Abstract The identification of novel molecular mechanisms underlying exhausted CD8+ T cells (TEX) represents a critical objective in advancing immunotherapeutic strategies for cancer and other diseases. Nevertheless, high-throughput analysis of in vitro TEX remains challenging. We developed a human in vitro model of CD8+ T cell exhaustion using antigen-specific or TCR-transgenic T cells subjected to chronic antigen stimulation. Repeated exposure to cognate peptide antigens induced hallmark exhaustion phenotypes, including co-expression of inhibitory receptors (PD-1, TIM-3 and LAG-3) and transcription factor TOX. Exhausted T cells exhibited diminished cytokine secretion (e.g. IFN-γ), reduced proliferation (e.g. Ki-67), and impaired cytotoxicity. These features closely mirror exhaustion observed in tumour-infiltrating lymphocytes and chronic infection settings. Checkpoint blockade was evaluated for functional rescue. Anti-PD-1 restored partial proliferation and modest IFN-γ production. However, neither agent fully reversed exhaustion, as other inhibitory pathways remained active. These findings align with clinical observations where monotherapy yields partial rescue. Our model thus enables quantitative assessment of exhaustion reversal and supports combinatorial checkpoint strategies. To generate sufficient antigen-specific T cells for high-throughput assays, we employed artificial antigen-presenting cells (aAPCs) derived from engineered K562 lines expressing diverse HLA alleles and co-stimulatory ligands. This platform supports expansion of rare endogenous T cells to 108 cells per donor, preserving native TCR expression. Functional validation included CMV-specific and MART-1-specific T cells, which demonstrated antigen-specific cytotoxicity and cytokine responses. This scalable and physiologically relevant assay system permits precise modelling of T cell exhaustion and facilitates systematic evaluation of immunotherapeutic interventions. It serves as a reliable platform for the quantitative screening of agents that inhibit or reverse T cell exhaustion, thereby supporting the rational development of advanced immunotherapies. Citation Format: Emilio Cosimo, Hannah Findlay, Robert Benson, Agapitos Patakas, . In vitro antigen-specific T cell exhaustion model for immunomodulators evaluation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2802.
Cosimo et al. (Fri,) studied this question.