Characterization of the cytolytic activity of CD4+ and CD8+ tumor-infiltrating lymphocytes in human renal cell carcinoma

Previously we showed that IL2 expanded tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma mediated non-major histocompatibility complex-restricted cytotoxicity. Phenotypic analysis showed that cultured TILs were composed mostly of T-lymphocytes with varying numbers of CD4+, CD8+, and CD56+ (Leu19+) populations. Here we compared the cytolytic activity of the two predominant TIL subsets, CD3+CD4+ and CD3+CD8+, to that of the CD56+ populations. Using magnetic beads coated with antibodies to either CD4 or CD8, CD3+CD4+, and CD3+CD8+ TILs were isolated in a highly enriched form (greater than 92%) and could be expanded for over 40 days in vitro with 1000 units/ml IL2. In a 4-h 51Cr release assay the CD4+ and CD8+ TILs showed minimal lytic activity, whereas unseparated cells exhibited significant levels of non-major histocompatibility complex-restricted cytotoxicity. The lytic activity seen in the 4-h assay with unseparated TILs appeared to be related to the presence of CD56+ populations. With one exception none of the purified CD4+ or CD8+ TILs expressed any significant levels of CD56, while the unseparated TILs contained varying numbers of CD3+CD56+ and CD3-CD56+ populations. Cell-sorting experiments verified that the CD56+ populations were responsible for most of the lytic activity in 4 h even though CD3+CD56- cells represented the predominant cell type. Although CD3+CD56- TILs were minimally lytic in 4 h, we show here that both CD3+CD4+ and CD3+CD8+ subsets displayed substantial cytotoxicity in long-term assays. In the 18-h 51Cr release assay 5 of 6 CD4+ and 2 of 3 CD8+ TILs were lytic for the autologous tumor. In two cases, restimulation with the autologous tumor induced augmented cytolytic activity of TIL subsets and in one case induced lytic activity in 4 h. The cytotoxic activity of TIL subsets was further examined using a 72-h assay in which TILs were cocultured with a confluent layer of tumor cells. The degree of cytotoxicity was quantitated by measuring the amount of crystal violet dye that was incorporated by tumor cells which remained after the incubation period. CD4+ and CD8+ TILs typically caused greater than a 50% reduction of tumor cells in 3 days and the level of reduction was increased when IL2 was added to the cultures. All the CD4+ and CD8+ subset preparations were cytotoxic in the 3-day assay even though some were not lytic for certain targets in the 18-h 51Cr release assay.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 autologous tumor-specific T cells in malignant melanoma

T cell lines and clones with autologous tumor-specific activity have been developed in malignant melanoma by stimulating peripheral blood lymphocytes (PBL), lymph node lymphocytes or tumor-infiltrating lymphocytes (TIL) with autologous melanoma cells in the presence of recombinant interleukin 2 (rIL2). T-cell lines and clones have been developed with specific cytotoxicity and/or proliferative responses for autologous melanoma targets but not for allogeneic melanoma tumor cells, autologous normal cells or natural killer (NK)-sensitive targets. The concentration of rIL2 is critical for the generation of autologous tumor-specific T-cell lines, with low rIL2 concentrations (up to 800 IU/ml) facilitating the growth of T-cell lines with tumor-specific activity. The T-cell receptor (TCR) and the CD3 antigen are involved in specific cytotoxicity and/or proliferative responses of these T-cell lines and clones. An oligoclonal pattern of -chain TCR gene rearrangements was observed on T-cell lines and clones with autologous tumor-specific cytotoxicity, suggesting that they are comprised of T cells that have undergone a clonal expansion in response to particular antigen. Autologous tumor-specific cytotoxic T cells are HLA-restricted and recognize on the melanoma tumor cells HLA Class I or possibly Class II antigens plus a tumor-specific determinant. TIL from patients with metastatic melanoma have unique characteristics in comparison with PBL and lymph node lymphocytes and they appear to contain substantial proportions of T cells that have been locally sensitized to autologous tumor cells. Single stimulation of TIL with autologous tumor cells in the presence of rIL2 is sufficient for the generation of T cell lines with autologous tumor-specific activity, whereas, multiple stimulation of PBL and lymph node lymphocytes was required to achieve the same purpose. TIL-derived T cell lines have been expanded in rIL2 in vitro by at least 1,500-fold without losing their activity. Approximately, 40% of the patients exhibited complete or partial responses to adoptive immunotherapy with melanoma TIL and rIL2.     

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.     

Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells

Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.     

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.     

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.     

Lymphocytes infiltrating human ovarian tumors: synergy between tumor necrosis factor alpha and interleukin 2 in the generation of CD8+ effectors from tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TIL) were isolated by enzymatic digestion and gradient centrifugation from 18 human ovarian carcinomas. These cells were cultured in a complete medium supplemented with recombinant interleukin 2 (IL2) alone or recombinant IL2 plus recombinant tumor necrosis factor alpha (TNF-alpha), and their growth and antitumor cytotoxicity were determined. TIL cultured in the presence of IL2 plus TNF-alpha (1000 units/ml each) for 6 days showed significantly higher cytotoxicity against fresh autologous tumor targets than did TIL cultured with IL2 alone (e.g., mean lytic units/10(7) cells for 8 TIL preparations were 290 versus 74; P less than 0.05). No differences in [3H]thymidine uptake or natural killer cell activity were observed among these TIL cultures. In titration experiments, optimal synergistic concentrations of IL2 and TNF-alpha were determined as 10(2) and 10(3) units/ml, respectively. Using these concentrations for culturing the TIL, effector cells developed which preferentially lysed autologous tumor and displayed a CD8+ phenotype (up to 75% positive). However, the autologous tumor cytotoxicity mediated by these cultured TIL on day 6 was short lived. By day 12, it was replaced by non-major histocompatibility complex-restricted, lymphokine-activated killer cell-like activity mediated by CD3-CD56+ effector cells. Simultaneously, the production of gamma-interferon and interleukin 1 decreased in these cultures. In contrast to TNF-alpha, anti-CD3 antibody synergized with IL2 to increase 2-3-fold TIL proliferation but not their cytotoxic activity against autologous tumor cell targets. These data suggest that TNF-alpha and IL2 synergize early in culture to induce tumor-reactive CD8+ effectors, some of which may be specific for autologous ovarian tumor cells. However, the conditions needed to sustain the specific autologous tumor responses in long-term cultures of human TIL remain to be determined.     

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.     

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.     

Proliferative and cytolytic potentials of purified human tumor-infiltrating T lymphocytes. Impaired response to mitogen-driven stimulation despite T-cell receptor expression

Using limiting dilution analysis (LDA) we have previously shown that in most instances, the frequency (F) of proliferative T lymphocyte precursors (PTL-P) was strikingly reduced in tumor-infiltrating lymphocytes (TIL). In this study involving 19 cases, we show that the impaired clonogenic potential of CD2+ TILs is primarily caused by an intrinsic defect rather than to suppressor T cells or to a direct effect of the tumor cells usually present in the culture system. This was demonstrated by experiments in which the F of PTL-Ps was quantitated both in highly purified CD2+ TILs (using a cell-sorter) and in non-purified TIL suspensions (still containing tumor cells), which originated from the same biopsy specimen. The F of PTL-Ps was virtually identical in either sorted or nonsorted suspensions and the data from LDA were always consistent with the single-hit Poisson model, indicating that no suppressor cells interfered with growth of CD2+ TIL. Stimulation of sorted CD2+ TIL in low-density cultures by either phytohemagglutinin or anti-CD3-monoclonal antibody (MAb) indicated that the antigen-dependent activation pathway was impaired, although structurally intact T-cell receptor (TCR) complexes were apparently expressed, as assessed by immunofluorescence. The depressed proliferative response of CD2+ TIL could not be reversed in vitro when phorbol-esters were used in combination with ionomycin, which bypass the TCR. Nevertheless, 180 clones obtained from 8 cases were analyzed for their cytolytic activity. The majority mediated specific lytic activity (against unknown antigens), as assessed by lectin-dependent cell cytotoxicity, whereas only 6% of them manifested lymphokine-activated killing on appropriate targets.     

Tumor lysate and IL-18 loaded dendritic cells elicits Th1 response,tumor-specific CD8+ cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor lysate-loaded dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumor cells in patients with malignant glioma. CTL from three of five patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts and were variably cytotoxic against the LAK-sensitive cell line Daudi. Also, DCs pulsed normal brain lysate failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two of five patients CD8+ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8+ T cells isolated during these ineffective primings secreted large amounts of IL-10, less amounts of IFN-γ as detected by ELISA, Type 2 bias in the CD8+ T cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor lysate-loaded DC can be an effective tool in inducing glioma-specific CD8+ CTL able to kill autologous glioma cells in vitro. However, high levels of tumor specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. Moreover, cytotoxic responses were augmented by transfecting DC with the gene for IL-18. For all five patients, CD8+T cells treated with IL18 transfected DC produced Th1 response. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs loaded with total tumor lysate and IL-18 may represent a method for inducing Th1 immunoresponses against the entire repertoire of glioma antigens.     

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.     

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.     

In vitro induction of cytotoxic activity against carcinomatous pleural or peritoneal lymphoid cells cultivated with cytokines, and an immunological phenotypic analysis of the effector cells

The functional and phenotypic characteristics of carcinomatous pleural or peritoneal lymphoid cells cultivated with either rIL 2 or TCGF have been investigated. The cultivation of the lymphoid cells with cytokines was initiated by a mixture of coexisting, viable carcinoma cells for 14 days. Results have indicated that cytokine-activated lymphoid cells from malignant pleural and peritoneal effusions showed considerable cytolytic activity against K562 and Daudi cells. The cell population responsible for LAK and/or CTL effector cells of TCGF-activated lymphoid cells were CD8+ CD11- cells. Further, in rIL 2-expanded cultures from pleural and peritoneal lymphoid cells, the CD4+ Leu 8- population was found to contain effector cells of cytotoxic activity against the tumor cells. It further was seen that the TCGF-activated CD8+ CD11- T cells possessed a more potent killing activity, in comparison to the rIL 2-activated CD4+ Leu8- T cells. However, rIL 2-activated lymphoid cells from ascites in liver cirrhosis (used for a control) showed a higher tumoricidal activity.     

Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

人细胞毒T淋巴细胞杀伤胃癌细胞及诱发凋亡的实验研究

目的　测定特异性细胞毒 T淋巴细胞 (CTL )对胃癌细胞的体外杀伤活性及观察形态学改变。方法　用分离胃腺癌患者的癌细胞经丝裂霉素 C处理后 ,作为抗原与抗CD3单克隆抗体、重组白细胞介素 - 2刺激患者自体外周血单个核细胞 ,体外诱导 CTL,用 MTT比色法测定 CTL 对自体癌细胞、人胃癌细胞株 (BGC- 82 3和 MGC- 80 3)的体外杀伤活性。将 CTL与 BGC- 82 3共同温育 ,通过透射和扫描电镜观察杀伤靶细胞的形态改变。结果　 CTL对自体胃癌细胞有特异性杀伤作用 ,可使靶细胞凋亡或溶解坏死。结论　体外混合细胞培养诱导出的 CTL,对靶细胞有明显的杀伤作用     

LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.     

Tumor‐specific cytokine release by donor T cells induces an effective host anti‐tumor response through recruitment of host naive antigen presenting cells

We recently reported that tumor eradication induced by immunotherapy (IT) in a congenic mouse model using tumor infiltrating lymphocytes (TIL) + recombinant interleukin‐2 (rIL‐2) is dependent on recruitment of naive host immune cells at the tumor sites. The recruitment of host immune cells was induced mainly through a local secretion of interferon‐γ (IFN‐γ) produced by donor T cells. We now further investigated how a non‐specific inflammatory response progresses to a host T‐cell‐mediated tumor‐specific response. In crossover experiments using MCA‐105 and MCA‐205 sarcoma tumors, pulmonary metastatic disease was eradicated only in mice treated with tumor‐matched TIL + rIL‐2. In vitro, TIL stimulated with the tumor of origin secreted relatively high levels of IFN‐γ and granulocyte‐macrophage colony stimulating factor (GM‐CSF) compared to TIL stimulated with mismatched tumor cells. In lungs of tumor‐bearing mice treated with matched TIL + rIL‐2, significant increases in the percentages of IFN‐γ, GM‐CSF and tumor necrosis factor‐α (TNF‐α) positive cells were detected, as well as of macrophages, natural killer (NK) cells and dendritic cells. Depletion of macrophages or NK cells did not inhibit the efficacy. In contrast, depletion of dendritic cells partially inhibited the efficacy of the treatment. Combined depletion of dendritic cells and macrophages abrogated more than 80% of the efficacy. Our data suggest that successful IT may require 3 steps: (1) release of inflammatory cytokines by donor TIL after restimulation by tumor cells; (2) infiltration of host immune cells in response to local cytokine production; and (3) activation of tumor‐specific host immune cells by dendritic cells and to a lesser extent by macrophages. Int. J. Cancer 80:308–314, 1999. © 1999 Wiley‐Liss, Inc.     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.