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.