Abstract

Introduction: Regulatory T cells (Treg) have a pivotal role in tolerance and immune homeostasis. In autoimmunity, Treg are destabilized and degenerate, losing Foxp3 expression and compromising their regulatory function, while gaining a T effector cell (Teff)-like phenotype. Upon Foxp3 loss-of-function, Treg development gives rise to a heterogeneous population of Treg cells (∆Treg). ∆Treg retain the core Treg transcriptome and epigenetic imprint and can be classified based on the expression of the high-affinity IL-2 receptor-alpha (IL2Ra or CD25) and Foxp3 locus activity (Zemmour D et al. 2021). We herein propose a stepwise degeneration driven by a decrease in IL2/CD25 signaling and subsequent loss of Foxp1 expression. Methodology: We first characterized CD25+ and CD25-∆Treg by flow cytometry, RNAseq and functional assays on ∆Tregs of Foxp3eGFP∆iCre mice. Subsequently, we studied the role of IL2 and Foxp1 by the generation Foxp3eGFP∆iCreR26STAT5bCA Foxp3∆EGFPiCreFoxp1∆/∆ strains and IL2 Complex treatment. Results and Discussion: CD25+∆Treg expression profile, as compared to the one of CD25– ∆Treg cells, presented increased levels of genes typically expressed in Treg, such as Il2ra and Il10. Consistently, CD25-∆Treg were biased toward Th1, presented impaired suppressive capacity, and downregulated Foxp1. Treatment with IL2/IL2 and STAT5b gain of function upregulated CD25, decreased Th1 bias and inflammation, supporting the role of IL2 signaling halting the passage from CD25+ to CD25-∆Treg. ∆Treg-specific depletion of Foxp1 led to a steep decrease in the frequency of ∆Treg, increased T-bet expression, and a reduction in the Foxp3 locus activity, suggesting that Foxp1 loss drives ∆CD25-∆Tregs into a terminally degenerated state. Our results indicate that IL2 signaling is sufficient to maintain ∆Treg regulatory capacity, while Foxp1 expression is necessary to stabilize their identity and prevent terminal degeneration. We are currently doing scRNAseq and ChIPseq to elucidate the molecular mechanisms by which IL2 signaling and Foxp1 maintain Treg identity in the absence of Foxp3. Acknowledgements: National Institutes of Health grant R01 #81368, Beth Israel Hospital Rodent Histology Core, Alexander Rudensky for kindly providing the R6STAT5bCA strain.