Advisor: Cheng Zhu, PhD (Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology) 

Committee Members:

Karmella Haynes, PhD (Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology)

John Blazeck, PhD (Department of Chemical Engineering, Georgia Institute of Technology)

Nicole Schmitt, PhD (School of Medicine, Winship Cancer Institute, Emory University)

Edward Botchwey, PhD (Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology)

CHOLESTEROL IMPAIRS T-CELL RECEPTOR ANTIGEN RECOGNITION IN TUMOR MICROENVIRONMENT THROUGH CONVERGING T-REG AND ADENOSINERGIC IMMUNOSUPPRESSANT PATHWAYS

Tumor killing by cytotoxic T cells is initiated after the T cell receptor (TCR) recognizes tumor antigen through direct physical contact and bond formation. T cells decipher antigenic peptides by forming bonds between TCRs and peptide loaded major histocompatibility complexes (pMHCs). CD8+ T cells reactive against highly immunogenic antigens have a diminished anti-tumor protective effect when exposed to the TME. The mechanisms driving TME mediated immunosuppression are not fully understood. Advancing the understanding of immunosuppression in cancer will uncover useful information towards the advancement of therapeutic strategies. Our lab has previously shown that melanoma TME suppresses TCR 2D affinity, endogenous pulling forces, and activation in CD8+ cytotoxic T cells. Here, we examine the mechanisms driving suppressed TCR-pMHC interactions. Three immunosuppressants implicated in TME immune system evasion were screened as candidates of TCR inhibition. Directly targeting cholesterol metabolism, regulatory T cell (Treg) function, and adenosinergic pathways elucidated a compartment specific response to each inhibitor. These inhibitory pathways converge by altering cholesterol membrane accumulation.

Treatment with lovastatin, an FDA approved HMG-CoA reductase inhibitor (cholesterol synthesis inhibitor), reversed TME effects on impaired TCR binding affinity, endogenous pulling forces, and calcium fluxing. The current study also uncovers impaired TCR binding affinity and recovery with lovastatin using the MOC1 head and neck squamous cell carcinoma (HNSCC) animal model. Results show a progressive reduction in TCR 2D binding affinity of CD8+ T cells exposed to TME in HNSCC, and recovery when cholesterol synthesis is inhibited, similar to melanoma. Treatment with lovastatin was also confirmed to mitigate the TME effects on TCR antigen recognition by targeting membrane cholesterol accumulation. Reduction of membrane cholesterol via cholesterol sulfate (CS) also recovered TCR binding affinity by reducing membrane accumulation and average TCR site density/μm2. This suggests that the immunosuppression of CD8+ T cells in TME is attributed to impaired TCR antigen recognition, due, in part, to increased accumulation of membrane cholesterol.

In connection to findings from targeting cholesterol metabolic pathways, we explored Treg immunosuppression in TME mediated TCR inhibition and its possible connection to cholesterol mediated TCR inhibition. Treg depletion eliminated the compartment specific differences in binding affinity between cells exposed to TME and those that were not. This suggests Tregs as a role player in TCR inhibition. Results show regulatory T cell depletion reduces cholesterol accumulation in the membrane of CD8+ T cells exposed to TME, implicating Tregs in the mechanism of cholesterol-mediated TCR inhibition.

A well-known mechanism of Tregs cytotoxic CD8+ T cell inhibition works through the adenosinergic pathway through increased ectonucleotidase expression. We determine that ex-vivo stimulation with an adenosine-like receptor agonist increases the membrane cholesterol accumulation in CD8+ T cells exposed to TME. This data supports the notion that in cancer, Tregs drive membrane cholesterol accumulation in CD8+ T cells through the adenosinergic pathway. Finally, data shows targeting regulatory T cells lowers the concentration of adenosine in tumor infiltrating lymphocytes and enhances adenosine A2A receptor expression of non-TME exposed cells when regulatory T cells are selectively depleted, further connecting alterations in these pathways to membrane cholesterol accumulation and TCR inhibition in the TME.