Rapamycin promotes the enrichment of CD4(+)CD25(hi)FoxP3(+) T regulatory cells from naïve CD4(+) T cells of baboon that suppress antiporcine xenogenic response in vitro

Abstract

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 peripheral blood mononuclear cell (PBMC) and interleukin (IL)-2, and have demonstrated the suppression of autologus CD4(+)CD25(neg) 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 nmol/L) using irradiated pig PBMCs and IL-2 to enrich the regulatory T cells. CD4(+)CD25(+)FoxP3(+) Treg cells were increased up to 2 times in the presence of rapamycin versus without rapamycin in vitro. However, a higher dose of rapamycin (> or = 10 nmol/L) 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 93% at a 1:1 ratio (Treg 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.

Figure 1

Flow cytometric analysis of FoxP3 staining on CD4⁺CD25⁺ T cells with and without rapamycin after 4 week expansion of CD4+ T cells in culture.

Fig: 2

Percent suppression of the proliferation of baboon CD4+CD25^neg cells. Naïve CD4+CD25^high Treg cells, ex-vivo expanded CD4+ T (5×10⁴) expanded without and with rapamycin and purified CD4+CD25^high obtained from ex-vivo expanded CD4+ T in presence of Rapamycin were co-cultured with autologus CD4+CD25^neg effector (Teff) cells (5×10⁴) and stimulated with irradiated pig PBMCs. Percent suppression of CD4+CD25^neg cell proliferation was assessed by using the above mentioned formula (p value *p<0.02, **P<0.03).