An in vitro T cell Dysfunction/Exhaustion Assay System that is partially reversible by pembrolizumab

Abstract The persistence of antigen and chronic stimulation of T cells results in T cells’ functional decay, referred to as T cell dysfunction or exhaustion (TEX). TEX is an essential part of the checks and balances of the immune system chronic T cell stimulation. Assays that model T cell dysfunction/exhaustion with functional readouts are urgently needed for identification and characterization of active drug candidates. Here, to recapitulate persistent of antigen as it happens in tumor microenvironment, we have optimized an in vitro stimulation set-up using PBMCs from various donors that models key features of T cell exhaustion/dysfunction including main markers and functional features of T cell exhaustion. Various stimulation conditions were assessed, with stimulation lasting up to four rounds, and optimal conditions were selected for anti-CD3/anti-CD28 stimulations of PBMCs that resulted in a) increased expression of the co-inhibitory molecules associated with exhausted T cells, PD-1, TIM3, LAG3, CD38, and CD39, and b) impaired T cell functions as demonstrated by reduced expression of IFN-γ, TNF-α, and reduced cytotoxic killing in an allogeneic setting, using both a 2D cancer cell line and a 3D tumoroid model. This in vitro T cell dysfunction/exhaustion platform is a candidate to test drug candidates for their ability to delay or partially reverse T cell exhaustion. Crown Bioscience internal R&D