Shared transcriptional trajectory of tissue tregs between tolerant-grafts and lymphoid organs in transplant tolerance
Haina Wang1, Zhuangzi Wang1, Yuanfei Zhao1, Leigh Nicholson1, Wayne J Hawthorne1, Elvira Jimenez-vera1, Brian Gloss2, Joey Lai2, Adwin Thomas1, Geoff Y Zhang3, Yuan Min Wang3, Natasha Rogers1, Guoping Zheng1, Shounan Yi1, Stephen I Alexander 3, Philip J O’Connell1, Min Hu1.
1Center for Transplant and Renal Research, Westmead Institute for Medical Research, Sydney NSW, Australia; 2Scientific Platforms, Westmead Institute for Medical Research, Sydney NSW, Australia; 3Centre for Kidney Research, Children's Hospital at Westmead, Sydney NSW, Australia
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.
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