Background: Blockade of the B7-CD28 and CD40-CD154 pathways induces tolerance towards porcine-islet-cell-cluster (NICC) xenografts in mice.
Aims: 1) Investigate CD4+Foxp3+Tregs in mouse-model of NICC xenograft-tolerance, 2) identify in situ immune-cell subtypes using imaging mass cytometry (IMC) and 3) assess the function of these Foxp3+Tregs.
Methods: C57BL/6-DEREG-mice with diphtheria-toxin(DT)-receptor/GFP attached to Foxp3-gene were transplanted with NICC under renal-capsule. Recipient-mice received CTLA-4-Fc and MR-1 mAb. Foxp3+Tregs were depleted by DT from day 3-17 (induction-phase), or 80-100 days post-transplantation (maintenance-phase). NICC-xenograft function determined by insulin/glucagon staining and serum porcine-c-peptide concentration. A 28-antibody panel for IMC was used to identify in situ cells and a bioinformatics pipeline was created for data analysis.
Results: More CD4+Foxp3+Tregs were seen in NICC-xenografts of treated mice on day-8 and 100 than day-20. Foxp3+ cells increased significantly in the draining-lymph-nodes of treated mice on day-8 (P=0.0010) and 100 (P=0.0128) when compared to the untreated/rejecting groups. CD4+Foxp3+Treg depletion during induction- or maintenance-phase led to rapid xenograft rejection. CD4+Foxp3+Tregs from lymphoid-organs of graft-tolerant DEREG-mice showed a significant increase in CD127, downregulated CD25, and reduced CD27 and CD62L expression compared to naïve/rejecting xenografts. IMC successfully quantified in situ immune-cell subsets (Fig.1), showing increased CD4+CD25-Foxp3+Tregs in tolerant compared to rejecting grafts, and localized within grafts at day-20. CD127+hiCD25+/lowCD44+hiCD62L-CD27-CD4+Foxp3+Tregs were transferred to NICC-transplanted Rag-\- mice and had a greater suppressive capacity than naïve-Tregs.

Conclusion: Foxp3+Tregs are essential for NICC-xenograft survival in this model. Importantly, CD127+hiCD44+hiCD62L-CD27-CD25+/lowCD4+Foxp3+Tregs were a subpopulation of Tregs that showed features of antigen-specific memory.