Background: Foxp3⁺Tregs have an essential role in allograft tolerance. Aim: To evaluate Foxp3⁺Tregs in the spontaneous tolerance of mouse renal allografts. Methods: A standard mouse renal transplant tolerance model DBA/2(H-2d) to C57BL/6(H-2b) was used. The recipients included C57BL/6, Foxp3^RFP, Foxp3^GFP and DEREG mice (all on C57BL/6 background), with a host nephrectomy was performed. DEREG mice were used for depletion of Tregs. DT was given to DEREG mice for 3 days prior to transplant. Foxp3^RFP, Foxp3^GFP allowed tracking of Tregs. Renal allografts were harvested from at day 7, 14 and ³69 post transplantation(PTX). Allograft I-Tregs(CD4⁺GFP⁺) were sorted by FACS and expression of cytokines, Blimp-1 and CXCR3 assessed. I-Tregs of day 14 PTX were adoptively transferred into Rag-/- mice to assess dominant tolerance. Results: Treg-depleted DEREG mice, rejected DBA/2 renal allografts (n=8) with a median survival time (MST) of 6.5 days. In contrast C57BL/6 background mice (n=39) showed long-term tolerance with a MST>100 days (p<0.0001) for DBA renal allografts. Tolerant renal allografts at day 7 and >100 days had localized interstitial infiltrates of mononuclear cells. GFP⁺ Tregs were found in tolerant allografts at day 7 and >100 PTX but not in rejecting allografts. The proportion of Tregs increased the renal allograft at day 7 and 14 PTX (p<0.05). Tregs were highest in the draining LN (DLN) compared to PB and control LN at day 7and >84 PTx in Foxp3^GFP mice (p<0.05). Allograft I-Tregs express elevated levels of TGF-Β, IL-10, Blimp-1, and CXCR3. Adoptively transferring I-Tregs into Rag-/- mice which were subsequently transplanted with DBA/2 skin grafts showed skin graft survival was greater than 100 days and remained intact following challenge with non-Tregs (CD4⁺GFP^–). Conclusion: Renal allografts require Tregs to develop tolerance in this model. This is associated with increased Blimp-1, and CXCR3 in allograft I-Tregs that can transfer dominant tolerance.

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