Background: Regulatory T cells (Tregs) are essential for maintaining immune homeostasis. Their use as an adoptive cellular therapy is under investigation as a method to reduce reliance on long-term pharmacological immunosuppression. However, a deeper understanding of Treg biology is necessary to understand how these cells will behave in hypoxic or inflamed environments. In this study, we assess the effects of manipulation of the molecular hypoxia-sensing pathway on Tregs.
Methods: Mice were generated expressing a doxycycline-dependent short hairpin RNA targetting prolyl hydroxylase domain (PHD)2, the major enzyme responsible for degradation of the hypoxia inducible factor alpha (HIFα) subunits. Mice were also generated bearing additional shRNAs against either HIF1-α or HIF2-α, the two principal subunits that regulate transcription of hypoxia-sensitive genes. Tregs from these mice were assessed using a skin transplant regulation model. In order to identify transcriptional targets, we performed single cell RNA sequencing on flow-sorted CD4⁺ T cells from control and PHD2 knockdown mice.
Results: PHD2 knockdown mice developed an autoimmmune phenotype typified by an increased number of leukocytes but especially of Tregs. This phenotype was rescued by concomitant knockdown of HIF2-α but not HIF1-α. Tregs from these mice were incapable of protecting skin allografts from rejection in vivo. Despite this apparent loss of function, Tregs maintained their expected phenotypic signature, including classical surface markers and TSDR demethylation of the Foxp3 locus. Single cell RNA sequencing of CD4⁺ T cells identified a unique population of Treg cells with a specific signature associated with PHD2 knockdown.
Conclusions: Exploring the pathways that regulate Tregs is of great relevance in transplantation. Here we show that a novel pathway regulated by HIF2-α controls Treg function, which may be relevant in hypoxic environments. This provides an opportunity for manipulation of Treg function, particularly with the current availability of small molecule inhibitors of the HIF pathway.