*Purpose: We have previously reported that DEPTOR activity within CD4⁺Foxp3⁺ Tregs increases immunomodulation following transplantation. DEPTOR functions by both stabilization of Foxp3 expression through epigenetic mechanisms and via the modulation of Treg metabolism. In a fully MHC mismatched heterotopic murine cardiac transplantation model, transgenic DEPTOR overexpression induces long-term allograft survival following transient costimulatory blockade.

*Methods: T cell subsets from Treg-specific DEPTOR KO mice (Foxp3-cre DEPTOR^lox/lox; ΔDEP-Foxp3) were profiled using FACS, and functional responses were evaluated using ELISPOT. Treg subset differentiation was evaluated in ΔDEP-Foxp3 fate tracker mice (Foxp3-cre Rosa26-lox-STOP-lox-GFP DEPTOR^lox/lox; ΔDEP-Rosa-Foxp3) using FACS and single cell RNAseq.

*Results: We observed that ΔDEP-Foxp3 mice develop skin lesions after 6-12 months that are associated with hair loss, mononuclear infiltration, and intradermal immunoglobulin depositions. Using FACS, we find that the spleens in diseased ΔDEP-Foxp3 mice were hyperplastic, had increased numbers of CD3⁺ T cells (10.2×10⁷/spleen vs. 3.2×10⁷/spleen; P<0.05), increased expression of T cell activation markers (CD69⁺ [within CD4⁺]: 27% vs. 13%; P<0.05), and expanded frequencies of CD4⁺Foxp3⁺ Tregs (Foxp3⁺[within CD4⁺]: 27% vs. 15%; P<0.05), all consistent with autoimmunity. We used a NP-KLH vaccination model to investigate the immunoregulatory function of DEPTOR deficient Tregs. By ELISPOT, we found that ΔDEP-Foxp3 mice had increased numbers of NP-specific IgG producing B cells vs. controls (65.5 vs. 5.5 spots/well; P=0.01) confirming that DEPTOR deficient Tregs lack suppressive function in vivo. Using our fate tracker mouse, we surprisingly failed to observe any differences in Treg dedifferentiation (Foxp3^negGFP⁺ cells, i.e. exTregs) in ΔDEP-Rosa-Foxp3 vs. WT controls (both <0.1% within CD4⁺) suggestive that DEPTOR may regulate Treg subset differentiation. To identify DEPTOR-dependent Treg subset(s) that modulate B cell responses, we sorted GFP⁺ cells from ΔDEP-Rosa-Foxp3 and WT controls, performed single cell RNAseq, clustered subsets based on their transcriptomes and evaluated the ratio of WT to KO cells per cluster. Initial analysis suggests that Tregs from KO mice have different Treg subset compositions indicating that DEPTOR is a central regulator of Treg subset differentiation and/or that it functions via its expression in novel subpopulations.

*Conclusions: We find that DEPTOR is a central modulator of Treg subset differentiation and that DEPTOR-dependent Treg subset(s) regulate T and B cell responses. We speculate that DEPTOR is a novel pharmacological target to modulate Treg specific interactions with both T and B cells of great significance for tolerance induction following transplantation.

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