HLA-A28 molecules loaded with the antigenic peptide. MUM-3 tetramers stained 1.2% of blood CD8 cells, a frequency that has never been reported for T cells directed against a strictly tumor-specific Ag. To confirm these results, the CD8 T cells that were clearly labeled with tetramers were restimulated in clonal conditions. About 90% of these cells proliferated, and all the resulting clones proved lytic and MUM-3 specific. By improving the conditions used for the in vitro restimulation of CTL precursors by the tumor cells, the same frequency could be obtained in limiting dilution analysis. These results show that some cancer patients have a high frequency of circulating CTL that are directed against a strictly tumor-specific Ag. These CTL are responsive to restimulation in vitro and are easily detected with tetramers. Such responses may therefore be an achievable goal for therapeutic vaccination with tumor-specific Ags.