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 (CD127highTreg, 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 CD127highTregs 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 CD127highTregs 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 CD127highTregs 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. National Health and Medical Research Council (NHMRC; Grants: GNT1013185 & GNT1125456). JDRF/Australian Research Council (Grant: 4-SRA-2016-265-M-B). Diabetes Australia (Grant: Y16G-HUMI). Westmead Scientific Platforms, Westmead Institute for Medical Research. Department of Animal Care at Westmead Hospital. Cancer Institute New South Wales. Ian Potter Foundation. Ramaciotti Facility for Human Systems Biology. Sydney Cytometry, University of Sydney.

Background: We previously identified xeno-antigen reactive human HLADR+CD27+Tregs with enhanced suppressive function in vitro and more capable of suppressing islet xenograft rejection in a humanised mouse model of neonatal porcine islet cell clusters (NICC) transplantation. Aims: 1) Phenotyping HLADR+CD27+Tregs and 2) evaluating possible pathways through which they exert a suppressive activity. Methods: Human CD4+CD25+hiCD127-Tregs isolated from peripheral-blood-mononuclear-cells (PBMCs) were co-cultured with irradiated porcine PBMCs for three subsequent cycles in the presence of IL-2/rapamycin and anti-CD3/CD28 beads. At day 21, phenotyping HLADR+CD27+Tregs of expanded Tregs was performed using flow cytometry for both surface- and intracellular expression (MFI) of the transmembrane proteins CD95, ICOS, CTLA-4, GITR; surface expression of CD39 and CD62L; and intracellular expression of FOXP3 and Helios compared to CD27-, HLADR-, HLADR-CD27-, HLADR+CD27-, HLADR-CD27+, depleted-HLADR+CD27+Treg subsets or all expanded Tregs. Results: There are no differences of FOXP3 and CD39 expression on HLADR+CD27+Tregs compared to all other subsets. CD62L increases significantly on HLADR+CD27+Tregs when compared to HLADR-CD27-, CD27- and HLADR-Tregs. HLADR+CD27+Tregs have an increasing trend of Helios expression compared to all other subsets, with a significant increase when compared to HLADR-CD27- and HLADR-Tregs. An increasing trend can also be observed in HLADR+CD27+Tregs CTLA-4 expression when compared to all other subsets. Furthermore, HLADR+CD27+Tregs show significant enhanced total-expression of CD95 (P <0.01) compared to all other subsets (except HLA-DR+CD27-Tregs), suggesting that the enhanced expression of CD95 is associated with HLA-DR expression. HLADR+CD27+ Tregs also show significant enhanced surface-expression of ICOS (P <0.05) compared to depleted-HLADR+CD27+Tregs subset. At the same time, both surface-expression and total-expression of GITR is decreased significantly in HLADR+CD27+Tregs compared to all other subsets. Conclusion: The enhanced suppressive function of xeno-antigen reactive HLADR+CD27+Tregs is associated with enhanced expression of CD95 apoptotic antigen and ICOS costimulatory molecule. HLADR and CD27 are the important immune checkpoints for xeno-antigen specific Tregs.

Aim: Determine differences in immunophenotype of paediatric kidney transplant recipients vs. healthy matching-age paediatric and adult controls by minimum blood volume. Methods: Absolute cell-count and leukocyte-profiling panels (46 fluorochrome-conjugated mouse-anti-human antibodies) for T cell, Treg, B, NK, DCs, and monocyte subsets were used to quantify immune-cell populations for 8 paediatric kidney transplant (PKTx) recipients, 5 healthy matching-age paediatric and 8 adult controls. Whole-blood sample (1050µl) were used for flow-cytometry analysis. Results: Absolute number of immune-cell subsets were in normal range for all groups. Age has correlation to CD45RO+memory T cells, but not to CD27+IgD-memory B cells. Healthy children had higher proportions of CD4-CD8-T (13.7±5.8 vs 7±4.3%, p=0.03) and γδT-cells (12±5.7vs 5.4±4.6%, p=0.04), CD4+CD25+CD127-Tregs (10±1.4 vs 7±1.6%, p<0.01), lower proportion of HLADR+CD45RA-CD4+ (0.03±0.04vs 7.42±5.64%, p<0.01), HLADR+CD45RA-CD8+ (0.0012±0.0018 vs 8±5.1%, p =0.04) T-cells compared to adults. HLA-DR was upregulated on both CD4+CD45RA- and CD8+CD45RA-T cells in PKTx, whilst CD183 (CXCR3) on CD4+CD45RO+ T-cells was downregulated. Compared to their healthy counterparts, PKTx had lower proportions of CD27+CD38lowclass-switch memory B-cells (38±7.6 vs 53±5.3%, p<0.01), non-classical monocytes (1.9±0.9 vs 5.8±1.1%, p<0.001), Tregs [CD4+CD25+CD127-Tregs (4.8±1.7% vs 10±1.4%, p<0.001) and CD4+FOXP3+Tregs (2.8±0.85% vs 6.3±1.3%, p<0.01)]. A small proportion of CD127+CD45RO+FOXP3 (2-5%) were observed in all groups. There was no difference in proportions of NK and DC between transplant vs control population. Conclusion: Immune profiling by multi-colour flow cytometry revealed differences by ages and after transplantation and offers valuable insight into unique cell subset changes present in the transplant population that could be targeted clinically or to monitor patient condition.

Background: We found previously that depletion of CD4+Foxp3+Tregs at early time (within 20 days) and later time (80 days) of transplantation abrogated pig-islet-xenograft tolerance in mice induced by short-term CTLA4-Fc/MR-1 treatment. We also identified memory-like CD127+/highCD4+GFP+/Foxp3+Tregs (CD127+/highTreg) in spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer model. Aims: 1) Further characterise tissue CD127+/high Tregs. 2) Investigate transcriptional profile of CD4+Foxp3+Treg and non-Foxp3 CD4+ subsets in transplant tolerance. Methods: We used DEpletion of REGulatory T cells (DEREG) mice, which carry the enhanced GFP transgene under Foxp3 promoter as recipients of NICC transplantation tolerance model. Cell-subsets were selected with FACS/Cell Sorter based on positive or negative expressions of CD4, GFP, and CD127 or CD45, CD4 and GFP. mRNA expression of Il-10, Tgf-β, Blimp-1, Ebi3 (reflecting IL-35) of CD127+/high Tregs was assessed using TaqMan® Gene Expression Assay. Bulk RNA-Seq revealed the transcriptional profiles of CD127+/highTreg, CD127-/low Treg, CD4+GFP-Foxp3+ Treg, non-Foxp3 CD4+, and CD45+CD4- subsets from spleens (sp), graft draining-lymphocytes (DLN/dln), or grafts in mice with 100-day tolerant-graft induced by CTLA4-Fc/MR-1 blockade or naïve DEREG-mice. Results: RT-PCR showed Ebi3, Il-10, Blimp-1 significantly increased in splenic CD127+/high Tregs compared to naïve-CD4+GFP-Foxp3+Tregs or non-Foxp3 CD4+T cells. The proportion of CD127+/high Tregs was higher in tolerant grafts (25.6±3.1%) than tolerant spleens (14.8±0.4%). 15 pairwise-comparisons identified 1740 differentially expressed genes (DEGs) (FDR<0.05) that clearly distinguished between CD45+CD4-, Foxp3-CD4+T, and Treg subsets; with no striking differences seen for CD45+CD4- cells (spleen) and mild differences in Foxp3-CD4+T cells (spleen) between naive and tolerant-groups; and diverse differences within Treg subsets. Next, 9 paired cross-comparisons between different Treg subsets identified 427 DEGs and showed large difference between graft-Treg and Treg subsets of spleen or DLN; moderate differences between spTreg and dlnTreg subsets; and minor differences within the three Treg subsets of spleen or DLN. Further, compared to naïve-Treg or CD127-/low Treg subsets, graft-Tregs shared many upregulated-DEGs across dlnCD127+/high Treg, and/or spCD127+/high Treg including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab1 (an MHC-II gene), Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features and high suppressive functions in both mice and/or humans. Conclusion: Tissue-Tregs (CD127+/high Tregs) developed in graft, spleen and DLN of transplant-tolerant mice share a transcriptional trajectory with other tissue/tumour Tregs.