Chronic anti-CD3/CD28 Dynabeads stimulation generates exhausted T cells with a distinct metabolic phenotype 2341

Abstract Description T cell exhaustion is a major challenge in cancer immunotherapy, as exhausted T cells lose essential effector function and persistence in the TME. Recent studies showed that impairment of mitochondria function plays a critical role in developing and maintaining T cell exhaustion. These observations prompted us to explore the potential use of metabolic profiling of T cells to assess relative T cell exhaustion. In our study, we induced exhaustion in memory T cells via in vitro chronic stimulation with anti-CD3/CD28 Dynabeads and used briefly stimulated (24-hour) counterparts as controls. Across all three donors, chronically stimulated T cells exhibited reduced proliferation, increased percentage of PD1+Tim3+ population, and decreased IL2 secretion upon restimulation, while TNFα and IFNγ secretion varied by donor. Importantly, when assessed using the Seahorse XF T cell Metabolic Fitness assay, these exhausted T cells displayed a distinctive metabolic phenotype, including a significant increase in glycolytic activity, with reduced mitochondrial spare respiratory capacity (SRC). In addition, these cells have a decreased capacity to further increase glycolytic activity upon acute restimulation. These findings suggest that metabolic profiling is a valuable tool for identifying metabolically fit T cells and designing strategies to improve T cell therapies’ efficacy, representing a great addition to the current matrix of parameters for assessing T cell exhaustion. Topic Categories Technological Innovations in Immunology (TECH)