Abstract 4745: Tim-3 and TIGIT mark NK and T cells susceptible to effector dysfunction in human bladder cancer
Authors: Farkas Adam M. , Audenet Francois , Anastos Harry , Oh William K. , Galsky Matthew D. , Sfakianos John P. , Bhardwaj Nina 
Published: August 2018

Abstract
Purpose: To characterize the evolution of Natural Killer (NK) and T cell exhaustion in peripheral blood and tumor tissue of patients representing the spectrum of human bladder cancer (BC), and to identify molecules for which therapeutic modulation enhances the anti-tumor immune response.
Experimental Procedures: PBMC and freshly resected primary tumors were analyzed by FACS to determine phenotype. NK function was assessed via activation with IL-2 or IL-15 followed by co-culture with K562 cells to induce cytokine production and degranulation. For blockade studies, X-Tim-3 and X-TIGIT mAbs were added prior to K562 stimulation.
Results: T cells undergo a process of exhaustion in the context of chronic inflammatory pathologies such as cancer that is characterized by diminished effector functionality and subsequent tumor outgrowth. Inhibition of signaling through certain mediators of this process (e.g. CTLA-4 and PD-(L)1) have shown clinical benefit in subsets of patients with a wide array of malignancies. However, it is unknown whether NK exhaustion occurs, and what molecules define it. We found that NK undergo an analogous process of exhaustion based on analysis of PBMC and tumor from 59 individuals with non, and muscle-invasive BC. NK exhaustion is marked by significant up-regulation of Tim-3 and TIGIT in both PBMC and tumor. T cells demonstrated a similar expression pattern but with a lower frequency of positive cells. The magnitude of NK Tim-3 expression is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients. Importantly, both molecules are expressed at similar frequencies on NK isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK in PBMC from BC patients are functionally comparable to NK 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 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 in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition.
Conclusions: NK and T cell acquisition of Tim-3 and TIGIT are indicators of BC and detectable in peripheral blood and tumor, but mark effector dysfunction only in tumor tissue. Blockade of Tim-3 enhances NK IFNγ/TNFα production in PBMC of BC patients and represents a new strategy to modulate innate, anti-tumor immunity.
Citation Format: Adam M. Farkas, Francois Audenet, Harry Anastos, William K. Oh, Matthew D. Galsky, John P. Sfakianos, Nina Bhardwaj. Tim-3 and TIGIT mark NK and T cells susceptible to effector dysfunction in human bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4745.