FOXP3+TREGS THAT EXPRESSED CD127 EXISTING MEMORY TREGS FEATURE IN A TOLERANT MOUSE MODEL AND IN HUMAN SKIN

Background: We have demonstrated that toleranceof porcine NICC xenografts in mouse recipients can be induced by costimulation-blockade with CTLA4-Fc and anti-CD154 mAb (MR-1). CD4+CD25+Foxp3+Tregs were shown to be essential for the induction and maintenance of tolerance. Aims: 1) To phenotype long lasting (>100 days) Foxp3+ Tregs in a mouse model of porcine-NICC xenografts tolerance; 2) To assess the capacity of these Foxp3+Tregs to provide antigen specific tolerance;and 3) To determine if a subset of human Tregs can be identified with a similar phenotype and function. Methods: Flow cytometric analysis was used to phenotype Foxp3+(GFP+) Tregs for CD25, CD27, CD39, CD44, CD62L, CD127, and MHC-II expressions. Foxp3+(GFP+) Tregs were isolated from Foxp3+(GFP)-micerecipients of porcine NICC that received CTLA4-Fc and MR1 treatment (tolerant-Tregs), those that received porcine NICC without treatment (rejection-Tregs), and those that had no treatment and no transplantation (naïve-group). The functional capacity of these Foxp3+Treg was assessed after adoptive transfer to Rag-/- mouse recipients of porcine-NICC-xenografts. Human CD3 T cells were isolated from the dermis of adult human skin, and FOXP3+Tregs were assessed for expression of CD127, CD27, CD25 CD39, CD45RO, CTLA-4, HLA-DR, CCR7, CD154. Results: Foxp3+(GFP+) mice made tolerant to NICC xenografts by CTLA4-Fc/MR-1 treatment, developed a population of Foxp3+(GFP+) Tregs with high expression of CD127, downregulated expression of CD25, andreduced expression of CD27 and CD62L compared to Tregs of naïve mice and Tregs from mice with rejected xenografts. In Foxp3+(GFP+) mice that had no treatment and rejected islet xenografts, Foxp3+Tregs revealed upregulated expression of MHC-II, low or dim expression of CD127, low levels of CD62L, and similar level of CD27 and CD25 compared to naïve-Tregs. The different expression of CD127, CD62L, CD27, CD25 and MHC-II on tolerant-Tregs and rejection-Tregs suggest that they may be useful marks to distinguish activated effector Tregs from antigen specific memory Tregs. Sorted CD127+hiCD25+/lowCD44+hiCD62L-CD27-CD4+Foxp3+Tregs expanded in Rag-\- mice with NICC-xenografts and had a higher suppressive capacitythan naïve-Tregs. Isolated FOXP3+Tregs from the dermis of human skin were CD127+CD27+CD25+CD39+CTLA4+CD45RO+ Tregs. The proportion of HLA-DR+CD45RO+ memory Tregs was 20% of FOXP3+Tregs. Conclusion: The expression of CD127 on human CD45RO+ memory-Tregs from skin and Tregs of long-term transplant tolerant mice suggested that CD127 is important antigen marker for identification of memory Tregs in both mouse and human.