Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

Proliferative response of lymphocytes from ovarian cancer patients to autologous tumor cells

Summary Peripheral blood lymphocytes (PBL) from 43 patients with histologically confirmed ovarian carcinoma were stimulated in mixed lymphocyte-tumor culture (MLTC) with purified autologous tumor cells. Positive results, assessed as lymphocyte proliferation, were observed in 21 cases (48.8%). Lymphoid cells associated with ascitic fluid or infiltrating solid masses were in general less reactive than PBL as only 3/11 cases had positive MLTC. Tumor cells isolated from peritoneal effusions showed no significant difference in stimulatory potential as compared to the primary tumor. These results suggest that in an appreciable proportion of ovarian carcinoma patients (approximately 50%), lymphocytes have the potential to react to autologous tumor cells. Comprehension of the immunological mechanisms of antitumor resistance may have direct practical relevance for more effective treatment of neoplasms.     

Inhibition of human killer cells by autologous lymphocytes.

Human antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells is shown in this study to be inhibited by autologous lymphocytes. Inhibitor activity resides in a population of lymphocytes lacking Fc receptors, i.e., depletion of Fc receptor-bearing lymphocytes on immobolized enriches for inhibition. A T cell-enriched population does not inhibit. The effect is not steric inhibition since addition of large numbers of sheep or chicken erythrocytes does not decrease ADCC. Spontaneous cytotoxicity mediated by NK cells in the absence of added antibody is not inhibited by the FcR-depleted population, indicating that K and NK cells differ from each other in this respect.     

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Cellular interaction against autologous tumor cells between IL-2-cultured lymphocytes and fresh peripheral blood lymphocytes in patients with breast cancer given immuno-chemotherapy

In patients with Stage II or III breast cancer and in patients with liver metastases from breast cancer, we examined cellular interaction in the cytotoxicity against autologous tumor cells by interleukin-2(IL-2)-cultured lymphocytes (CL) and fresh peripheral blood lymphocytes (FPBL) treated with immunochemotherapy including OK-432 and cyclophosphamide. In flow cytometric analysis, CD8 + CD11b+ and CD16+ cells significantly decreased after immuno-chemotherapy in both groups of patients. A protocol study in Stage II or III breast cancer patients showed suppressive activity of FPBL on the cytotoxic activity of CL in 3/9 of the non-treatment group but no suppressive activity and enhancing activity in 3/7 in the immuno-chemotherapy group. Moreover, in 19 patients with liver metastases from breast cancer treated with immuno-chemotherapy including adoptive immunotherapy, FPBL in 6/19 showed enhancing activity, and in 8/19 suppressive activity in the lysis of autologous tumor cells. In assaysin vitro using autologous and allogeneic tumor cells, FPBL showed a partial specificity in cellular interaction against autologous tumor cells. CD4-depleted FPBL inhibited cytotoxicity of CL, while CD8-depleted FPBL enhanced cytotoxicity of CL in patients with liver metastases. These results suggest that immuno-chemotherapy eliminates the suppressive population in FPBL and may induce tumor regression if combined with adoptive immunotherapy using CL.Abbreviations IL-2 interleukin-2 - CL IL-2-cultured lymphocytes - FPBL fresh peripheral blood lymphocytes - AIT adoptive immunotherapy     

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.     

Phagocytosis of autologous lymphocytes by cervical preneoplastic and neoplastic cells

Eighty-nine random Pap smears of the uterine cervix were examined to evaluate the phagocytic abnormal cells (PACs) of atypical, dysplastic and neoplastic tissues against infiltrated blood cells. The results revealed that none of the PACs have been identified in atypical (II) and mild dysplastic cells (IIIa). Low levels (1.2%) of PACs were initially demonstrated in patients with moderate dysplasia (IIIb) that increased 1.7-fold in subsequent severe dysplasia (IIIc) and further increased 2.8-fold in invasive carcinomas of the cervix (V). In addition, the data showed age-related responsiveness toward the development of PACs, where 82% of old patients have developed phagocytic activity in moderate dysplastic cells compared with 27% of young patients. However, the difference became less significant at the subsequent classes of the disease. These data further demonstrated that PACs are class-dependent and it may explain one mechanism by which precancerous cells escape immunosurveillance.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

Destruction of autologous human lymphocytes by interleukin 2-activated cytotoxic cells

Human and murine lymphocyte populations differentiate into lymphokine activated killer (LAK) cells after in vitro or in vivo exposure to interleukin 2 (IL 2). LAK cells mediate destruction of neoplastic tissue in vitro and have been reported to spare normal tissue. However, systemic toxicity is observed in mice and patients receiving IL 2 infusions. Some aspects of this toxicity are similar to that seen in graft-vs-host disease, suggesting that IL 2 may cause an immune-mediated destruction of normal tissues. We have evaluated this issue by examining the destructive potential of fresh human lymphocytes cultured in media containing highly purified recombinant human IL 2. In the absence of any exogenous antigen or allogeneic stimulating cells, strong proliferative responses were induced after 6 days of exposure to IL 2. Lymphocytes harvested from these 6-day cultures were highly cytotoxic to K562 and Daudi target cells. These IL 2-activated cells were also cytotoxic against autologous and allogeneic normal lymphocyte target cells. This autologous lymphocyte destruction was detected in media containing autologous serum and was directly dependent on the concentration of IL 2 added to the cultures. These studies demonstrate that populations of IL 2-activated lymphocytes, containing LAK activity, can mediate low-level but significant destruction of normal lymphocytes in vitro.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Skin tests with autologous cholesteryl hemisuccinate treated tumour cells in cancer patients

Summary Skin tests with autologous irradiated tumour cells were performed in 20 malignant melanoma, 7 breast and 6 ovarian cancer patients. In the majority of cases evident reaction was noted with cholesteryl hemisuccinate (CHS)-treated cells while the reaction with untreated cells was mostly negative.No correlation was found between this reactivity and the ability of patients to be sensitized to DNCB and to their reactivity to PPD. No correlation was found between reactivity to CHS-treated tumour cells and the stage and course of the disease.     

CD8+ lymphocytes suppress HIV production by autologous CD4+ cells without eliminating the infected cells from culture

Studies of separated peripheral blood mononuclear cell subsets have indicated that the CD8+ lymphocyte is the primary cell type responsible for suppressing human immunodeficiency virus (HIV) replication by infected CD4+ cells. The effect of this antiviral activity is dose-dependent and does not involve killing of the infected cell. These observations indicate that this response is distinct from the anti-HIV cytotoxic mechanisms also described for human CD8+ cells.     

Lysis of autologous tumor cells by blood lymphocytes tested at the time of surgery

Summary Lymphocyte-mediated lysis of autologous tumor cells (autologous lymphocyte cytotoxocity ALC) was tested at the time of surgery in 108 patients (46 squamous cell carcinomas of the lung, 25 adenocarcinomas of the lung, 19 soft tissue sarcomas and 18 osteosarcomas). The clinical course of these patients in relation to the test results has been published previously. The group was evaluated again after an extended observation time, now with a mean of 80.2 months (range 36–108). The test was rarely positive in patients with metastasis (2 out of 28 experiments).There was a correlation between the ALC results and the postsurgical clinical course for patients without detectable metastasis in that (1) a negative test was invariably a bad prognostic sign, i.e., all 32 patients with negative ALC died within 3 years (mean survival time 16.1 months). (2) The remission and survival times were longer for the ALC positive patients (p<0.001). (3) All 37 individuals who are alive at present without recurrence belong to the reactive group.The ALC results correlated with the clinical course in 88% of patients. The correlation was highest for the groups of soft tissue sarcoma and adenocarcinoma of the lung. There was no correlation between killing of K562 cells and ALC, or between lymphoproliferative response to PHA and ALC reactivity.