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.     

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.     

Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes

We studied the susceptibility of autologous and allogeneic tumors to lysis by human tumor infiltrating lymphocytes (TIL) after pre-incubation of the tumors with human rIFN-gamma and human rTNF-alpha. Preincubation of the tumor lines with IFN-gamma or TNF enhanced susceptibility to lysis significantly; the combination of both cytokines was more effective than either alone. Pretreatment for at least 24 h was required to enhance lytic susceptibility and maximal lysis was observed after pretreatment for 48 to 72 h. Highly specific TIL lysed only their autologous tumor targets and failed to lyse cytokine pretreated allogeneic tumor cells. In TIL populations with varying specificity, cytokine pretreatment of targets enhanced autologous lysis as well as allogeneic lysis. This cytokine-mediated effect could also be observed in a lectin-dependent cytotoxicity assay and did not correlate directly with enhanced expression of MHC class I Ag or the adhesion molecules LFA-3 and ICAM-1. These results suggest that enhancement of lysis may occur at a postbinding stage by making the target cell more sensitive to the cytotoxic factors delivered by the killer cell. The fact that lysis of cytokine treated targets by cells with LAK activity was not enhanced suggests that cells with lymphokine-activated killer activity and tumor-derived T cells kill tumor targets via different mechanisms.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

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.     

Rejection of allogeneic tumor is not determined by host responses to MHC class I molecules and is mediated by CD4-CD8+ T lymphocytes that are not lytic for the tumor

In previous studies, the murine SaI (A/J derived, KkDd) sarcoma was transfected with the allogeneic MHC class I H-2Kb gene, and expressed high levels of H-2Kb antigen. Contrary to expectations, the tumor cells expressing the alloantigen (SKB3.1M tumor cells) were not rejected by autologous A/J mice. Because these results contradict the laws of transplantation immunology, the present studies were undertaken to examine the immunogenicity of SKB3.1M and SaI cells in allogeneic hosts. Similar to SKB3.1M, SaI cells are lethal in some allogeneic strains, despite tumor-host MHC class I incompatibilities. Tumor challenges of SKB3.1M and SaI cells, however induce MHC class I-specific antibodies and CTL in both tumor-resistant and -susceptible hosts. Although the tumors induce specific CTL, tumor cells are not lysed in vitro by these CTL, suggesting that the tumor cells are resistant to CTL-mediated lysis. Since growth of these tumors does not follow the classical rules of allograft transplantation, and because the tumor is not susceptible to CTL-mediated lysis, we have used Winn assays to identify the effector lymphocyte(s) responsible for SaI rejection. Depletion studies demonstrate that the effector cell is a CD4-CD8+ T lymphocyte. Collectively these studies suggest that the host's response to MHC class I alloantigens of SKB3.1M and SaI cells does not determine tumor rejection, and that effector cells other than classically defined CTL, but with the CD4-CD8+ phenotype, can mediate tumor-specific immunity.     

Detection of simian immunodeficiency virus (SIV)-specific T-cell-mediated cytotoxicity in the peripheral blood from infected cynomolgus monkeys

We have previously demonstrated that peptide immunization restimulates the memory CD4 T-cell response, but fails to induce cytotoxic T lymphocyte (CTL) in cynomolgus macaques. To examine the nature of protective immunity to simian immunodeficiency virus (SIV) in this study, freshly isolated peripheral blood mononuclear cells (PBMC) from four infected juvenile cynomolgus macaques and from three uninfected control macaques were assessed for CTL activity monthly for 9 consecutive months, beginning 1 month after detection of infection. Target cells consisted of major histocompatibility (MHC) haploidentical parental PBMC which were stimulated with mitogen and then pulsed with heat-killed SIVcyn. CTL activity was demonstrated in PBMCs from all four infected animals. The effector cells are T cells which mediate cytotoxicity against SIVcyn-pulsed target cells in an MHC-restricted manner. Furthermore, the cytotoxicity is virus specific and predominantly, if not exclusively, mediated by CD8+ T cells; it is also MHC class I restricted. Incubation of target cells with pepstatin A during antigen pulsing prior to the cytotoxic assay inhibited target cell generation, suggesting that viral antigens are processed via an endocytic pathway.     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

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.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

Membrane structures involved in auto-tumor recognition

Tumor patients' blood lymphocytes have the capacity to recognize autologous tumor cells in vitro. A consequence of this recognition is the proliferation of small-size, high-density, resting T cells. Both helper (CD4+) and cytotoxic/suppressor (CD8+) T lymphocytes proliferate in the mixed lymphocyte-tumor cell cultures. In contrast to the autologous mixed lymphocyte cultures, both the auto-erythrocyte rosetting and non-rosetting (AE+ and AE-) T cells participate in the auto-tumor response. In contrast to stimulation by virus-infected or hapten-modified cells, DR antigen expression is not essential for stimulation by autologous tumor cells. In a proportion of cancer patients, blood lymphocytes have the capacity to lyse the patients' own tumor cells in vitro. There are two populations of lymphocytes with auto-tumor cytotoxic function. The first is characterized by low buoyant density and by non-adaptive cytotoxicity. In contrast to the recognition of hapten-modified or virus-infected target cells by the CTL, recognition of autologous tumor cells by the cytotoxic LD cells occurs even when the MHC class I antigens are blocked by mAb. The CD3 complex is also not involved in LD-mediated lysis. The other population with auto-tumor cytotoxic function comprises high-density, resting T cells. Recognition of autologous tumor cells by cytotoxic HD lymphocytes shares the characteristics of CTLs, i.e., their function is abrogated by pretreatment of the effectors with mAbs directed to the T3 receptor complex and by preincubation of the targets with mAb to the MHC class I antigens. Cytotoxicity of HD cells is restricted to the autologous tumor cells. This selectivity and the characteristics shared with CTL suggest that the auto-tumor reactivity of HD lymphocytes reflects an immune response against the autologous tumor.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Tolerance induction by transcutaneous immunization through ultraviolet-irradiated skin is transferable through CD4+CD25+ T regulatory cells and is dependent on host-derived IL-10

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4(+)CD25(+) T regulatory (T(reg)) cells. These Ag-specific T(reg) cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of T(reg) cells through UV-irradiated skin as IL-10-deficient T(reg) cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated T(reg) cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced T(reg) cells mediate their effects in part through the modulation of IL-10.     

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.     

Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes

Cell-based antitumor immunity is driven by CD8(+) cytotoxic T cells bearing TCR that recognize specific tumor-associated peptides bound to class I MHC molecules. Of several cellular proteins involved in T cell:target-cell interaction, the TCR determines specificity of binding; however, the relative amount of its contribution to cellular avidity remains unknown. To study the relationship between TCR affinity and cellular avidity, with the intent of identifying optimal TCR for gene therapy, we derived 24 MART-1:27-35 (MART-1) melanoma Ag-reactive tumor-infiltrating lymphocyte (TIL) clones from the tumors of five patients. These MART-1-reactive clones displayed a wide variety of cellular avidities. alpha and beta TCR genes were isolated from these clones, and TCR RNA was electroporated into the same non-MART-1-reactive allogeneic donor PBMC and TIL. TCR recipient cells gained the ability to recognize both MART-1 peptide and MART-1-expressing tumors in vitro, with avidities that closely corresponded to the original TCR clones (p = 0.018-0.0003). Clone DMF5, from a TIL infusion that mediated tumor regression clinically, showed the highest avidity against MART-1 expressing tumors in vitro, both endogenously in the TIL clone, and after RNA electroporation into donor T cells. Thus, we demonstrated that the TCR appeared to be the core determinant of MART-1 Ag-specific cellular avidity in these activated T cells and that nonreactive PBMC or TIL could be made tumor-reactive with a specific and predetermined avidity. We propose that inducing expression of this highly avid TCR in patient PBMC has the potential to induce tumor regression, as an "off-the-shelf" reagent for allogeneic melanoma patient gene therapy.     

Adoptive immunotherapy of advanced melanoma patients with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes selected in vitro with low doses of IL-2

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogeneic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8×10⁹, range: 0.35 × 10⁹–20 × 10⁹) and IL-2 (mean dose: 130 × 10⁶ IU, range: 28.8 × 10⁶–231 × 10⁶ IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL.