The CD4+CD25+FoxP3+ regulatory T (Treg) cells play an important role in regulating the immune response. These Treg cells are present in peripheral blood and lymphoid organs and have a high potential for immunotherapy in clinics. Adoptive Cell transfer therapy using CD4+CD25+ cells has been shown to prevent autoimmune diseases and has also induced transplant tolerance in mice. Treg cells low frequency in peripheral blood will necessitate its ex-vivo expansion to enable adaptive immunotherapy. Recently, it has been reported that rapamycin, an immunosuppressive agent, inhibits T cell proliferation while selectively increasing the number of Treg cells. Based on this additional mode of action, rapamycin can be used to expand Treg cells for ex-vivo cellular therapy in T-cell-mediated diseases and in transplantation. We have reported the ex-vivo expansion of baboon Treg cells, using irradiated pig PBMC and IL-2, and have demonstrated the suppression of autologus CD4+CD25neg T-cell proliferation in response to pig PBMCs. In the present study, we have expanded baboon CD4+ T cells in the presence or absence of rapamycin (0.1-10 nM) using irradiated pig PBMCs and IL-2 to enrich the regulatory T cells. CD4+CD25+FoxP3+ Treg cells were increased up to two times in the presence of rapamycin as compared without rapamycin in-vitro. However, a higher dose of rapamycin (≥10nM) considerably decreases the number of Treg cells. Furthermore, purified CD4+CD25+ Treg cells enriched from CD4+ cells in the presence of rapamycin were able to suppress the baboon anti-porcine xenogeneic immune responses in vitro up to 90% at a 1:1 ratio (T regulatory Cells: T effector cells) and suppression ability exists even at a 1:256 ratio whereas freshly isolated natural Treg cells suppress only 70% at 1:1 and lose their suppressive ability (>50%) at 1:16. Our results demonstrate that the addition of rapamycin to the culture enriches the Treg phenotype and induces functional regulatory T cells. This method may allow the production of large numbers of regulatory cells for the preclinical testing of Treg cell therapy in a non human primate model.
CD4+CD25+ Regulatory T (Treg) cells play an important role in regulating and/or suppressing immune responses by controlling Ag-driven T-cell proliferation. These cells are a low frequency subpopulation of CD4+ cells, representing 1-2% of total lymphocytes. Treg cells that are thymic derived are characterized as naturally occurring regulatory T cells and are believed to control autoreactive T cells, maintain homeostasis, and induce peripheral tolerance. Treg cells also express forkhead box protein 3 (FoxP3), a Forkhead family transcription factor that converts CD4+CD25neg cells into suppressive CD4+CD25hi T cells. As most T cells express CD25 after stimulation, FoxP3 is considered to be a specific marker of Treg cells. Experiments in small animal models suggest that the adoptive transfer of natural Treg cells can prevent autoimmune diseases. Edinger et al reported that CD4+CD25+ regulatory T cells prevent graft-versus-host disease (GVHD) without affecting graft versus tumor (GVT) activity after bone marrow transplantation (BMT) in mice(1). For clinical Treg immunotherapy, high numbers of cells are needed. Successful ex-vivo expansion of naturally occurring human and murine CD4+CD25+ T cells is achieved after TCR stimulation in the presence of T cell growth factors such as IL-2, IL-7 and IL-15(2). Ex-vivo expanded cells retain suppressive function in both in-vitro and in-vivo animal models and induce experimental transplantation tolerance(3). Furthermore, other studies have demonstrated that fresh and ex-vivo expanded CD4+CD25+ regulatory T cells can also prevent the development of GVHD and inhibit cardiac graft rejection in murine models(4, 5).
Rapamycin is an immunosuppressive agent which blocks the intracellular signaling in response to T cell growth factor, favors expansion of CD4+CD25+ T cells in-vitro and in-vivo(6). Recently, we have identified the phenotype and expanded ex-vivo baboon naturally occurring CD4+CD25+ Treg cells(7). The aim of the present study was to evaluate the effect and utility of rapamycin on Treg cell induction and expansion of unmanipulated baboon CD4+ T cells.