Suppressive and Gut Reparative Functions of Human Type 1 T-regulatory Cells Cook, Laura; Stahl, Martin; Han, Xiao; Nazli, Aisha; MacDonald, Katherine N.; Wong, May Q.; Tsai, Kevin; Dizzell, Sara; Jacobson, Kevan; Bressler, Brian; et al.
Background & Aims. T-regulatory (Treg) cells suppress immune responses to maintain homeostasis. There are two main subsets of Treg cells, FOXP3-positive Treg cells that do not produce high levels of effector cytokines and Type 1 Treg (Tr1) cells that are FOXP3-negative and secrete interleukin 10 (IL10). IL10 is an important anti-inflammatory cytokine, therefore Tr1 cells might be uniquely suited for development as a treatment for inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. Methods. We obtained blood samples and colon biopsies from patients with Crohn’s disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples, stimulated with anti-CD3/CD28 beads and Tr1 cells purified using an IL10 cytokine capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3/CD28 beads and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colonic organoid cultures were established then cultured with Tr1 or FOXP3-positive Treg cell culture supernatants and analyzed by immunofluorescence and flow cytometry. T84 cells (human colonic adenocarcinoma epithelial cells) were incubated with Tr1 or FOXP3-positive Treg cell culture supernatants and trans-epithelial electrical resistance was measured to determine epithelial cell barrier function. Results. Phenotypes of Tr1 cells isolated from healthy subjects or patients with Crohn’s disease or ulcerative colitis did not differ significantly following expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1beta (IL1B) and TNF from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy subjects and IBD patients secreted IL22, which regulated repair of the epithelium and promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. Conclusion. Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (innate immune response). They also secrete IL22 to regulate repair of the epithelium and promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.
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