Functional Studies Of DIgR2, A Novel Immune Inhibitory Receptor Expressed On Mouse Dendritic Cells

As the most potent antigen-presenting cells (APC) in the immune system, dendritic cells (DCs) play critical roles in the initiation of immune responses and tolerance induction. 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. Among the immune receptor families known to be expressed by DCs, immunoglobulin superfamily (IgSF) seems to be unique in terms of their structural features and their roles in the regulation of DCs development and function. IgSF receptors are mostly type I membrane proteins, characterized by one or several immunoglobulin-like domains in the extracellular regions, and these domains are pivotal in cell-cell recognition and interaction. IgSF receptors are abundant in immune system, and widely distribute in lymphoid and myeloid cells including T cells, B cells, monocytes, DCs and NK cells. It is well recognized that a vast quantity of IgSF molecules are involved in the shape of synapse formed between T cells and APCs, regulating various processes ranging from Ag recognition, cell-cell interaction to signal transduction.Many immune molecules belonging to the IgSF have been recently isolated and defined as key players of 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 theinhibitory receptors serve as scaffolds for recruitment of protein-tyrosine phosphatases to mediate negative signaling, maintaining adequate thresholds for cell activation to avoid irrelevant cell activation and induction of autoimmunity. Many inhibitory receptors expressed by DCs have been identified while the new members and their functions need further investigation.Human CMRF35-A, previously isolated by recognition with the CMRF-35 monoclonal antibody, is a surface molecule with an IgV-like domain. Chromosome mapping and gene database querying locate CMRF35-A on human chromosome 17, where CMRF35-A and at least six other genes constitute an Ig family. Members of this family are involved in multiple facets of the of immune regulation, such as modulation of mast cell activation, regulation of NK and eosinophils activities. Moreover, single nucleotide polymorphisms (SNP) in this cluster linked with psoriasis, atopic dermatitis and psoriatic arthritis, underscored the importance of CMRF35-A-like molecules in the regulation of immune response and immunological pathogenesis.In this study, by searching mouse EST database against human CMRF35-A, we identified a mouse homologue and its full-length cDNA (accession No AF 251703) was isolated from mouse DCs, which contains a single open reading frame encoding a transmembrane protein of 330 amino acids with a predicted molecular mass of 37 kD. The amino acid sequence begins with a hydrophobic signal peptide of 21 amino acids followed by an extracellular region composed of one IgV domains. The putative cytoplasmic domain is characterized by the presence of two tandem ITIMs spaced by one Tyr-x-x-Met motif, suggesting that an inhibitory signal might be transduced via these motifs. Therefore, this molecule is a member of the expanding of Ig-like inhibitory receptors family, and was designated as dendritic cell-derived Ig-like receptor2 (DIgR2) besides DIgRI identification.Both the DIgRI, 2 genes were mapped to the proximal end of mouse chromosome 11 in a region corresponding to the human chromosome 17 the CMRF-35A gene is oriented. Additionally, a DIgR2-containing gene cluster containing several related genes was identified in mice chromosome HE, which was designed as CLM1-9. Comparativeanalysis indicated that DIgR2 is closely related to some other Ig-families, including CLMs (81% identity with CLM3), human CMRF-35s (48% identity with CMRF35-A6 or IgSF13) and TREMs (Triggering receptors expressed by myeloid cells, 25% identity with mTREM-1).Then we detected the expression of DIgR2 by Northern and RT-PCR. DIgR2 mRNA was widely distributed in mouse tissues, including heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis, with highest level in heart. RT-PCR analysis of cell lines and freshly isolated cells demonstrated that DIgR2 mRNA was preferentially expressed in monocytes, DCs and B cells, but not in T cells, and an elaborate real-time quantitative PCR assay further revealed that DIgR2 levels increased during DC culture in vitro, and induced by LPS and IL-10 but down-regulated upon CD40L stimulation.Next we defined biochemical characteristics of DIgR2. We first carried out immunoprecipitations from DIgR2-transfected NIH3T3 cells, and detected a prominent band of 60 kD in transfected cells under both nonreducing and reducing conditions, indicating that the mature protein did not form a disulfide-linked dimer despite of several cysteine residues within its extracellular region. The discrepancy between the apparent molecular mass (~60kDa) and deduced mass (-37 kDa) resultes from the glycosylation since molecular mass was reduced to approximate 37 kDa upon PNGase treatment. Furthermore, we stained DIgR2-transfected 3T3 cells with anti-DIgR2 pAb and examined its localization by confocal microscope. The uniform fluorescence exhibited in the surface of NIH3T3 cells confirmed DIgR2 as a surface molecule.Since DIgR2 contains two ITIMs in its cytoplasmic region, we investigated whether DIgR2 could associate with tyrosine phosphatase SHP-1, SHP-2, a general way of inhibitory receptors to transmit their signals. We treated DIgR2-transfected L929 cells with sodium pervanadate, and found that DIgR2 can be phosphorylated on Tyr residue and could associate with SHP-1, indicating that DIgR2 is a structurally inhibitory IgSF receptor and potentially transduce inhibitory signals.The next step to investigate the functions of DIgR2 is to identify its potential ligand(s).Since preferential expression of DIgR2 on APCs, especially inducible expression on DCs suggested the possible involvement of DIgR2 in APC/T cell interaction, we began with T lymphocytes to search for the cells expressing its putative ligand(s). Using DIgR2-Fc fusion protein we identified CD3+ T cells as the candidate, because the fusion protein showed to strongly bind CD3+ T cells, while no binding of human immunoglobin (hlg) to T cells occurred and excessive GST-DIgR2 fusion protein could abolish the binding of DIgR2-Fc to T cells. Specific binding of DIgR2-Fc to T cells suggested involvement of DIgR2 in DC-T cell interactions. To further explore this possibility, we added overdose DIgR2-Fc fusion protein into the allogenic MLR system, and found that the proliferation of T cells was significantly promoted by DCs when DIgR2 was blocked with soluble DIgR2-Fc while no increase found in the presence of hlg. To exclude direct effects of DIgR2-Fc on T cells in the test above, a small interfering RNA (siRNA) that specifically downregulates DIgR2 expression was also utilized to block DIgR2 expression on DCs. Similarly, the proliferation of T cells stimulated with DIgR2-siRNA-DCs was significantly increased, compared with DIgR2-mut-siRNA-DCs or control DCs, strengthening the evidences that DIgR2 is a functional molecule involved in DC-T interaction.Then, to further investigate the role of DIgR2 in antigen-specific immune response, we used CD4+ T cells purified from DO11.10xC57BL/6 Fl mice as responders and DIgR2-siRNA-DCs, DIgR2-mut-siRNA-DCs or control DCs from C57 BL/6xBalb/c Fl mice as stimulators in MLR assay. After 3 days, DIgR2-silenced DCs showed significant increase in their ability to promote Ag-specific responses, as determined by the increased proliferation of DC-stimulated DO11.10 T cells. More importantly, DIgR2-siRNA-DCs stimulated DO 11.10 T cells to produce significantly higher levels of IFN-y and IL-2, as measured by ELISA assay and intracellular staining, indicating that DIgR2 expression on DCs not only affected proliferation of T cells, but also activation and polarization of T cells. Furthermore, we tested the ability of DIgR2-silenced DCs to prime an Ag-specific response in vivo, by adoptively transferring peptide-pulsed and DIgR2-silenced DCs into the recipient mice preinjected with DO11.10 T cells. Remarkable increase of DO11.10 (KJl-26+) T cells in the spleen of DIgR2-siRNA-DCs mice provided further in vivoevidence that DIgR2 signaling could negatively regulate the abilities of DCs to prime Ag-specific T cell responses.The data described here establish an important role of DIgR2 in the fine-tuning of immune responses. How does DIgR2 execute its effects in immune response? Is it that engagement of DIgR2 with putative ligand(s) on T cells delivers a signaling to regulate DCs function? In the above experiments, when the phenotypes and cytokine production of DIgR2-silenced DCs were compared with those of control DCs, we found a profound increase of IL-12p70 by DIgR2-silenced DCs. In view of the critical role of IL-12p70 in promoting DC-initiated T cell activation and Thl polarizaton, this augmented production of IL-12p70 may be responsible, at least in part, for subsequent increased T cell proliferation and enhanced IFN-y production.To better understand the contribution of DIgR2 in negative regulation of DC-initiated T cell responses in vivo, we also evaluated the immune response elicited by DIgR2-siRNA-DCs immunization. Firstly we constructed adenoviral siRNA expressing vectors, an efficient gene silencing approach for DCs, to block the expression of DIgR2. Then AV-transduced, OVA protein-pulsed and LPS-matured DCs were used to immunize C57BL/6 mice, and two weeks later, splenocytes from immunized mice were collected and subjected to immune analysis. Results showed that more potent Thl response was induced by immunization with DIgR2-silenced DCs, as more IFN-y was produced when splenocytes from AV-DIgR2-siRNA mice were restimulated with OVA II peptide (322-339). Consistently, more robust CTL response was elicited by AV-DIgR2-siRNA mice, evidenced by increased percentages of OVA- tetramer+ CD8, and IFN-7+ CD8 cells in these mice. And also, the cytotoxicity of splenocytes against OVA-specific target EG7.OVA cells was significantly increased in mice immunized with DIgR2-silenced DCs, as compared to that of the mice immunized with control DCs or AV-DIgR2-mut -siRNA-DCs.Immunization with antigen-pulsed DCs can effectively activate antigen-specific CTLs, however, their efficacy in inducing antitumor immunity in vivo is far satisfactoryand has to be further improved. We wondered whether blockade of DIgR2-medaited inhibitory signals in DCs could promote Ag-specific immunity to protect mice from tumor challenge. C57BL/6 mice preimmunized twice with "silencer AV"-transduced, OVA-pulsed, ex vivo matured DC vaccines were burdened with EG7.0VA lymphoma one week after the final immunization. It was shown that DIgR2-siRNA-DC vaccine resulted in more significantly suppression of tumor growth in the immunized mice as shown by the tumor volume 24d after tumor inoculation. Moreover, more than half (60%) of the mice immunized with DIgR2-siRNA-DCs remained tumor free 60 days after tumor inoculation, whereas mice treated with DIgR2-mut-siRNA-DCs and control DCs only gained 20% complete prevention from tumor challenge.In summary, here we functionally characterized DIgR2 as a novel representative of a family of inhibitory receptors belonging to the immunoglobulin superfamily. We show that DIgR2 contains two ITIMs within its cytoplasmic region and associates with SHP-1 when phospherated. Blockade of DIgR2 on DCs by pretreatment with DIgR2-Ig fusion protein or by silencing with specific small interferencing RNA enhances DC-initiated T cell proliferation and antigen-specific T cell responses both in vitro and in vivo. Furthermore, immunization of mice with antigen-pulsed, DIgR2-silenced DCs elicits more potent antigen-specific CD4+ and CD8+ T cell responses, thus protecting the vaccinated mice from tumor challenge more effectively. Our data suggest that DIgR2 is a functionally inhibitory receptor and define a novel regulatory pathway involved in DC-initiated T cell responses.