Wednesday, April 2, 2025
4/2/2025
Functional consequences of high glucose culture on adoptive T cell therapies for cancer - PROJECT SUMMARY/ABSTRACT Adoptive cell therapy (ACT) is a form of immunotherapy in which T cells are isolated from a patient, genetically modified, expanded in vitro and then transferred back to the patient to initiate antitumor immunity. Chimeric antigen receptor T cells (CAR-T) is a form of ACT where the T cells are modified to contain a tumor specific receptor (CAR). CAR-T cells have demonstrated clinical success particularly in hematological malignancies, however, efficacy in solid tumors has remained poor and many patients ultimately relapse. The in vitro expansion step of CAR-T cell therapy is critical for generating enough cells to transfer back to the patient, but the transfer of cells from a patient to an in vitro environment and then back to the patient requires T cells to be metabolically flexible. Cell culture media has been well demonstrated to induce metabolic dysfunction on T cells and alter their effector function. Healthy blood glucose is approximately 5mM, however, the in vitro expansion media contains nearly 10x blood glucose levels to favor proliferation. Preliminary data suggests that T cells cultured in high levels of glucose are less responsive to T cell receptor (TCR) stimulation demonstrating a reduction in IFNγ and TNFα when stimulated with αCD3/αCD28. Additionally, adoptive transfer of tumor-specific T cells cultured in 55mM glucose into B16F10 tumor-bearing mice demonstrate reduced tumor control compared T cells cultured in lower glucose concentrations (11mM). Increases in both intracellular and extracellular glucose have been shown to affect glycosylation of cellular proteins. Glycosylation is an important post-translational modification that impacts transport, signaling and localization of proteins. Thus, we wondered if intracellular glycosylation mark, O-linked β-N-acetylglucosamine (O-GlcNAc) would be changed following culture in higher levels of glucose. Indeed, we see elevated proteome wide O-GlcNAc as we increase glucose in culture media. O-GlcNAc is added to serine and threonine residues by the enzyme O-GlcNAc transferase (OGT). Given the role of serine’s and threonine’s in phosphorylation, we assessed whether T cell signaling would be altered by exposure to high levels of glucose in culture. We restimulated the cells using a time course to assess phosphorylation of downstream T cell signaling intermediates. Our preliminary data suggests delayed induction and reduced maximal induction of p-Erk, p-S6 and p-Akt. Thus, we hypothesize that high levels of glucose in cell culture media alter T cell signaling pathways by blocking critical phosphorylation sites of signaling proteins with O-GlcNAc. We will test this hypothesize by (1) identifying what cellular proteins are differentially O-GlcNAcylated and whether inhibiting O- GlcNAcylation restores cytokine production, antitumor immunity and T cell signaling and (2) whether these findings translate to human models using CAR-T cells. Findings from these studies will provide mechanistic understanding of how excess glucose in the culture media contributes to protein glycosylation and whether this is linked to decreased effector function. We will translate these findings to CAR-T cells. The insights from this study will provide rationale for optimizing culture media formulations to enhance patient response to adoptive cell therapies.
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