Introduction: CD4⁺Foxp3⁺regulatory T-cells (Tregs) are essential for islet xenotransplant tolerance. We identified a novel memory-like CD127^+/highTreg subset in the spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer mouse model. Our aims were to: (1) Further characterise CD127^+/high Tregs. (2) Investigate transcriptional profile of CD4⁺Foxp3⁺Treg and non-Foxp3 CD4⁺ subsets in transplant tolerance.

Methods: DEpletion of REGulatory T-cells (DEREG) mice carrying a GFP transgene under a Foxp3 promoter were used as recipients in a porcine NICC xenotransplant model. Cell-subsets were selected with BD Influx Cell Sorter based on expression of CD4, GFP, and CD127 or CD45, CD4 and GFP. Bulk RNA-Seq and flow-cytometry profiled the transcriptomes and phenotypes of Treg subsets (CD127^highTreg, CD127^-/lowTreg, and all Treg) from the spleen (sp), draining lymph node (DLN) and grafts of tolerant-mice (day-100) compared to non-transplant mice. Quantitative PCR (qPCR) was used to assess expression of Il10/Tgfb1/Ifnγ/Il2/Il7//Il18//Il33/Ctla4 in spleen, ALN, DLN and graft cells of transplant mice (CTLA4-Fc/MR-1-treatment), rejection mice (no treatment) at day-8/100 and non-transplant mice. Imaging mass cytometry (17 antibodies) was used to evaluate the graft-infiltrating immune cells in tolerant-(day-8, 20, 100) and rejection-(day-8, 20) groups.

Results: A high proportion of CD127^highTregs was observed in tolerant-grafts compared to tolerant-spleens [25.6±3.1% vs 14.8±0.4%]. Ebi3(IL-35), Il-10 and Blimp-1 expression were upregulated in splenic CD127^highTregs of day-100 transplant-mice compared to naïve-Tregs. We identified 1740 differentially expressed genes (DEGs)(FDR<0.05) from 15 pairwise-comparisons that distinguished between CD45⁺CD4^-, Foxp3^-CD4⁺T-cells, and Treg subsets with: similar expression patterns between naive and tolerant CD45⁺CD4^- cells; minor differences in Foxp3^-CD4⁺T cells; and notable differences across Treg subsets where transcriptional profile was not uniform. Between different Treg subsets, 9 paired cross-comparisons identified 427 DEGs. In tolerant mice, both sp- and DLN-CD127⁺Tregs showed an effector/memory Treg profile. Compared to naïve-Treg or CD127^-/lowTreg subsets, graft-Treg and CD127^highTregs displayed upregulated DEGs including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab, Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features.  Additionally, measured by qPCR, increased Il7 expression and decreased Il2 expression in day-100 vs day-8 tolerant-graft suggest activated Tregs may preferentially utilise IL-7 or IL-35 over IL-2 (more broadly acting).

Conclusion: Memory-like CD127^+/highTregs are critical for maintaining tolerance and may share a transcriptional trajectory with other tissue/tumour Tregs.