Modular Antigen-Specific T Cell Suppression Assay For In Vitro Drug Screening

Abstract T cell exhaustion (TEX) describes a T cell differentiation continuum of hierarchical progressive diminution of effector function. Many factors have been attributed to this including expression of multiple co-inhibitory receptors, altered transcription factor expression, epigenetic rewiring and dysregulated metabolism. Antigen persistence is necessary for driving TEX maintenance in both chronic viral infection and cancer settings. In addition to persistent antigen exposure, tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment (TME) encounter immunosuppressive metabolic byproducts, suppressive cytokines, hypoxia and cellular debris which converge to suppress T cell function. These suppressed T cells are incapable of mounting an optimal anti-tumor response due to dampened proliferation, metabolic fitness and/or cytolytic capability. Our team has previously deployed usage of the recall antigen assay to screen immune checkpoint inhibitor (ICI) drug candidates. We have shown that in some donor PBMCs, ICI drugs such as pembrolizumab are able to boost antigen-specific T cell recall. We adapted this antigen recall assay to study TME-mediated T cell suppression. We show application of this TME-based recall assay by addition of adenosine which suppresses antigen-specific T cell expansion without altering T cell viability. Interestingly, T cell suppression was partially reversed with addition of CMV lysate. This assay is modular in that TME metabolites can be used alone or in combination, with the potential to screen for donors who are in vitro “responders” and “non-responders” to various drug classes.