Abstract 465: Characterization of NK cell exhaustion in melanoma at different stages.
Authors: Silva Ines Pires Da , Gallois Anne , Osman Iman , Bhardwaj Nina 
Published: August 2014

Abstract
BACKGROUND: The concept of CD8+ T cell exhaustion in the context of metastatic cancer has been reinforced by the recent success of immunotherapies targeting the exhaustion markers CTLA-4 and PD-1 in advanced melanoma. Exhausted T cells are characterized by: 1) an over expression of inhibitory receptors or exhaustion markers such as CTLA-4, PD-1 and Tim-3; 2) a loss of function (cytotoxicity, cytokine production and proliferation); 3) a down-regulation of cytokine receptors, rendering them refractory to cytokine stimulation; and 4) changes in transcription factors, including downregulation of T-bet expression. T cell exhaustion has been extensively studied in the context of chronic infectious diseases and different types of cancer, however little is known about exhaustion of NK cells in the same background. NK cells, innate immune cells that eliminate tumors through cytotoxicity and IFN-γ production, have a key role in immune surveillance of tumors, including melanoma. In this study, we characterize and compare the phenotype of NK cells from patients with melanoma of different stages (I, II, III, IV) and healthy donors.
METHODS: We compared NK cells from 10 patients with melanoma at each stage of presentation (I, II, III, IV) and from 20 healthy donors as it relates to membrane expression of activating (NKG2D and NKp46) and inhibitory receptors (KIRB1 and KIRNKAT2), function (cytotoxicity, IFN-γ production and proliferation) and the intracellular expression of the transcription factor T-bet. NK cells’ cytotoxicity was measured by Lamp-1 expression using K562 cells as target cells. IFN-γ production was measured after 4h stimulation with rhIL-12. Proliferation was quantified by CFSE after 6 days in the presence of rhIL-2.
RESULTS: As the stage of the melanoma advanced we observed a pattern of gradual increase of inhibitory receptors (with a higher difference between stage II and stage III) and a concomitant decrease of activating receptors (with a higher difference between stage III and stage IV). With regard to the three most important NK cell functions, IFN-γ production and proliferation were impaired beginning with stage I and advancing to stage IV, while cytotoxicity was reduced only at stage III and stage IV. Similarly, T-bet expression also began to decrease at stage III.
CONCLUSIONS: These data suggest that NK cells from melanoma patients gradually develop a phenotypic and functional profile consistent with exhaustion, beginning with stage I and advancing to stage IV. The exhaustion phenotype begins with initial loss of IFN-γ production and proliferation ability, and finally cytotoxicity. Therefore, the concept that NK cells also become exhausted make them an interesting population to target therapeutically. Furthermore, as the exhaustion phenotype is acquired progressively through the stages, and not exclusive to stage IV, it may be possible to intervene early in the course of tumor progression.
Citation Format: Ines Pires Da Silva, Anne Gallois, Iman Osman, Nina Bhardwaj. Characterization of NK cell exhaustion in melanoma at different stages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2013-465