Recombinant CD200 Does Not Bind Activating Closely Related to CD200 Receptor

This information is current as Deborah Hatherley, Holly M. Cherwinski, Mehrdad Moshref of September 25, 2021. and A. Neil Barclay J Immunol 2005; 175:2469-2474; ; doi: 10.4049/jimmunol.175.4.2469 http://www.jimmunol.org/content/175/4/2469 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Recombinant CD200 Protein Does Not Bind Activating Proteins Closely Related to CD200 Receptor1

Deborah Hatherley,* Holly M. Cherwinski,† Mehrdad Moshref,† and A. Neil Barclay2*

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 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 M) 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 recom- binant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating , 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, Downloaded from 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. The Journal of Immunology, 2005, 175: 2469–2474.

ell surface molecules containing Ig-like domains are par- dently for CD200R3 (16). Thus, these proteins are expected to give http://www.jimmunol.org/ ticularly common in mammalian genomes (1). They are activating signals and, hence, are similar to the many examples of C often involved in the intercellular communication of leu- paired receptors where closely related genes contain members that ϭ kocytes, and their interactions are typified by low affinities (Kd can give inhibitory signals and those that give activating signals 1–100 ␮M) when measured in the monomeric state (2). CD200 through adaptors such as DAP12. In some cases, the ligands for the (OX2) is a widely distributed cell surface protein that interacts inhibitory forms do not bind the activating forms, such as with the with a receptor (CD200R) that is highly expressed on myeloid signal regulatory proteins (SIRPs)3 (17, 18). The NKAT2 inhibi- cells. Both proteins contain two extracellular Ig-like domains, but tory p58 binds the HLA-C peptide complex with much higher the receptor differs in that it has a longer cytoplasmic tail contain- affinity than the activating p50 (19); however, both activating ing known signaling motifs (3). Studies with the CD200 knockout Ly49H and inhibitory Ly49I can bind the same m157 mouse CMV by guest on September 25, 2021 (4, 5) and in vitro analysis (6–9) indicated that CD200-CD200R protein, although affinities have not been measured (20). Both interactions are involved in the control of myeloid cellular func- paired Ig-like type 2 receptor (PILR)␣ (inhibitory) and PILR␤ tion. The broad tissue distribution of CD200 and changes in its (activating) bind a CD99-related protein (21). With regard to level of expression provide a mechanism for locally regulating CD200, it was reported that CD200-Fc fusion proteins did not bind myeloid cellular activity at appropriate sites, such as inflamed tis- to CD200RLa and CD200RLb (14), but a contradictory report sug- sue (10, 11). The CD200-CD200R regulatory mechanism is an gested that similar reagents bound all the activating proteins as attractive target for immunomodulation, because its manipulation well as the inhibitory form (15). Clearly this has important func- can induce either immune tolerance or autoimmune diseases. tional implications and possibilities for therapies using CD200-Fc CD200-Fc fusion proteins have been shown to provide beneficial fusion proteins. immunomodulatory effects in models of arthritis and allograft re- In this study we critically examine binding of CD200-Fc and jection (12, 13). other CD200 fusion proteins to CD200RL, proteins expressed at In addition to the inhibitor CD200R, several related genes have the cell surface and directly at the protein level, to determine the been identified in the mouse. These were termed CD200RL (for affinities of the interactions at 37°C. This establishes that CD200 is receptor like) (14), CD200R1, CD200R2, etc. (15, 16). These are not a significant ligand for four CD200R-related proteins. predicted to be associated with DNAX activating protein (DAP)12, and this has been shown for two of the mouse genes, CD200RLa Materials and Methods (CD200R4) and CD200RLb (CD200R3) (14), and also indepen- Construction, expression, and purification of soluble recombinant proteins *Sir William Dunn School of Pathology, University of Oxford, Oxford, United King- A fusion protein consisting of the extracellular domain of mouse (m) dom; and †DNAX Research Institute, Palo Alto, CA 94304 CD200 fused to the Fc domain of mouse IgG1 mutated in the CH2 domain Received for publication March 28, 2005. Accepted for publication June 6, 2005. (D265A) to inhibit binding to FcRs (CD200-Fc) and control mutant IgG (cFc) were generated as previously described (22). The inability of the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance fusion proteins to bind FcRs was confirmed by staining the FcR-positive with 18 U.S.C. Section 1734 solely to indicate this fact. cell line J774 (data not shown). The clonings of CD200RLa and CD200RLb were described previously (14). Full-length CD200RLc was 1 This work was supported by the Medical Research Council and the Cellular Immu- nology Unit hybridoma fund. DNAX is supported by Schering Plough Corp. 2 Address correspondence and reprint requests to Dr. A. Neil Barclay, Sir William 3 Abbreviations used in this paper: SIRP, signal-regulatory protein; cFc, control mu- Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, U.K. tant IgG; RU, response unit; DAP, DNAX activating protein; PILR, paired Ig-like OX1 3RE. E-mail address: [email protected] type 2 receptor.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 2470 MOUSE CD200R not obtained in that study. The complete extracellular region of CD200RLc Measurement of affinities of interactions of CD200 using surface was isolated by PCR from cDNA purified from mouse peritoneal exudate plasmon resonance cells, and the extracellular region had the same sequence as that previously published (accession no. NM_206535). CD200RLe (accession no. Affinity and kinetic data were collected using a Biacore 2000 (Biacore) at BAC40774) was isolated by PCR from cDNA prepared from bone marrow 37°C as previously described (3). Briefly, ϳ2500 response units (RU) of mast cells derived from CD-1 mice (Charles River Laboratories) as pre- streptavidin was coupled to a CM5 research grade chip using amine cou- viously described (14). The mCD200R 5Ј primer (5Ј-gtgcttaaccagattc pling. Biotinylated mCD200RCD4d3ϩ4-related proteins and control cactcc-3Ј) and 3Ј primer (5Ј-tgttttcttgtattgtgtcatcagac-3Ј) were used. Fusion CD4d3ϩ4 were each immobilized at ϳ1000 RU. For kinetic analysis, proteins for mouse CD200R, CD200RLa, CD200RLb, and CD200RLc and serially diluted monomeric CD200CD4d3ϩ4 purified proteins were CD200RLe with domains 3 and 4 of rat CD4 plus a biotinylation signal injected at the indicated active concentrations over all four flow cells con- Ϫ Ϫ were prepared as previously described (14, 23). The boundary between the nected in series. The extinction coefficient, 53,590 M 1cm 1, was calcu- CD200RLa part and CD4 was MTTPRSTSIT, for CD200RLb TSILP lated. Kd values were obtained by both nonlinear curve fitting and STSIT, for CD200RLc TTPSTSIT and for CD200RLe QGSTSIT (CD4 Scatchard transformations to the binding data. The following were then linker is underlined). Mouse CD200CD4d3ϩ4 (4) was produced in passed over all four flow cells sequentially: cIg, CD200-Fc, specific CHO.K1 cells using the expression vector pEE14 and was subsequently CD200R mAb, and OX68 mAb recognizing CD4d3ϩ4 common to all purified from spent tissue culture medium by immunoaffinity chromatog- proteins (all at 20 ␮g/ml). raphy using an OX68 mAb-Sepharose 4B column (3, 24). Before BIAcore analysis, the purified CD200 protein was fractionated by gel filtration on a Results Superdex S-200 column (Pharmacia Biotech) to exclude larger protein ag- gregates that are known to influence binding measurements (25). Analysis of the interaction of CD200 with CD200R and related proteins expressed at the cell surface Flow cytometry

Fig. 1 shows the similarity in the amino acid sequences of the Downloaded from Ba/F3 cells expressing mouse CD200R, CD200RLa, and CD200RLb and extracellular regions of mCD200R and related proteins. Ba/F3 mAb recognizing mCD200R (DX109), mCD200RLa (DX87), and cells were transfected with constructs for CD200R, CD200RLa, or mCD200RLb (DX116) were generated as previously described (14). Sur- face expression of the CD200 receptors was analyzed by flow cytometry CD200RLb, and the three receptors were expressed at high levels, using the specific CD200 receptor mAb described above, followed by goat as shown by binding of three specific mAb (Fig. 2, A–C). A anti-rat PE-conjugated secondary Ab (Caltag Laboratories). CD200-Fc or DAP12 construct was cotransfected with CD200RLa and 5 cFc (2 ␮g/5 ϫ 10 cells) was incubated on ice for 30 min at 4°C in PBS CD200RLb, because previous studies had shown that this was nec- containing 1% BSA and 0.05% sodium azide. Cells were washed, and Fc http://www.jimmunol.org/ was detected using a PE-conjugated rat anti-mouse IgG1 secondary Ab essary to obtain expression of CD200RLa and CD200RLb at the (clone A85-1; BD Biosciences). Analyses were performed on a FACScan cell surface (14). Soluble fusion proteins consisting of the extra- flow cytometer (BD Biosciences). cellular domains of mCD200 fused to the Fc binding domain of by guest on September 25, 2021

FIGURE 1. The mCD200R and related sequences. The NH2 terminus is based on protein data for rat CD200R. The superscript bars predict the extent of the ␤ strands characteristic of the Ig fold by comparison with solved structures. Additional mouse genes include two forms isolated through their association with DAP12 (mCD200RLa and mCD200RLb; accession no. XM156177 and XM148097). A sequence found in some mouse strains, but not the C57BL genome (BAC40774), is named CD200RLe. This and mCD200RLc (NM_206535) are predicted to be associated with DAP12. Residues identical in four or more sequences are boxed (three for cytoplasmic regions). An additional sequence, termed CD200RLd, is not shown, because it is incomplete and may not give an expressed protein (14). The Journal of Immunology 2471 Downloaded from http://www.jimmunol.org/

FIGURE 2. Flow cytometry showing binding of CD200-Fc to CD200R, but not to CD200RLa and CD200RLb. Ba/F3 cells expressing mCD200R (A and D), Ba/F3 cells expressing DAP12 and CD200RLa (B and E), and Ba/F3 cells expressing DAP12 and CD200RLb (C and F) were stained with specific mAb (A–C) or CD200-Fc (D–F). All three constructs were expressed at high levels (A–C), and CD200-Fc fusion protein gave strong specific binding to CD200R (D), but not to CD200RLa (E) or CD200RLb (F). Controls for mAb labeling (cIgG; A–C) and Fc fusion protein (cFc; D–F) gave no labeling. by guest on September 25, 2021

mouse IgG gave very strong binding to cells expressing CD200R produce anomalous binding. The resultant preparation was passed and not to cells expressing the activating receptors CD200RLa and over the four proteins. Clear binding to CD200R was obtained, but CD200RLb (Fig. 2, D–F). No binding could be obtained for these not to CD200RLa, CD200RLb, or the control CD4d3ϩ4 (Fig. 3A), proteins using a variety of CD200-Fc fusion protein concentra- confirming the data on binding to cells (Fig. 2). The interactions tions, labeling times, and temperatures, indicating that CD200 is a were weak as expected with rapid dissociation over a few seconds. ligand for CD200R, but not for the related activating gene prod- Thus, high concentrations of mCD200 were used, which gave rise ucts, CD200RLa and CD200RLb. to a bulk effect as the protein passed over the chip. For the control protein, CD4d3ϩ4, this was seen as signal that quickly reached Analysis of interaction of CD200 with CD200R and related proteins at the protein level equilibrium and was washed out rapidly. The specific binding of CD200 to CD200R showed an increased signal compared with the The failure of CD200-Fc protein to bind to CD200RLa and control, which also quickly reached equilibrium and then dissoci- CD200RLb is unlikely to be due to a lack of sensitivity in the ated rapidly. CD200RLb gave the same signal as the control pro- assay, because very high levels of expression of all three receptors tein, indicating that there was no specific binding to CD200; trace were obtained (Fig. 2, A–C), and there was strong binding of binding was observed with CD200RLa, and this was quantitated CD200-Fc to the control CD200R (Fig. 2D). However, analysis further (see below and Fig. 4A). using purified proteins allowed a quantitative comparison of inter- The dimeric CD200-Fc fusion protein used in cell binding as- acting proteins to be determined. CD200R, CD200RLa, says was passed over the flow cells with similar results, namely CD200RLb, and two gene products, CD200RLc and CD200RLe (see below), for which specific mAb were not available, were ex- good binding to CD200R, but not to CD200RLa and CD200RLb pressed as chimeric proteins consisting of the extracellular regions (Fig. 3B). The fusion protein was at a much lower concentration ϳ ␮ of these proteins together with two domains of rat CD4 as an ( 0.2 M) than the monomeric CD200 protein in Fig. 3A and, as antigenic tag and a sequence that allows biotinylation. The proteins expected, gave more avid binding, with a slower on rate; equilib- were expressed transiently, biotinylated, concentrated, and bound rium was not reached, and dissociation occurred over ϳ5 min. to streptavidin-coated BIAcore chips along with a control protein, The conditions of protein production and immobilization permit CD4d3ϩ4, consisting of the two domains of CD4 present in each the minimum of handling and are unlikely to cause denaturation. construct. Mouse CD200CD4d3ϩ4 was expressed in Chinese Three mAb specific for the three proteins were passed over the hamster ovary cell lines and purified by affinity chromatography, flow cells, showing specific binding of DX109 to CD200R and of followed by gel filtration to remove any aggregates that might DX116 to CD200RLb (Fig. 3, C and E). The DX87 mAb did not 2472 MOUSE CD200R Downloaded from http://www.jimmunol.org/

FIGURE 3. Analysis by surface plasmon resonance shows mCD200 interacts with mCD200R, but not with related proteins. A, CD200CD4d3ϩ4 (40 ␮M) was injected at 20 ␮l/min at 37°C over four flow cells in sequence that had been coupled with CD200RCD4d3ϩ4-biotin (1167 RU) or CD200RLaCD4d3ϩ4-biotin (1064 RU), CD200RLbCD4d3ϩ4-biotin (1145 RU), and CD4d3ϩ4-biotin (1278 RU) immobilized on BIAcore chips to which streptavidin (2500 RU) had been previously coupled. The monomeric CD200CD4d3ϩ4 binds well to CD200R, trace amounts bind to CD200RLa, and does not bind to CD200RLb or the control, where the signal is due to the bulk effect of the high protein concentration. B, CD200-Fc fusion protein binds to CD200CD4d3ϩ4, binds well to CD200R, but does not bind to CD200RLa, CD200RLb, or the control, giving a similar bulk effect to the control Fc. C,

DX109 mAb binds only to CD200R. D, DX87 mAb gives minimal binding to CD200RLa (see text). E, DX116 mAb binds only CD200RLb. F, CD200R, by guest on September 25, 2021 CD200RLa, CD200RLb, and control CD4d3ϩ4 protein bind OX68 mAb recognizing the CD4 tag present in all proteins, giving additional evidence for the presence of well-folded chimeric proteins on the chip. The bars above the trace indicate the duration of the injection of the protein named. label CD200R and CD200RLb as expected, but only gave mar- not known whether this protein is expressed on normal cells (14). ginal labeling of CD200RLa (20 RU; Fig. 3D). However, this mAb This gene product also gave no significant binding to CD200 (Fig. did give very good labeling of CD200RLa expressed at the cell 4C), whereas CD200R gave values similar to those shown in Fig. surface (Fig. 2). It is possible that this mAb reacts better with cell 4A. One CD200R mAb, OX110, cross-reacted weakly on this pro- surface-expressed protein that might have different glycosylation tein (data not shown). compared with the soluble extracellular region of the fusion pro- tein. (This phenomenon has been observed for another highly gly- A new CD200R-like gene (CD200RLe or CD200R5) present in cosylated cell surface protein, SIRP␥ (18).) This protein and in- some strains of mice deed all the CD200Rs were reactive with the OX68 mAb that This gene (accession no. BAC40774) was characterized as an ex- binds the CD4 tag present in the chimeric proteins (Fig. 3F), sug- pressed sequence tag from the NOD mouse, noted in database gesting that all the proteins are correctly folded. In the unlikely searches (16), and termed CD200R5 (16). No mRNA expression event that the CD200RLa was completely inactive, the data from was found in C57BL/6 mice (16), and we found no evidence of this Fig. 2, B and E, clearly showed no CD200 binding to antigenically gene in searches of the mouse genome (C57BL/6J; ͗www. active CD200RLa expressed at the cell surface. ensembl.org/Mus_musculus/͘), through established sequence tag The experiment was repeated with a range of concentrations of searches, or through functional analysis. However, it was ex- CD200-CD4d3ϩ4 protein, and the equilibrium binding levels are pressed in the CD1 mouse strain. The extracellular region of this plotted in Fig. 4A. The affinity for CD200 binding to CD200R was protein was expressed as described above, and binding to CD200 ϭϳ ␮ calculated (Kd 4 M at 37°C) from curve fitting (Fig. 4A) and was analyzed. This protein also did not bind CD200 (Fig. 4C). We Scatchard analysis (Fig. 4B) and was comparable to that deter- term this gene product CD200RLe (RL stands for receptor like), ϭ ␮ mined for the equivalent interaction in rats (Kd 1 M at 37°C) rather than CD200R5, because it is not a receptor of CD200. It ϭ ␮ (3) and humans (Kd 0.5 M at 37°C) (14). The binding to should be noted that it is probably not present in all mouse strains, CD200RLa was too weak to get accurate values, but extrapolation and likewise, the expression of all the other gene products has not Ͼ ␮ of the data would indicate a Kd 500 M at 37°C, which is un- been tested in this mouse. likely to be significant physiologically. There was no detectable binding of CD200-CD4d3ϩ4 to CD200RLb. Discussion Comparable experiments were conducted for CD200RLc, al- We present clear evidence that CD200 does not bind CD200RLa, though there are no specific mAb recognizing this protein, and it is CD200RLb, CD200RLc, or CD200RLe, but gives good binding to The Journal of Immunology 2473 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 4. Equilibrium binding data for CD200 binding to CD200R, CD200RLa, CD200RLb, CD200RLc, and CD200RLe. A, A series of concen- trations of CD200CD4d3ϩ4 was passed over the proteins as described in Fig. 3A. The amount of CD200CD4d3ϩ4 that bound at each concentration was calculated as the difference between the responses at equilibrium in the CD200RCD4d3ϩ4 and control flow cells. The curved lines in the main plot are ϭ ␮ nonlinear curve fits to the data and correspond to Kd 4.8 M for CD200 binding CD200R. No binding was obtained to CD200RLb, and trace amounts Ͼ ␮ of binding to CD200RLa indicate a Kd of 500 M. C, Comparable experiment showing that CD200 did not bind CD200RLe, and there was only trace ϭ ␮ binding to CD200RLc. A similar affinity (Kd 3.8 M) was found for CD200 binding CD200R as in A. B and D, Scatchard analysis of data in A and C, showing good linearity and comparable Kd values to curve fitting for CD200 binding to CD200R.

CD200R. CD200RLa and CD200RLb are known to be expressed being observed. DAP12 is a small disulfide-bonded homodimer on normal leukocytes (14). These data are in agreement with and that is structurally similar to the Fc⑀RI ␥-chain and the TCR extend those reported previously (14), but disagree with a recent ␨-chain (26, 27) that contains a negatively charged residue in its paper by Gorczynski et al. (15), who found that CD200RLa, transmembrane that allows pairing with a variety of cell surface CD200RLb, and CD200RLc bound CD200 in addition to receptors with positively charged amino acid residues in their CD200R. In the latter, it is argued that the CD200RLb used (14) transmembrane regions. The cytoplasmic domain of DAP12 con- was not full length, because they identified a Met further upstream. tains a consensus ITAM motif, which, when phosphorylated, re- In fact, the construct used previously (14) and in this paper also cruits protein tyrosine kinases (26–29). There is a charged residue included this Met, so there is no difference in the protein, just the in all the CD200RL gene products, and they probably all interact interpretation of which Met might be used as an initiator. By anal- with DAP12 and have the potential to produce activating signals. ogy with the other genes, the earlier Met is more likely to be used, The current analysis uses well-characterized mAb that are highly as suggested by Gorczynski et al. (15). Similar Fc fusion proteins specific for the gene products and recognize the proteins on normal are used to recognize CD200R and related genes transfected into cells, whereas in the study by Gorczynski et al. (15) the Abs are cell lines. The data we present show that there is no reaction at the less well characterized antipeptide reagents. cell surface using similar reagents, and virtually no binding at the Analysis of the sequence similarities of the extracellular regions protein level using carefully controlled reagents. The mostly likely of the receptors shows that CD200R is most similar to the strain- explanation for the discrepancy centers on the fact that we (14) specific CD200RLe (91% amino acid sequence identity), but is have shown that the activating CD200RLa and CD200RLb were also similar to CD200RLa (84%) and CD200RLc (83%, although only expressed in the presence of DAP12, and others (16) have this lacks part of the N-terminal region), and is less similar to shown the same for CD200RLb (CD200R3). In the study by Gorc- CD200RLb (39%). Mutagenesis analysis of the human CD200R zynski et al. (15) this does not seem to have been done, and it has identified residues in predicted ␤ strands C and F important for seems unlikely that specific binding of CD200 to these products is binding human CD200 (30). Analysis of this region in the mouse 2474 MOUSE CD200R shows that it is highly conserved between the binding CD200R and immunosuppressant that prolongs allo- and xenograft survival. J. Immunol. 163: the nonbinding CD200RLe. However, a single residue change can 1654–1660. 14. Wright, G. J., H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, eliminate human CD200R-CD200 binding (30), and this sensitivity of M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, et al. 2003. Char- the binding site to small changes in sequence may be important in the acterization of the CD200 receptor family in mice and humans and their inter- evolution of activating receptors from inhibitory receptors. actions with CD200. J. Immunol. 171: 3034–3046. 15. Gorczynski, R., Z. Chen, Y. Kai, L. Lee, S. Wong, and P. A. Marsden. 2004. CD200R resembles other gene families, such as SIRP, killer cell CD200 is a ligand for all members of the CD200R family of immunoregulatory Ig-like receptor, NKG2A/C, LY49, dendritic cell immunoreceptor/ molecules. J. Immunol. 172: 7744–7749. dendritic cell immunoactivating receptor, and PILR, that have both 16. Voehringer, D., D. B. Rosen, L. L. Lanier, and R. M. Locksley. 2004. CD200 receptor family members represent novel DAP12-associated activating receptors activating and inhibitory forms (31–35). The numbers of genes can on and mast cells. J. Biol. Chem. 279: 54117–54123. vary considerably between species and strains, and this is also ob- 17. Seiffert, M., P. Brossart, C. Cant, M. Cella, M. Colonna, W. Brugger, L. Kanz, ␣ ␣ served with CD200R, for which there are several related genes in the A. Ullrich, and H. J. Buhring. 2001. Signal-regulatory protein (SIRP ) but not SIRP␤ is involved in T-cell activation, binds to CD47 with high affinity, and is mouse, but only one in the human, and the number in mice may vary. expressed on immature CD34ϩCD38Ϫ hematopoietic cells. Blood 97: Like some other paired receptors, the inhibitory CD200R binds a host 2741–2749. 18. Brooke, G., J. D. Holbrook, M. H. Brown, and A. N. Barclay. 2004. Human protein. In addition, there is an example where a CD200 homologue lymphocytes interact directly with CD47 through a novel member of the signal (K14) in the HHV8 virus binds the inhibitory receptor (9). The ligands regulatory protein (SIRP) family. J. Immunol. 173: 2562–2570. of CD200RLa, CD200RLb, CD200RLc, and CD200RLe and the 19. Vales-Gomez, M., H. T. Reyburn, R. A. Erskine, and J. Strominger. 1998. Dif- ferential binding to HLA-C of p50-activating and p58-inhibitory natural killer functional and biological significance of these receptors remain un- cell receptors. Proc. Natl. Acad. Sci. USA 95: 14326–14331. known; however, it is possible that these receptors have evolved to 20. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier. 2002. interact with bacterial or viral components in a manner similar to that Direct recognition of cytomegalovirus by activating and inhibitory NK cell re- Downloaded from recently described for a DAP12 pairing member of the Ly49 family ceptors. Science 296: 1323–1326. 21. Shiratori, I., K. Ogasawara, T. Saito, L. L. Lanier, and H. Arase. 2004. Activation and mouse CMV m157 protein (20, 36). of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor. J. Exp. Med. 199: Acknowledgments 525–533. 22. Cherwinski, H. M., C. A. Murphy, B. L. Joyce, M. E. Bigler, Y. S. Song, We are grateful to Marion H. Brown and Dan Gorman for helpful S. M. Zurawski, M. M. Moshrefi, D. M. Gorman, K. L. Miller, S. Zhang, et al. discussions. 2005. The CD200 receptor is a novel and potent regulator of murine and human http://www.jimmunol.org/ mast cell function. J. Immunol. 174: 1348–1356. Disclosures 23. Brown, M. H., K. 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