Murine CD4(+)CD25(-) cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo

Background: Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance. Methods: Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4(+)CD25(-) cells we generated in vitro functional CD4(+)CD25(+) iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model. Results: TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 beta and higher levels of colonic IL-17 when compared to diseased control group. Conclusions: This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo