Preferential lymphocyte-mediated cytotoxicity of syngeneic target cells in chickens bearing tumors induced by avian sarcoma virus

Splenic lymphocytes from chickens bearing tumors induced by avian sarcoma virus are able to cause the specific killing of cultured avian sarcoma cells. This cytotoxicity appears to follow classical patterns of syngeneic restriction. Little or no specific killing of tumor targets occurred when spleen cells from one inbred line of chickens were tested against allogeneic targets, although syngeneic killing proceeded relatively efficiently. Other patterns of immune reactivity did not appear to be syngeneically restricted. Namely, sera from tumor-bearing hosts were equally reactive in indirect immunofluorescence assays with syngeneic and allogeneic target cells. And, peripheral blood lymphocytes from sensitized hosts could be stimulated equally well by tumor cell culture fluids of allogeneic or syngeneic origin.     

Cell-mediated cytotoxicity to moloney sarcoma in syngeneic and allogeneic rats

Cell-mediated cytotoxicity in the primary immune response to Moloney sarcoma tumor (MST) in allogeneic and syngeneic rats was found to be predominantly T-cell-dependent. A minor non-T-cell cytotoxic activity may also have been detected. CMC was presumably directed against tumor and viral related antigens in the syngeneic host and primarily against alloantigens in the allogeneic host. CMC was more vigorous in the allogeneic recipient than in the syngeneic host. This may be due to differences in quantities or immunogenicities of the various antigens involved. Two peaks of T-cells in effector populations were observed during a 20-day post-inoculation period. The first peak corresponded to peak T-cell-mediated cytotoxicity on day 8 and the second peak occurred on days 13 or 14 when CMC was minimal or undetectable.     

Disease-related lymphocyte cytotoxicity in rats bearing transitional cell carcinoma of bladder. effect of immunomodulators

Blood lymphocytes from rats bearing transitional cell carcinoma (TCC) of the bladder were studied for their cytotoxicity in vitro against xenogeneic YAC-1 target and against syngeneic TCC cells. Control lymphocytes were obtained from age and sex-matched syngeneic rats. The following differences were observed: (I) lymphocytes from TCC-bearing rats were cytotoxic to syngeneic TCC target cells whereas those from control rats were not; (2) lymphocytes from TCC-bearing and control rats were cytotoxic to NK sensitive YAC-I cells; however, NK cells from TCC-bearing rats were more adherent to nylon wool-columns than NK cells from control rats. The adherent and non-adherent cells from TCC-bearing rats were both cytotoxic to syngeneic TCC target cells. Levamisole treatment of effector cells from TCC-bearing rats did not affect the NK activity, yet it increased the cytotoxicity of non-adherent cells on TCC target cells. Treatment of the adherent cells with poly-l:poly-C increased slightly their NK activity on YAC-I cells and their anti-TCC cytotoxicity. However, a marked increase in the cytotoxicity by both adherent and non-adherent cell fractions was observed on TCC target cells pretreated with poly-l:poly-C. A disease-related cytotoxicity of lymphocytes from rats bearing TCC has been observed. Treatment of TCC target cells with poly-l:poly-C increased their susceptibility to lysis by the activated effector cells.     

Natural cytotoxicity of mouse, rat, and human lymphocytes against heterologous target cells.

Natural cell-mediated cytotoxicity by a particular subpopulation of lymphocytes [designated natural killer (NK) cells] in NIH Swiss nude and CBA/N mice, WF rats, and humans was demonstrated against tumor cells in 4-hour 51Cr release assays. In most studies, only reactivity against target cells of the homologous species was examined. In the present studies, mouse NK activity also was found against a rat lymphoma tissue culture cell line and against human tissue culture lines. Rat NK cells reacted not only against syngeneic tumor cells but also against heterologous tumor cell lines. In contrast to the heterologous NK activity in rodents, no significant NK activity of human peripheral blood lymphocytes against heterologous targets was found in the present studies. In mice and rats the effector cells that mediated the cytotoxicity against heterologous target cells were indistinguishable from NK cells, the effector cells being nonadherent and nonphagocytic. In addition, the mouse effector cells for heterologous activity as well as mouse NK cells were sensitive to repeated treatment with anti-Thy 1.2 serum plus complement. The specificities of these reactions were indicated by a cold target inhibition assay. The results indicated a sharing of specificities between homologous and heterologous tumor cells recognized by mouse and rat NK cells. In contrast, only the human cell lines were able to appreciably inhibit the cytotoxicity of human peripheral blood lymphocytes.     

Dissociation of antiviral and antitumor immunity in resistance to Marek's disease.

Immunization of chickens either with gluteral-dehyde-inactivated chicken kidney cells infected with Marek's disease (MD) virus or with glutaraldehyde-inactivated cells of MD lymphoma-derived continuous lymphoblastoid cell lines protected against MD. The former type of immunity was associated with an immunologic suppression of virus replication and virus antigen production after challenge with virulent virus, but lymphocytes specifically cytotoxic to cells bearing MD tumor antigens were not detected. In the latter type of immunity, virus multiplication was not affected; some evidence of the stimulation of cell-mediated antitumor immunity was found. The results supported the view that immunity to MD may be directed against either virus-specific or tumor-specific antigens and that in natural resistance to MD both mechanisms may be operative.     

Human tumor-lymphocyte interaction in vitro. VI. Specificity of primary and secondary autologous lymphocyte-mediated cytotoxicity.

A T-cell-enriched lymphocyte subset of samples from 15 tumor patients was tested for primary cytotoxicity against autologous tumor cell preparations and against 1-3 different allogeneic tumor cell preparations from biopsy material. Allogeneic cytotoxicity occurred in only 1 of 10 patients with autologous reactivity. The lymphocytes of 14 patients were cultured with autologous cells from biopsy material for 6 days. These lymphocytes killed autologous targets, but only 1 patient's lymphocytes were cytotoxic against 1 of the 4 allogeneic tumors tested. Cocultivation with allogeneic cells from biopsy specimens generated cytotoxicity toward the sensitizing allogeneic cells in 3 of 9 test combinations. In 2 of 3 instances the effectors were also active against the autologous tumor cells. Cytotoxicity in primary and secondary tests occurred thus only rarely against allogeneic targets. This indicated either the presence of individual tumor-related antigens on the cells from biopsy material or reflected the histocompatibility restriction of T-cell-mediated cytotoxicity.     

In vitro induction of tumor-specific immunity by highly purified VSV grown in SV40-transformed cells and tumor glycolipids incorporated into liposomes

Cytotoxic effector lymphocytes (CL) were induced by in vitro immunization of spleen cells from normal Syrian hamsters to syngeneic tumor cells, either SV40-transformed (EH-SV) or spontaneously transformed (EH-N). The lymphocyte reactivity was measured in a direct 51Cr release cytotoxicity assay performed with EH-SV- and EH-N-labelled targets. A specific cytotoxic effect against tumor cells carrying the sensitizing antigens was observed. Cytotoxic effector lymphocytes were also induced by in vitro immunization of hamster spleen cells to highly purified vesicular stomatitis virus (VSV) grown either in syngeneic SV40-transformed fibroblasts or in "normal" fibroblasts. Purified virus possessing an intact envelope or virus subparticles devoid of their glycoprotein spikes stimulated the cellular immune responses against host tumor antigens present within the viral envelope. Cytotoxicity assays have revealed two tumor-specific antigens (TSA), one induced by SV40 and present in SV40-transformed cell lines and the other present in "normal" cells. CL were also induced by in vitro sensitization of spleen cells from normal hamsters to liposomes containing the polar glycolipid fraction from EH-SV and/or EH-N cells. A specific cytotoxic effect against tumor cells that have supplied the glycolipid extract was observed, suggesting specific recognition of glycolipid antigens characteristic for each tumor line. This study supports the view that surface glycolipids act as tumor-specific antigens implicated in the destruction of SV40-induced tumors in Syrian hamsters.     

Development of cell-mediated immunity to Marek's disease tumor cells in chickens inoculated with Marek's disease vaccines.

Chickens inoculated with herpesvirus of turkeys or with apathogenic or attenuated vaccine strains of Marek's disease virus (MDV) developed a T-cell-mediated immune response to Marek's disease (MD) tumor cells. This immune response was detected in a 4-hour 51Cr-release assay in which effector cells obtained from spleens of vaccinated chickens were reacted with 51Cr-labeled target cells of an MD lymphoblastoid cell line (MSB-1). The cytotoxic effector cells generated by the vaccine viruses had characteristics similar to those noted previously for anti-MSB-1 effector cells generated by MDV. The immune response was specific to MSB-1 cells, because another target cell line (TLT) antigenically unrelated to MSB-1 cells was not lysed by the effector cells nor did the unrelated target cells inhibit the cytotoxicity of effector cells against MSB-1 target in a cold-target inhibition assay. Because MSB-1 cells contain MD tumor-associated surface antigen, we postulated that the immune response detected in the vaccinated chickens may be directed against this antigen and that the antitumor antigen immunity may play a role in the mechanism of vaccine protection against lymphoma development by pathogenic MDV.     

Cytotoxicity of guinea-pig lymphoid cells against guinea-pig hepatoma cells in tissue culture.

The cytotoxic effect of guinea-pig lymphoid cells on guinea-pig hepatoma cell lines in tissue culture was investigated, using the microplate technique of Takasugi and Klein (1970). The effect of lymphoid cells from guinea-pigs immunized against tumor cells was compared to that of cells from normal controls. Several ratios of effector to target cells (10 : 1, 50 : 1, 150: 1, 250 : 1) were used. In Hartley guinea-pigs immunized with allogeneic tumour cells, peripheral blood lymphoid cells from 14/16 animals showed significant cytotoxicity against that tumour in culture. In a syngeneic tumour/host system, 7/13 animals showed cytotoxicity. Spleen cells gave less consistent results in both systems. The cytotoxic activity of subpopulations of immune lymphocytes against tumour cells in vitro was investigated. It was found that although both T-cell-enriched and T-cell-depleted cell populations exhibited cytotoxicity against tumour cells, the unfractionated cell population was the most effective. This suggests that some degree of cell cooperation may be involved in the cytotoxicity. Antibody-dependent cellular cytotoxicity was also obtained. A T-cell-depleted population of normal cells was shown to be cytotoxic to tumour cells in the presence of serum from immune animals. This type of cytotoxicity could be obtained concomitantly with cell-mediated cytotoxicity in the same animals.     

Retinoic acid stimulation of the induction of mouse killer T-cells in allogeneic and syngeneic systems.

The ability of retinoic acid (RA), a potent antitumor agent, to stimulate cell-mediated cytotoxicity (CMC) in mice was investigated. Low doses of RA (5-300 micrograms/mouse/day) administered ip into C57BL/6 mice for 5 days daily or for 1--3 months three times a week before immunization in vivo or in vitro with allogeneic BALB/c S194 myeloma cells led to an enhanced cytotoxic activity of their spleen effector cells. Similarly, in a syngeneic situation injection of RA into C57BL/6 or BALB/c mice before in vitro challenge with EL 4 (C57BL/6) or S194 (BALB/c) tumor cells strongly stimulated CMC. The enhanced cytotoxic activity was effected by thymus-derived lymphocytes (T-cells) and specific for the H-2 histocompatibility antigens in the case of the allogeneic sensitization or specific for tumor antigens in the case of the syngeneic sensitization. Because RA had no effect on the effector step of CMC, RA likely enhanced the induction step of T-CMC. The action of RA was antigen-dependent, and it is therefore a true adjuvant rather than a nonspecific stimulator or polyclonal activator of cytotoxic T-cells.     

Studies on a gross-virus-induced lymphoma in the rat. III. Optimisation, specificity and applications of the in vitro immune response.

Conditions are described for optimal stimulation in vitro of lymphocytes from rats primed in vivo with syngeneic MuLV-G leukaemia cells. Cultures generate effector cells which show high cytotoxic activity against leukaemic cells. Specific stimulation can be obtained with leukaemic cells and with disrupted MuLV. Leukaemic cells, normal spleen cells and the viron internal protein p30 competitively inhibit effector cells, but purified virus exerts non-specific inhibition. These findings are interpreted as supporting the hypothesis that virion antigens constitute a major target of anti-tumour cytotoxic immunity.     

Induction of cytotoxic activity in human lymphocytes against autologous and allogeneic melanoma cells in vitro by culture with interleukin 2

The influence of interleukin 2 (IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured for 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with non-melanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and changes in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by natural killer cells. These findings appear to have important implications both in the understanding of tumour host relationships and for the use of IL2 in therapy.     

Transplantable Marek's disease lymphomas. II. Variable tumor immunity induced by different lymphoblastoid cells

The effectiveness of cells from 6 different Marek's disease (MD) lymphoblastoid cell lines to induce immunity to syngeneic transplantable MD lymphomas was investigated in 2 related inbred lines of White Leghorn chickens (lines G-B1 and G-B2) that have different major histocompatibility complex (MHC) genotypes. Cells from 2 line G-B1 lymphomas (MDCT-NYM1 and MDCT-UG1) and 1 line G-B2 lymphoma (MDCT-UG2) were used for challenge. Three of the lymphoblastoid cell lines tested were developed from these lymphomas. Growth of palpable lymphomas was lowest among G-B2 chickens immunized with syngeneic lymphoblastoid cells. Protection against the early lethal effects of the highly virulent transplantable lymphomas was greatest in both lines of chickens when the lymphoblastoid cells were syngeneic with the hosts. Lymphoblastoid cells of unknown MHC type either failed to induce immunity to the lymphomas in both lines or protected some line G-B2 chickens challenged with syngeneic MDCT-UG2 lymphoma cells.     

Cellular and humoral immunity against human malignant melanoma

The humoral and cellular immunological responses of melanoma patients against tumor-associated antigens were studied by means of the indirect membrane immunofluorescence test and a microassay for cell-mediated cytotoxicity. Melanoma target cells obtained from 22 different surgical specimens were used. A total of 15 different sera were tested in 27 fluorescence assays. Humoral antibodies were found in all the 13 autochthonous sera tested: eight out of ten sera cross-reacted with one or two allogeneic melanoma cells. The 31 microassays performed with lymphocytes from 16 patients showed that effector cells specifically killed the melanoma target cells in seven out of 12 autochthonous tests and in 14 out of 19 allogeneic tests. No cytotoxicity was found when melanoma-patient lymphocytes were tested against control cells or when control lymphocytes were tested against melanoma cells. The observed cross-reactivity pattern indicates that more than one tumor-specific antigen may be present in melanoma cells.     

Restricted autologous lymphocytotoxicity in lung neoplasia.

Blood lymphocytes from 47 patients with lung carcinoma have been tested for cytotoxicity against cells isolated from the autologous tumour. Significant cytotoxic potential was found in 15 cases. The effectors were also tested against allogeneic tumour targets from lung and other sites. Reactions were only rarely detected (2/32 positive against lung and 1/13 positive against non-lung cells). The restriction of cytotoxicity to the autologous combination was also apparent in in vitro-generated effectors. Blood lymphocytes were co-cultivated with autologous tumour and subsequently tested against autologous or allogeneic targets. Cytotoxicity was found in 13/17 lung tumours against autologous tumour, with no reactions recorded against allogeneic tumour targets, but one case positive against the K562 cell line. These data suggest either the expression of individually distinct antigens on human pulmonary neoplasms, or the requirement for histocompatibility between target and effector in cytotoxicity reactions in man, and therefore differ from previously described patterns of lymphocytotoxicity against human tumours.     

Significance of the proline assay in the study of anti-MSV cell-mediated immune reactions.

The cytolysis of 3H-proline-labelled tumour cells growing in monolayer by syngeneic immune lymphocytes has been studied in the murine sarcoma virus (MSV) system. Results show that the proline assay (PA) is a convenient way to reveal the activity of cytolytic T lymphocytes against FMR-like antigens. Using the same effector and target cells, the classical chromium-release test (CRT) fails to reveal any cytolytic activity, and the visual microcytotoxicity assay as well as several derived isotopic methods are known to reveal mainly non-specific reactions due to non-T effector cells. The PA, therefore, appears to be a useful method for testing an antitumour reaction against tumour cells in monolayer. The results are, however, different from those obtained in the CRT using the same effector cells but lymphoma cells in suspension as targets, the major discrepancies being the following: (a) the PA does not provide truly quantitative data, due to the very high lymphoid: effector cell ratios needed in this test; (b) unexpected patterns of antigenic specificities are sometimes detected in PA; (c) a non-specific natural killer activity of non-T cells is frequently detected in the PA, masking at low lymphoid: target cell ratios the T-dependent specific cytolysis; (d) the H-2 restriction of the cytolytic T-cell activity is poorly detected in PA, whereas the role of H-2 antigens is clearly shown by blocking experiments using anti-H-2 antibodies.     

Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity.

Lymphoid cells from many normal mice of a variety of inbred strains were found to have reactivity, in a 51Cr release cytotoxicity assay, against several syngeneic and allogeneic tumors. Very high reactivity was seen with effector cells from athymic nude mice, which was consistent with other evidence that the reactivity was not T-cell dependent. Target cells susceptible to lysis included tumors induced by oncogenic type-C viruses but also tumors induced by other means and expressing endogenous type-C viruses. The levels of natural reactivity were influenced by age, with highest cytotoxicity produced by cells from 5- to 8-week-old mice. Lymph-node cells, spleen cells, peritoneal exudate cells and peripheral blood lymphocytes all had cytotoxic reactivity. The specificity of the reactions was analyzed in detail by ana inhibition assay. Evidence was obtained for natural reactivty against several different antigens, each apparently associated with expression of murine endogenous type-C viruses.     

Macrophage-mediated in vitro sensitization of T-lymphocytes. I; Detection of murine leukemia virus-associated antigens.

Cytotoxic effector T-lymphocytes were produced in vitro by sensitization of spleen cells on monolayers of syngeneic macrophages that had been fed with radiation leukemia virus-containing cell extracts or with supernatants of virus-producing cell cultures. The sensitized lymphocytes were cytotoxic to cell lines that expressed viral antigens. Secondary mouse embryo fibroblasts were little affected. Sensitization via macrophages appeared to be a useful system for identification of viral antigens on surfaces of various target cells, as well as for tests of the protective effect of such lymphocytes against tumor growth in vivo.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. IV. Antigenic differences between a metastasizing variant and the parental tumor line revealed by cytotoxic T lymphocytes.

The syngeneic cytotoxic T-cell response against a metastasizing murine lymphoma variant was investigated and compared with the response against the non-metastasizing parental tumor line Eb. Anti-tumor cytotoxicity was not detectable in a 4-h 51Cr release assay in spleens taken directly from tumor-bearing animals (primary CMC). After restimulation in vitro (secondary CMC) however, high anti-tumor cytotoxic activity was detected. This activity was mediated by immune T lymphocytes as shown by its sensitivity to treatment with anti-Thy 1.2 serum and complement. Ten cells of the metastasizing tumor ESb, inoculated subcutaneously, were sufficient to raise a local tumor and metastases and to induce cytotoxic T memory cells in the spleens. In contrast, about 104 cells were required to raise a local tumor and to induce splenic cytotoxic T memory cells, when the parental tumor Eb was tested. The specificity studies of the anti-tumor cytotoxic activity demonstrated that cytotoxic T cells could distinguish unrelated, chemically induced syngeneic tumors and also recognize antigenic differences between the parental tumor Eb and its variant ESb. Eb and ESb tumor cells were recognized as carrying distinct antigens at the responder cell level, the stimulator cell level and the target cell level. The in vivo significance of these findings is discussed.     

Selective and non-selective lymphocytotoxicity in human melanoma: observation on the effect of long-term culture and fetal bovine serum on target-cell sensitivity to lymphocytes.

In vitro cell mediated cytotoxicity (CMC) assays have been conducted in a human melanoma system with a 3H-proline retention technique. Melanoma target cells from long-term cultures ("cell lines") are found to exhibit increased susceptibility for lymphocyte cytotoxicity in comparison to the same target cells from short-term culture. The higher sensitivity of the "cell line" derived target cells is seen with lymphocytes, irrespective of diagnosis of the donor. In parallel experiments with the target cells grown in medium supplemented with fetal calf serum (FCS) and AB+ human serum (from a normal male doner), the melanoma target cells grown with FCS do not show any enhanced cytotoxicity, suggesting no causal relationship of such enhanced sensitivity of "cell line"-derived target cells to "heterologous melanoma antigens" that might have been acquired by the target cells following the use of FCS in tissue culture. In controlled assays of in vitro CMC, lymphocytes from melanoma patients (14/44) exhibited selective cytotoxicity (destruction of only one target-cell type) against the melanoma target cells, whereas only 3/97 control lymphocytes (other malignancies and normal donors) showed such melanoma-selective cytotoxicity. This difference is statistically significant at p less than 0.001. Non-selective cytotoxicity (destruction of two or more unrelated target cell types) was seen with lymphocytes from 9/44 melanoma patients, 13/51 patients with other malignancies and 8/46 normal donors. No correlation of selective cytotoxicity could be established with donors' age, sex, stage of disease, therapy or history of blood transfusion. Such a correlation may emerge as our series becomes larger. Despite the lack of any correlation between selective cytotoxicity and disease status, our study reaffirms the existence of selective cytotoxicity by melanoma patients' lymphocytes against melanoma target cells.