PO-372 IL-2 and IL-15 cytokine in vitro treatments induce NKG2D, CD158a and CD158b receptor expression on T, NKT- like and NK cell lymphocyte subsets from regional lymph nodes of melanoma patients

Abstract

IntroductionRegional lymph nodes (LN)s are important immunological barriers in spreading of malignant tumours but also represent the most frequent early metastatic site in melanoma. Considering that cytokine therapy has shown substantial toxicity, there is a growing need for further in vitro testing of these agents to enlighten aspects of their regional application in malignancies. The aim of this study was to investigate the effect interleukin (IL)−2 and IL-15, cytokines with similar immune-enhancing effects, on the expression of activating (NKG2D) and inhibitory (CD158a,CD158b) receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients.Material and methodsMononuclear cells (MNC)s were purified from regional LNs of 35 melanoma patients (clinical stages II-IV) and in vitro cultured for 7 days in cell culture medium RPMI1640 (CM) alone, CM with 200 IU/ml rhIL-2 and CM with 25 ng/ml IL-15. Expression of NKG2D, CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cells was estimated by flow cytometry. Statistical significance between the values obtained with cytokine compared to control CM treatments was evaluated by Wilcoxon signed rank test. NK cell cytotoxicity was evaluated by standard 51Cr release assay.Results and discussionsOur results show that IL-2 and IL-15 in vitro treatments significantly increase expression of activating NKG2D receptor on lymphocytes and their CD8+ T, NKT-like and NK cell subsets. Regarding investigated inhibitory receptors, the significant increase was obtained for CD158a only after IL-15 treatment on lymphocytes and their CD8+ T and NKT- like subsets. However, the expression of CD158b receptor significantly increased after IL-2 cytokine treatment on lymphocytes, CD8+ T and NK cells, and after IL-15 treatment on all investigated lymphocyte subsets.Both cytokines augmented NK cell antitumor cytotoxicity that positively correlated (p<0.01, Spearman signed rank test) with the expression of NKG2D activating receptor on NK cells from regional LNs of melanoma patients, irrespective of LN involvement. Conversely, the expression of inhibitory receptors did not correlate with NK cell cytotoxicity except for CD158b that after control CM treatment showed negative correlation.ConclusionAntitumor potential of immune cells from regional LNs induced with IL-2 and IL-15 cytokine treatments may indicate the importance of this immune cell population in the development of therapeutic strategies that involve local cytokine application.