Abstract B157: Modulation of natural killer cell dysfunction in human bladder cancer
Authors: Farkas Adam M. , Audenet Francois , Anastos Harry , Galsky Matthew , Sfakianos John P. , Bhardwaj Nina 
Published: February 2019

Abstract
T-cells undergo a progressive program of dysfunction termed “exhaustion” during chronic inflammatory pathologies such as cancer. T-cell exhaustion is characterized by diminished effector functionality and inefficient tumor immunosurveillance. However, it is unknown whether an analogous program undermines the contribution of innate lymphocytes like natural killer cells (NK) to tumor-surveillance, and what phenotype might define it. Therefore, we examined expression of inhibitory receptors and the corresponding functional potential of NK and T-cells present in peripheral blood mononuclear cells (PBMC) and tumor tissue of 59 individuals representative of the clinical spectrum of human bladder cancer (BC). PBMC and cell suspensions from freshly dissociated primary tumors were analyzed by flow cytometry to measure changes in the expression of the inhibitory receptors Tim-3, TIGIT, and PD-1, and to determine the composition of hematopoietic lineages in each tissue. NK effector function was assessed via activation with IL-2 or IL-15, followed by co-culture with Class I HLA-deficient targeT-cells to measure cytokine production and degranulation. Additionally, we determined the effect on NK cell function of ex vivo blockade of Tim-3 and TIGIT by the addition of monoclonal antibodies prior to functional assays. We found that NK cells significantly up-regulate expression of Tim-3 and TIGIT, but not PD-1, in both the PBMC and tumors of BC patients. T-cells demonstrate a similar pattern of expression. but with a far lower frequency of positive cells. The magnitude of Tim-3 expression by NK cells is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients independently of tumor stage. Importantly, both molecules are expressed at similar frequencies on NK cells isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK cells in PBMC from BC patients are functionally comparable to NK cells from healthy donors in their ability to produce IFNγ/TNFα and degranulate in response to targeT-cells, while tumor NK are refractory to both stimuli. NK cells from tumor tissue are not terminally exhausted as effector functions are restored after “resting” ex vivo prior to stimulation. Ex vivo blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK and T-cells from BC patients, but is ineffective for NK cells in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK cells from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition. Overall, our data suggest that both NK and T-cells from patients with BC are enriched for expression of the shared inhibitory receptors Tim-3 and TIGIT, and that expression in the peripheral blood mirrors expression in the tumor. However, tissue-specific cues present in bladder tumors, but not in the periphery, ultimately confer effector dysfunction. Furthermore, because blockade of Tim-3 enhances IFNγ and TNFα production by NK cells present in the peripheral blood of BC patients, it may represent a new strategy to modulate innate, antitumor immunity while simultaneously conferring benefit in the adaptive compartment.
Citation Format: Adam M. Farkas, Francois Audenet, Harry Anastos, Matthew Galsky, John P. Sfakianos, Nina Bhardwaj. Modulation of natural killer cell dysfunction in human bladder cancer [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 B157.