Solid-phase anti-CD3 antibody activation and cryopreservation of human tumor-infiltrating lymphocytes derived from epithelial ovarian cancer.

The effect of solid-phase anti-CD3 antibody activation and cryopreservation was evaluated on thirteen samples of tumor-infiltrating lymphocytes (TILs) derived from epithelial ovarian cancer. Seven preparations of TILs were cultured with or without solid-phase anti-CD3 antibody in addition to 100 units/ml of recombinant interleukin-2 (rIL-2). The proliferation rate of all of the seven TIL preparations stimulated by anti-CD3 antibody on the fourth or fifth day of culture was 3.4 to 9.8 times greater than that of lymphocytes cultured with rIL-2 alone. Furthermore, in an experiment with five TIL samples activated with anti-CD3 antibody, three of them showed augmented cytotoxic activity against autologous fresh tumor cells. The population of CD3+/CD8+ TILs was increased after 4-5 weeks of cultivation and CD8+ lymphocytes amounted to over 70% in all of seven preparations tested, whereas two of seven preparations not activated by anti-CD3 antibody were CD3+/CD4(+)-dominant. In addition, nine preparations of TILs cultured with rIL-2 were cryopreserved for several weeks; after recovery from cryopreservation, no major change was observed in cell surface markers, in growth rate or in cytotoxic activity. These results suggest that cryopreserved and/or anti-CD3 antibody-activated lymphocytes could conveniently be employed in a clinical trial of adoptive immunotherapy employing TIL.     

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.     

Antitumor activity and surface phenotypes of human glioma-infiltrating lymphocytes after in vitro expansion in the presence of interleukin 2

The present study describes a method for in vitro expansion and characterization of antitumor-reactive lymphoid cells isolated from human malignant astrocytomas. Glioma-infiltrating lymphocytes were separated from 24 glioma specimens and cultured in medium containing interleukin 2 (50 to 2000 units/ml). Within 20 to 42 days after the initiation of culture, 20 of 24 cultures of glioma-derived lymphocytes expanded with a substantial increase in cell numbers, of at least 5 x 10(8) cells up to 5 x 10(9), with a simultaneous elimination of contaminating autologous glioma cells. The expanding glioma-derived lymphocytes consisted of 90 +/- 8% (SD) CD3+ T-cells including both CD4+ and CD8+ subpopulations. CD16 was expressed on 4 +/- 5% of the cells and three cultures studied exhibited 14% +/- 1 of Leu-19-positive cells. After 4 to 8 weeks of proliferation, interleukin 2 receptor expression decreased from 36 +/- 28% to less than 10% and the lymphocytes ceased to grow in all cultures. Glioma-derived effector lymphocytes could lyse almost all the autologous tumor targets as well as allogeneic glioma cells. The cytotoxic activity of long-term cultured peripheral blood lymphocytes obtained from the same patients appeared to be similar to that of glioma-derived lymphocytes in killing autologous tumor cells. In summary, glioma-derived lymphocytes expanded in bulk culture with high concentrations of interleukin 2 (2000 units/ml) consisted predominantly of T-lymphoblasts with the ability to kill autologous glioma cells. The tumor-infiltrating lymphocytes could be expanded to sufficient numbers for possible use in the adoptive immunotherapy of malignant gliomas.     

Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization, and antitumor activity

We have described a method for the generation, from fresh human renal cell cancers, of lymphoid cells that are capable of exhibiting significant antitumor reactivity when tested in short term 51Cr release assays. Tumor cell suspensions obtained from 37 consecutive fresh human renal cell cancer specimens (35 patients) could be separated by using enzymatic techniques and culturing in medium containing recombinant interleukin 2 (IL-2). The total cell recovery was 1.5 X 10(9) +/- 2.2 (SE) per tumor with a range of 1 X 10(8) to 5 X 10(9) cells. The percentage of tumor cells in the suspension ranged from 6 to 75% with a mean of 39.1 +/- 3.3%. The remaining cells were predominantly lymphocytes. Viability of mononuclear cells was greater than 90%. Activated tumor-infiltrating lymphocytes (TIL) within these tumors expand and by 10 to 14 days after initiation of culture a 5- to 15-fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. Lymphocytes were recultured in fresh medium containing IL-2 and continued to expand between 2- and 10-fold every 4 to 6 days for an average of 33.7 +/- 4.5 days, resulting in greater than 50,000-fold increase in the total number of lymphocytes. The average number of splits was 4.9 +/- 0.8, with a range of 0 to 21. In 11 of 11 cases tested, TIL exhibited a far better expansion capability in vitro compared to that of peripheral blood lymphocytes obtained from the same patient and grown under identical conditions. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). With continued in vitro expansion (up to 50 days) there was a concomitant increase in the helper T (Leu 3+) and pan T populations (Leu 4+) and decrease in Leu 2+ and HLA-DR+ cells. Compared with expanded peripheral blood lymphocytes, these cells demonstrated higher levels of IL-2+ receptors and HLA-DR+ antigens. Renal TIL effectors expanded in IL-2 could lyse almost all autologous tumor targets in 4-h chromium release assays. Allogeneic renal as well as nonrenal targets were equally lysed. TIL lysis of cultured tumor targets K562 and Daudi was significantly better than lysis of autologous, allogeneic-renal, and nonrenal targets. No statistically significant difference in the cytotoxic activity of renal TIL or peripheral blood lymphocyte effectors in killing autologous or allogeneic targets could be demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors.

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.     

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.     

Lysis of fresh human tumor cells by autologous large granular lymphocytes from peripheral blood and pleural effusions

Human lymphocytes and their subpopulations from the peripheral blood and pleural effusions of cancer patients were tested for cytotoxicity against fresh tumor cells isolated from carcinomatous pleural effusions of the same patients. Fresh tumor cells were relatively resistant to lysis by autologous unseparated lymphocytes in a 4 h Cr-release assay. Positive reactions were recorded in 10 of 38 blood samples and 10 of 37 effusion specimens. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation resulted in enhancement of cytotoxic activity against autologous tumor cells and K562 cells, with no reactivity in LGL-depleted small T-lymphocyte populations. Significant lysis of effusion tumor cells by autologous LGL was observed in 15 of 22 blood specimens and 15 of 21 effusion samples. Further depletion of high-affinity sheep erythrocyte rosetting cells from Percoll-purified LGL populations gave an increase in autologous tumor-killing activity. Depletion of LGL/K562 conjugates from LGL populations decreased lysis of autologous tumor cells and K562 cells. Effusion tumor cells that were susceptible to lysis by allogeneic normal LGL were also killed by autologous LGL, and effusion tumor cells resistant to lysis by allogeneic NK cells were not lysed by autologous LGL. In a single-cell cytotoxicity assay in agarose, 4-26% LGL bound autologous tumor cells and 0.2-5% LGL killed these target cells, while 12-45% LGL bound K562 cells and 2-20% LGL lysed them. These results indicate that cytotoxic potential for autologous effusion tumor cells is present in the peripheral blood and pleural effusions of cancer patients; it is strongly associated with a minor proportion of LGL and restricted to the cell population that can lyse NK-sensitive K562 cells.     

Solid-phase anti-CD3 antibody activation of murine tumor-infiltrating lymphocytes

Seventeen consecutive s.c. murine tumors, derived from a sarcoma and a colon adenocarcinoma, were cultured in the presence of recombinant interleukin 2 (rIL-2) for growth of tumor-infiltrating lymphocytes (TIL). Identical cultures were activated by solid-phase monoclonal antibody directed against the murine CD3 epsilon-chain, in conjunction with rIL-2. Forty-eight h later, cells were replaced in rIL-2 alone. Proliferation of anti-CD3-stimulated cultures was 1- to 17-fold greater than those cultured with rIL-2 alone (P less than 0.05). Both culture conditions yielded TIL which stained greater than 80% Thy-1.2+/Lyt-2+ (P greater than 0.05), less than 7% Thy-1.2+/L3T4+ (P greater than 0.05). Regardless of culture condition, longitudinal studies of in vitro cytotoxicity generated from 10 TIL preparations revealed no significant differences between the ability of TIL to lyse the murine natural killer-sensitive line YAC or heterologous or autologous tumor (P greater than 0.05). In vivo antitumor activity of TIL was tested by the adoptive transfer of suboptimal doses of TIL plus systemic rIL-2 to mice with pulmonary micrometastatic disease. No difference in tumor regression was noted between the TIL cultured with anti-CD3 plus rIL-2 or with rIL-2 alone (P greater than 0.05). Anti-CD3 stimulation of murine TIL cultures significantly increases lymphocyte cell yield without alteration of their phenotype, in vitro tumoricidal activity, or in vivo therapeutic effect.     

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.     

Infiltration of interleukin-2-inducible killer cells in ascitic fluid and pleural effusions of advanced cancer patients

Using ascitic fluid or pleural effusion obtained from 13 ovarian or metastatic breast cancer patients, we separated tumor cells from effusion-associated lymphocytes (EAL) with Percoll density centrifugation. Lymphocytes were incubated with recombinant interleukin 2 (IL-2) for 3-4 days and then assessed for tumoricidal activity in a 51chromium-release assay. The IL-2-activated EAL were found to lyse autologous fresh tumor cells, as well as allogeneic fresh tumor cells and FMEX tumor cells, a melanoma cell line which is resistant to natural killer cell activity but is sensitive to lysis by lymphokine-activated killer cells. There was little or no tumoricidal activity seen in freshly isolated EAL or in EAL which were cultured in medium without IL-2. Phenotypically, the IL-2-activated EAL were largely CD3-, although some cytolytic activity was found in CD3+ populations. Also, most activity was found in cells positive for CD2 (OKT11) and CD16 (Leu 11b), and negative for the monocyte marker Leu M3. These results indicate that the activated cell types found in EAL were predominantly natural killer/lymphokine-activated killer-like with a small contribution from T-cells. Finally, EAL were readily activated by IL-2 in medium containing autologous effusion fluid, indicating that in situ activation of tumoricidal activity by IL-2 can occur in the face of potentially inhibitory substances or cells that may exist in the effusions. Direct introduction of IL-2 may therefore be a potential therapeutic modality of effusion-forming cancers.     

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.     

Lymphokine-activated killer-cell function of lymphocytes from peripheral blood, regional lymph nodes and tumor tissues of patients with oral cancer

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I+ and CD-25+ cells upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8+ cells, in addition to CD25+ and HNK-I+ cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL. In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8+ cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and tumor-infiltrating lymphocytes activated by PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10-100 micrograms/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN) alpha or IFN gamma. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with alpha-chain or beta-chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractionation experiments revealed that CD3-CD16+ large granular lymphocytes (LGL) and/or CD3+CD16- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.     

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.     

Clonal and functional analysis for the augmentation of tumour-infiltrating lymphocytes by interleukin 4.

In the adoptive immunotherapy for cancer, the amounts of induced effector cells play a major role in improving therapeutic efficacy. We have already demonstrated that interleukin 4 (IL-4) augments proliferation of tumour-infiltrating lymphocytes (TILs) without altering the cytotoxic activity against autologous tumour cells. The present study is designed to investigate how IL-4 augments TILs by using established TIL clones in terms of IL-2/IL-2 receptor system. CD4+, CD8+ and CD4+ CD8+ (double positive) TIL clones were established from cancer patients. At clonal level, IL-4 augmented the proliferation of IL-2-activated TIL clones irrespective of phenotypes. In order to clarify the mechanism of IL-4 at clonal level, the blocking assay by anti-IL-2 receptor alpha and beta chain and binding assay of IL-2 on the cell surface and the measurement of the internalisation of IL-2 in the cell were performed. It was clarified that IL-4 up-regulated the IL-2 receptor and then augmented the action of IL-2 molecule on the cell surface stimulated by IL-4. Furthermore, binding IL-2 internalised rapidly into the cells. Thus, it is suggested that signal transduction is augmented and proliferation of TILs is enhanced by IL-4 via the action of IL-2/IL-2 receptor system.     

Altered CD94/NKG2A and perforin expression reduce the cytotoxic activity in malignant pleural effusions

CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism.This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A.The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n = 19) and congestive heart failure (CHF) (n = 11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n = 10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay.Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression.In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space.     

Solid-phase Anti-CD3 Antibody Activation and Cryopreservation of Human Tumor-infiltrating Lymphocytes Derived from Epithelial Ovarian Cancer

The effect of solid-phase anti-CD3 antibody activation and cryopreservation was evaluated on thirteen samples of tumor-infiltrating lymphocytes (TILs) derived from epithelial ovarian cancer. Seven preparations of TILs were cultured with or without solid-phase anti-CD3 antibody in addition to 100 units/ml of recombinant interleukin-2 (rIL-2). The proliferation rate of all of the seven TIL preparations stimulated by anti-CD3 antibody on the fourth or fifth day of culture was 3.4 to 9.8 times greater than that of lymphocytes cultured with rIL-2 alone. Furthermore, in an experiment with five TIL samples activated with anti-CD3 antibody, three of them showed augmented cytotoxic activity against autologous fresh tumor cells. The population of CD3⁺/CD8⁺ TILs was increased after 4–5 weeks of cultivation and CD8⁺ lymphocytes amounted to over 70% in all of seven preparations tested, whereas two of seven preparations not activated by anti-CD3 antibody were CD3⁺/CD4⁺-dominant. In addition, nine preparations of TILs cultured with rIL-2 were cryopreserved for several weeks; after recovery from cryopreservation, no major change was observed in cell surface markers, in growth rate or in cytotoxic activity. These results suggest that cryopreserved and/or anti-CD3 antibody-activated lymphocytes could conveniently be employed in a clinical trial of adoptive immunotherapy employing TIL.     

Generation of CD4+ cytotoxic T lymphocytes stimulated by immobilized anti-CD3 monoclonal antibody and interleukin-2 in cancer patients

The proliferation of autologous tumor-reactive cytotoxic T lymphocytes (CTL), induced by autologous mixed lymphocyte tumor-cell culture, was remarkably enhanced by activation with immobilized anti-CD3 monoclonal antibody (MAb) and interleukin-2 (IL-2), as compared with IL-2 alone. The activated CTL exhibited high cytotoxicity against autologous tumor cells. Cytotoxicity against autologous tumor cells was inhibited by anti-HLA-DR MAb. In negative selection with immunomagnetic beads, cytotoxicity against autologous tumor cells was inhibited by the elimination of CD4⁺ cells. The major cell-surface antigens of the activated CTL were CD3⁺, CD4⁺, CD25⁺, CD45RO⁺and CD45RA^-, suggesting helper T cells, and the activated CTL produced IL-2. It is concluded that the CTL activated by immobilized anti-CD3 MAb and IL-2 were CD4 cells that had both killer and helper functions. Our findings indicate that adoptive immunotherapy using these activated CTL would be effective in cancer patients. © 1995 Wiley-Liss, Inc.     

Autologous mixed lymphocyte-tumor reaction and autologous mixed lymphocyte reaction. II. Generation of specific and non-specific killer T cells capable of lysing autologous tumor

The specific and non-specific nature of autotumor cytotoxicity induced in autologous mixed lymphocyte-tumor culture (AMLTC) and autologous mixed lymphocyte culture (AMLC) was studied in patients with carcinomatous pleural effusions. Small- and medium-sized blood lymphocytes that were isolated by centrifugation on discontinuous Percoll gradients did not lyse autologous, freshly isolated effusion tumor cells. In vitro activation of the small lymphocytes, but not of the medium lymphocytes, with autologous tumor cells generated cytotoxic potential restricted to autologous tumor. When stimulated with autologous non-malignant non-T cells, the medium lymphocytes, but not small lymphocytes, were triggered to cytotoxicity that acted not only on autologous tumor cells but also on allogeneic tumor cells, T blasts, and tumor cell lines. Experiments using monoclonal antibodies (MAb) and complement (C') showed that both types of killer cells were CD2+ CD3+ CD16- T cells. Autotumor cytotoxicity developed in AMLTC was mediated by the CD4- CD8+ T cell subset in 6 of 9 cases and the CD4+ CD8- subset in the other 3 cases. In contrast, cytotoxicity induced in AMLC was exerted exclusively by the CD8+ subset. The enrichment of blasts from cultured T cells on discontinuous density gradients enhanced autotumor killing activity, with no reactivity recorded for blast-depleted, resting T cells. Addition of mitomycin-C-treated large granular lymphocytes (LGL) to AMLTC abolished the induction of autotumor killer cells, whereas non-specific killer cells were generated in AMLC irrespective of the presence of LGL. These results indicate that stimulation of autoreactive T cells in AMLTC and in AMLC could induce 2 distinct types of autotumor killer cells.     

Tumor-reactive T-cells accumulate in lung cancer tissues but fail to respond due to tumor cell-derived factor.

The purpose of this study was to elucidate a possible immune response to tumor cells mediated by tumor-infiltrating lymphocytes (TIL) in lung cancer. In flow cytometry, the majority of T-cells of TIL were CD45RA-, CD45RO+, and CDw29high, and expressed HLA-DR. The expression of interleukin 2 receptor beta chain increased in both CD4+ and CD8+ TIL compared with both types of T-cells in peripheral blood. These results indicate that the major population of TIL is activated memory T-cells. The TIL preparation, which was usually contaminated with 5 to 10% tumor cells, did not exhibit any response in autologous mixed lymphocyte-tumor culture even in the presence of interleukin 2 (IL-2) in all five cases tested. Although purified T-cells from TIL showed the positive response in only 1 of 10 cases tested without addition of IL-2, it occurred in 7 of 10 cases in the addition of a low concentration of IL-2. The IL-2-dependent response to irradiated autologous tumor cells was suppressed when nonirradiated autologous tumor cells were added to the culture. Culture supernatants of four lung cancer cell lines and freshly prepared lung cancer cells obtained from 6 cases exhibited suppressive activity against anti-CD3 antibody-induced mitogenesis of peripheral blood mononuclear cells from healthy donors. We suggest that, taken together, (a) the major population of TIL in lung cancer are activated memory T-cells, and they include tumor-reactive ones, and that (b) the function of the TIL is impaired by unavailability of IL-2 and/or by suppression due to lung cancer cell-derived factor(s).