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

Abstract

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.

Clearance rates and systemic effects of intravenously administered interleukin 2 (IL-2) containing preparations in human subjects

“…The dose of IL-2 given to the subjects in this study was selected so that an initial level of at least 2 u/ml in plasma would be obtained as this was the level used in vitro for induction of cytotoxic T cell activity (Hersey et al, 1981). Based on an expected plasma volume of -4% of body weight and assuming no destruction or absorption of the IL-2 it would be expected that 14,000 u in Patient 1 would give an initial value of 7.3 u ml' (calculated plasma volume 1.921) and 17,000 u in Patient 2 an initial value of 5.8 u ml-1 (calculated plasma volume 2.921).…”

“…He failed to respond to treatment with several courses of DTIC (Dacarbazine) (Flow Laboratories) culture medium (without foetal calf serum) for 24-36h. Further details of the procedures are described elsewhere (Hersey et al, 1981). After membrane chromatography on YM30 membranes (Amicon) in 300-ml capacity Amicon diaflo cells to remove PHA, the volume of the supernatant was reduced to 60-100ml by ultrafiltration on YM5 (Amicon) membranes.…”

“…Assays of IL-2 in supernatants were carried out as previously described (Hersey et al, 1981). Normal blood lymphocytes which had been maintained in IL-2 for 7 days after stimulation by PHA for 3 days were used as the target cell populations.…”

“…The 5"Cr release assays and target cells used in studies on the effect of fresh serum on IL-2 induced cytotoxic activity are fully described elsewhere (Hersey et al, 1981).…”

“…We have recently substantiated these studies by showing that culture of lymphocytes from patients with melanoma in IL-2 containing supernatants resulted in the induction of cytotoxic cells in vitro against autologous melanoma cells and a variety of allogeneic target cells (Hersey et al, 1981). Culture of the lymphocytes with tumour cells was not necessary for the induction of cytotoxicity against the autologous melanoma cells.…”