Foxp3⁺ regulatory T cells (Tregs) are critical mediators of immune tolerance and are absolutely required for allograft tolerance in animal models. In addition, clinical trials for ex vivo-expanded Tregs are underway, using either bulk, nonspecific Tregs or alloantigen-expanded Tregs. However, the fate of endogenous, naturally occurring allospecific Treg populations remains to be determined. Here, we take advantage of a recently developed peptide:MHC tetramer (2W:I-A^b)-based system to study an endogenous allospecific Treg population in a mouse transplantation model in which donor cells transgenically express the model antigen 2W. 2W⁺ BALB/c x C57BL/6 F1 donor hearts were transplanted into C57BL/6 recipients either with no treatment or in combination with anti-CD154 and donor splenocytes to induce tolerance. Compared to naïve controls, tolerant animals had a significantly higher percentage (~30% vs 10%) and total number of splenic donor (2W:I-A^b)-specific Tregs at day 7 and 30 after transplantation. The enrichment of 2W:I-A^b-reactive Tregs among 2W:I-A^b CD4⁺ cells was even more dramatic in the tolerant graft and was significantly increased relative to the percentage of bulk Tregs (~70% vs 25%). We determined that the increased Treg percentages was due to the inhibition of donor-specific Tconv expansion and a modest increase in Treg numbers. We next tested if the accumulation of endogenous donor-specific Tregs in the spleen and graft was due to the expansion of preexisting Tregs by quantifying the expression of the proliferation marker Ki67 at 7-30 days after transplantation. Increased percentages of 2W:I-A^b-specific Foxp3⁺ Tregs expressed Ki67 in tolerant recipients, compared to naïve or acutely rejecting recipients. To test whether conversion of 2W:I-A^b Tconvs into induced iTregs also contributed to increased 2W:I-A^b Treg percentages, we transferred Foxp3^–CD44^–CD4⁺ T cells from Foxp3-GFP transgenic mice into transplant recipients at the time of tolerance induction. A small but detectable percentage of these transferred cells differentiated into Foxp3⁺ Tregs in the spleen and graft. Taken together, we show for the first time that the increased percentages of endogenous donor-specific Tregs observed in tolerance is achieved through the inhibition of donor-specific Tconv expansion and a modest increase in Treg numbers that is dependent on proliferation of Tregs and conversion of Tconvs into iTregs.

CITATION INFORMATION: Young J., Yin D., Alegre M-.L., Chong A. Enrichment of Endogenous Donor-Reactive Tregs in the Spleen and Graft during Transplantation Tolerance is the Result of Treg Proliferation and Conversion from Tconv Am J Transplant. 2017;17 (suppl 3).

« Back to 2018 American Transplant Congress