The ever-increasing disparity between the lack of organ donors and patients on the transplant waiting list is increasing worldwide. For the past several decades xenotransplantation has led the way to correct this deficit and remains clearly the only feasible option to provide a means to meet the demand for patients in need of an organ transplant. Xenotransplantation's ability to provide a specifically designed unlimited supply of organs, suited to treat the various needs for transplant organs and cells, has recently been championed by successful pre-clinical trials that have run long-term in non-human primate studies.In this review we show how these improvements have come about due to longterm dedicated research and recent advances in biomedical engineering technology, such as genome editing tools including zinc finger nucleases, TALEN, and CRISPER/ Cas9 which have paved the way for significant breakthroughs in improving xenograft outcomes through genetic modifications to the donor source pig. Other novel approaches include the development of decellularized porcine tissue, such as corneas which can now be transplanted into patients with the minimal need for immunosuppression or other side effects. Further genetic variants of the porcine genome are also now being optimized to abrogate rejection. The emergence of new modalities such as; mesenchymal stem cells, donor thymic vascularization, in vivo bioreactors, chemokine and cytokine therapies have come to show improvements in xenograft outcomes. Furthermore, new studies confirm the safety status of using porcine xenografts, verifying that with current technologies and approaches, the issue of PERV transmission is a moot point. These breakthroughs and technological advancements push the reality of xenotransplantation one step closer to the clinic. K E Y W O R D S complement regulation, CRISPR, Mesenchymal stem cells, zoonosis
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 +/high CD4 + GFP + / Foxp3 + Tregs (CD127 +/high Treg) 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 +/high Treg, CD127 -/low Treg, CD4 + GFP -Foxp3 + Treg, non-Foxp3 CD4 + , and CD45 + CD4subsets 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 + CD4cells (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,
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