Tumor-infiltrating Lymphocytes Cultures Research Articles

Patterns of Cytokine Release of Unselected and CD8+ Selected Renal Cell Carcinoma Tumor-Infiltrating Lymphocytes (TIL): Evidence for Enhanced Specific Killing of Tumor Necrosis Factor-Secreting/IL-6 Nonsecreting TIL in Vitro and Correlation with Complete Response in Vivo

Murine systems have demonstrated improved anti-tumor efficacy when tumor-infiltrating lymphocytes (TIL) are combined with interleukin-2 (IL-2). One goal of human adoptive immunotherapy is to identify and expand TIL with specific activity against autologous tumor. In this study we attempted to isolate and characterize such cells by phenotype and cytokine expression pattern. Fourteen unselected (bulk) TIL and 5 CD8+ selected cultures were established from primary renal cell carcinoma. All cultures were grown in the presence of IL-2; triplicate cultures of each TIL culture were grown in IL-2 alone or were intermittently cocultured with irradiated allogeneic or autulogous tumor. The in vitro cytotoxicity, phenotype, and cytokine expression pattern as defined by the secretion of tumor necrosis factor-α (TNF-α), interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-4, IL-6, and IL-12 were evaluated for each culture. While there was significant heterogeneity among the cultures under differing culture conditions as characterized by cytotoxicity, phenotype, and cytokine secretion pattern, we identified 8 TIL cultures which demonstrated specific enhanced lysis of only autologous tumor upon exposure to irradiated autologous tumor in vitro. These cultures demonstrate a decreased proportion of cells expressing the phenotype CD11b+. More importantly, these cultures were defined by the cytokine secretion phenotype TNF(+)/IL-6(-) upon exposure to irradiated autologous tumor. When utilized in vivo in adoptive immunotherapy of metastatic renal cell carcinoma together with IL-2, complete resolution of all metastatic tumor has only been achieved in patients who received TIL with the cytokine profile TNF(+)/IL-6(-). These findings suggest that tumor-specific renal TIL with enhanced tumor-specific cytotoxicity can be generated in vitro and can be characterized by a specific pattern of cytokine secretion. In addition, patients who receive TIL characterized by the cytokine profile TNF(+)/IL-6(-) may have an improved outcome when receiving immunotherapy.

T-Cell Receptor Repertoire in Tumor-Infiltrating Lymphocytes. Analysis of Melanoma-Specific Long-Term Lines

Cytotoxic T-lymphocytes (CTLs) can be isolated from human melanoma biopsies that specifically lyse autologous melanoma in vitro and can be effective therapeutic agents for patients with advanced disease. Recent evidence indicates that HLA-A2-restricted, melanoma-specific tumor-infiltrating lymphocytes (TILs) recognize melanomas obtained from different HLA-A2+ patients, suggesting the presence of one or more common melanoma antigens. Furthermore, T-cell receptor (TCR) repertoire analysis by other groups of TILs from fresh melanoma biopsies suggests that there is limited TCR V gene usage in TILs. One serious limitation in analyzing the TCR repertoire in fresh tumors has been the inability to correlate TCR usage with immune function. Therefore, the TCR repertoire was determined in long-term TIL cultures that specifically lysed autologous melanoma in vitro and in many cases mediated in vivo regression of metastatic cancer in patients with advanced disease. The TCR repertoire in cultured melanoma-specific TILs was diverse, with each TIL containing an average of 9.5 +/- 5.7 of the 23 V alpha and 11.2 +/- 5.9 of the 23 V beta subfamilies. Despite the large diversity observed, several V alpha and V beta genes (V alpha 1, V alpha 2, V alpha 22, V beta 13, V beta 14, and V beta 18) are very commonly found in melanoma-specific TILs. No statistically significant associations were observed between the presence of a TCR V gene subfamily in TILs and clinical response, HLA haplotype, or age of the culture. Even though the results in this study suggest that certain TCR V gene segments may be involved in immune responses to human melanoma, we were unable to demonstrate functionally that a particular T-cell clonotype recognizes melanoma tumor-associated antigens. Only the analysis of melanoma-specific CTL clones can determine which clonotypes are important in lysis of human melanoma.

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 γ (IFNγ). Following IFNγ 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.

Isolation and characterization of tumor-infiltrating lymphocytes from cervical carcinoma.

The evidence that virus-induced tumors generally elicit T-cell responses prompts the notion that HPV-related cervical carcinoma would be amenable to treatment by T-cell-mediated adoptive therapy. Therefore, we cultured and cloned tumor-infiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor-cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL-2), followed by limiting dilution, T-cell clones were generated in the presence of 20 U/ml rIL-2 and irradiated autologous tumor cells, PBLs and EBV-transformed B-cell lines. Phenotypically, all clones were CD3/CD8-positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK-sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor-cell lines or tumor-cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti-CD3, anti-CD8 and anti-ICAM1 but not by anti-HLA class-1 (W6/32, B9.12.1), anti-allele-specific HLA determinants and anti-LFA-3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3-associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients.

T-CELL RECEPTOR V-ALPHA AND V-BETA GENE USAGE IN INTERLEUKIN-2-CULTURED TUMOR-INFILTRATING LYMPHOCYTES FROM PATIENTS WITH BREAST-CANCER

Tumor-infiltrating lymphocytes (TIL) are often found in malignant breast tumors, and have been claimed to be of prognostic value. It has been proposed that TIL may represent an enriched population of tumor-specific cytotoxic lymphocytes, reacting with antigenic determinants on the tumor cell surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells. The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between phenotypes and expression of any particular V segments were found. Neither was there any correlation between the expression of specific V alpha/V beta segments and cytotoxicity against allogeneic tumor cells.

Reactivation of murine tumour-infiltrating lymphocytes with solid-phase anti-CD3 antibody: in vitro cytokine production is associated with in vivo efficacy

Previously we described the use of solid-phase anti-CD3 monoclonal antibody (mAb) to stimulate murine tumour-infiltrating lymphocytes (TIL) and their subsequent expansion in recombinant interleukin 2 (rlL-2). In a pulmonary metastases model using the methyl-cholanthrene-induced sarcoma MCA-105 anti-CD3 activated TIL were capable of eradicating disease similar to TIL cultured in rIL-2 only. Here we extend these observations by characterizing the biological effects of sequential solid-phase anti-CD3 activation. TIL from MCA-105 tumour activated with solid-phase anti-CD3 on day 1 were reactivated on day 14, or day 26, or both and compared to TIL grown in rIL-2 only or TIL activated with anti-CD3 once on day 1. Reactivation enhanced in vitro proliferation 1.8-to 4-fold compared to TIL activated once with anti-CD3 ( P < 0.05). In addition, the total lytic capacity of the cultures was enhanced after reactivation without changing the phenotype of the TIL cultures. Reactivation resulted in a greater in vivo efficacy when the TIL were administered within 72 h of reactivation. In contrast, TIL activated with anti-CD3 on day 1 and day 14 were least effective of all TIL cultures ( P < 0.05). This correlated with in vitro cytokine production. The most effective TIL cultures in vivo produced 4- to 100-fold higher amounts of cytokines, especially interferon gamma (IFNγ) and granulocyte macrophage colony stimulating factor (GM-CSF), than the other cultures. On the other hand, the least effective in vivo TIL culture, TIL activated with anti-CD3 on day 1 and 14, produced little or no cytokines. These data suggest that in vitro production of cytokines is indicative of in vivo efficacy of anti-CD3 activated TIL.

Proliferative, phenotypic and functional and molecular characteristics of tumour-infiltrating lymphocytes obtained from unselected patients with malignant melanomas and expanded in vitro in the presence of recombinant interleukin-2

Tumour-infiltrating lymphocytes (TIL) derived from several histotypes, including melanoma, can be expanded in vitro in the presence of recombinant interleukin-2 (rIL-2) and infused as part of experimental adoptive immunotherapy protocols. Several authors have described the isolation and the expansion of TIL, but little is known about the actual proportion of unselected melanoma biopsies that give rise to 'positive' TIL cultures. We evaluated the proliferative, phenotypic, functional and molecular characteristics of cultured TIL obtained from 26 patients with metastatic melanomas, eligible for inclusion in a protocol of adoptive immunotherapy. Sixteen proliferating cultures were obtained. All TIL belonged to the T cell lineage and were characterized by a wide range of cytolytic activity against autologous and, to a lesser extent, allogeneic melanoma cells. Molecular analysis of the constant region of T cell receptor-beta (TCR-beta) showed an oligoclonal population of TIL in the majority of expanded samples, indicating that a selected subset of lymphocytes present in the tumour site could be expanded in vitro. These features, together with the high number of proliferating samples, make these populations of TIL suitable for infusion into patients with advanced disease.

Association of HER2/neu expression with sensitivity to tumor-specific CTL in human ovarian cancer.

To study potential sources of tumor-associated Ags in human ovarian cancer, we have established two ovarian tumor cell lines (OvS1 and OvA2) from two ovarian cancer patients, which express the cellular oncogene HER2/neu. Corresponding tumor infiltrating lymphocyte cultures have also been established and display an autologous tumor-specific pattern of cytotoxicity that is HLA-A2 restricted. To determine the potential relationship between HER2/neu expression and CTL-mediated cytolysis, we first established tumor cell clones from OvS1. These were categorized as high or low expressors of HER2/neu (cOvS1+ or cOvS1-, respectively), and cOvS1+ clones displayed a significantly higher sensitivity to CTL killing as compared with cOvS1- clones. To modulate the expression of HER2/neu, ovarian cancer cells were treated with IFN-gamma. After this exposure, HER2/neu expression was significantly decreased, whereas the expression of HLA Class I was significantly increased. Despite the increase in HLA Class I molecules on the cell surface, CTL-mediated cytolysis of both OvS1 and OvA2 was significantly decreased. IFN-gamma treated cOvS1+ clones displayed a similar decrease in sensitivity to CTL killing, whereas IFN-gamma treated cOvS1- clones displayed an increase or no change in sensitivity to CTL. To confirm this apparent association between HER2/neu expression and CTL recognition, melanoma tumor cell lines that were insensitive to ovarian tumor-specific CTL were transfected with the HER2/neu gene. An HLA-A2+ HER2/neu-transfected melanoma cell line was made sensitive to HLA-A2 restricted ovarian tumor-specific CTL but not to HLA-A2 unrestricted CTL, whereas an HLA-A2- HER2/neu-transfected melanoma remained insensitive to HLA-A2 restricted CTL. These results demonstrate that the sensitivity of ovarian epithelial tumor cells to CTL-mediated lysis is associated with the level of expression of HER2/neu, suggesting that this oncogene product may serve as a source of tumor-associated Ags or as an inducer of such peptides. This is the first time in a human tumor system that oncogene expression has been related to the induction of antigenicity. These results prompt us to approach new strategies for immunotherapy of 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 I 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 gamma (IFN gamma). Following IFN gamma 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.

The use of interleukin-6 to generate tumor-infiltrating lymphocytes with enhanced in vivo antitumor activity.

Tumor-infiltrating lymphocytes (TIL) are cytotoxic T cells isolated from solid tumors and expanded in vitro in recombinant interleukin-2 (rIL-2). TIL have antitumor effects in murine models and in some patients with melanoma. In an effort to generate murine TIL with enhanced in vivo therapeutic efficacy, viable tumor cells were coinjected with a collagen matrix plus recombinant human IL-6 (rIL-6) subcutaneously into syngeneic mice to achieve sustained local concentrations of rIL-6 at the tumor site from which TIL were derived. In five separate experiments, single cell suspensions of tumors were admixed with either (a) Hanks' balanced salt solution (HBSS), (b) 2% (20 mg/ml) collagen matrix only, (c) 250 micrograms rIL-6 only, or (d) 250 micrograms rIL-6 in a 2% collagen matrix (prolonged release) before subcutaneous inoculation. These tumors were subsequently resected and TIL were isolated and expanded in vitro. TIL generated from tumors admixed with matrix plus rIL-6 were significantly more effective than TIL expanded from tumors admixed with HBSS (four of five experiments), TIL from tumors admixed with matrix only (five of five experiments), and TIL from tumors admixed with rIL-6 only (three of four experiments) in an established tumor treatment model. In no experiment was any other TIL culture superior to TIL grown from tumors augmented with collagen matrix plus rIL-6. These results suggest that strategies designed to increase the local concentrations of cytokines at tumor sites may lead to the generation of more potent TIL for clinical administration.

Tumor-Infiltrating Lymphocytes Derived From Select B-Cell Lymphomas Secrete Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor-α in Response to Autologous Tumor Stimulation

Tumor infiltrating lymphocytes (TIL) were cultured from 17 B-cell lymphoma specimens derived from patients with predominantly low-grade malignancies. Specimens included 15 lymph-node biopsies, 1 malignant pleural effusion, and PBL from 1 patient with circulating lymphoma cells. The phenotypic and proliferative characteristics of TIL cultured in interleukin-2 (IL-2) were studied, as well as cytolysis and cytokine secretion in response to autologous tumor. Flow cytometry of fresh tumor suspensions showed that 50% of cells (median) were malignant B cells and 36% were infiltrating T lymphocytes. After culture for approximately 1 month, TIL were 75% +/- 8% CD3+ (mean +/- SEM), 47% +/- 8% CD4+ and 35% +/- 7% CD8+. TIL proliferation was modest in most cases: the median maximum expansion was 32-fold in 25 days. Lysis of autologous tumor in 4-hour 51Cr release assays was mediated by 2 of 12 TIL studied, but was nonspecific. However, these same two TIL, when cocultured with various tumor stimulators, preferentially secreted tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor after autologous tumor stimulation; unstimulated TIL secreted undetectable or barely detectable levels of these cytokines. In one TIL culture, cytokines were secreted by purified CD4+ TIL but not by CD8+ cells, and secretion was completely abrogated by the anti-major histocompatibility complex (MHC) class II antibody IVA12. Thus, although specific cytokine secretion by lymphoma TIL in response to autologous tumor was observed, it occurred in fewer than 20% of patients studied.

Tumor-infiltrating lymphocytes derived from select B-cell lymphomas secrete granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha in response to autologous tumor stimulation

Tumor infiltrating lymphocytes (TIL) were cultured from 17 B-cell lymphoma specimens derived from patients with predominantly low-grade malignancies. Specimens included 15 lymph-node biopsies, 1 malignant pleural effusion, and PBL from 1 patient with circulating lymphoma cells. The phenotypic and proliferative characteristics of TIL cultured in interleukin-2 (IL-2) were studied, as well as cytolysis and cytokine secretion in response to autologous tumor. Flow cytometry of fresh tumor suspensions showed that 50% of cells (median) were malignant B cells and 36% were infiltrating T lymphocytes. After culture for approximately 1 month, TIL were 75% +/- 8% CD3+ (mean +/- SEM), 47% +/- 8% CD4+ and 35% +/- 7% CD8+. TIL proliferation was modest in most cases: the median maximum expansion was 32-fold in 25 days. Lysis of autologous tumor in 4-hour 51Cr release assays was mediated by 2 of 12 TIL studied, but was nonspecific. However, these same two TIL, when cocultured with various tumor stimulators, preferentially secreted tumor necrosis factor-alpha and granulocyte-macrophage colony- stimulating factor after autologous tumor stimulation; unstimulated TIL secreted undetectable or barely detectable levels of these cytokines. In one TIL culture, cytokines were secreted by purified CD4+ TIL but not by CD8+ cells, and secretion was completely abrogated by the anti- major histocompatibility complex (MHC) class II antibody IVA12. Thus, although specific cytokine secretion by lymphoma TIL in response to autologous tumor was observed, it occurred in fewer than 20% of patients studied.

Differential Effects of Inteleukin-2 and CD3 Triggering on Cytokine Gene Transcription and Secretion in Cultured Tumor Infiltrating Lymphocytes

The therapeutic potential of tumor-infiltrating lymphocytes (TIL) is currently under investigation. TIL have been reported to display characteristic functional defects, including impairments of activation mechanisms. In animal models, therapeutic efficacy of adoptive TIL transfer has been correlated with their capacity to produce cytokines rather than with their in vitro cytotoxic potential. In this work we assayed cytokine gene transcription and protein production in eight cultured TIL populations stimulated with recombinant human IL-2 (rhIL-2), solid-phase-bound anti-CD3 monoclonal antibodies (mAb), or a combination of the two. By using a sensitive reverse polymerase chain reaction technique, we observed that transcription of IL-5, GM-CSF, and TNF-α could be detected in unstimulated, IL-2-starved TIL from all cultures, while IL-4 and IFN-γ genes were found to be transcribed in two cultures out of eight. A 50 U/ml dose of rhIL-2 was sufficient to induce IL-4 and IFN-γ gene transcription in the remaining six cultures and in four more TIL populations, respectively. In contrast, rhIL-2 could not induce IL-2 gene transcription in five of eight TIL cultures. Furthermore, it could not induce IL- 10 gene transcription in any TIL population. Anti-CD3 mAb triggering, however, induced transcription of all these cytokine genes. On the other hand, IL-6, IL-7, or TGF-β 2 gene transcription could not be induced by any of the stimuli used, including the combination of anti-CD3 and rhIL-2. Despite detection of their gene transcripts, GM-CSF, IFN-γ, or TNF-α was never detectable, at the protein level, in unstimulated TIL. Stimulation by rhIL-2 induced GM-CSF secretion, albeit to different extents, in all TIL populations. In contrast, rhIL-2 induced IFN-γ and TNF-α production in three and one TIL population, respectively. CD3 triggering however, induced cytokine production in all TIL populations. Addition of rhIL-2 significantly increased production of GM-CSF, but not of IFN-γ and TNF-α. These data underline that stimulation with a moderate dose of rhIL-2 reveals considerable heterogeneity of TIL populations regarding cytokine gene transcription and secretion. On the other hand, triggering of the CD3-T-cell receptor complex induces transcription of an extended panel of cytokine genes in all TIL populations and secretion of GM-CSF, IFN-γ and TNF-α in virtually all cultures. Thus, if stimulated by properly presented antigenic peptides, TIL are capable of mounting an efficient response in terms of cytokine gene expression and protein production.

T cell killing of human colon carcinomas by monoclonal-antibody-targeted superantigens.

The bacterial superantigen staphylococcal enterotoxin A (SEA) induces T cell activation as well as directing activated T cells to kill major-histocompatibility-complex-class-II-expressing tumours such as freshly prepared leukemia cells. We now report that conjugates of SEA and the colon-carcinoma-reactive mAb C215 mediate T-cell-dependent killing of freshly isolated cells obtained from surgical specimens of human colon carcinomas. Cytotoxicity was observed at nanomolar concentrations of conjugate while no or very low effects were seen with the mAb C215 or SEA alone. Tumour-infiltrating lymphocytes (TIL) did not exert any cytotoxicity against conjugate-treated tumour cells immediately after isolation. In vitro culture of TIL with interleukin-2 and SEA resulted in SEA-mAb-conjugate-dependent killing of freshly isolated tumour cells. This suggests that mAb-SEA conjugates may be of potential use to target T lymphocytes, including TIL, against colon carcinoma cells in vivo.

Functional and molecular characterization of tumor-infiltrating lymphocytes transduced with tumor necrosis factor-alpha cDNA for the gene therapy of cancer in humans.

TNF is effective in causing the regression of selected murine tumors when administered at high concentrations. Therapeutic levels in humans cannot be obtained systemically, however, because of dose-limiting toxicity. The development of immunotherapy with IL-2 and tumor-infiltrating lymphocytes (TIL), which can accumulate at tumor sites in some patients, and of efficient retroviral techniques for gene transfer into eukaryotic cells has allowed new therapeutic approaches using TNF. We have retrovirally transduced human TIL with the gene for TNF in an attempt to deliver high concentrations of TNF to the tumor site without dose-limiting systemic toxicity. Successful gene insertion was confirmed by Southern hybridization in 16 of 16 transduced and selected TIL cultures from 15 different patients, with an estimated 28 to 93% transduced cells within each culture. Transduced selected TIL cultures produced greater amounts of TNF, compared with nontransduced controls, in 11 of 16 cultures evaluated. However, overall production of TNF was > 30-fold lower, compared with a transduced and highly selected tumor cell line control (MEL-TNF). In addition, steady state levels of vector-derived transcript in nine of 10 transduced selected TIL cultures were < 14% of the amount seen in the MEL-TNF control line. In an attempt to increase TNF production, TIL were transduced with a mutated form of TNF containing the IFN-gamma signal peptide in place of the transmembranous region, to enhance secretion into the endoplasmic reticulum. By using this vector, TNF production increased by an average of fivefold. These studies demonstrate that TIL can be genetically modified to express and secrete a protein for use in targeted cancer therapy but that partial expression blockades exist that prevent maximal cytokine production by introduced genes in TIL.

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) sensitized in 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.

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: analysis by cytokine secretion.

Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28,000-fold) over 49 culture days (range 26-76 days). By phenotype, mature cultures were 69%-99% CD3+ (mean 93%) and contained mixed populations of CD4+ and CD8+ cells (CD4 > CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term 51Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon gamma, and tumor necrosis factor alpha. Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4+ and CD8+ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.

Regulatory effects of interleukin-4 on tumor-infiltrating lymphocytes derived from human renal cell carcinoma

We have studied the effects of interleukin-4 (IL-4) on the expansion, proliferation, phenotype, and antitumor activity of tumor-infiltrating lymphocytes (TIL) derived from human renal cell carcinoma. Cultures were obtained from three primary renal tumors and one group of tumor-invaded, regional lymph nodes. IL-4 induced a significant increase in lymphocyte expansion and proliferation, but the response was dependent on the concurrent dose of IL-2 in culture. Increased growth activity was only observed in those cultures receiving low doses (20 U/ml) of IL-2 (average increase of fold expansion of 6.5, P < 0.01) with no changes in growth activity in the high dose (1000 U/ml) cultures. The combination of low dose IL-2 and IL-4 (200 U/ml) promoted lymphocyte growth significantly better than high dose IL-2 alone, the current standard growth regimen for in vitro expansion of TIL. TIL grown in the presence of IL-4 significantly reduced the level of nonspecific, non-major histocompatibility complex-restricted antitumor activity ( P < 0.01 for allogeneic renal, nonrenal, and NK-sensitive K562 cells), while exhibiting no effect on the level of autologous killing. This is in contrast to previous findings of significant enhancement of autologous antitumor activity using IL-4 on tumor-specific, melanoma-derived TIL. We also evaluated the effects of irradiated autologous tumor stimulation (TIL:tumor ratio, 10:1) on TIL cultures. Addition of autologous tumor cells increased expansion and proliferation of all cultures regardless of concurrent lymphokines present in the culture media (average increase fold expansion of 2.21, P < 0.05). In addition, significantly enhanced levels of autologous killing were observed in some cultures after the addition of autologous tumor cells (3.4 and 5.9 lytic units without and with autologous tumor stimulation, respectively, P < 0.02). Therefore, the greatest total fold expansion was seen in those cultures containing the combination of IL-4, low dose IL-2, and irradiated autologous tumor cells, which when compared to the standard TIL growth condition of 1000 U/ml of IL-2 alone exhibited a 5.22 ± 2.44 greater fold expansion. Our results suggest that IL-4 and autologous tumor stimulation are effective growth factors, when used in combination with a low dose IL-2 regimen, and may be of significant benefit for growing activated TIL from patients with renal cell carcinoma (RCC). Furthermore, the different biologic properties and responses to exogenous lymphokines of TIL derived from RCC versus other histologically distinct neoplasms suggests the need for an individualized approach in determining the optimal regimen for the expansion and activation of these cells.

Immunotherapy with a tumor-infiltrating lymphocyte clone, soluble antigen, and cyclophosphamide.

A tumor-specific cytotoxic T-lymphocyte clone derived from bulk cultures of tumor-infiltrating lymphocytes attenuated the outgrowth of methylcholanthrene (MCA)-induced fibrosarcomas in C3H/HeJ mice. In 4-hour chromium 51-release assays, bulk cultures of tumor-infiltrating lymphocytes showed nonspecificity for MCA-induced tumors (MCA-F, MCA-D, MCA-SP), YAC-1, and EL-4. In contrast, a cytotoxic T-lymphocyte-cloned line specifically killed MCA-F, but not MCA-D or MCA-SP. Cytotoxic T-lymphocyte line 8 protected syngeneic hosts in local and systemic adoptive transfer assays. Hosts bearing 4-day-established MCA-F tumor cells received single agents or combinations of isoelectrophoretically purified butyl alcohol-extracted tumor-specific transplantation antigen (1 microgram/wk), cyclophosphamide (20 mg/kg on days 4 and 11), and/or cytotoxic T-lymphocytes (1 x 10(7) cells on days 7 and 14). While the mean tumor diameter was 11.6 +/- 1.3 mm in untreated hosts or after single treatments, the combination of tumor-specific transplantation antigen and cyclophosphamide along with the cytotoxic T-lymphocyte clone resulted in tumor diameters of 1.8 +/- 0.5 mm. The triple combination prolonged host survival from 39.6 +/- 1.6 to 57.2 +/- 4.7 days compared with antigen and cyclophosphamide treatment.

Expansion of major histocompatibility complex-restricted antimelanoma cytotoxic T-cell lymphocyte clones with identical T-cell receptor from tumor-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) were isolated from a subcutaneous metastasis of melanoma and cytotoxic T-cell lymphocyte (CTL) lines were obtained by sensitizing in vitro four separate aliquots of TILs with autologous tumor cells and recombinant interleukin-2. All CTL lines were predominantly WT31+, CD3+, and CD8+ and displayed a preferential cytotoxic activity against the autologous tumor. T-cell receptor (TCR) composition was analyzed by using the polymerase chain reaction with 5' variable region (V alpha or V beta)-specific primers and 3' constant (C alpha or C beta) primers. The entire repertoire of the V alpha and V beta gene families tested was present in fresh TILs and in the CTL lines, although, in the latter, consistent quantitative variations in transcripts of several V alpha and V beta occurred. CTL clones that exhibited CD3-dependent and major histocompatibility complex-restricted killing of the autologous melanoma were isolated from the four TIL cultures. TCR analysis indicated that, independently from the culture of origin, only two combinations of V alpha and V beta gene families were present in the majority of these CTL clones. These V alpha and V beta gene families were not found in a panel of CTL clones that did not lyse the autologous tumor. This study indicates that recognition of melanoma antigens can strongly select for certain types of TCR-bearing T-lymphocytes.