Abstract
NK1.1-CD4+NKG2D+ cells exert their immune-regulatory function in tumor as an unconventional regulatory T cell subset through the production of TGF-β1; however, the molecular mechanisms involving with the activation of nuclear factors for TGF-β1 transcription remain unclear. Here we determined that the PI3K-p85α subunit was specifically activated in NK1.1-CD4+NKG2D+ cells following an 8-hour stimulation by sRAE-1 or α-CD3/sRAE-1, subsequently leading to the activation of PI3K-p110, Akt, and JNK. On the contrary, α-CD3/α-CD28 stimulation did not induce the activation of PI3K-p85 and JNK. Consequently, activation of the nuclear transcription factor AP-1 as a consequence of JNK activation regulated TGF-β1 expression in NK1.1-CD4+NKG2D+ cells. Furthermore, activation of NF-κB in NK1.1-CD4+NKG2D+ cells resulted from both protein kinase C activation downstream of TCR/CD3 signaling and PI3K activation induced by NKG2D engagement. The STAT3-Y705 phosphorylation, as activated by PI3K, under stimulations of the sRAE-1 or α-CD3/sRAE-1 also contributed to the TGF-β1 expression in NK1.1-CD4+NKG2D+ cells. Moreover, ChIP assay confirmed that STAT3 was capable of binding with the promoter regions of TGF-β1. In conclusion, our data showed that the TGF-β1 transcription in NK1.1-CD4+NKG2D+ cells induced by sRAE-1 or α-CD3/sRAE-1 was involved with the AP-1, NF-κB, and STAT3 signaling pathways; therefore, regulation of AP-1, NF-κB, and STAT3 activation may play important roles in the development and function of NK1.1-CD4+NKG2D+ cells.