*Purpose: The role of regulatory CD4⁺Foxp3⁺ T cells (Treg) suppressor function within allografts in tolerance remains unclear. Thus, we sought to directly address: 1) whether Treg can directly suppress within allograft, and 2) whether Treg suppression within allograft is required for tolerance induction.

*Methods: First, we used a model where immune reactions are restrained to the graft only. Effector T cells (Teff) alone, or with Treg (2-3×10⁶ each) were transferred to B6 mice lacking secondary lymphoid organs (SLO-; splenectomized LTβR^-/-) 5 days after islet allograft (Balb/c). Second, given that S1PR1 signaling is necessary for lymphocyte exit from SLO, we prevented Treg migration to allografts at the time of tolerance induction by removing S1PR1 expression on endogenous Treg specifically. Tamoxifen-fed B6.Foxp3^{EGFP-Cre/Ert2}.S1PR1^fl/fl (S1PR1KO-Treg) or B6.S1PR1^fl/fl (control; WT-Treg) mice received allogeneic Balb/c islets and were left untreated or treated with the tolerogenic anti-CD45RB antibody.

*Results: In the model where immune reactions are restrained to the graft only, SLO- untreated recipients failed to reject islet allografts (>100d MST). Adoptive transfer of Teff alone induced acute rejection (10d MST). In contrast, Teff + Treg co-transfers significantly prolonged graft survival, with 65% of recipients surviving long-term (>100d MST; p<0.05 vs. Teff alone). Interestingly, Treg did not affect Teff proliferation or accumulation within allografts, but significantly reduced MHC-II expression on DCs and IFN-γ in Teff.

Next, we addressed whether Treg suppression within allograft is required for tolerance induction using mice where Treg migration to allografts is impaired. Untreated mice rejected allografts acutely (18d MST). Interestingly, while anti-CD45RB treatment induced tolerance in WT-Treg control mice (95d MST), all S1PR1KO-Treg mice rejected their graft (30d MST; p<0.05 vs WT-Treg). In addition, these results correlated with a 50% decrease in Treg infiltrating islet allografts and a correlative 90% increase in Treg in lymph nodes on day 10 in S1PR1KO-Treg compared to WT-Treg recipients.

*Conclusions: Overall, our data demonstrate that Treg can suppress rejection by Teff within allografts without prior activation in SLO, and that Treg suppressor function within allografts is required for tolerance induction.

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