Interferon-α (IFNα) signaling is essential for antiviral response via induction of IFN-stimulated genes (ISGs). Through a non-biased high-throughput RNAi screening of 711 known epigenetic modifiers in cellular models of IFNα-mediated inhibition of HBV replication, we identified methyltransferase SETD2 as a critical amplifier of IFNα-mediated antiviral immunity. Conditional knockout mice with hepatocyte-specific deletion of Setd2 exhibit enhanced HBV infection. Mechanistically, SETD2 directly mediates STAT1 methylation on lysine 525 via its methyltransferase activity, which reinforces IFN-activated STAT1 phosphorylation and antiviral cellular response. In addition, SETD2 selectively catalyzes the tri-methylation of H3K36 on promoters of some ISGs such as ISG15, leading to gene activation. Our study identifies STAT1 methylation on K525 catalyzed by the methyltransferase SETD2 as an essential signaling event for IFNα-dependent antiviral immunity and indicates potential of SETD2 in controlling viral infections.

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IntroductionThe Th1 cellular immune response is crucial for antitumor and antimicrobial immunity, 1,2 and powerful immunomodulatory adjuvants that induce Th1 polarization are an important facet of vaccination strategies. 3,4 While traditional adjuvants, such as aluminum salts and oil emulsions, mainly evoke Th2 responses, characterized by production of antibodies specific for conformational antigenic determinants, 5 new generation adjuvants, including CpG-rich motifs, monophosphoryl lipid A (MPA), and quil a saponin (QS21) promote Th1 polarization. 6 These effects are thought to result from the activation of antigen-presenting cells (APCs), in particular, dendritic cells (DCs). 7,8 In recent years the molecular chaperone heat shock protein (HSP) has been revealed to interact with APCs, and its considerable capacity to induce antigen-specific CD8 ϩ cytotoxic T lymphocyte (CTL) and Th1 responses has attracted much attention. 9,10 Extracellular HSPs can interact with APCs, exhibiting potent adjuvant functions in stimulating the host immune response. 10,11 This interaction triggers a cascade of events, including re-presentation of the chaperoned peptides by the major histocompatibility complex (MHC), secretion of proinflammatory cytokines, and maturation of DCs. [12][13][14] These properties combine to make HSPs a potent adjuvant, eliciting significant immune responses against associated antigens.The Hsp70 subfamily is one of the most important HSP subfamilies. Hsp70 prepared from tumor cells or virus-infected cells are capable of eliciting potent antigen-specific CD8 ϩ CTL responses 10,15 ; these responses are CD4 ϩ T-cell-independent and have been attributed to antigenic peptides bound to the HSP. 16 It also has been shown that Hsp70 complexed with ovalbumin (OVA) antigen-specific epitopes can activate DCs and induce OVAspecific CTL responses. 17 Hsp70 can bind to CD91, CD14, and TLR2/4 receptors on the surface of APCs, activating APCs and facilitating the re-presentation of HSP-associated antigen via a TAP-dependent or TAP-independent route. 13,14,18,19 These findings demonstrate the adjuvant effects of Hsp70 and encourage the potential application of Hsp70 in vaccination.We report here a new member of the Hsp70 subfamily cloned from a human DC cDNA library, Hsp70-like protein 1 (Hsp70L1). Recombinant Hsp70L1 can bind DCs, resulting in DC maturation and activation. While Hsp70L1 shares common receptors (CD91, TLR2, TLR4) on DCs with Hsp70, there are functional differences between Hsp70L1 and Hsp70. Hsp70L1 can induce DCs to secrete IP-10, a cytokine vital for Th1 polarization, but Hsp70 cannot. Moreover, Hsp70L1 is more potent than Hsp70 in the stimulation of IL-12p70, MIP-1␣, MIP-1␤, RANTES, CCR7, and CXCR4 expression by DCs. Hsp70L1 may therefore polarize Th1 responses more effectively than Hsp70. We used OVA as a model antigen to evaluate the adjuvant effects of Hsp70L1 in vivo, finding that Hsp70L1-OVA 257-264 hybrid peptide immunization could strongly Materials and methods Cell linesE.G7-OVA, an OVA-express...

IntroductionDendritic cells (DCs) play critical roles in the initiation of immune responses and induction of tolerance. 1 Balanced signaling transmitted via different activating and inhibitory receptors in DCs can regulate the functional status of DCs, thus determining duration and magnitude of T-cell responses. 2,3 Among the immune receptor families known to be expressed by DCs, the immunoglobulin superfamily (IgSF) seems to be unique in terms of structural features and roles in the regulation of DC development and function. 4 IgSF receptors are mostly type I membrane proteins, characterized by 1 or more immunoglobulin-like domains in the extracellular regions, which are pivotal in cell-cell recognition and interaction. IgSF receptors are abundant in the immune system and are widely distributed in lymphoid and myeloid cells including T cells, B cells, monocytes, DCs, and natural killer (NK) cells. It is well recognized that a vast quantity of IgSF molecules are involved in shaping the synapse formed between T cells and antigenpresenting cells (APCs), regulating various processes ranging from antigen (Ag) recognition and cell-cell interaction to signal transduction. 5 Many immune molecules belonging to the IgSF have recently been isolated and defined as key players in the innate and adaptive immune response, some of which act as inhibitory receptors with profound influence on immune responses. Inhibitory receptors are characterized by the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic regions. Upon engagement with ligands, ITIMs contained in the inhibitory receptors serve as scaffolds for recruitment of protein-tyrosine phosphatases to mediate negative signaling, 6 maintaining adequate thresholds for cell activation to avoid irrelevant cell activation and induction of autoimmunity. 7 Considering the critical roles of DCs in the immune response, much attention has been focused on the isolation and functional analysis of receptors on DCs, especially on inhibitory receptors. Immunoglobulin-like transcript 3 (ILT3) and ILT4, 2 Ig superfamily receptors containing ITIMs, can negatively regulate antigen presentation, T-cell costimulation, and cytokine production by DCs. Up-regulation of ILT3/ILT4 by suppressor T cells is responsible for the tolerogenecity of monocytes and DCs. 8,9 Similarly, paired immunoglobulin-like inhibitory receptor B (PIR-B), a murine homologue of ILT4, has also been defined as a key negative regulator of DC functions, and impaired maturation of DCs and imbalanced T helper 1 (Th1) and Th2 immune responses occurred in PIR-B Ϫ/Ϫ mice. 10,11 Despite the fact that many inhibitory receptors belonging to Ig superfamily have been identified, unidentified members expressed by DCs remain to be isolated and investigated, and the identification of novel inhibitory immunoreceptors should further elucidate the sophisticated interactions between DCs and T cells. 13 where CMRF35-A and at least 6 other genes constitute an Ig family. Members of this family are involved ...