CD4+Foxp3+ Tregs play a critical role in transplant tolerance. Foxp3 does not initiate thymic differentiation of natural Treg (nTreg), but is essential for later differentiation and regulatory function. By transferring CFSE-labeled Foxp3+ (Treg) vs. Foxp3- (Tconv) CD4 cells from Foxp3-reporter mice into replete congenic recipients, we showed that nTreg exhibit very high constitutive homeostatic proliferation (HP) compared to Tconv (∼50% vs. <10% proliferate over 10d, respectively). High HP has important therapeutic implications, since HP can be specifically manipulated to expand nTreg in vivo. Foxp3, itself, might regulate Treg HP. To test this, CFSE-labeled nTreg from Foxp3-reporter mice expressing Foxp3 at wt levels (FIR) or reduced levels (FILIG; 90% reduction) were adoptively transferred into replete congenic mice. Whereas 50% of nTreg with wt Foxp3 underwent HP, <10% of Filig nTreg underwent HP (similar to Tconv). Transduction of Filig nTreg cells with Foxp3 retrovirus (rv) to restore wt Foxp3 levels, restored high rates of HP (40%) vs. empty vector controls (12% HP). This suggests that Foxp3 is necessary for high HP by nTreg. However, when Foxp3 was expressed in Tconv cells by rv transduction, these induced Tregs (iTregs) exhibited similar HP to control Tconv cells transduced with vector control rv (17% vs. 16%, respectively). Thus, Foxp3 is required, but is not sufficient for increased HP. Peripheral transfer of transduced T cells (above) was next compared to endogenously selected T cells generated after transplantation of transduced Filig (Thy1.2) bone marrow (BM) into lethally irradiated Thy1.1 hosts. Filig BM was infected with Foxp3 vs. control rv, allowing “color coding” (GFP= endogenous Foxp3; RFP= transduced Foxp3). Surprisingly, in this setting, Tconv cells (no endogenous Foxp3) transduced to constitutively express Foxp3, underwent higher HP compared to control (or nontransduced) Foxp3- counterparts (32% vs. 12%). Thus, forced Foxp3 expression during thymic ontogeny augments HP capacity, not seen when Foxp3 is acquired by mature Tconv in the periphery. One possibility is that forced thymic Foxp3 expression allows selection of a higher affinity (autoreactive) TCR repertoire conducive to high HP. This is now being evaluated by deep sequencing TCRs from BM chimeras (Tconv ± transduced Foxp3 vs. nTreg). These findings provide new insight into the regulation of HP by Treg, the role of Foxp3 expression in Treg homeostasis, and highlight additional distinctions between induced and natural Treg.

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