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Induced T regulatory (iTreg) cells suppress T and B cell immune responses

Avneesh Singh, Caleb Seavey, Keith Horvath, Muhammad Mohiuddin

Cardiothoracic Surgery Research Program, NHLBI / NIH, Bethesda, MD, United States

The CD4+CD25+FoxP3+ regulatory T (Treg) cells play an important role in regulating immune response. These Treg cells are present in peripheral blood and lymphoid organs and have a high potential for immunotherapy in clinics. Due to their high potency for immune suppression, these cells have been expanded in-vitro by different methods using IL-2 and TCR stimulation. Ex-vivo expanded Treg (iTreg) cells exhibit similar characteristics to the natural occurring Tregs and have a high therapeutic potential for immunotherapy. The aim of our study is to investigate the effect of iTreg cells on to effector T and B cells. In this study we have purified and successfully expanded naturally occurring baboon CD4+CD25+ Treg cells. Highly purified CD4+CD25Hi Tregs cells and CD4+CD25neg cells were expanded for 4 weeks in presence of IL-2 and dynal IgG beads coated with anti monkey CD3 and CD28 antibodies. After 4 weeks CD4+CD25Hi expanded more than 400X and such iTreg cells retained the high expression of FoxP3. To demonstrate the suppression of T cells, mixed lymphocyte reaction (MLR) was done by using CD4+CD25neg cells and irradiated pig APC along with iTreg cells. iTreg cells suppress the effector T cells effectively (> 90%) in presence of xenogeneic immune response. Further, effect of iTreg on to B cells was examined by co-culturing them together with PMA and Ionomycin. To monitor the B cell proliferation, B cells were purified from baboon peripheral blood and labeled with CFSE and iTreg cells were irradiated. By using FACS analysis in co-culture experiment, we found that B cells proliferation was inhibited by more than 60-70% in presence of CD4+CD25Hi iTregs cells in 5 days. However, ex-vivo expanded CD4+CD25neg cells did not inhibit the PMA and Ionomycin induced B cell proliferation. We propose that this versatile capacity of ex-vivo expanded CD4+CD25Hi iTregs might be important to efficiently limit T and B cell responses that might help to prevent the hyperacute and delayed rejection in xenotransplantation.