Long-term interleukin 2-dependent growth and cytotoxic activity of tumor-infiltrating lymphocytes from human squamous cell carcinomas of the head and neck

Tumor-infiltrating lymphocytes (TIL) from 16 squamous cell carcinomas of head and neck (SCCH&N) and four nonsquamous cell carcinomas were studied. By immunoperoxidase staining in situ, the tumors studied were found to be infiltrated mainly by CD2+CD3+ cells, and 30-50% of the T-lymphocytes were HLA-DR positive and transferrin-receptor positive. They also contained scarce NKH1+ cells. When TIL as well as autologous peripheral blood lymphocytes (A-PBL) were cultured in 1,000 U/ml of recombinant interleukin 2 (rIL2), TIL proliferated in all but three cases, and A-PBL proliferated in all but two cases. Frequently, but not always, TIL expanded better than A-PBL. The median expansion for TIL was 100-fold and that for A-PBL was 31-fold in long-term cultures maintained for up to 88 days. TIL obtained from untreated primary SCCH&N were initially delayed for up to 20 days in their proliferative response to rIL2, but then grew well. In contrast, TIL and A-PBL from metastatic SCCH&N either did not proliferate or were delayed in their proliferative response for up to 40 or 50 days. A-PBL, when tested early (days 10-20 in culture), showed the highest cytotoxic activity against cultured and fresh tumor-cell targets, whereas TIL were most active later in culture (days 20-30). On a per culture basis, TIL achieved higher antitumor cytotoxicity than A-PBL. By day 80, lytic activities of most TIL cultures declined to undetectable levels. CD3+Leu19- T-lymphocytes were the major expanding cell population in most TIL cultures. However, these cells were poor mediators of antitumor cytotoxicity in TIL or A-PBL cultures as shown in cell sorting experiments. The antitumor effector cells expressed CD3-Leu19+ and/or CD3+Leu19+ phenotypes. On Giemsa-stained smears, these two types of IL2-expanded effector cells had the morphology of large granular lymphocytes. Our results indicate that TIL from human SCCH&N could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL2.     

Functional and phenotypic analysis of tumor-infiltrating lymphocytes isolated from human primary and metastatic liver tumors and cultured in recombinant interleukin-2

Tumor-infiltrating lymphocytes (TIL) were isolated from 40 of 51 consecutive human liver tumor samples (primary hepatocellular carcinoma, 16 of 18; metastatic, 23 of 29; benign, one of four). Functional and phenotypic characteristics of fresh and recombinant interleukin-2 (rIL-2)-expanded TIL were evaluated. The expansion of TIL from hepatic tumors in the presence of 1000 units/ml of rIL-2 was possible in 60% of cases. In comparison to TIL from metastatic liver tumors, TIL obtained from primary liver tumors expanded faster and better in rIL-2 cultures. Expanded TIL from primary tumors had significantly higher cytotoxicity against K562 targets, but not Raji targets, than those from metastatic tumors. Cytotoxicity against fresh autologous tumor targets was detected in seven of eight cultures tested. TIL from primary tumors retained antitumor reactivity significantly longer in culture. The optimal in vitro cytotoxicity was achieved between days 20 and 60 of culture in the presence of rIL-2. Antitumor activity was associated with the increase in these TIL cultures of a cell population expressing the Leu19 antigen with or without the CD3 antigen. The frequency of the CD3+Leu19+ population showed a bimodal distribution during culture: the first peak of CD3+Leu19+ cells occurred between days 30 and 60 and was associated with the increased antitumor activity; the second peak occurred after day 60 and was not associated with activity. These findings demonstrate that TIL from most human hepatic tumors can be successfully isolated, cultured in rIL-2, and enriched in Leu19+ effectors. In addition, these TIL upon IL-2 activation in vitro are capable of lysing fresh autologous and/or allogeneic tumor targets.     

Dominance of CD4+ alpha/beta T-cells and inferior role of innate immune reaction in liver metastases

BACKGROUND: Tumor infiltrating lymphocytes (TIL) are frequently present in human tumors with CD8+(-)T-cells as effector and CD4+ T-cells as helper cells. Despite the well established knowledge about primary tumors, only little is known about metastatic disease, especially for liver metastases. The role of the innate immune system in the tumor defence is still enigmatic. MATERIALS AND METHODS: We performed a subtyping of TIL in 20 liver metastases. Using immunohistochemistry, CD20+, CD3+, CD56+, CD4+ and CD8+ lymphocytes, gamma/delta-T-cells and alpha/beta-T-cells in the tumor, the peritumoral region, portal tracts and lobules were investigated. RESULTS: The immune response was highly accentuated in the surroundings of the metastases with only few lymphocytes in the tumor itself. There was a dominance of CD3+(-)CD4+(-)alpha/beta-T-cells with a lower number of CD8+(-)T-cells. The CD4+/CD8+ ratio was 6:1. CD56+(-)NK/NKT-cells and gamma/delta-T-cells were rare. No differences were found between metastases from different primaries or according to the number or diameter of the metastases. CONCLUSION: TIL are part of an interaction between the metastatic tumor and the liver. Among them CD4+ T-cells seem to have a unique independent function in tumor response. The localization of the immune response in the tumor periphery might be a reason for insufficient tumor defense. A defect in the innate immune system could be a reason for the escape of the metastatic tumor cells from tumor surveillance.     

Clonal analysis of lymphocytes from tumor, peripheral blood, and nontumorous kidney in primary renal cell carcinoma

The immunological properties of lymphocytes from tumor, peripheral blood (PBL), and nontumorous kidney from 16 patients with renal cell carcinoma were characterized at the clonal level with respect to their clonogenic efficiency, phenotypic expression, and cytotoxicity against autologous and allogenic tumor cells. The objectives were to delineate: (a) the quantitative differences in the immunological properties of tumor-infiltrating lymphocytes (TIL) from patient to patient; and (b) the qualitative differences in immunological properties between TIL and lymphocytes from peripheral blood or nontumorous kidney from a single patient. A total of 926 clones were characterized for phenotype expression, and 465 clones were characterized for cytotoxicity. The clonogenic efficiency of TIL varied with individuals: high in one patient; relatively high to moderate in seven patients; low in seven patients, and extremely low in the remaining one patient. The levels of autologous tumor cell lysis by TIL clones also varied with individuals. More than one-third of the TIL clones established in 4 of 13 patients displayed significant (greater than or equal to 10%) lysis against autologous tumor cells, and in each of the four patients the average percentage of lysis in the total TIL clones was higher than 10%. In two patients, 5 of 26 or 3 of 13 TIL clones were cytotoxic, but averages of percentage of lysis in the total clones were less than 10%. One 1 or 2 TIL clones of 10-27 total clones were cytotoxic in each of 4 patients, while no cytotoxic TIL clones were found in the remaining 3 patients. Clonogenic efficiency did not correlate with the level of cytotoxicity, and TIL from no tumors displayed both high proliferation and high cytotoxicity at the clonal level. In a majority of patients (12 of 13), most cytotoxic TIL clones against autologous tumor cells also lysed allogenic tumor cells. In contrast, TIL clones lysed only autologous tumor cells in the remaining one patient (patient 2). The clonogenic efficiency of TIL was lower than that of PBL in 6 of 12 patients, while the opposite was true in the remaining 6 patients. The level of cytotoxicity in the PBL clones of these 12 patients primarily correlated with that of the TIL clones. With one exception (patient 2), most cytotoxic PBL clones against autologous tumor cells also lysed allogenic targets in a majority of patients. CD4+CD8-T-cell clones (70-85%) predominated in all patients regardless of the different lymphocyte sources.(ABSTRACT TRUNCATED AT 400 WORDS)     

A correlation between the expression of CD 8 antigen and specific cytotoxicity of tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TIL) from six gynecologic malignant tumors (two uterine cervical cancers, two ovarian serous cystadenocarcinomas, and two uterine corpus cancers), cultured in the presence of recombinant interleukin 2, were assayed for their cytotoxic activities against various fresh tumor cells including autologous tumors. A clear correlation between phenotype and cytotoxic activity of TIL was observed. Four of six TIL preparations exhibited strong cytotoxic activity against autologous fresh tumor target cells, and were all CD8+. In contrast, cytotoxic activity was not detected in any of the CD4+ TIL preparations. The cytotoxic activities of the CD8+ TIL preparations were highly specific; only autologous fresh tumor cells were lysed. This result is consistent with the notion that TIL are of a different cell lineage from lymphokine-activated killer cells which are antigen-nonspecific and CD8-. Instead, TIL appear to be of cytotoxic T cell lineage that is highly antigen-specific and CD8+. To explore the potential for clinical use, we have attempted to augment the cytotoxic activities of these CD8+ TIL by treatment of the target tumor cells with gamma interferon (IFN) in vitro, hoping that elevated expression of MHC class I gene products on the cell surface would enhance their recognition. It was observed that brief treatment of freshly prepared tumor cells in vitro with gamma-IFN resulted in augmentation of the expression of MHC class I gene products, and the treated tumor cells were more susceptible to lysis by TIL than untreated cells.     

MHC class I-restricted recognition of a melanoma antigen by a human CD4+ tumor infiltrating lymphocyte

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.     

Differential regulation by interleukin-4 and interferon-gamma of an autologous melanoma-specific cytotoxic T-cell clone and the tumor-infiltrating lymphocytes from which it was established

To investigate the specificity of human tumor-infiltrating lymphocytes (TIL) against autologous tumors, TIL from five metastatic melanoma patients were expanded with rIL-2 and assessed for cytotoxicity in chromium release assays. TIL from a patient showing preferential cytotoxicity against autologous melanoma cells were further analysed. TIL were cloned by limiting dilution. Four out of 27 clones showed substantial cytotoxicity against autologous melanoma and one clone, designated as No. 8a-5 (CD3+, CD4-, CD8+, CD56-), selectively killed autologous melanoma but did not kill six different allogeneic melanoma, K562, or autologous or allogeneic Con A lymphoblast targets. Cytotoxicity of No. 8a-5 cells was inhibited by anti-HLA class I MAb (w6/32), by anti-beta 2-microglobulin MAb, and by anti-CD3 (OKT3) MAb, suggesting that the specific cytotoxicity was HLA class I-restricted and that the clone utilized the T-cell receptor complex for recognition of targets. Pretreatment with rIFN-gamma increased the sensitivity of autologous melanoma targets to lysis by No. 8a-5 cells. Exogenous rIL-4 enhanced [3H]TdR incorporation by these TIL. In contrast, rIFN-gamma reduced the sensitivity of the autologous melanoma to lysis by uncloned TIL, and rIL-4 suppressed the cytotoxicity and cell proliferation of uncloned TIL. These results indicate that both specific and non-specific cytotoxic cells can be developed from the same TIL and that these can be differentially regulated.     

MHC class-I-restricted auto-tumor-specific CD4+CD8- T-cell clones established from autologous mixed lymphocyte-tumor-cell culture (MLTC).

Autologous mixed lymphocyte-tumor cell cultures (MLTC) were initiated with cytokine (IFN gamma and TNF alpha)-treated ex-vivo tumor cells of lung, ovarian, breast and stomach carcinomas. The cytokine-treated tumors expressed class-I but not class-II molecules. Although the proportion of CD8+ lymphocytes increased in the bulk culture of MLTCs, in 5/7 experiments the majority of the established T-cell clones were CD4+. Among the CD8+ clones a high proportion (77%) was cytotoxic, while the proliferative response was more frequent among the CD4+ clones (70%). In 4/26 cytotoxic T-lymphocyte (CTL) clones (3/17 CD4+ and 1/9 CD8+), derived from a patient with class I+ class II- stomach carcinoma, lysis was restricted to the autologous tumor cells. These auto-tumor-specific clones did not lyse the autologous ConA blasts, the 5 allogeneic ex-vivo tumors, the NK-sensitive K562 or the relatively sensitive Daudi cells. The cytotoxicity of these clones was inhibited by pre-incubation of the tumor cells with W6/32 (alpha-class I) MAb, or by preincubation of the lymphocytes with OKT3 (alpha-CD3) MAb. The alpha-CD4 (OKT4) MAb had only a marginal effect on the CD4+ clones, while the lytic function of the CD8+ clone was inhibited by the alpha-CD8 (OKT8) MAb. The 3 CD4+ CTL clones also responded with proliferation to the autologous tumor cells. This proliferative response was inhibited by the presence of W6/32 MAb. Our results indicate that the auto-tumor lysis exerted by CD4+ CTL clones was restricted by the class-I antigens, and that the CD4 molecules of the clones were not essential for the lytic interaction.     

Clinical application of adoptive immunotherapy by cytotoxic T lymphocytes induced from tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TIL) were cultured with interleukin 2 (IL-2) to induce the cytotoxic T lymphocytes possessing autologous tumor-killing activity from 21 cancer patients (11 with solid tumor and 10 with malignant peritoneal or pleural effusions), and transferred into 7 patients as IL-2-activated TIL adoptively. The clinical application of activated TIL by adoptive transfer could result the complete regression of malignant pleural effusions in a patient with pancreatic cancer, and the nearly complete regression of malignant ascites in a patient with gastric cancer. The autologous tumor cells were isolated at the purity of more than 90% by Ficoll-Hypaque and Percoll discontinuous gradients, and then the TIL were cultured with IL-2 until 4 weeks. The optimal concentration of IL-2 was 1,500 IU/ml to obtain maximum proliferation and autologous tumor killing activity. The cytotoxic activities of activated TIL at 3 weeks-incubation was 72 +/- 15, 42 +/- 26, 27 +/- 21 and 22 +/- 15% against K562, Daudi, KATO-III and autologous tumor, respectively. By negative selection method, it was clarified that the killer cells recognizing autologous tumor consisted of CD4 or CD8 positive T lymphocyte in 43% of patients. The CD8 positive cells and CD56 positive cells increased, the CD4 positive cells and CD16 positive cells decreased by flow cytometry. The activated TIL could lyse not only cultured tumor cell lines, also other autologous tumor cells. The CD56+ cells were isolated by the Panning method, these cells could not lyse autologous tumor cells. Thus, it was indicated that the cytotoxic T lymphocytes recognizing autologous tumor could be generated from TIL and the adoptive immunotherapy of activated TIL was effective in cancer therapy.     

Tumor‐reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor‐reactive T‐cell subset in metastatic colorectal cancers

High level of T‐cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor‐reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor‐reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4⁺CD8αβ⁺ (DP) αβ T cells. Interestingly, a high proportion (16–20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class‐I, proliferated poorly and secreted higher amounts of IL‐4 and IL‐13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor‐reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL‐4 and IL‐13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.     

TIL from renal-cell carcinoma: restimulation with tumor influences proliferation and cytolytic activity

TIL were cultured from human renal-cell carcinoma (RCC) in 1,000 U/ml rIL2 and restimulated with autologous tumor in efforts to establish which conditions would best expand the number of lymphocytes cytotoxic for autologous tumor. Greater cell yields resulted from multiple restimulations of TIL with autologous tumor. In most instances, these TIL lysed autologous tumor better than TIL grown in rIL2 alone. Enhanced proliferation was seen also after restimulation of TIL with allogeneic RCC as well as with tumor cells of non-renal origin. Although in some instances lysis of autologous tumor appeared to be specific, restimulation with autologous tumor did not consistently result in the generation of specific cytolytic T cells. Attempts to culture more specific cytolytic T cells by using 50 U/ml rIL2 were successful in expanding TIL with enhanced lytic activity; however, this activity was not specific for autologous tumor. The phenotype of the tumor-restimulated TIL generally did not change. In most of the TIL cultures, CD3+CD4+ cells were predominant with low numbers of CD3+Leu19+ cells and minimal numbers of CD3-Leu19+ cells. Thus, the cytotoxic response to tumor was mediated by T cells and not NK cells. Overall, our data indicate that restimulation of TIL with autologous tumor may be beneficial for growing larger numbers of cells which have increased lytic activity and for prolonging the presence of lytic activity among the expanded TIL.     

Common tcr-Beta chain rearrangements in short-term cultured lymphocytes infiltrating ovarian-tumors

We have recently reported that tumor reactive T cell lines and clones can be developed from lymphocytes infiltrating ovarian malignant tumors and ascites (TIL/TAL). These cells are currently used for immunotherapy of ovarian cancer. In this study we investigated the presence of common non-germline rearrangements at TCR Cbeta1 among freshly isolated and cultured ovarian TIL/TAL. The cultured ovarian TIL/TAL show a characteristic T cell phenotype (>95% CD3+) and mediate tumor target lysis. Our results show that non-germline rearrangements can be detected in cultured ovarian TAL isolated from ascites but not in freshly isolated TAL. Certain of rearranged bands were common among cultured TAL. In contrast, in samples of freshly isolated TIL from solid ovarian tumors localized at different organ sites, common rearrangements were found among fresh and cultured TIL. The results suggest that the observed polyclonality of T cells isolated from freshly collected ascitic tumors may be due, at least in part, to either dilution by passenger lymphocytes, or the lack of an in vivo oligoclonal response to autologous tumor, and indicate the usefulness of molecular analysis of TCR V gene segments in T cells used for immunotherapy.     

Immunotherapy of bovine ocular squamous cell carcinoma: isolation, culture and characterization of lymphocytes present in the tumor

Bovine ocular squamous cell carcinoma (BOSCC) is sensitive to intralesional immunotherapy with BCG or recombinant human IL-2 (rhIL-2). The mechanism of tumor regression is as yet unclear. Alterations in the concentration of IL-2 (and possibly other factors) in the tumor, due to regional injection or induction by BCG, may induce killer cell activity and thus tumor regression. To investigate this, lymphocytes were isolated by mechanical fractionation of biopsies of BOSCC. Growth, phenotypical, and functional characteristics were studied. TIL could be isolated and grown from all biopsies of BOSCC. An estimated increase in cell number of 50-150 fold was observed during 5-7 weeks of culture. FACS analysis of a limited number of the TIL cultures showed a characteristic shift in phenotypes until day 28 of culture. CD2+ cells (50-70%), and as a consequence of this CD2- cells, remained stable in number. The number of CD8+ cells increased. CD4+ cells were detected in low numbers by day 28. Prolonged culture resulted in an increase of CD2- gamma delta + cells, CD2+4-8- cells, and occasionally of both CD8+ and CD2+ cells. In 51Cr release assays TIL showed cytotoxicity for BOSCC-derived tumor cell lines in general, which increased transiently by cocultivation with tumor cells. Killing of YAC-1, and P815 was far less efficient. Preferential killing of autologous cell lines was not seen. In conclusion, TIL from bovine ocular squamous cell carcinomas can be cultured in the presence of rhIL-2, which induces cytotoxic activity for BOSCC-derived tumor cells. Cells responsible for killing in vitro and potentially for regression of the tumor after immunotherapy with BCG or rhIL2 cannot yet be identified. Depletion and blocking experiments are being conducted in order to identify the cells (CD2+8+, CD2-gamma delta + or other CD2 +/-) responsible for killing.     

Peripheral blood and tumor infiltrating lymphocytes in non-small cell lung cancer: analysis at the population and clonal level

Tumor infiltrating (TIL) and peripheral blood lymphocytes (PBL) were isolated from 18 patients with non-small cell lung cancer undergoing radical surgery. Surface marker analysis revealed that TILs and PBLs mainly consisted of CD3+ T cells and that TILs generally displayed a lower CD4/CD8 ratio. Differences were found in the expression of CD25 (IL-2 receptor) and DR (MHC class II) antigens, which were increased in TILs, and in the percentage of CD16+ natural killer (NK) cells, which was reduced in TILs as compared to PBLs. Accordingly, the NK activity of TILs was lower than that of PBLs, whereas neither TILs nor PBLs expressed spontaneous cytolytic activity against fresh autologous tumor cells, melanoma cells and the "NK-resistant" A549 lung carcinoma cell line. After 4 days of culture in medium with recombinant-interleukin-2 (rIL-2), TILs and PBLs acquired cytolytic activity against all cell targets, but TILs expressed higher levels of cytotoxicity than autologous PBLs only in 3 patients out of 16 tested. More importantly, both TILs and PBLs displayed similar levels of cytotoxic activity against autologous tumor cells. TILs and PBLs from 8 patients were also analyzed by a limiting dilution microculture system. Cloning efficiency was remarkably lower in TILs, and surface marker analysis of T cell clones confirmed that an accumulation of CD8+ lymphocytes, which displayed cytolytic activity in a lectin-dependent assay, occurred at the tumor site. The non-MHC-restricted cytolytic activity of TIL- and PBL-derived T cell clones against K562, A549, and allogeneic melanoma cells and the cytolytic activity against autologous tumor cells showed no significant differences. Only 53% of TIL clones released IL-2 in response to PHA + TPA stimulation, whereas 68% of PBL-derived clones were IL-2 producers. Moreover, most PBL- and TIL-derived clones released tumor necrosis factor alpha in response to mitogen stimulation.     

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 tumorinfiltrating 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-ICAMI but not by anti-HLA class-1 (W6/32, B9.12.I), 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. © 1994 Wiley-Liss, Inc.     

Human melanoma-mediated inhibition of autologous CD4+ helper tumor-infiltrating lymphocyte growth in vitro.

Tumor-infiltrating lymphocytes (TIL) were isolated from a human melanoma metastatic to the abdomen. The TIL were 99% CD3+ and 99% CD4+ and CD8-. They were dependent on interleukin-2 (IL-2) for growth, as measured in a thymidine uptake assay, and were not cytotoxic to autologous or allogeneic melanoma or K562. When co-cultured with irradiated autologous tumor cells, or tumor cell supernatants, the TIL not only did not respond, but the IL-2-dependent growth was inhibited significantly. Inhibition occurred during the first 24 hours of co-culture and persisted as long as the tumor was present. After being washed free of inhibitory tumor cells, the TIL again were able to grow in the presence of IL-2, indicating that the inhibition was not caused by irreversible toxicity mediated by the tumor. Addition of excess IL-2 did not reverse the inhibitory effect, but addition of indomethacin, an inhibitor of cyclooxygenase and prostaglandin synthesis, partially blocked the inhibition. These data show melanoma-mediated inhibition of induction and expansion of human T-cells in vitro, which may reflect one of the mechanisms of inhibition of cellular responses in vivo. These results stress the need to examine the techniques for optimal in vitro expansion of tumor-specific TIL or cytotoxic T-cells for adoptive immunotherapy.     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

A Correlation between the Expression of CD 8 Antigen and Specific Cytotoxicity of Tumor-infiltrating Lymphocytes

Tumor-infiltrating lymphocytes (TIL) from six gynecologic malignant tumors (two uterine cervical cancers, two ovarian serous cystadenocarcinomas, and two uterine corpus cancers), cultured in the presence of recombinant interleukin 2, were assayed for their cytotoxic activities against various fresh tumor cells including autologous tumors. A clear correlation between phenotype and cytotoxic activity of TIL was observed. Four of six TIL preparations exhibited strong cytotoxic activity against autologous fresh tumor target cells, and were all CD8^±. In contrast, cytotoxic activity was not detected in any of the CD4^± TIL preparations. The cytotoxic activities of the CD8^± TIL preparations were highly specific; only autologous fresh tumor cells were lysed. This result is consistent with the notion that TIL are of a different cell lineage from lympholdne-activated killer cells which are antigen-nonspecific and CD8^-. Instead, TIL appear to be of cytotoxic T cell lineage that is highly antigen-specific and CD8^±. To explore the potential for clinical use, we have attempted to augment the cytotoxic activities of these CD8^± TIL by treatment of the target tumor cells with gamma interferon (IFN) in vitro , hoping that elevated expression of MHC class I gene products on the cell surface would enhance their recognition. It was observed that brief treatment of freshly prepared tumor cells in vitro with gamma-IFN resulted in augmentation of the expression of MHC class I gene products, and the treated tumor cells were more susceptible to lysis by TIL than untreated cells.     

Interleukin 2 activation of cytotoxic T-lymphocytes infiltrating into human metastatic melanomas

Tumor infiltrating lymphocytes (TIL) were isolated from 22 tumors obtained from 15 patients with metastatic melanoma. In 18 of the 22 tumors, a substantial number of lymphocytes was isolated with an average lymphoid cell:tumor cell ratio of 1.26. The TIL were predominantly cytotoxic/suppressor T-lymphocytes with an average of 87% Leu4+, 61% Leu2a+, and 18% Leu3a+ cells. There were less than 2% natural killer cells, B-cells, or macrophages. An average of 3.8% (range, less than 0.1 to 8.6%) of freshly isolated TIL bound to autologous tumor cells. Prior to culture, none of the tumor-binding cells (TBC) was cytotoxic as judged by trypan blue exclusion. The frequency of TBC increased to 11.6% after 2 days of culture, and 10% of these TBC developed cytotoxic activity. When interleukin 2 was added to cultures, the frequency of TBC increased, and the frequency of cytotoxic TBC was 2-fold higher compared to control cultures. After 10 days of culture with interleukin 2, TIL increased in number with a concomitant disappearance of tumor cells, whereas there were severe decreases of lymphocytes and no decrease of tumor cells in control cultures. TIL were cultured for 8 to 10 days with recombinant interleukin 2 and tested for cytotoxicity against autologous and allogenic tumor cells and K562 targets in a 4-h 51Cr release assay. rIL2-cultured TIL from all nine patients tested exhibited the highest levels of lysis against autologous tumor cells. Of the nine TIL samples, five exhibited an apparent specificity for autologous melanoma, while four specimens killed both allogenic and autologous melanoma. The ability of TIL to kill K562 targets appeared to parallel the ability to kill allogenic targets. For comparison, recombinant interleukin 2-cultured peripheral blood mononuclear cells from the same patients were assayed for cytotoxic activity against autologous and allogenic melanomas. Unlike some TIL, none of the peripheral blood mononuclear cells exhibited specificity for autologous tumor cells. In summary, TIL isolated from metastatic melanoma patients were predominantly cytotoxic T-lymphocytes with the ability to recognize and kill autologous tumor cells after in vitro culture; interleukin 2 induced proliferation of TIL and augmented their cytotoxic activity such that they eliminated autologous tumor cells.