TIGIT+ iTregs elicited by human regulatory macrophages control T cell immunity

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based, adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+ induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs (miTregs) relies on multiple, non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-β, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct pathway Tregs. To better characterise the phenotype of human miTregs, whole genome gene expression profiling studies by microarray were performed. Mregs and IFN-γ Mφ were generated from CD14+ monocytes of 5 healthy donors. Seven days later, CD3+ T cells were isolated from 5 unrelated healthy donors and split into two lots: From the first lot, a pure population of fresh CD4+ T cells was obtained by flow-sorting and total RNA was extracted; the second lot of T cells was added into direct co-culture with allogeneic Mregs or IFN-γ Mφ at a 1:1 ratio for 5 days, or were cultured alone without stimulation for 5 days. After coculture, CD4+ and CD8+ T cells were flow-sorted and total RNA was extracted. Microarray analysis was performed on the Agilent Whole Human Genome Oligo Microarray 8x60K platform. Hence, a dataset was generated comprising eight sample groups (S1-S8) representing four CD4+ T cell populations and four CD8+ T cell populations from 5 independent donors produced by single-colour hybridisations.