Abstract
IntroductionCD4+ T cells with regulatory function co-expressing Foxp3 and RORγt are linked to the development of oral tolerance towards innocuous food antigens in mice. This study aimed to discern the role played by IL-6 and retinoic acid (RA) in the in vitro generation of Foxp3+RORγt+ T cells and to investigate whether such cells have suppressive properties.MethodsCD4+CD25- T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-β and different concentrations of IL-6 and/or RA. Percentage of Foxp3+, RORγt+, IL-17+, Foxp3+RORγt-, Foxp3+RORγt+, and Foxp3-RORγt+ T cells within the total CD4+ T cell population, production of cytokines (IL-10 and IL-17A) and gene expression (Foxp3, Rorc, Tgfb1, Il6, Il10, and Il17) were assessed at different time points. The phenotype and ability of cells generated from CD4+CD44-CD62L+ cells in the presence of RA to suppress effector T cell proliferation was assessed.ResultsTGF-β plus IL-6 induced the generation of Foxp3+ and double positive Foxp3+RORγt+ T cells to a higher extent than TGF-β alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-β, increased Foxp3 and Rorc expression and Foxp3 and RORγt transcription and promoted the differentiation of Foxp3+RORγt- and Foxp3+RORγt+ cells that expressed and secreted IL-17. Foxp3+ T cells generated in vitro in presence of RA were functionally suppressive.ConclusionsUnder the influence of IL-2 and TGF-β, suppressive Foxp3+RORγt+ T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype.
Highlights
CD4+ T cells with regulatory function co-expressing forkhead box P3 (Foxp3) and RORgt are linked to the development of oral tolerance towards innocuous food antigens in mice
As expected, following 96 h of culture, TGF-b induced the expansion of Foxp3+ cells from CD4+CD25- cells, while TGF-b plus IL-6 increased the percentage of RORgt+ and IL-17+ cells, and TGF-b plus IL-6 and retinoic acid (RA) led to intermediate levels of these cells (Figure 1A)
While Foxp3+RORgt+ cells had been previously induced in vitro from mouse naïve cells under T cell receptor (TCR) stimulation in the presence of TGF-b [2, 13], we initially used purified murine spleen CD4+ cells depleted of CD25+ cells that, in addition to CD44-CD62L+ naïve T cells, contain CD44+CD62L+ central memory and CD44+CD62L- effector memory cells, basically because naïve cells readily convert into Foxp3+ cells independently of factors relevant for Foxp3+ and RORgt+ cell generation, such as RA [17]
Summary
CD4+ T cells with regulatory function co-expressing Foxp and RORgt are linked to the development of oral tolerance towards innocuous food antigens in mice. Intestinal homeostasis relies on the development of tolerance mechanisms that recognize symbiotic microorganisms and innocuous food antigens In this respect, induced regulatory T (Treg) cells are considered essential for establishing peripheral tolerance by counteracting the activity of the different effector T helper (Th) cell subsets. While the mechanisms underlying their generation are not fully clear, involvement of antigens and metabolites derived from the gut microbiota, which stimulate epithelial production of retinoic acid (RA), has been proposed [3, 6] In this respect, effective oral immunotherapy with peptides was found to cause an increase in RORgt+ Treg cells through the enhancement of vitamin A metabolism [7, 8]
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