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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 fro 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 nonmelanoma 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 change in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by nature killer cells. These findings appear to have important implications both in the understanding of tumor host relationships and for the use of IL2 in therapy.

Immunoperoxidase localization of monoclonal antibodies in sections of melanoma has been used to identify histological features which may be of prognostic importance in melanoma, in particular whether certain structures on melanoma cells may determine the degree and nature of lymphoid infiltrates and whether these may be related to prognosis. Monoclonal antibodies to lymphocyte subpopulations were used to identify and quantitate lymphoid infiltrates in 15 primary melanomas and 8 cutaneous metastases. These were correlated with histological features identified in routine sections. There was a wide variation in the numbers of mononuclear cells associated with both primary and metastatic melanoma. T cells and to a lesser extent macrophages accounted for the majority of the cells, B cells and natural killer (NK) cells for only 2% of the infiltrates. The Leu 3a (helper) T cell subpopulation predominated in primary tumours, OKT8 positive (suppressor/cytotoxic) cells in metastases. Infiltrates in which OKT8 cells predominated were associated with ulceration, a high mitotic rate and thick primary tumours. The converse applied to Leu 3a infiltrates. Infiltrates with a high proportion of Leu 3a positive cells tended to be associated with Thy-1 positive, DR antigen negative tumours. Thy-1 antigens were predominantly expressed on primary tumours and rarely on metastases whereas the converse applied to expression of the acute lymphoblastic leukemia antigen (CALLA) on melanoma cells. These findings suggest that certain melanoma antigens may be related to the nature of the lymphoid infiltrate associated with melanoma and possibly with the behaviour of the tumour in the host. They further suggest that identification of cell surface structures and lymphoid infiltrates by these techniques may be a valuable extension of routine histopathological assessment of prognosis in melanoma.

The effector cells responsible for cytotoxic activity induced by phytohaemagglutinin, (PHA) pokeweed mitogen (PWM) target cells complexes with IgG antibody has been investigated using cell separation techniques based on rosette formation and separation through Hypaque-Ficoll mixtures. It was shown the PHA-induced cytotoxicity is predominantly a function of T cells and that Fc receptor-bearing cells are not involved to any major extent. Antibody-dependent killing is conversely a function of Fc receptor-bearing cells among which two subtypes can be distinguished. One of these has receptors for activated complement while the other bears Fc receptors only and has no detectable receptors for complement. PWM appears to induce cytotoxicity in both T- and non-T-cell populations but the major cell type involved appears to be Fc receptor-bearing cells similar to those mediating antibody-dependent killing. It is concluded that PHA and antibody-dependent killing are the two most useful assays for discriminating between the cytotoxic activity of T and non-T cell in clinical studies.

Previous studies in vitro have shown that monoclonal antibodies (MAbs) against gangliosides GD3 and GD2 potentiate lymphocyte responses to a variety of stimuli. The purpose of the present study was to determine by immunohistological techniques whether GD3 and GD2 was expressed on lymphoid cells in vivo around melanoma cells. Studies on metastases in lymph nodes indicated that the lymphoid infiltrate around the margins of the metastases was predominantly CD4+ T cells, which were shown by dual labelling techniques to express mainly GD2 and to a lesser extent GD3. CD4+GD3+ T cells were detected more frequently in cortical regions of the lymph nodes. CD8+ T cells were less numerous than CD4+ T cells and expressed both GD3 and GD2. Expression of GD2 was also prominent on CD4+ T cells, B lymphocytes and dendritic reticular cells in germinal centres, whereas GD3 was mainly expressed on T cells in the margins of the follicles. In contrast to the predominance of CD4+ T cells in lymph nodes, CD4+ and CD8+ T cells were in approximately equal proportions about primary melanoma and metastases in skin. GD2 was largely undetectable on lymphocytes at these sites. In contrast, GD3 was detected on both CD8+ and CD4+ lymphocytes but not on B lymphocytes. The absence of GD2 on CD4+ T cells in skin suggested the latter were a different subpopulation to those in lymph nodes. There appeared to be no clear correlation, however, with subsets of CD4 T cells defined by the 2H4 and Leu 8 MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood lymphocytes and rectal plasma cells have been studied in patients with ulcerative colitis taking part in a double-blind trial of treatment with azathioprine. Treatment for 1 year resulted in a modest fall in blood lymphocyte count, with little change in neutrophils or platelets. There was no major change in the proportions of circulating T and B lymphocytes, suggesting that the number of such cells per millilitre of blood fell in proportion to the change in lymphocyte count. The number of plasma cells in the rectal lamina propria was reduced to a mean less than half that of the control patient group. Blood K-cell cytotoxic activity fell at least 25-fold after 1 year's treatment. PHA-induced cytotoxicity was also reduced, but less consistently. Reduced K-cell activity is interpreted as reflecting depletion of effector cells from the circulation. The fall in lymphocyte count, K-cell activity and gut plasma cells was slow, indicating continuous inhibition of lymphopoiesis or differentiation throughout the trial period. Thus, azathioprine has some immunosuppressive effects which develop only after prolonged treatment. The clinical results of the trial did not show a major beneficial effect of azathioprine in the treatment of ulcerative colitis, nor were there clear correlations between the results of lymphocyte assays and clinical response in individual patients.

Normal non-adherent mononuclear cells were shown to inhibit colony formation by normal human marrow cells cultured for 7 d in semi-solid agar. Inhibition was the same using cells from the marrow donor or from an unrelated normal subject, and was shown to be dose-dependent over the range of 4 X 10(5) to 6 X 10(3) mononuclear cells per 1 X 10(5) marrow cells plated. Inhibition was not seen in 14 d cultures, and it is postulated that colony-forming cells sensitive to lymphocyte inhibition belonged to the population known to give rise to colonies after 7 d in culture. Cell fractionation studies showed that inhibition was due to non-B non-T lymphocytes, purified B cells or T cells being neither inhibitory nor stimulatory. Inhibition was only shown with intact viable lymphocytes and it was not possible to extract inhibitory activity from the cells, or to produce inhibition by media conditioned by lymphocytes. The effect was apparently due to a direct action on colony-forming cells in the marrow and was not due to inhibition of colony stimulating activity (CSA) production, or to absorption or inactivation of CSA. These results emphasize the need to include appropriate controls when looking for possible cell-mediated inhibitors in disease states, particularly when 7 d cultures are used.

Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD3 may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD3 may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD3. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4+ and T8+ clones. The potentiation of these responses was not specific for M.Abs to GD3 but was also seen with M.Abs to GD2 and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD3 during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD3 but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo.

We have previously shown that tumour cells may release soluble factors which inhibit the division of a variety of cells. These factors had marked immunosuppressive activity in vitro as shown by their ability to Inhibit lymphocyte mitogenic responses to lectins. allogeneic lymphocytes and pokeweed mitogen-induced immunoglobulin production. It was postulated that these factors may play an important role in determining the outcome of tumour host interactions in humans and be the source of immunosuppressive factors in the circulation of patients with cancer. In the present study, characterization of these mitogenic inhibitory factors released into the supernatants of cultured tumour cells was carried out by a number of sequential separation procedures involving affinity chromatography on wheat-germ lectin (WGL). gel filtration on PL-agarose and electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate (PAGE-SDS). The factors were identified throughout the separation procedure using inhibition of phytohaemagglutinin (PHA) stimulation of normal lymphocytes as an index of inhibitory function. By these techniques the inhibitory activity was shown to be associated with glycoproteins containing N-acetyl-β-D-glucosamine (NAcGlu) and having molecular weights (mol. wt) of approximately 140.000 and over 200,000 daltons. A further small mol. wt peak of 70,000 daltons was observed on PAGE-SDS. pi values of the fractions were 7.4 and 4.2. The activity was destroyed by heating, low pH and repeated freeze-thawing but was resistant to proteolytic enzymes, deoxyribonuclease, ribonuclease and neuraminidase. The active fractions were not associated with proteolytic activity and the inhibition of mitogenic responses was irreversible in nature. These results form the basis of studies to detect this factor in biological fluids and further evaluate its role in the tumour host relationship. [ABSTRACT FROM AUTHOR]

A number of previous reports have described the presence of factors which inhibit the response of lymphoid cells to phytohaemagglutinin. The present study describes an inhibitor of cell division synthesized and released directly from cultured tumour cells which appears to have similar immunosuppressive effects in vitro. This factor(s) was detected in a wide variety of cultured tumour cells and some cultures of normal foetal tissue. It inhibited the mitogenic response of lymphocytes to PHA and also the spontaneous cell division of a variety of cultured cells including the cells producing the factor. Pulse cytophotometry showed that cells were inhibited in the G1 phase. Production of the factor increased with time and was related to the number of cells in culture. Its synthesis was inhibited by cycloheximide. DNA and RNA synthesis was not required for its production. The factor appeared to have a high degree of biological activity m terms of the number of cultured cells required for production and in regard to the number of cells inhibited. The biological significance of this factor in vivo is unknown but in vitro it was shown to inhibit immunoglobulin and mitogen-induced responses which suggests it may play an important role in suppression of the immune response against tumour cells in the host. [ABSTRACT FROM AUTHOR]

This report investigates functions and number of different lymphocyte types in patients with testicular and ovarian tumours before, during and following therapeutic irradiation. Irradiation causes loss of T cells, B cells and K cells at similar rates. The long term depletion of T cells, which is well recorded, is due to delayed and incomplete repopulation of a substantial proportion of this cell type. B cells and K cells, however, return to levels very close to normal by 6 and 3 months respectively after irradiation. Long-term depletion of T cells occurs even when the thymus is not irradiated. [ABSTRACT FROM AUTHOR]

Rat lymphoma cells sensitized with antibody and incubated with normal macrophages were shown to become resistant to damage from cytotoxic antibody and complement or cytotoxic lymphocytes. Both lymphoma specific antibody and normal macrophages were required to produce protection. The cytotoxic effect of lymphocytes or antibody with complement and the protective effect of antibody with macrophages was assessed by 51Cr release and by loss of capacity of iymphoma cells to transfer the disease to syngeneic rats. No cytotoxic effect could be attributed to macrophages from sensitized allogeneic rats. Inhibition of phagocytosis by Cytochalasin B did not destroy the capacity of macrophages to protect lymphoma cells. Sodium fluoride, however, did block the protective effect. It is envisaged that this protective phenomenon may be of importance in understanding some aspects of growth of tumours in vivo despite the presence of immune cytotoxic mechanisms. [ABSTRACT FROM AUTHOR]