FcRγ presence in TCR complex of double-negative T cells is critical for their regulatory function

TCRαβ⁺CD4^- CD8^- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8⁺ and CD4⁺ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRγ was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRγ-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRγ- sufficient DN T cells, FcRγ-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRγ, DN Treg cell clones also express higher levels of TCRβ, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRγ associates with the TCR complex and that both FcRγ and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRγ expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRγ deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRγ plays a role in mediating TCR signaling in DN Treg cells.