Dysregulated regulatory T cell responses expand fibroblasts and lead to chronic rejection after heart transplantation
Chronic rejection after organ transplantation manifests as immunosuppressant-resistant graft vascular remodeling and fibrosis, which remains the dominant driver of mortality after the first year of heart transplantation. Single-cell RNAseq analysis of MHCII-mismatched heart transplants developing chronic rejection identified graft IL-33 as a stimulator of tissue repair pathways in infiltrating myeloid cells and regulatory T cells (Tregs). Using IL-33-deficient donor mice, it was revealed that graft fibroblast-derived IL-33 potently induced Amphiregulin (Areg) expression by recipient Treg. Areg is an epidermal growth factor secreted by multiple immune cells to shape immunomodulation and tissue repair. In particular, Areg is proposed to play a major role in Treg-mediated muscle, epithelium, and nerve repair. Assessing recipient mice with Treg-specific deletion of Areg demonstrated that this pathway surprisingly contributes to chronic rejection. Specifically, heart transplants from recipients with Areg-deficient Tregs exhibited less vasculopathy and vessel-associated fibrotic niches infiltrated by recipient T cells. Mechanistically, we show that Areg does not impact Treg suppressive function, but that IL-33 mediated Treg-secretion of Areg increases fibroblast proliferation and migration. In total, these studies identify how a dysregulated repair response involving interactions between IL-33+ fibroblasts in the allograft and recipient Treg contributes to the progression of chronic rejection. Overall design: The overall purpose of this study was to elucidate how local IL-33 supports Treg-mediated tissue repair responses and the ways that these processes become dysregulated following transplant. We hypothesized that the recipient immune responses to persistent MHC differences will instigate immune-mediated injuries that disrupts effective repair of the damages sustained during transplantation. On day 14 post-transplantation we applied scRNA-seq with cell hashing to immune cells (lympholyte-M seperation) from BM12 chronic murine heart rejection transplant model that were compare to  BM12 heart grafts deficient in IL-33 (n=3/group), enabling us to understand the full scope of IL-33 in regulating immune remodeling of graft tissue. IL-33 modulated over 700 genes in heart-infiltrating Tregs, including the growth factor amphiregulin (Areg), yet when B6 Foxp3YFP-CrexAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 hearts were assessed at day 100 (n=6-10), the deletion of Areg in Tregs protected against chronic rejection. Our analyzed data reflects bioinformatic computational tools, in vitro assays, and immunofluorescence. Age and sex matched mice were randomly assigned to experimental groups and the studies were not blinded. HTO Barcode Hashtagged Sample TotalSeq-A0301 ACCCACCAGTAAGAC BM12 WT 1 TotalSeq-A0302 GGTCGAGAGCATTCA BM12 WT 2 TotalSeq-A0303 CTTGCCGCATGTCAT BM12 WT 3 TotalSeq-A0304 AAAGCATTCTTCACG BM12 IL33 KO 4 TotalSeq-A0305 CTTTGTCTTTGTGAG BM12 IL33 KO 5 TotalSeq-A0306 TATGCTGCCACGGTA BM12 IL33 KO 6