An Investigation of the Interactions Between Lymphokine Activated Killer Cells and T-Lymphocytes In Vitro

Lymphokine activated killer (LAK) cells are efficient mediators of tumour destruction in experimental models of metastatic cancer, but their mode of action in vivo is poorly understood. The general efficacy of LAK cell therapy in humans has been disappointing, although notable individual successes were observed in clinical trials, particularly in patients with melanoma or renal cell carcinoma. However, the mechanism by which LAK cells mediate tumour regression in these patients remains undefined. Although LAK cells are capable of directly killing tumour cells in vitro, evidence that they localise at the site of the tumour in vivo is sparse: it has been shown that following administration, LAK cells do not accumulate at or infiltrate into metastatic lesions. Intravenous infusion of LAK cells into the peripheral circulation results in LAK cell trafficking via the lungs to the liver and spleen for clearance. Since LAK cells in vivo appear to infiltrate the spleen, which contains a lymphoid reservoir of T-cells and monocytes, the opportunity for LAK cells to interact with the hosts' immunocytes may exist. Consequently, any effects which LAK cells exert on tumour regression may be indirect, via the local secretion of secondary cytokines and/or their interactions with other cells of the immune system. This project therefore investigates the hypothesis that regulatory cytokines, known to be produced by LAK cells, may augment a cytotoxic T-lymphocyte mediated anti-tumour response, assuming that T-cells primed to the tumour are present in the spleen. The premise for this hypothesis is based on previous observations in a murine model, which demonstrated that the systemic administration of interleukin-2 (IL-2) to mice enhanced the delayed hypersensitivity reaction to a contact allergen. It was subsequently shown that the effects of IL-2 observed on this T-cell mediated response could be modulated by LAK cells, suggesting that the presence of LAK cells altered the effects of IL-2 in the spleen. It therefore appeared that LAK cells could interact with activated T-cells within the immune system and thus, modulate their function. This is particularly interesting since LAK cells can secrete secondary cytokines which may be capable of regulating T-cell activity in vitro. To address the proposal that LAK cells could modulate T-cell function in vitro, through the release of secondary cytokines, LAK cells were induced with either recombinant human IL-2 or soluble monoclonal anti-CD3. By varying both time in culture and dose of stimulus, the production of cytokines (tumour necrosis factor alpha, interferon gamma and interleukin-1) and the cytotoxic capacity of various LAK cell populations were compared. Both stimulants induced LAK activity although the LAK activity of these effector cells showed poor correlation with the cytokines produced. To determine whether LAK cell-derived supernatants had the capability of regulating T-cell cytotoxicity in vitro, modulatory effects on a cytotoxic T-cell line were studied. The ability of these cells to kill an EBV transformed target cell line, of appropriate HLA haplotype, was not enhanced by any of the supernatants generated over a five hour period. Increasing exposure of both the T-cell and target cell lines to the supernatants to twenty-four hours produced complications: exposure of target cells to supernatants alone for a prolonged period induced lysis. A few, random LAK supernatants augmented cytotoxicity when the T-cell line was preactivated with supernatant for nineteen hours prior to incorporating target cells into the assay. However the enhanced killing observed was not considerably different to the lytic activities obtained in the initial five hour cytotoxicity assay. Furthermore, for those supernatants involved, no predictive pattern could be determined when considering cytokine levels within the supernatant itself or the parameters under which the supernatant was generated. Thus, in this system, secondary cytokines produced by LAK cells were unable to enhance T-cell cytotoxicity in vitro, with only a few exceptions. The ability to regulate T-cell function by increasing T-cell proliferation was also addressed. Peripheral T-cells proliferated in response to the IL-2 induced supernatants over seventy-two hours. However, stimulation with these supernatants plus exogenous, soluble anti-CD3 enhanced the response considerably. The proliferation stimulated by the anti-CD3 induced supernatants was more pronounced, although the addition of exogenous anti-CD3 had marginal effect on the proliferative response observed. In conclusion, recombinant IL-2 and soluble monoclonal anti-CD3 were both capable of inducing LAK activity and cytokine secretion in normal peripheral blood mononuclear cells. However, the secondary cytokines produced by these LAK populations had only marginal effects on T-cell cytotoxicity and/or proliferation in vitro within this system. These results imply that secondary cytokine production by LAK cells would fail to modulate T-cell function in vivo, but further study would be required to confirm whether this is the case