*Purpose: We have previously reported that the intracellular protein DEPTOR stabilizes Foxp3 expression, enhances suppressive Treg function and inhibits Treg dedifferentiation. Overexpression of DEPTOR within Tregs is potent to inhibit alloimmune responses, prolong cardiac allograft survival and induce operational tolerance in combination with co-stimulatory blockade.

*Methods: To investigate the function of Treg DEPTOR, we generated Foxp3-specific DEPTOR knock-out (KO) mice (dDEP-Foxp3).

*Results: Phenotyping of KO mice was initially normal, but they developed patchy skin lesions with hyperkeratosis, local inflammatory infiltrates and IgG deposits after 9 months that were associated with splenomegaly and local lymph node hypertrophy. dDEP-Foxp3 mice with skin lesions had massive expansion of splenic CD3⁺ T cells (10.2×10⁷/spleen vs. 3.2×10⁷/spleen; P<0.05) which expressed higher levels of T cell activation markers (CD69⁺: 27% vs. 13%; P<0.05). Numbers of CD4⁺Foxp3⁺ Tregs also increased in dDEP-Foxp3 mice (within CD4⁺ cells: 27% vs. 15%; P<0.05) with lower expression levels of CD25 (mean fluorescence intensities: 75.2 vs. 113.5; P<0.05), suggesting a high turnover and/or proliferative compensation for inferior functional activity. Preliminary analysis suggests that CD138⁺ plasma cells also expand within lymph nodes of dDEP-Foxp3 mice with skin lesions (27.1% vs. 1.73% in WT mice). To test the biology of Treg DEPTOR in vivo, we vaccinated dDEP-Foxp3 mice with NP-KLH and measured B cell priming against NP in ELISPOT assays in vitro (vs. WT controls). We found that B cells from KO mice produced higher levels of NP-specific IgG (65.5 vs. 5.5 spots/well; P=0.01) compared to B cells isolated from WT mice. Additionally, by flow cytometry, we found higher levels of GL7 expression (48% vs. 33%; P<0.01) and class switch to IgG producing (7.9% vs. 2.5%; P<0.001) B cells in KO vs. WT mice. To understand the molecular mechanisms of DEPTOR function, we performed RNA-seq on FACS-sorted dDEP-Foxp3 and WT Tregs and observed intrinsic hyperactivities of mTOR, STAT3 and STAT5 pathways in KO cells. Additionally, transcriptomic analysis suggested that DEPTOR deficient Tregs have a decreased lineage commitment due to intrinsic hyperactivity of proinflammatory signaling.

*Conclusions: In summary, these findings suggest that DEPTOR modulates multiple signaling pathways in CD4⁺Foxp3⁺ Tregs and acts as an intrinsic regulator to coordinate both T cell and B cell activation responses. Our findings further establish DEPTOR as a central modulator of immunoregulation and lead to the intriguing possibility that pharmacologics that target DEPTOR degradation will serve as powerful immunosuppressants following transplantation.

« Back to 2020 American Transplant Congress