NK cells from malignant melanoma patients exhibit an exhausted phenotype characterized by i) up-regulation of inhibitory receptors (TIM-3, KIRB1 and KIRNKAT2), ii) down-regulation of activating receptors (NKG2D and NKp46), IL-2R and transcription factors (T-bet and Eomes) and iii) loss of IFN-γ production, proliferation and cytotoxicity (Cancer Immunol Res 2014). This exhaustion can be partially reversed by checkpoint blockade of Tim-3. The mechanisms underlying the induction of the development of exhausted NK cells and its reversal are still not well defined. We hypothesize that reversal of exhaustion in NK cells should contribute towards the full restoration of immune responses that are required to eliminate melanoma cells. For that reason it is essential to more fully appreciate the mechanisms underlying immune dysregulation in the TME.
It is known that the metastatic melanoma microenvironment is characterized by the overexpression and production of different ligands or receptors such as i) galectin-9/HGMB-1/CEACAM-1 for Tim-3 ii) MICA for NKG2D iii) CD155 for DNAM-1 /TIGIT/CD96 iv) GD3 for Siglec7/9 that fully contribute to induction of NK-cell exhaustion. The aim of this project is to use transcriptome and CyTOF analysis for comparing human healthy donors (HD) and melanoma donors (MD) patterns to identify the factors and signaling mechanisms that promote immune cell exhaustion in the Tumor Microenvironment (TME).
Our preliminary results show that priming NK cells from HDs with IL-2 increases expression levels of epigenetic modifiers (HIST1H1C, HIST1H4I, ZNF286B, HIST2H2BE) whereas IL-2/IL-12 stimulated NK cells up regulated mRNA encoding cytokines (IFN-γ, IL-1β, IL-6 and TNF) and chemokines (CCL3, CXCL1, CXCL3). This is consistent with an activation phenotype that can also be observed by CyTOF analysis where only IL-2/IL-12 stimulated NK cells are able to produce the following cytokines and chemokines: IFN-γ, TNFα, CCL3, CCL4 and CXCL1. We also found that STAT and Lck proteins are important intermediaries for the priming and activation process on NK cells. STAT4 and STAT5 are involved in IFN-γ production which is important for early control of tumors; and Lck is involved in modulating inhibitory signals mediated by Tim-3 (Nat Med 2012).
Data on MD samples are being evaluated in order to define the cellular mechanisms dysregulated during NK cell exhaustion. These studies should lead to the discovery of additional checkpoint molecules and signaling pathways that can be modulated to restore both innate and adaptive immunity in vivo.
Citation Format: Elena Gonzalez-Gugel, Shaheen Ensanyat, Adiel Munk, Nina Bhardwaj. Mechanisms underlying the induction of natural killer cell exhaustion in metastatic melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B161.
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