Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor

CD200 (OX2) is a cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed mainly on myeloid cells and is involved in regulation of macrophage and mast cell function. In mouse there are up to five genes related to CD200R with conflicting data as to whether they bind CD200. We show that mouse CD200 binds the inhibitory receptor CD200R with a comparable affinity (Kd = 4 microM) to those found for the rat and human CD200 CD200R interactions. CD200 gave negligible binding to the activating receptors, CD200RLa, CD200RLb, and CD200RLc, by direct analysis at the protein level using recombinant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating gene, CD200RLe, found in only some mouse strains also did not bind CD200. Thus, the CD200 receptor family consists of both activatory and inhibitory members like several other paired ligand receptors, such as signal regulatory protein, killer cell Ig-like receptor/KAR, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and paired Ig-like type 2 receptor. Although the ligand for the inhibitory product is a widely distributed host protein, the ligands of the activating forms remain to be identified, and one possibility is that they are pathogen components.     

Heterogeneity in the CD200R paired receptor family

Paired receptors are groups of closely related membrane proteins that have the potential to either inhibit or activate. The CD200R family consists of one inhibitory member, CD200R and various numbers of activating genes according to species with three defined in C57BL/6 mice. A genomic PCR strategy was used to examine the repertoire of genes in both laboratory and wild-derived mice. Most mouse strains tested (18/22) had three activating genes, and 16 of these had either the combination of CD200RLa, Lb, and Lc or CD200RLa, Lb, and Le. The Lc and Le genes were mutually exclusive and were equally common (10 strains). Wild-derived mice varied more with one example of strains with one, two, and four activating genes. An inhibitory CD200R gene was detected in each mouse strain, although two slightly different sequences were found in both laboratory and wild-derived mice. This diversity is probably being driven by pathogens but is less extensive than for many NK paired receptors such as KIR and Ly49. It is possible that myeloid paired receptors are involved in immune regulation of responses against pathogens rather than directly killing infected cells as for NK cells and, hence, under less intense evolutionary pressure.     

The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.     

The CD200 receptor is a novel and potent regulator of murine and human mast cell function

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.     

Molecular mechanisms of CD200 inhibition of mast cell activation

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.     

Modulation of the murine CD8 gene complex following the targeted integration of human CD2-locus control region sequences

The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.     

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.     

Chronic infection drives expression of the inhibitory receptor CD200R, and its ligand CD200, by mouse and human CD4 T cells

Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man.     

Mast cells and basophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner

Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via FcepsilonRI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.     

Down-regulation of basophil function by human CD200 and human herpesvirus-8 CD200

Human and rodent CD200 are recognized by the inhibitory CD200R, and these molecules play an important role in the regulation of the immune system. Several viruses, such as human herpesvirus-6 (HHV-6), HHV-7, and HHV-8, possess a CD200 homologue, suggesting that these viruses regulate the immune response via CD200R. In this study, we analyzed the effect of human CD200 and the viral CD200 homologues on human CD200R-expressing cells. We found that human CD200R is predominantly expressed on basophils in amounts higher than on other human peripheral blood leukocytes. Furthermore, the viral CD200 homologues as well as human CD200 were recognized by human CD200R, and the activation of basophils was down-regulated by these CD200 proteins. These results suggested that CD200R is an important regulatory molecule of basophil activation. In addition, the presence of CD200 homologues on several viruses suggests a potentially unique relationship between basophil function and viral infection.     

The mouse CD7 gene: identification of a new element common to the human CD7 and mouse Thy-1 promoters

HumanCD7 (CD7) is a 40000M _r member of the immunoglobulin gene superfamily that is expressed early in natural killer (NK) and T-lymphocyte development.CD7 is involved in lymphocyte activation, as ligation ofCD7 activates NK and TCRγδ T lymphocytes, and ligation ofCD7 on TCRαβ T lymphocytes induces a non-mitogenic calcium flux. We have previously cloned and characterized the gene for humanCD7 (hCDT) and have described its expression in transgenic mice. Recently a mouse cDNA homologous tohCD7 was reported, which we mapped to the corresponding mouse chromosomal location ashCD7. We now report the identification and characterization of a mouseCD7 (mCDT) genomic clone. We demonstrated that themCD7 gene was similar both in size and structural organization tohCD7. Comparison of the 5′ flanking sequences of themCD7 andhCD7 genes revealed two regions of sequence similarity. Electrophoretic mobility shift assay confirmed both of these regions to be sites of tissue-restricted protein binding in vitro. The more 3′ similarity region also shared sequence with a region in the mouseThy-1 gene 5′ flanking region, suggesting that this sequence may be a cis-acting regulatory element common to all three genes. Thus, the promoter regions and exonic organization were similar in the humanCDT, mouseCDT, and mouseThy-1 genes. The nucleotide sequence data reported in thts paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U23462     

CD33/Siglec-3 binding specificity,expression pattern,and consequences of gene deletion in mice

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to alpha2-3- or alpha2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.     

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.     

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain.

The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals.     

Detection of a Soluble Form of CD109 in Serum of CD109 Transgenic and Tumor Xenografted Mice

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is expressed at high levels in some human tumors including squamous cell carcinomas. As CD109 is reportedly cleaved by furin and its soluble form is secreted into culture medium in vitro, we hypothesized that CD109 could serve as a tumor marker in vivo. In this study, we investigated CD109 as a novel serum tumor marker using transgenic mice that overexpress mouse CD109 (mCD109-TG mice) and tumor xenografted mice inoculated with human CD109 (hCD109)-overexpressing HEK293 cells. In sera and urine of mCD109-TG mice, mCD109 was detected using western blotting. In xenografted mice, hCD109 secreted from inoculated tumors was detected in sera, using western blotting and CD109 ELISA. Concentrations of tumor-secreted CD109 increased proportionally as tumors enlarged. Concentrations of secreted CD109 decreased notably by 17 h after tumor resection, and became undetectable 48 h after resection. The half-life of tumor-secreted CD109 was about 5.86±0.17 h. These results indicate that CD109 is present in serum as a soluble form, and suggest its potential as a novel tumor marker in patients with cancers that express CD109.     

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200^−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.     

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development unitl their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (MCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in inter-specific backcross progeny revealed linkage of mCd19 with hemoglobin (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere — Hbb — mCd19 — H19 — Int-2 —telomere. The genetic distance between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C polypeptide.     

Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (K(D) = 0.5 microM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.