In Vitro Expansion Of Human Alloantigen-specific CD4~+CD25~+Regulatory T Cells

CD4+CD25+Foxp3+regulatory T cells (Tregs) play an important role in the maintenance of immune tolerance and treatment of allograft rejection and autoimmune diseases. Since the specific marker and the limited numbers of Tregs that are available hamper their clinical applications. Expanded Tregs have been reported to be more therapeutically effective than primary Tregs. Currently, human naturally occurring Tregs can be expanded polyclonally, by using anti-CD3and anti-CD28antibody stimulation in combination with IL-2or in an alloantigen-specific manner with allogeneic APCs. However, there are only few antigen-specific Tregs in the population of the polyclonal Treg, and the large-scale in vitro expansion of alloantigen-specific Tregs is difficult in an alloantigen-specific manner with allogeneic APCs. Adoptive transfer of antigen-specific Tregs has been shown to more effective in preventing and treating autoimmune diseases than polyclonal Tregs.Taken these current studies together, we established method to ex vivo expand a higher amount of functionally active human alloantigen-specific Foxp3+CD4+regulatory T cells from allogeneic B cells:in the primary stimulation cycle, magnetic bead-sorted CD4+CD25’Foxp3+T cells were cultured with allogeneic B cells at B-to-T cell ratios of4:1in the presence of exogenous IL-2and costimulatory anti-CD28Abs; to obtain the highest number of Tregs with direct alloantigen-specificity, the B cell-expanded Tregs were expanded polyclonally by using anti-CD3and aini-CD28antibody in combination with IL-2in the presence or absence of Rapamycin. After the primary and secondary expansion, the naive Tregs could be expanded1X103-fold into alloantigen-specific Foxp3+CD4+regulatory T cells in the absence of Rapamycin, the percentage of B-expanded Tregs>80%; However, an0.8X103-fold and the percentage of B cell-expanded Tregs>90%could be obtained in the presence of Rapamycin; B cell-expanded Tregs cultured in the presence of Rapamycin expressed higher level of Foxp3, CTLA4and CD39as compared to B cell-expanded Tregs cultured in the absence of Rapamycin. But the level of HLA-DR were expressed equally whether expansion culture contained Rapamycin or not.B celleexpanded Tregs culturCd in the absence of Rapamycin exprcssed Iowlevel of IL-2, IL.-17, IL-4and IFN-γ. However, B cell-expanded Tregs cultured in the presence of Rapamycin almost did not express IL一2, IL-17,IL-4and IFN-γ.The former remained partially anergic,and the Iatter remained anergic.B cell-expanded Tregs were capable of imposing potent inhibit alloprolif-eration of responder T cells elicited by specifjc alloantigens,and Iess potential to inhibit nonSpecific immmune response.Highest number of functionally active human alloantigen-specinc CD4+CD25+Foxp3+regulatory T cells could be obtained after stimulated with allogeneic B cells in Vitro and then expanded by using anti-CD3and anti-CD28antibod y. Rapamycin could increase the percentage of B-expanded Tregs and suppressive abilit y.B ceIl-expanded Tregs were enriched for alloantigen specincity. Our results also provide a theoretical basis for using third-party Treg cells for therapeutic applications.Tregs can be generated through specific metabOlic patllways.The elevation of intracellular cAMP in vitro with PDE inhibitors not only inhibits T cell prolif.eration and interleukin-2(IL-2) secretion but also generates populations of Tregs. CD4+CD25+Foxp3+Tregs were generated by stimulation of CD4+CD25-Foxp3-T cells in vftro with allogenieic tolerogenic dendritic cells(tDCs)combined with Cilo stamide in the presence of different cytOkines.The data showed that adding exogenous IL-2could promote to.nduce CD4+CD25+Foxp3+Tregs by Cilostamide.In addition.adding TGF-p COuld enhance the level Of Foxp3in the presence of IL-2and Cilostnmide.Therefore. Cilostamide combined with tDCsinVitro can generate Tregs in the presence of IL-2and TGF-β.The result will be of help for thefurther research on how to effectively Hse Cilostamide tO generate functional Tregs cells in vitro.