Impact of activated invariant natural killer T cells on the expansion of regulatory T cell precursors in murine thymocytes in vitro

Abstract

Tolerance induction is a goal of clinical transplantation to prevent graft rejection without the lifelong use of immunosuppressive drugs. In a series of mouse studies, we previously reported that the establishment of mixed chimerism by treatment with a ligand for invariant natural killer T (iNKT) cells with CD40 signal blockade makes it possible to prevent allograft rejection without immunosuppressants, and this approach fails in thymectomized recipient mice. In this study, we showed that iNKT cells in murine thymocyte cultures are indispensable for the expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells as well as CD4⁺CD25⁺Foxp3^- cells, which contained precursor Tregs (preTregs). After the culture of BALB/c mouse-derived thymocytes in the presence of α-galactosylceramide (α-GalCer), a representative ligand for iNKT cells, the ratio of CD4⁺CD25⁺Foxp3^- preTregs to total CD4⁺CD8^- T cells was much higher than that of CD4⁺CD25⁺Foxp3⁺ Treg cells, regardless of anti-CD40 L mAb treatment. The proliferation of CD4⁺CD25⁺Foxp3^- cells, but not Treg cells, was significantly augmented, and the stability of Treg cells was not affected by α-GalCer. The expansion of thymocyte-derived Tregs was not inhibited by cytokine neutralization. However, in vitro thymus-derived CD4⁺CD25⁺Foxp3^- cells expressed Foxp3 after IL-2 stimulation in a dose-dependent manner. These results collectively suggest that in vitro thymus-derived Treg cell expansion by α-GalCer treatment was caused by the proliferation of CD4⁺CD25⁺Foxp3^- preTregs but not existing Treg cells.

Keywords: Invariant natural killer T cell; Regulatory T cell; Regulatory T cell precursor; Thymus; Tolerance; α-galactosylceramide.