Tumor-specific cytolysis by lymphocytes infiltrating human melanomas

Tumor infiltrating lymphocytes (TIL) were grown in IL-2 from single cell tumor suspensions of 14 human melanomas resected from 12 patients. As a function of time in culture, 4 of 14 TIL cultures eventually expressed highly specific cytolytic activity against fresh autologous melanoma targets in short term chromium release assays, failing to lyse multiple allogeneic tumors or autologous normal cells. These highly specific TIL were identified as CTL by phenotype (CD3+/CD4-/CD8+/Leu7-) and by function (lysis inhibited by antibodies directed against CD3 and MHC class I molecules). Cell separation experiments using immunomagnetic beads identified a highly tumor-specific CTL subpopulation within a nonspecific TIL culture, suggesting that the lytic activity of tumor-specific CTL may be diluted by the nonspecific killer activity present in heterogeneous TIL cultures. These studies provide evidence for specific MHC-restricted human immune responses against autologous tumor in cancer-bearing patients, and may be of importance to ongoing clinical trials using TIL in the immunotherapy of advanced malignancies.     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Specific release of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IFN-gamma by human tumor-infiltrating lymphocytes after autologous tumor stimulation

Tumor-infiltrating lymphocytes (TIL) have been cultured from a variety of human tumors, and some melanoma TIL have demonstrated specific, MHC-restricted recognition of autologous tumor in short term lysis assays. The current study investigates cytokine release by TIL as an indicator of specific tumor recognition. We have identified two of four melanoma and one of seven breast carcinoma TIL cultures that specifically release granulocyte-macrophage-CSF, TNF-alpha, and IFN-gamma after autologous tumor stimulation. The other cultures either do not secrete cytokine or secrete cytokine in a nonspecific fashion. The amount of specific cytokine released is directly related to the number of TIL and stimulating tumor cells. Studies of TIL, from two melanoma patients, separated into CD4+ and CD8+ populations revealed that CD8+ cells were responsible for virtually all of the specific cytokine secretion, although both populations released cytokines when activated by immobilized anti-CD3 antibody. Specific cytokine release by CD8+ TIL was inhibited by anti-MHC class I mAb. Specific cytokine release was also detected from a CD4+ breast cancer TIL culture, and this was inhibited by anti-MHC class II mAb. The clinical significance of this specific mode of immune antitumor reactivity is currently under investigation.     

Phenotypic characterization of murine tumor-infiltrating T lymphocytes.

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR alpha beta+, with a smaller population of TCR gamma delta+ cells. The TCR alpha beta+ cells expressed a broad distribution of V beta phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR alpha beta+/gamma delta-. Individual long term TIL populations initially expressed multiple V beta products, but rapidly restricted their V beta expression, frequently expressing a single dominant V beta. The identity of this dominant V beta varied among different TIL lines, but the overall representation of V beta phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of V beta usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple alpha- and beta-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.     

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.     

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.     

Enhancement of in vitro tumor-infiltrating lymphocyte cytotoxicity by heteroconjugated antibodies.

Tumor-infiltrating lymphocytes (TIL) were obtained from a mouse melanoma cell line (CL 62) transfected with the gene for the human melanoma Ag p97. TIL were cultured with anti-CD3 antibody and IL-2 for up to 38 days. Flow cytometry identified these TIL as Thy-1.2 + ve/CD4-ve/CD8 + ve cells. A heteroconjugated antibody 500A2 x 96.5, specific for both the CD3 Ag on TIL and the p97 Ag on CL 62 melanoma cells, was prepared using N-succinimidyl-3-(2-pyridyldithio)-propionate as a linking agent. TIL alone demonstrated low levels of cytotoxicity against autologous CL 62 tumor and also against the parental K1735 tumor and an allogeneic murine melanoma (B16). The addition of 500A2 x 96.5 heteroconjugated antibody enhanced TIL-mediated lysis of CL 62 tumor, but not of the K1735 or B16 tumors. This enhanced cytotoxicity was elicited at E:T ratios as low as 0.4:1, and in TIL cultured for 7 to 38 days. These results suggest that hetero-conjugated antibody may enhance the anti-tumor effect of TIL in vivo.     

Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction

CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.     

A ras-mutated peptide targeted by CTL infiltrating a human melanoma lesion

Ags derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient's HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55-64(Q61R) was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101(+) melanoma cell lines. This peptide and its wild-type homolog (55-64(wt)) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes.     

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.     

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells.

CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA-1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer.     

Recognition of neuroectodermal tumors by melanoma-specific cytotoxic T lymphocytes: evidence for antigen sharing by tumors derived from the neural crest

Melanomas from different patients have been shown to express shared tumor antigens, which can be recognized in the context of the appropriate MHC class 1 molecules by cytolytic T cells. To determine if T-cell-defined melanoma antigens are expressed on other tumors of neuroectodermal origin, four melanoma-specific cytotoxic T lymphocyte (CTL) cultures derived from tumor-infiltrating lymphocytes (TIL) were tested for lysis of a panel of 23 HLA-A2⁺ neuroectodermal tumor cell lines of various histologies, including retinoblastoma (1), neuroblastoma (8), neuroepithelioma (6), astrocytoma (2), neuroglioma (1), and Ewing's sarcoma (5). Low expression of MHC class I and/or ICAM-1 molecules was found on 22 of 23 neuroectodermal tumor lines, and could be enhanced by treatment with interferon (IFN). Following IFN treatment, three Ewing's sarcoma lines were lysed by at least one melanoma TIL culture, and levels of lysis were comparable to melanoma lysis by these TIL. Lysis could be inhibited by monoclonal antibodies directed against MHC class I molecules and against CD3, indicating specific immune recognition of tumor-associated antigens. None of the other neuroectodermal tumors tested were lysed by TIL, but they could be lysed by non-MHC-restricted lymphokine-activated killer cells. This demonstration of immunological cross-reactivity between melanomas and Ewing's sarcomas, two tumors of distinct histological types with a common embryonic origin, has implications for the developmental nature of these CTL-defined tumor antigens. It also raises the possibility that specific antitumor immunotherapies, such as vaccines, may be reactive against more than one form of cancer.     

Induction and large-scale expansion of CD8+ tumor specific cytotoxic T lymphocytes from peripheral blood lymphocytes by in vitro stimulation with CD80-transfected autologous melanoma cells.

Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.     

High expression of adhesion molecules/activation markers with little interleukin-2, interferon γ, and tumor necrosis factor β gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4⁺ and CD8⁺ subsets and extensively studied. Three CD4⁺ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor when stimulated by autologous tumor and not by a large panel of stimulators (24–34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4⁺ TIL. These findings demonstrate that MHC-class-II-restricted CD4⁺ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.     

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

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

Autologous tumour-specific cytotoxic T-cell clone established from tumour-infiltrating lymphocytes (TIL) of malignant ascites in the absence of recombinant interleukin 2(rIL2): Activation by autologous tumour cell alone

Mixtures of tumour infiltrating lymphocytes (TIL) and tumour cells collected from malignant ascites of a patient with pancreatic cancer were cultured using a microplate without recombinant interleukin 2(rIL2). TIL rapidly proliferated from 21–51 days after the initiation of culture in 20 out of 30 wells tested. Cytotoxicity was examined in 5 out of the 20 TIL-growing wells. One CD8⁺TIL (well-1) displayed autologous tumour-specific cytotoxicity. Repeated stimulation with autologous tumour cells, in the absence of rIL2, was required for further propagation in long-term (60 days) culture of TIL. Four clones were established from well-1 by limiting dilution without rIL2. Surface phenotypes of the 4 clones were the same as those of well-1, i.e., CD8⁺, CD16, CD25⁺, HLA-DR⁺. And autologous tumour cells were required for continuous proliferation of these CD8⁺ T-cell clones. Both well-1 and the 4 clones displayed similar degrees of cytotoxicity restricted to autologous tumour cells. These results indicate that TIL from malignant ascites may contain precursor cytotoxic T-lymphocytes (CTL) sensitizedin vivo to autologous tumour cells, and that TIL are able to grow for several weeks or more with substantial increases in cell numbers in the absence of rIL2.     

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8⁺ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8⁺ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8⁺ T cells, on the yield, phenotype, and functional activity of expanded CD8⁺ T cells during the REP. We found that CD8⁺ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG₄ (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8⁺ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8⁺ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8⁺ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996