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BACKGROUND & AIMS T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin 10 (IL10). IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in treatment of 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 and anti-CD28 beads, and Tr1 cells were 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 and anti-CD28 beads and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells and trans-epithelial electrical resistance was measured to determine epithelial cell barrier function. RESULTS Phenotypes of Tr1 cells isolated from controls vs 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 patients with Crohn's disease or ulcerative colitis 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. CONCLUSIONS Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate 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.