A high-throughput assay using dysfunctional T cells for phenotypic screening immune checkpoint modulators.

During a prolonged response to virus infection or tumorigenesis, effector T cells are persistently stimulated and become dysfunctional. Dysfunctional T cells overexpress inhibitory regulators, exhibit decreased effector cytokine production, and lose robust effector functions, leading to the failure of antigen/cancer elimination. In the present study, we established a protocol to generate dysfunctional T cells from human peripheral blood mononuclear cells (PBMCs) and developed a fully automated high-throughput assay in 384-well format to assess immune checkpoint modulators. Specifically, PBMCs were maintained with interleukin-2 (IL-2) and Phaseolus Vulgaris Leucoagglutinin (PHA-L) to induce dysfunctional T cells. Cell populations were subsequently examined and quantified using flow cytometry, and validated T cells were used for the further assay development. To assess compound activity, cells were pre-incubated with testing compounds for 24 h, followed by a stimulation with anti-CD3 and anti-CD28. The cell supernatants were then harvested for cytokine measurements (e.g., IL-2 and interferon γ (IFN-γ)) using AlphaLISA assays to evaluate the activation of T cell receptor signaling. Cell lysates in the same assay plate were used for a viability assay to assess cytotoxicity. This workflow was implemented for assessing 15 compounds selected based on cellular pathways they modulate, of which 2 compounds promoted IL-2 and IFN-γ production in dysfunctional T cells upon activation, suggesting potential therapeutic values against T cell dysfunction.