A HIF-INDEPENDENT PROGRAM SUSTAINS TUMOR TREG FUNCTION IN OXYGEN-DEPRIVED ENVIRONMENTS

The immune-suppressive tumor microenvironment (TME) is both hypoxic and enriched with Treg. Their function in hypoxia has been poorly characterized. Definition of Treg responses to hypoxia would enable their selective targeting in cancer therapy, bypassing potential systemic adverse effects. We showed TME hypoxia and Treg infiltration correlated in a public RNA-seq cohort and refined this finding by immunohistochemistry. We then demonstrated that TI-Treg expanded in 3% O2 but not in ambient air. Our epigenomic atlas of Treg showed reduced hypoxia-inducible factor (HIF) signaling in TI- Treg, so we explored alternative responses. We performed time-course RNA-seq of Treg in either oxygen condition and analyzed transcription factor motifs in differential cis-regulatory elements, finding oxygen sensor RBPJ to be enriched in enhancers, which we validated by ChIP. We observed RBPJ upregulation in TI- as opposed to NAT- and PB- Treg by scRNA-seq and cytometry. Inhibiting RBPJ prevented a staggering proportion of oxygen-induced gene upregulation, including genes involved in immune suppression and bioenergetics. Similarly, flow cytometry showed OX40 and CTLA4 upregulation at protein level in 3% O2, reverted to baseline by RBPJ inhibition. Suppression assay showed Treg suppressive function was accordingly dependent upon RBPJ activity in 3% O2. We validated our findings in tumor explants, a patient-derived TME model. We observed concordant modulation of multiple markers, including CTLA4 and OX40. We propose that Treg function in the hypoxic microenvironment hinges on RBPJ orchestrating a novel hypoxia program. Derailing it has the potential to modulate TI-Treg exclusively, yielding immunotherapy that only affects anti-tumor immunity.