Induction and Potential Reversal of a T Cell Exhaustion-Like State: In Vitro Potency Assay for Functional Screening of Immune Checkpoint Drug Candidates

Abstract

Tumor infiltrating lymphocytes (TIL) entering the tumor microenvironment (TME) encounter many suppressive factors resulting in a spectrum of possible differentiation paths, many with overlapping characteristics. This differentiation spectrum includes T cell anergy, unresponsiveness, quiescence, tolerance, dysfunction/suppression, and exhaustion, each with many subtypes. Immune checkpoint blockade (ICB) cancer drug therapies have focused on reinvigorating exhausted T cells (T_EX) and dysfunctional T cells (herein referred to as T_EX). Many factors have been attributed to as causative to this exhausted state including sustained surface expression of multiple co-inhibitory receptors, altered transcription factor expression, epigenetic rewiring, and dysregulated metabolism. Antigen persistence is necessary for driving T_EX maintenance in both the chronic viral infection setting and cancer. In addition to persistent antigen exposure, TILs within the TME encounter numerous tumor-mediated immunosuppressive metabolic by-products, suppressive cytokines, hypoxia, and cellular debris which converge to suppress T cell function and uniquely alter its transcription factor profile. These suppressed or dysfunctional T cells are incapable of mounting an optimal anti-tumor response in part due to lack of fitness in competing for glucose and oxygen. This chapter describes two different potency assays: (a) first we describe optimization of a core recall antigen-based in vitro potency assay for screening of immunopotentiating drug candidates; (b) the second assay bundles the core assay with further addition of immunosuppressive agents derived from the TME making a customizable T cell exhaustion-like assay. Our recall antigen assay is being used as a tool for function-based screening of ICB drug candidates. In this assay healthy human Peripheral Blood Mononuclear Cells (PBMCs) are stimulated with peptide(s) and grown in culture for 1 week. Day 4 supernatants are functionally assayed by ELISA for IFN-γ secretion, and cells are assayed on day 7 by flow cytometry for CD8⁺ or CD4⁺ T cell expansion by using a single or cocktail of pMHC tetramers. We have shown that in roughly 30% of donor PBMCs, ICB drugs such as pembrolizumab are able to boost both IFN-γ secretion and antigen-specific recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory receptor downstream signaling is ameliorated with ICB drugs releasing these T cells from the repressive effects of these co-inhibitory receptors. We have further enhanced our recall assay to more closely resemble the TME by including metabolites and other suppressive agents at concentrations not normally encountered by T cells in a healthy environment. We show one example of this whereby addition of adenosine to the culture results in suppression of antigen-specific T cell expansion without affecting cell viability. Further, we screened several drug candidates on their ability to counteract the negative effects of adenosine and found that one of the drugs was indeed able to restore antigen-specific T cell expansion. Hence, this TME-based recall antigen assay allows for dissection of the effects of TME-based factors on donor-specific T cell response as well as drug candidate screening.