Mechanisms involved in regulation of MHC class I molecules in murine embryonic stem cells

Major histocompatibility complex (MHC) is at the center of immune responses that support survival, fitness and adaptation of mammalian species to the environment. These molecules are not only crucial for adaptive and innate immune responses against microorganisms and cancer cells but also play an important role in reproduction process and development of embryo during the preimplantation period. In the present study, we use murine embryonic stem cells (ESCs) as model to dissect the molecular mechanism involved in the regulation of MHC class I molecules during differentiation in vitro. MHC class I molecules are expressed at very low levels on murine ESCs and they are not induced by the immunomodulatory cytokine interferon gamma (IFN) despite the presence of IFN receptors on their cell surface. First, we showed that removal of leukemia inhibitory factor (LIF), a standard component of murine ES cell culture media required for the maintenance of a pluripotent state, did not result in up regulation of MHC class I expression in murine ESCs, presumably due to incomplete inactivation under these experimental conditions of STAT3 signaling pathway, which is used by LIF. However, the addition of LIF to differentiated cells in embryoid bodies strongly suppressed the expression of these molecules. Down regulation of STAT3 in undifferentiated ESCs cultured in the presence of LIF significantly increased the expression of MHC class I molecules and this was further enhanced by IFN treatment. Flow cytometric analysis revealed that STAT1 is phosphorylated by IFN in STAT3 knockdown (KD) ESCs, whereas there was only weak or no phophorylation detected in mock siRNA 647-treated ESCs exposed to IFN. Luciferase reporter assay also indicated that GAS promoter responded to IFN much strongly in STAT3-depleted ESCs than in intact cells, suggesting that ESCs do not respond to IFN at least partially due to inhibitory effects of STAT3-signaling components on STAT1-phosphorylation. Moreover, the down regulation of suppressor of cytokine signaling 3 (SOCS3) in STAT3 KD ESCs increases the possible regulation of STAT1 phosphorylation by SOCS3. No effect in MHC class I molecules induction was observed in STAT3 KD ES-derived cardiomyocytes (ESCMs) indicating the differential regulation of these molecules during the course of differentiation from undifferentiated stage (ESCs) to differentiated stage (ESCMs). An additional mechanism by which STAT3 regulates MHC class I expression may involve epigenetic modification of MHC class I gene expression since Dnmt knockout (KO) murine ESCs showed upregulation of MHC class I molecules. Chromatin modifying gene Eed1 but not Dnmt1 and Jmjd1 was significantly downregulated in STAT3 KD murine ESCs. Additionally, murine ESCs showed increased MHC class I expression and enhanced response to IFN after treatment with the DNA-methyltransferase inhibitor 2,5-azacytidine and histone deacetylase inhibitor Trichostatin A. Modulation of MHC class I expression by STAT3 KD in ESCs reduced their lysis by activated syngeneic NK cells and increased their lysis by cytotoxic T cells compared to mock siRNA 647-treated ESCs. These data indicate that STAT3 pathway plays a dual role in modulating the MHC class I expression in ESCs. Interfering with the inhibitory pathways that suppress MHC class I expression in pluripotent ESCs may help to control teratoma formation from contaminating ESCs in therapeutic cell transplants and may also help to eradicate cancer cells and virus-infected cells that are known to frequently evade immune recognition by down regulating the MHC class I expression.