Abstract A191: Novel approaches to the study of NK cell exhaustion in humans
Authors: Gonzalez-Gugel Elena , Sadanala Keerthi Caroline , Rahman Adeeb , Blumberg Richard Stephen , Horowitz Amir , Bhardwaj Nina 
Published: February 2019

Abstract
The engagement of activating or inhibitory receptors and production of immunosuppressive factors in the tumor microenvironment can modulate NK cell activities, such as cytotoxicity and cytokine production. In certain instances, NK cells acquire a dysfunctional state, resembling the “exhaustion phenotype” described for T-cells characterized by (i) overexpression of inhibitory receptors such as PD-1, CTLA-4, LAG-3, TIGIT and TIM-3, (ii) down regulation of cytokine receptors, rendering them refractory to cytokine stimulation; (iii) loss of function (cytotoxicity, abnormally low cytokine production, and proliferation); and (iv) down regulation of transcription factors T-bet and Eomes. The mechanisms underlying NK cell exhaustion in cancer remain undefined. We hypothesized that the interaction between immunogenetics, education and environment may play an important role during induction of NK cell exhaustion. Checkpoint molecules and NK cell receptors such as Tim-3, TIGIT, inhibitory KIRs and NKG2A among others are key candidates for mediating the exhaustion profile. The aim of this project was to use a selected collection of healthy donors who are HLA and KIR genotyped, and whose CMV and EBV status are defined, to i) clarify the steady state distribution of checkpoint molecules on NK cells between individuals and ii) determine how these profiles change with exposure to immune suppressive environments. In order to do so we applied Mass Cytometry (CyTOF) technology for detailed analysis. In addition, we developed an in vitro NK cell exhaustion assay used to evaluate gradual changes in NK cell activity determined by their cytotoxicity and cytokine production capacities. Purified NK cells from healthy individuals were cultured for short- and long-term cultures with completed media supplemented with recombinant human (rh)-IL-2 or rhIL-15 in the presence of increasing TGF-β concentrations (from 0.05, 0.5, 5 to 50ng/ml); or co-culture with m44, Gmel and Skm147 melanoma cell lines at a ratio of 1:10. Analysis of NK cell dysfunction was determined by quantifying degranulation and IFN-γ production. Our preliminary results confirmed that both immunosuppressive factors and NK cell ligands from the tumor microenvironment are necessary to completely shut down NK cell function. Interactions between NK cell ligands and their specific NK cell receptors are crucial for the induction of NK cell exhaustion in vitro by reducing NK cell activity nearly 80% measured by degranulation and IFN-γ production, whereas addition of increasing concentrations of rhTGF-β1 into the culture gradually drives NK cells to a process of exhaustion by reducing their cytotoxicity and cytokine production capacities by 40-50%. TGF-β1 immunosuppressive effect on NK cells reaches a maximum at 6 days of in vitro culture by noncanonical TGF-β signaling. More than that, we are able to confirm differences in the expression of activating or inhibitory NK cell receptors presented by each individual based on host-genetics and NK cell education in response to immunosuppressive cytokine stimulation. In summary, these results should help us to predict NK-cell response to therapies based on the presence or absence of unique NK cell subsets.
Citation Format: Elena Gonzalez-Gugel, Keerthi Caroline Sadanala, Adeeb Rahman, Richard Stephen Blumberg, Amir Horowitz, Nina Bhardwaj. Novel approaches to the study of NK cell exhaustion in humans [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A191.