Abstract 4856: Chronic anti-CD3/CD28 stimulation during cell expansion generates exhausted T cells with a distinct metabolic and cytotoxic phenotype

Abstract T cell exhaustion is one of the major causes of cancer immune evasion and contributes to the failure of immunotherapies targeted to solid tumors. Reversing T cell exhaustion is a promising strategy in cancer immunotherapy, as it can improve patients’ responses to immune checkpoint inhibitors and adoptive T cell therapies. Recent publications showed that impairment of mitochondria function plays a critical role in developing and maintaining T cell exhaustion. This prompted us to explore the possibility of using metabolic profiling of T cells to assess relative T cell exhaustion as a tool to optimize T cell therapy product development. In our study, we induced exhaustion in previously expanded T cells via chronic stimulation with anti-CD3/CD28 Dynabeads and used briefly stimulated (24-hour) counterparts as controls. Across all three donors tested, chronically stimulated T cells exhibited reduced net proliferation, increased percentage of PD1+Tim3+ population, and decreased IL-2 secretion upon restimulation, while TNFα and IFNγ secretion varied by donor. We performed Xcelligence RTCA impedance-based, acute killing assays using Epcam-BiTEs to engage T cells to the Epcam expressing T47D cells. Surprisingly, we found that in some donors, cells exposed to chronic anti-CD3/CD28 Dynabeads, despite secrete significantly less cytokines upon antigen restimulation, display similar acute cytotoxicity compared to its non-exhausted counterparts. Importantly, when assessed using Seahorse XF assays, these exhausted T cells displayed a very distinctive metabolic phenotype, including decreased maximal respiration and spare respiratory capacity (SRC), increased glycolytic activity, and reduced capacity to further increase glycolytic activity upon acute reactivation. These findings suggest that metabolic profiling can represent a valuable tool for identifying metabolically fit T cells and is a great addition to the current matrix of parameters for assessing T cell exhaustion. Citation Format: Guoxiao Wang, Xiaoyu Zhang, Natalia Romero. Chronic anti-CD3/CD28 stimulation during cell expansion generates exhausted T cells with a distinct metabolic and cytotoxic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4856.