Cell-mediated cytotoxicity in carcinoma of the human urinary bladder

Summary Lymphocytes from patients with tumors of the bladder or other unrelated tissues or with non-malignant genitourinary (GU) conditions and from normal subjects were tested in a microcytotoxicity assay against long- and short-term cultures (T24 and BT) derived from bladder carcinoma and several other target cell types, to determine the validity of the hypothesis that cell-mediated cytotoxicity (CMC) in bladder cancer patients is a specific disease-related phenomenon. At the effector cell level, lymphocytes from bladder cancer patients displayed uniformly greater cytotoxicity for T24 and BT cells than those from normal donors. This proclivity was shared by the lymphocytes of patients with non-GU cancers but not of those with non-malignant GU disorders. At the target cell level, CMC was observed less frequently against non-bladder tumor targets than against T24 and BT cells and the CMC of bladder cancer patients did not differ significantly from that of the other groups. The pattern of CMC observed against the bladder tumor-derived targets was thus one of target cell sensitivity rather than tumor-specificity and disease-related only to the extent that the CMC of patients with cancer was greater overall than of healthy subjects. Abrogation of CMC by passage of lymphocytes through immunoglobulin-coated columns indicated that the effector cells were principally of non-T type, bearing a superficial resemblance to those in normal individuals which induce non-disease related CMC.     

Disease-related lymphocyte cytotoxicity in vitro in patients with transitional cell carcinoma of the urinary bladder. Comparisons with other malignancies,acute cystitis,and healthy donors

Summary Blood lymphocytes from 100 patients with transitional cell carcinoma of the urinary bladder (TCC-bladder) were studied for their cytotoxicity in vitro against a panel of allogeneic tissue culture cell lines. Of the TCC-bladder patients, 45 were untreated for their disease, while 55 had been treated with local radiotherapy up to 12 years before testing. Control lymphocytes were obtained from (1) 45 untreated, age- and sex-matched patients with other neoplastic diseases, mainly urogenital cancers; (2) 19 patients with acute cystitis; and (3) 45 healthy donors. Lymphocytes from individual donors within all five groups were frequently cytotoxic to any one of the target cells. However, the lymphocytes from each of the two TCC-bladder groups were markedly more cytotoxic to two different bladder tumor targets than to control targets derived from normal bladder epithelium, from colon carcinoma, or from malignant melanoma. Similar comparisons made within each of the three control donor groups did not show this. The results indicate that the two bladder tumor targets were not more susceptible to lymphocyte-mediated lysis than the control targets. The mean cytotoxicity displayed by the lymphocytes from both TCC-bladder groups to the bladder tumor targets was significantly higher than that of the cancer control group and that of the healthy donors. No such elevation was seen when the cancer control group or the cystitis patients were compared with healthy donors. Although untreated TCC-patients with a larger tumor burden (stages T3–T4) appeared to be slightly less cytotoxic to all target cells than those with a smaller tumor burden (T1–T2), these differences were not statistically significant. On the other hand, among the treated TCC-patients, in the main those tested more than 1 year and up to 5 years after therapy exhibited a significantly elevated mean cytotoxicity to the bladder tumor targets. Within all five donor groups, the overall cytotoxicity to the bladder tumor targets and the normal bladder targets showed a statistically highly significant correlation. However, while there was no correlation for the untreated TCC-bladder patients and the clinical controls between cytotoxicity to the bladder tumor targets on one hand and non-bladder targets on the other, the cytotoxicity to the bladder tumor targets of the treated TCC-bladder patients was also correlated with that to the colon carcinoma and the melanoma targets. The results indicate that cytotoxicity in both TCC patients and controls reflects recognition by the lymphocytes of a variety of antigens, shared to different degrees by different groups of target cells. Furthermore, in TCC-bladder patients there is a superimposed cytotoxicity, which is related to their disease and which probably reflects reactions against one or several tumor-associated antigens.     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Membrane structures involved in auto-tumor recognition

Tumor patients' blood lymphocytes have the capacity to recognize autologous tumor cells in vitro. A consequence of this recognition is the proliferation of small-size, high-density, resting T cells. Both helper (CD4+) and cytotoxic/suppressor (CD8+) T lymphocytes proliferate in the mixed lymphocyte-tumor cell cultures. In contrast to the autologous mixed lymphocyte cultures, both the auto-erythrocyte rosetting and non-rosetting (AE+ and AE-) T cells participate in the auto-tumor response. In contrast to stimulation by virus-infected or hapten-modified cells, DR antigen expression is not essential for stimulation by autologous tumor cells. In a proportion of cancer patients, blood lymphocytes have the capacity to lyse the patients' own tumor cells in vitro. There are two populations of lymphocytes with auto-tumor cytotoxic function. The first is characterized by low buoyant density and by non-adaptive cytotoxicity. In contrast to the recognition of hapten-modified or virus-infected target cells by the CTL, recognition of autologous tumor cells by the cytotoxic LD cells occurs even when the MHC class I antigens are blocked by mAb. The CD3 complex is also not involved in LD-mediated lysis. The other population with auto-tumor cytotoxic function comprises high-density, resting T cells. Recognition of autologous tumor cells by cytotoxic HD lymphocytes shares the characteristics of CTLs, i.e., their function is abrogated by pretreatment of the effectors with mAbs directed to the T3 receptor complex and by preincubation of the targets with mAb to the MHC class I antigens. Cytotoxicity of HD cells is restricted to the autologous tumor cells. This selectivity and the characteristics shared with CTL suggest that the auto-tumor reactivity of HD lymphocytes reflects an immune response against the autologous tumor.     

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Serum IgG fractions from a large and homogeneous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the 'non-selective' CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

Suppressor cell regulation of the spontaneously induced in vitro secondary antibody response.

The cytotoxic effect of peripheral blood lymphocytes from patients with chronic active hepatitis (CAH) on Chang liver cells was studied using a chromium-51 release assay. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of glass-adherent mononuclear cells, indicating that the major effector cell is not a classical monocyte. Lymphocytes from 12 of 18 patients with CAH were cytotoxic, and the cytotoxic cells were contained within T-cell-depleted fractions. Further studies revealed that these effector cells were K cells, not B cells. K cells were identified as Fs- and complement receptor-bearing cells without detectable surface immunoglobulin. Serial studies in CAH showed continuous cytotoxicity in contrast to transient cytotoxicity in acute viral hepatitis, suggesting that the cytotoxic activity of lymphocytes is responsible for persistence of the disease.     

The effect of IL-2 and IFN-beta on autologous tumor cell kill by Percoll separated LGL fractions

Low density fractions of Percoll density gradient centrifugation of peripheral mononuclear cells contained the majority of large granular lymphocytes (LGL). LGL were used for 5-hr 51Cr release cytotoxic assay against autologous tumor cells in 20 patients with hematological malignancies (9AML, 4ALL and 7NHL). Mean % cytotoxicity (% CTX) was 6.0%, and the addition of IFN-beta and IL-2 in the medium induced the significant increase of % CTX to 15.0% and 26.1%, respectively. When LGL cultured in medium containing IFN-beta and IL-2 were assessed for cytotoxicity daily for 8 days, the enhancement of % CTX by IFN-beta was declined in a few days, while the enhancement by IL-2 was sustained for more than 8 days. The pretreatment of LGL with anti Leu-11 (CD16) plus complement abrogated the enhancing effect by IFN-beta or IL-2, but not with anti Leu-1 (CD5) plus complement. When this treatment was done on day 8 of IL-2 cocultivation, anti Leu-11 plus complement suppressed cytotoxicity significantly, and anti Leu-1 plus complement also induced mild suppression. The phenotypic characteristics of cells revealed the significant increase of anti Leu-19+ cells in IL-2 stimulated day 8 cells. High density fractions of Percoll gradient contained mostly T lymphocytes and showed no cytotoxicity against autologous tumor cells. However, cocultivation with IL-2 for 8 days induced the cytotoxicity, associated with increased number of anti Leu-19+ cells. These results suggested that IL-2 induced cytotoxic activity against autologous tumor cells might be related to the increase of anti Leu-19+ cells.     

Stage-related, cell-mediated cytotoxicity with effector cell analysis and computation of cytotoxic activity of T cells and non-T cells.

Cell-mediated cytotoxicity (CMC) by lymphocytes from patients with oral squamous cell carcinoma, as well as from nonmalignant control donors, was tested by a microcytotoxicity assay against a cultured cell line derived from an oral squamous cell carcinoma. In terms of the degree of CMC, stage-related cytotoxicity was observed. A further study of the effector cell analysis revealed that the cytotoxic effects of lymphocytes from both patients and control donors were largely attributable to non-T cells. However, the effectors were also stage related, and in early stage patients, T-cell-mediated cytotoxicity was super-imposed on non-T-cell-mediated cytotoxicity. This conclusion was further supported by the evidence of elevated cytotoxic activity of T cells in early stage patients, which was computed from simultaneous equations proposed in the present paper for computing the cytotoxic activity of T cells (CTA_T) and non-T cells (CTA_non-T).     

Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses.

Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice

It is assumed that CD8(+) cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4(+) helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL-resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft-induced macrophages (AIM) were cytotoxic against the allograft. Bacterially-elicited macrophages also exhibited cytotoxic activity (approximately 1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially-elicited macrophages was completely inhibited by the addition of donor (H-2(d))-type lymphoblasts, suggesting H-2(d)-specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three-dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord-like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.     

Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.     

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Summary Serum IgG fractions from a large and homogenous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the non-selective CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus.

Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.     

Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients

Summary Cytotoxicity was induced in lymphocytes (CL) from 10 out of 15 patients by autologous mixed lymphocyte tumor cell culture and further cultivation with recombinant interleukin-2. In cells from 3 of the 10 patients, cytotoxicity was suppressed by more than 50% when autologous peripheral blood mononuclear cells (PBMC) from the patients with large tumors were added to the autologous killing system. The cells responsible for suppressing the cytotoxicity in the effector phase were adherent or nonadherent to plastic depending on the patient examined. The T cell fraction from 1 patient significantly suppressed the cytotoxic activity, and this suppression was seen only in the autologous system. On the other hand, plastic adherent cells but not T cells from PBMC of 2 subjects suppressed the cytotoxic activity of CL. The reason why the main cell population suppressing the CL activity differed among the patients is unclear. However, the findings that the suppression was mostly abrogated following resection of the tumor mass suggested that suppressor cells, either of macrophage lineage or T cells, are induced in patients with a large tumor mass. This speculation is supported by the finding that the PBMC from a patient with tumor recurrence regained the suppressive activity.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Cytotoxic lymphocytes induced by soluble factors derived from the medium of leukocyte cultures.

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45 degrees C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered. The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.     

Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use

Adoptive immunotherapy with antitumor effector cells is an attractive therapeutic approach in metastatic renal cell carcinoma (RCC). The aim of the work was to enhance in vitro activation of lymphocytes with optimal cytotoxic activity against tumor cells. We evaluated a procedure based on the use of dendritic cells (DCs) loaded with irradiated tumor cells (DC-Tu) to stimulate lymphocytes. Experimental conditions were established with cells from healthy donors and melanoma cell lines. Procedures were then applied to cells from RCC patients. A total of 30 tumor biopsies, 14 proximal lymph nodes, and 17 peripheral blood samples from 30 patients were used. When lymphocytes were stimulated in vitro with DC-Tu, they responded to tumor cells with an increased cytolytic activity for all the assays with donor cells (n=18). For RCC patients, DC-Tu stimulation improved the final cytotoxic activity in only half of the assays (16/31). When significantly enhanced (>10%, n=8), responder cells resulted in a final 43% cytotoxicity against autologous RCC cells. Mechanism of lysis was at least in part class I mediated. Effector cells have no lytic activity against normal renal cells. Percentage of cells with regulatory T-cell phenotype was not found to be enhanced in the DC-Tu stimulated lymphocytes. Individual differences were observed in the characteristics of DCs generated from RCC patients in contrast to that observed in donors and could explain why lymphocyte stimulation was not improved by DC-Tu in half of the RCC assays. T-cell spreading was suitable for a therapeutic use (>10⁹ cells) irrespective of the procedure (with or without DC-Tu stimulation) or the tissular origin of lymphocytes from patients. Data show that precursors of selective antitumor effector cells are present in patients with RCC and can be amplified in vitro either with or without DC-Tu stimulation. One of these populations could be chosen for an adoptive transfer immunotherapy.This work was supported by grants from the Comité Grand Ouest de La Ligue Contre le Cancer and from the Faculté de Médecine de Rennes.     

Phorbol esters enhance spontaneous cytotoxicity of human lymphocytes, abrogate Fc receptor expression, and inhibit antibody-dependent lymphocyte-mediated cytotoxicity

Phorbol esters with tumor promoter activity enhance the spontaneous cytotoxicity of human lymphocytes against a variety of target cell lines, with an efficiency that correlates with their potency as tumor promoters or skin irritants. Analysis of surface marker expression of the lymphocytes cytotoxic after treatment with phorbol ester identified the cytotoxic cell subset as that containing natural killer cells. Although gamma-interferon (IFN gamma) is produced by T cells treated with phorbol esters, IFN gamma is probably not the mediator of enhancement of natural killer cell activity, because anti-IFN gamma antibodies failed to block this enhancement. Spontaneous cell-mediated cytotoxicity is inhibited when phorbol esters are present during the cytotoxic assay, but is enhanced when the effector cells are pretreated with these agents. On the other hand, antibody-dependent cytotoxicity mediated by lymphocytes is inhibited by phorbol ester pretreatment of the effector cells or by phorbol esters present during the cytotoxic assay. Treatment of lymphocytes with phorbol esters at 37 degrees C, but not at 4 degrees C, completely abrogates in 1 to 2 hr the expression of the receptor for the Fc fragment of IgG, as detected by rosette formation with IgG-sensitized erythrocytes and by reactivity with anti-Fc receptor antibodies. The inhibition of antibody-dependent cytotoxicity by phorbol esters is probably secondary to their effect on the Fc receptor.