Introduction: Previously we demonstrated CTLA-4Fc/MR-1 antibodies induced tolerance towards porcine islet cell cluster (NICC) xenografts in mice, and Foxp3Tregs from these mice showed dominant and specific tolerance to NICC. In this study, we are verifying the Tregs from long-term tolerance mice contain the memory-Tregs, distinguished memory-Tregs from activated-Tregs phenotypically in the NICC transplant model, and assessing the suppressive function of memory-Tregs from spleens into islet transplanted Reg KO mice.
Methods: C57BL/6-DEREG mice who have diphtheria toxin receptor-GFP attached to their Foxp3 gene were transplanted with 4000 IEQ NICC under the renal capsule, and treated IP with CTLA-4Fc (500ug) and MR-1 (500ug) at day 0, 2, 4 and 6. The phenotype of Foxp3 Tregs in the lymph nodes, draining lymph node and spleen were analyzed by flow-cytometry. CD4+GFP+ Tregs and highly selected (CD4+GFP-CD44+CD127+CD62L-) Tregs (>2×105) were isolated from spleens of naïve (n=5), rejected (n=3) and tolerant (n=8) DEREG mice, all the tolerant mice over 100-days post-transplant. On day 25, those Tregs were injected into the immune-deficient transplanted Rag KO mice into the tail vein separately. On day 45, injected transplanted Rag KO mice were challenged by CD4+GFP- T cells at a ratio of 1:3 from the Naïve C57BL/6-CD45.1GFP mice by tail vein. Transplanted Rag KO mice with no cells injected (n=2) and with only CD4+GFP- T cells injected (n=2) as positive and negative control groups. All mice would be sacrificed at day 100 after adoptive transfer.
Results: NICC xenograft tolerance was confirmed in recipient Foxp3-GFP mice, and rejected in control group. In phenotype, upregulations of CD44, MHC-II, CD39, especially CD127 (85.8% VS 4.3%) (p=0.002) in splenic CD3+CD4+Foxp3+Tregs occurred on the day 70 or more than 100 days after transplantation, and downregulations of CD27 (p < 0.0001) and CD62L (p = 0.0025) were found in the mouse with tolerant NICC grafts (5.6%, 3.7%), compared to Tregs of rejected group (73.8%, 36.6%) and naïve-Tregs of non-transplanted mice (78.8%, 46.1%). Additionally, in pooled draining lymph nodes from the transplanted mice more than 100 days, tolerant-Tregs also showed the increased CD127 (71.3% vs 12.5%) and decreased of CD27 (15.2% vs 74.7%) and CD62L (14.6% vs 49.3%), compared to rejected-Tregs. However, the up and down in long-term pooled lymph nodes were only found in CD127 and L-selection of the comparison between rejected-Tregs and tolerant-Tregs, which from 19.4% to 43.9%, and 51.2% to 38.4% respectively. For suppressive function, the sorted adoptive-transferred Tregs and T cells can be detected separately on 88-day by flow after adoptive transfer in Rag KO mice.  
Conclusion: Tolerant-Treg induced by costimulation-blockade by CTLA-4Fc and anti-CD154 antibodies in mice with NICC grafts may contain the memory-Tregs at the long-term time point, but function needs to be examined by histology further when they up to 100-day.