25Determining the impact of HIF2α inhibition on T cell function in clear cell renal cell carcinoma

Abstract Background Immune checkpoint blockade, which is standard-of-care therapy in the high-risk adjuvant and first-line metastatic-clear cell renal cell carcinoma (ccRCC) setting, relies on re-invigorating T cells for anti-tumor immunity. Belzutifan, a HIF2α inhibitor, was recently approved as a treatment for refractory, sporadic advanced RCC, and is under investigation in combination with pembrolizumab in the adjuvant (LITESPARK-022) and first-line metastatic (LITESPARK-012) setting. While preclinical murine studies suggest that HIF signaling enhances CD8+ T cell cytotoxicity, the impact of pharmacologic HIF2α inhibition on human T cell function remains unknown. We aimed to determine how HIF2α inhibition affects human T cell effector function (proliferation and cytokine production) under hypoxic conditions mimicking the RCC tumor microenvironment. Methods T cells were isolated from fresh human ccRCC tumors (ie, tumor-infiltrating lymphocytes) or from healthy donor peripheral blood mononuclear cells (PBMCs) and stimulated with anti-CD3/CD28 beads for 48 hours under normoxic (21% O2) or hypoxic (1% O2) conditions. HIF2α knockout (KO) T cells were generated using CRISPR-Cas9 electroporation. RT-qPCR and bulk RNA sequencing were used for transcriptomic analysis. For functional assays, T cells were treated with DMSO (vehicle), tacrolimus (control for effective inhibition of T cell function), or HIF2α inhibitors (belzutifan or PT2399). Cytokine production (IFNγ, IL-2, TNFα) was measured via intracellular flow cytometry, and proliferation was assessed using a flow cytometry-based dye dilution assay (CellTrace Violet). Results HIF2α mRNA expression increased ∼70-fold in T cells upon stimulation under hypoxic conditions. Differential gene expression analysis of bulk RNA sequencing revealed that IL-2 and IFNγ were among the most significantly downregulated genes in HIF2α knockout T cells, with log2 fold changes of –4.08 and –2.13, and adjusted p-values of 3.1 × 10-¹¹ and 5.5 × 10-²˚, respectively. Despite these transcriptional changes, pharmacologic treatment with belzutifan or PT2399 did not significantly alter cytokine production at the protein level in CD8+ T cells from either healthy donor PBMCs or ccRCC tumor-infiltrating lymphocytes. Similarly, proliferation assays revealed no significant difference in division index upon treatment with belzutifan or PT2399 compared to DMSO control. Of note, hypoxia alone reduced CD8+ T cell proliferation (P = .0048) and enhanced CD4+ T cell proliferation (P = .0034) compared to normoxia, but HIF2α inhibition did not modulate these effects. Conclusions Although HIF2α is strongly upregulated under hypoxia and its genetic deletion reduces cytokine gene expression, short-term pharmacologic inhibition does not impair effector cytokine production at the protein level or alter proliferation of human CD8+ T cells in vitro. In the settings examined, HIF2α inhibition did not impair T cell function, suggesting that combination with immune checkpoint inhibitors remains a viable therapeutic strategy pending results of ongoing clinical studies. Ongoing laboratory-based assessment will assess the impact of prolonged HIF2α inhibition on T cell phenotype, evaluate its effects on direct cytotoxicity using antigen-specific RCC killing assays, and examine potential synergy with anti–PD-1 therapy.