Date of Award

January 2026

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

David A. Braun

Abstract

Background/Scientific premise: Clear cell renal cell carcinoma (ccRCC)accounts for 75-80% of all kidney cancers and is characterized by von Hippel- Lindau (VHL) gene inactivation, leading to constitutive stabilization of hypoxiainducible factor 2-alpha (HIF-2α). Belzutifan, a selective HIF-2α inhibitor, received FDA approval in December 2023 for advanced ccRCC and is now being evaluated in combination with immune checkpoint inhibitors (ICIs) in multiple phase III trials. However, the effects of HIF-2α inhibition on anti-tumor T cell immunity remain incompletely understood. Preclinical studies using genetic HIF-2α manipulation in murine models suggest HIF-2α may support CD8+ T cell effector function, raising concerns that pharmacologic HIF-2α inhibition could impair T cell-mediated tumor control. Research Aims and Question: This study aimed to comprehensively characterize the effects of pharmacologic HIF-2α inhibition with belzutifan on human T cell phenotype and function across multiple clinically relevant T cell sources including peripheral blood mononuclear cells (PBMCs) from healthy donors and ccRCC patients, as well as tumor-infiltrating lymphocytes (TILs). We sought to determine whether pharmacologic HIF-2α inhibition has transcriptional or functional effects on T cells that could impair anti-tumor immunity. iii Methods/approach: For phenotypic characterization, we performed bulk RNA sequencing to evaluate transcriptional changes and 23-color spectral flow cytometry for surface marker profiling of activation, differentiation, exhaustion, and co-stimulatory molecules. For functional assessment, we measured T cell proliferation using CellTrace Violet dilution assays, evaluated cytokine production (interferon-gamma, tumor necrosis factor-alpha, and interleukin-2) through intracellular cytokine staining with analysis of polyfunctional responses, and assessed direct tumor cell killing using antigen-specific cytotoxicity assays with NY-ESO-1-specific T cells paired with engineered 786-O ccRCC target cells. To characterize HIF-2α-regulated transcriptional programs, we generated HIF-2α knockout T cells using CRISPR-Cas9 gene editing. We then treated healthy donor PBMCs, patient-derived PBMCs, and TILs with the HIF-2α inhibitors belzutifan or PT2399 compared to DMSO vehicle control under normoxic and hypoxic conditions. Clinical validation utilized paired pre- and post-treatment peripheral blood samples from seven ccRCC patients receiving HIF-2α inhibitor therapy. Results: Genetic HIF-2α knockout resulted in significant downregulation of genes encoding effector cytokines and TNF receptor superfamily members, with pathway analysis revealing effects on T cell activation and proliferation pathways. In contrast, pharmacologic HIF-2α inhibition with belzutifan or PT2399 produced minimal transcriptional changes across all T cell sources tested. Pharmacologic HIF-2α inhibition did not impair T cell proliferation, cytokine production, or polyfunctional capacity in CD4+ or CD8+ T cells from any source under either normoxic or hypoxic conditions. In antigen-specific killing assays, belzutifaniv treated T cells demonstrated slightly improved cytotoxic function compared to control. Spectral flow cytometry demonstrated preservation of T cell activation, differentiation, and exhaustion marker profiles with belzutifan treatment. Under hypoxic conditions in vitro, belzutifan treatment resulted in modest reductions in regulatory T cell frequency. Analysis of paired peripheral blood samples from patients treated with HIF-2α inhibitors revealed no significant changes in T cell phenotype or regulatory T cell frequency. Statement of scientific impact and relevance: This work demonstrates that pharmacologic HIF-2α inhibition with belzutifan preserves T cell proliferation, cytokine production, and cytotoxic capacity across multiple clinically relevant T cell sources, providing mechanistic support for the safe combination of belzutifan with immune checkpoint inhibitors in ongoing clinical trials for ccRCC. The comprehensive toolkit developed for assessing T cell phenotype and function provides a framework for evaluating immunologic effects of targeted therapies.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 07/14/2028

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