of April 9, 2018.This information is current as

Regulatory Protein (SIRP) FamilyCD47 through a Novel Member of the Signal Human Lymphocytes Interact Directly with

Neil BarclayGary Brooke, Joanna D. Holbrook, Marion H. Brown and A.

http://www.jimmunol.org/content/173/4/2562doi: 10.4049/jimmunol.173.4.2562

2004; 173:2562-2570; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/173/4/2562.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Human Lymphocytes Interact Directly with CD47 through aNovel Member of the Signal Regulatory Protein (SIRP)Family1

Gary Brooke,* Joanna D. Holbrook,† Marion H. Brown,* and A. Neil Barclay 2*

Two closely related proteins, signal regulatory protein� (SIRP�; SHPS-1/CD172) and SIRP�, have been described in humans.The existence of a third SIRP protein has been suggested by cDNA sequence only. We show that this third SIRP is a separate genethat is expressed as a protein with unique characteristics from both� and � genes and suggest that this gene should be termedSIRP�. We have expressed the extracellular region of SIRP� as a soluble protein and have shown that, like SIRP�, it binds CD47,but with a lower affinity (K d, �23�M) compared with SIRP� (Kd, �2 �M). mAbs specific to SIRP� show that it was not expressedon myeloid cells, in contrast to SIRP� and -�, being expressed instead on the majority of T cells and a proportion of B cells. Theshort cytoplasmic tail of SIRP� does not contain any known signaling motifs, nor does it contain a characteristic lysine, as withSIRP�, that is required for DAP12 interaction. DAP12 coexpression is a requirement for SIRP� surface expression, whereasSIRP� is expressed in its absence. The SIRP�-CD47 interaction may therefore not be capable of bidirectional signaling as withthe SIRP�-CD47, but, instead, use unidirectional signaling via CD47 only. The Journal of Immunology, 2004, 173: 2562–2570.

T he expression of signal regulatory protein� (SIRP�)3

(CD172) in humans and its homologues in other mam-mals has been observed on myeloid, neuronal, and endo-

thelial cells. SIRP� has been cloned several times in different spe-cies leading to a variety of names, including SIRP�, SHPS-1,MyD-1, P84/BIT, and MFR (1–6). The role of SIRP� is generallyassumed to be an inhibitory one, mainly because of its interactionvia ITIM motifs in its cytoplasmic tail with the Src homology 2domain-containing phosphatase 1 (Shp1) and Shp2 protein ty-rosine phosphatases (1, 2, 7, 8), and there are functional data sup-porting this (9). SIRP� can also have negative effects on the ex-pression of inflammatory cytokines, especially TNF-� (10, 11).The extracellular ligand for SIRP� is CD47, an unusual five-passtransmembrane protein with a single Ig-like domain (12, 13).CD47 itself has been ascribed a wide variety of functions and isubiquitously expressed. It interacts in acis manner with cell sur-face integrins and was originally termed integrin-associated pro-tein (14, 15). There is evidence that it affects cell behavior throughan interaction with heterotrimeric G proteins (16, 17). CD47 hasbeen shown to have effects on integrins, migration, phagocytosis,IL-12R expression, and T cell activation and conversely on anergyor cell death (18–26). Surprisingly, considering its ubiquitous ex-pression and the multiple effects that CD47 ligation can involve,CD47�/� mice are viable and healthy. An obvious phenotype is

only apparent when mice are shown to succumb to bacterial in-fection more quickly than their wild-type relatives. This seems tobe due to defects in neutrophil migration (27). CD47 has also beenpostulated to act as a marker of self, as CD47�/� RBC are rapidlyphagocytosed when injected into wild-type mice (28).

The role of CD47/SIRP� is further complicated by the findingthat thrombospondin-1 has been shown to interact with CD47 (29)via the C-terminal region. However, many of the effects seen mayalso be mediated by thrombospondin-1 adhesion with integrins orsimultaneous ligation with integrins and CD47 (30). It is thereforeimportant to discover other protein interactions in this system andtheir affinities and tissue distribution. In contrast to SIRP�, SIRP�appears to exert activatory stimuli by virtue of interacting with theDAP12 adapter protein via a charged lysine in the SIRP� trans-membrane region. DAP12 is thought to function via the bindingand activation of Src family kinases such as Syk through ITAMmotifs (31). However, despite the sequence similarity betweenSIRP� and SIRP�, SIRP� � Fc fusion proteins do not bind toCD47 expressed on the cell surface (32). A third SIRP-relatedprotein has been suggested at the cDNA level (33). In this study weshow that SIRP� arises from a unique gene that, at the amino acidlevel, is approximately equally conserved to both SIRP� andSIRP�. However, although the expressed protein has a truncatedcytoplasmic tail, it is unlike SIRP� in that it does not requireDAP12 for surface expression and binds to CD47. Specific mAbshow that it also has different cellular distributions to SIRP� and-�. Taken together, we suggest that the protein be named SIRP� todifferentiate it from SIRP� and SIRP�, but still indicate that itbelongs to the same closely related protein family. Finally, we alsoshow that the differences in affinity of interaction with CD47 maycontrol the functional outcome of an interaction between differentSIRP� and SIRP�.

Materials and MethodsConstruction, expression, and purification of human SIRP� andSIRP� � CD4 soluble fusion proteins

The sequence representing the three Ig-like domains that comprise theextracellular region of the human SIRP� was amplified by PCR using

*Sir William Dunn School of Pathology, University of Oxford, Oxford, United King-dom; and† GlaxoSmithKline UK Ltd., Uxbridge, United Kingdom

Received for publication June 17, 2003. Accepted for publication June 10, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Medical Research Council, the Arthritis Rheuma-tism Council, and GlaxoSmithKline Pharmaceuticals.2 Address correspondence and reprint requests to Dr. A. Neil Barclay, Sir WilliamDunn School of Pathology, University of Oxford, South Parks Road, Oxford, U.K.OX1 3RE. E-mail address: neil.barclay@path.ox.ac.uk3 Abbreviations used in this paper: SIRP, signal regulatory protein; 7-AAD, 7-ami-noactinomycin D; EST, established sequence tag; RU, response unit; Shp1, Src ho-mology 2 domain-containing phosphatase 1.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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human peripheral blood leukocytes cDNA as a template. The oligonucle-otides ATGATTCAGCCTGAGAAG (sense) and TCAGGTCTTCTGCTTCCAG (antisense) were designed using the known SIRP� sequence(EMBL accession no. AB042624). For soluble SIRP� the antisense primerused was GTAGCATCTGAGCTCTGG. The products were blunt end li-gated into pCR2.1 (Invitrogen Life Technologies, Carlsbad, CA). For chi-meric soluble proteins, the SIRP sequence was ligated into a pEFBOS-XBexpression vector containing rat CD4 Ig-like domains 3 and 4 (henceforthreferred to as CD4) and a biotinylation motif (34, 35). For construction ofsurface-expressed SIRP� bearing the FLAG epitope, the N terminus of thethree extracellular Ig-like domains of SIRP� was ligated (SalI-BamHI) intoa CD4Lflag-pEFBOS construct (using a rat CD4 leader (CD4L) ending . . .VVTTQG, followed by the FLAG epitope DYKDDDDKST). Protein ex-pression was detected with anti-FLAG-M2 mAb (Sigma-Aldrich, Poole,U.K.). For chimeric constructs containing additional leader peptides, eitherhuman SIRP� (SIRP�L) or rat CD4L sequences were ligated onto the 5�end of SIRP�. The resulting SIRP�L chimera had the following sequenceat the join site: EEELQMIQP (SIRP�L sequence underlined). To producethe SIRP�L/SIRP� construct, the SIRP� signal peptide was joined toSIRP� using an endogenous SIRP� PstI site (the PstI site was inserted intothe SIRP� sequence using PCR). Soluble recombinant proteins with the ratCD4 domains were purified by OX68 mAb affinity chromatography (34).

Characterization of protein interactions by surface plasmonresonance

Surface plasmon resonance measurements were obtained using a BIAcore2000 biosensor instrument (BIAcore, Stevenage, Herts, U.K.) using CM5research grade chips. The streptavidin was immobilized directly via aminecoupling in 10 mM sodium acetate, pH 4.5. Equilibrium affinity and kineticmeasurements were conducted in 10 mM HEPES (pH 7.4), 150 mM NaCl,3 mM EDTA, and 0.005% surfactant P20 at 37°C using short injectiontimes of 3 s (5 �l at 100 �l/min) to minimize the contribution of anyaggregated material. For equilibrium binding, immediately before the ex-periment, purified CD47 � CD4 was size fractionated to exclude any ag-gregated protein. Increasing and decreasing concentrations of monomericCD47 � CD4 were passed over SIRP�, SIRP�, or control CD4 (all coatedon chip at 1000 response units (RU)). For off-rate determinations,CD47 � CD4 (40 �M) was passed over immobilized SIRP� � CD4 (at 1600and 800 RU), or control CD4 (1600 RU). Kd values were obtained by bothnonlinear curve fitting of the Langmuir binding isotherm and Scatchardtransformations of the binding data (Origin software, OriginLab,Northampton, MA). koff values were determined by fitting a first-orderexponential decay curve to normalized data after subtraction of the nega-tive control values.

Generation of mAb

Six-week-old male BALB/c mice were immunized s.c. with 10–20 �g ofpurified SIRP� � CD4 in CFA and then with IFA. A mouse generating goodimmune responses to the immunogen was boosted, and 4 days later thespleen was removed, and hybridomas were generated using standard pro-cedures by fusing with the NS-1 cell line. Hybridoma supernatants wereinitially screened by ELISA, using SIRP� � CD4, human SIRP�.Fc (13),human SIRP� � Fc, or CD4 to eliminate hybridomas with cross-reactivityto rat CD4, human SIRP�, and SIRP�. The remaining hybridoma super-natants were screened for the ability to stain SIRP�-transfected 293T cells.Four hybridomas recognizing SIRP� were named OX116–OX119 andwere recloned. All mAb were of the mouse IgG1 isotype.

Cells

PBMC were purified from blood of healthy volunteers on Ficoll-Paquedensity gradient (Amersham Biosciences, Arlington Heights, IL) and cul-

tured in tissue culture medium (RPMI 1640, 10% FCS, 2 mM glutamine,50 �M 2-ME, 100 U/ml penicillin, and 100 �g/ml streptomycin). 293Tcells were transiently transfected with human SIRP� � pEFBOS, humanSIRP� � pcDNA6 (gift from M. Colonna, Washington University School ofMedicine, St. Louis, MO) and human FLAG-tagged DAP12 � pREP10 (giftfrom J. Sedgwick, DNAX, Palo Alto, CA) using FuGene transfection re-agent (Roche, Indianapolis, IN) and the manufacturer’s protocol. Surfaceexpression of transfected cells was analyzed after 48 h in culture.

Flow cytometric analyses

Cells were labeled by indirect immunofluorescence at 4°C in the presenceof 10 mM sodium azide according to standard procedures and were ana-lyzed by flow cytometry on a FACScan (BD Biosciences, Mountain View,CA). All mAb, unless otherwise stated, were obtained from BD Pharmin-gen (San Diego, CA) and were directly conjugated to FITC or PE. An-nexin-FITC (Roche)/propidium iodide (Sigma-Aldrich) staining was per-formed according to the manufacturer’s protocol. Two-color staining wasperformed using biotinylated SIRP� mAb OX116 or OX119 with strepta-vidin-PE and a second, directly FITC-conjugated mAb (CD3, CD4, CD8,CD19, and CD25). Isotype-matched biotinylated or fluorochrome-labeledmAb were used as controls. Generation of multivalent SIRP reagents usingavidin-coated fluorescent beads (Sphero beads; Biotechnologie, www.kisker-biotech.com) and subsequent binding assays were previously de-scribed (36, 37). For lymphocyte activation, cells were resuspended in 200�l of RPMI 1640, 10% FCS, and 50 �M 2-ME with antibiotics in flat-bottom, 96-well tissue culture plates at 106/ml. At the indicated time points,the cells were frozen in 10% DMSO/90% FCS at �80°C. All samples werethawed and resuspended in PBS with azide before analysis as describedabove.

Apoptosis assay

Jurkat or U937 cells at 4 � 105/well in 96-well plates were cultured for 3 hwith the indicated mAb or fusion proteins bound to avidin-coated fluores-cent beads (Sphero) in tissue culture medium. Ten microliters of beads(streptavidin-coated, preincubated with 20 �l of biotinylated fusion pro-tein) or mAb at 5 �g/ml (final concentration) were added per well. ThemAb used were control mAb (OX2), CD3 (mAb OKT3), CD47 (mAb1796; Cymbus Biotechnology (Southampton, Hants, U.K.) or mAbMCA911 (Serotec, Oxford, U.K.)), and CD51/61 (BD Pharmingen). Forblocking assays soluble CD47 � CD4 or CD4 alone as a control was addedat 100 �g/ml before addition of beads. Cells were pelleted and washed inbinding buffer (10 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM KCl,1 mM MgCl2, and 1.8 mM CaCl2). Cells were then incubated with an-nexin-PE/7-aminoactinomycin D (7-AAD) according to the manufacturer’sprotocol (BD Pharmingen) and analyzed on a FACScan (BD Biosciences,Mountain View, CA).

Immunoprecipitation

PBMC were isolated by Ficoll density gradient, surface-biotinylated, andlysed at 2 � 107/ml in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.4), 1 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 50 mM benzamidine,1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin) for 30 min,and insoluble material was spun down (13 krpm, 10 min). For all preclear-ing and immunoprecipitate steps, 107 goat anti-mouse conjugated Dynalbeads (Dynal Biotech, Great Neck, NJ) preincubated with 3 �g of mAb/2 � 107 cells were used. After immunoprecipitation, Dynal beads wereresuspended in reducing sample buffer by boiling for 5 min and loaded ontoa 4–12% gradient polyacrylamide gel (NOVEX, San Diego, CA). Afterelectrophoresis, the gel was blotted onto polyvinylidene difluoride, whichwas then blocked in 1% BSA before incubation with extra-avidin conju-gated to peroxidase and developed with ECL reagents (AmershamBiosciences).

Table I. Genomic structure of SIRPa

ExonExon Size

(bp)Intron Size

(bp)Present inVariant 1

Present inVariant 2

AcceptorSequence

DonorSequence

1 (noncoding) 114 8234 Yes Yes TTACAGgtg2 (coding) 356 12547 Yes Yes cagAAGTGG TGGGTGgtg3 (coding) 317 589 Yes No tagCCAAAC TCCGAGgta4 (coding) 332 4961 Yes No cagTTCCAC CCCCTGgtg5 (coding) 847 586 Yes Yes cagGCCCGG CTGACTgta6 (noncoding) 484 Yes Yes cagCTCCTT

a Splice donor acceptor dinucleotides are underlined.

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ResultsSIRP� is closely associated with other SIRP genes in the humangenome

Analysis of the genomic sequence using the University of Cali-fornia Santa Cruz human genome browser (http://genome.ucsc.edu) showed that SIRP� is present on chromosome 20p13 andhas six possible exons (Table I). This also shows a predicted signalpeptide that shows a high degree of homology with SIRP� peptide(Fig. 1a). There are at least two transcript variants of SIRP� shownby established sequence tags (ESTs). One form has exons 1–6(variant 1), whereas the other has exons 1, 2, 5, and 6 (variant 2).Both variants 1 and 2 are predicted to have the same transmem-brane domain. Variant 1 consists of an N-terminal, Ig-like V do-main and two Ig-like C domains, whereas variant 2 only containsthe N-terminal, Ig-like V domain (Fig. 1b). The existence of bothforms (at least at the mRNA level) was confirmed using PCR oncDNA from PBLs (data not shown). However, transient expressionof constructs containing variant 2 failed to produce surface protein,and immunoprecipitation data with available mAb (see below) didnot indicate the presence of a protein corresponding to the pre-dicted m.w. of variant 2. Therefore, surface expression of this pro-tein by leukocytes seems unlikely. SIRP� variant 1 is highly re-lated to SIRP� and SIRP�, as shown by the comparison of aminoacid sequences in Fig. 1a. Over the Ig-like regions, there is anequal level of conservation (79%). Very low levels of conservationwere seen in the transmembrane regions and cytoplasmic tail witheither SIRP� or SIRP�.

Analysis of the human genomic sequence also showed thatSIRP�, SIRP�, and SIRP� are situated in close proximity to eachother, over a combined distance of �377 kb. There are three otherputative genes in the SIRP cluster (Fig. 2a). A comparison of do-main structure of all SIRP genes is shown in Fig. 2b. Two of thesegenes show a relationship with the SIRP genes and therefore ap-pear to be other members of the SIRP gene family, although moredistantly related. Accession number AAH33502 represents the firstand is reported as an expressed cDNA. This has a deduced proteinsequence of 197 aa, which includes a leader sequence and onepredicted Ig-like domain. It has 48% amino acid identity withSIRP� in the Ig-like domain. The second is represented byGeneScan prediction NT_011387.32. This is not well supported byEST data, but does consist of an open reading frame predicted toencode a peptide of 651 aa. This has a putative leader sequence andfive predicted Ig-like domains. Most of the conservation withSIRPs resides in the most C-terminal, two Ig-like domains, whichboth have �60% amino acid identity with the first N-terminal,Ig-like domain of SIRP�. There are EST data for mouse and rathomologues (SWISSPROT: Q8BJ958 and XP_230596.1) thatshow closest similarity to NT_011387.32 and whose predictedtransmembranes are similar to SIRP�, including a single lysineresidue that may indicate DAP10 or DAP12 association. Theamino acid identities of the other NT_011387.32 Ig-like domainswith SIRP� are much lower (�30%). The third putative gene isrepresented by five expressed sequences, namely, BX096358,

FIGURE 1. A, Alignment ofamino acid sequences of humanSIRP� (SWISSPROT accession no.P78324), SIRP� (SWISSPROT ac-cession no. Q9P1W8), and SIRP�(SWISSPROT accession no.O00241) using GCG (49) andGeneDoc software (http://psc.edu/biomed/genedoc/). B, Alignment ofhuman SIRP� long form with humanSIRP� short form (-v2, middle) andbovine SIRP� (EMBL accession no.AJ563808). Conserved residues areindicated by dots, lack of sequence isshown by a dash, and predictedleader and transmembrane regionsare underlined.

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AA398753, AA292852, AA398753, and AA292852, and does notshare any sequence conservation with the other SIRP genes.

SIRP� conservation in mammals

Human SIRP� showed a high degree of conservation (78% aminoacid identity) with a partial cDNA from another large mammal,cattle (Fig. 1b). Although encoding a stop codon, the cDNA doesnot encode a transmembrane region and may encode a solubleprotein. Database searches for genomic and EST data have shownno evidence yet of SIRP� in mice or rats.

Cloning and expression of human SIRP�

The published SIRP� sequence appeared truncated at the N ter-minus (amino acid sequence commenced MIQP..) (33). SIRP� wasamplified by PCR from leukocyte cDNA using primers based onthis sequence, but protein could not be expressed (data not shown).A leader sequence from SIRP� or CD4 was inserted, and SIRP�was expressed both at the cell surface, as detected by the FLAGlabel, and with SIRP mAb B1D5 (gift from H. G. Buhring, Uni-versity of Tubingen, Tubingen, Germany), and as a soluble chi-meric construct with rat CD4 domains 3 and 4 (data not shown).Recently, a full-length protein sequence for SIRP� has been de-scribed (SWISSPROT accession no. Q9P1W8) that confirmed thatthe original cDNA sequence lacked a leader peptide. This se-quence also confirmed that the constructs we produced with theSIRP� signal peptide did not confer any SIRP�-specific aminoacids to the predicted mature SIRP� protein that could have af-fected the data.

Human SIRP� is a CD47 ligand

Given the sequence similarity between SIRP� and SIRP�, wewanted to establish whether there is also a direct interaction be-tween SIRP� and CD47. The binding of purified soluble recom-binant CD47 � CD4 to immobilized SIRP� was analyzed using sur-face plasmon resonance with a BIAcore. Similar amounts ofbiotinylated SIRP� � CD4, SIRP� � CD4, SIRP� � CD4, and con-trol CD4 were immobilized on separate flow cells via binding tocovalently attached streptavidin. Fig. 3 shows that SIRP�, likeSIRP�, does bind to CD47, but the lower equilibrium bindingindicates that it interacts with a lower affinity than SIRP�. SIRP�gave no signal above the control CD4 background level, indicatingthat it does not react with CD47, in agreement with cell binding

data (32) (E. Vernon Wilson, D. Voulgaraki, M. H. Brown, A. N.Barclay, and G. Brooke, unpublished observations).

To measure the affinity of the interaction, purified humanCD47 � CD4 was fractionated by gel filtration to ensure that it wasmonomeric. As shown in Fig. 3, CD47 � CD4 bound specifically toSIRP� in a dose-dependent manner. Nonlinear curve fitting of thedata collected at 37°C produced a Kd of 23 �M (Fig. 3). This iswithin the range of normal leukocyte cell surface interactions be-tween proteins, which range from 0.1–100 �M (38). Scatchardanalysis gave a similar result (Fig. 3). For comparison,CD47 � CD4 was also passed over SIRP� � CD4 bound to the chip,which produced a Kd of 2 �M, also shown on the same Scatchardplot (Fig. 3). Kinetic analysis of the dissociation rate with twodifferent levels of bound SIRP� � CD4 at 37°C showed a rate con-stant (koff) of 3.1 s�1 for the higher level of bound SIRP� � CD4(1600 RU) and a koff of 5.3 s�1 for the lower level (800 RU; Fig.3). This increase in the apparent koff most likely reflects the exis-tence of rebinding effects after dissociation, which result in under-estimation of the true koff. Thus, the true koff is �5.3 s�1. This fastoff-rate is comparable to other low affinity interactions, such as theCD2-CD58 interaction (39), and explains why the affinity is rela-tively low for SIRP�. From the dissociation rate constant koff andthe Kd, the association rate constant was calculated to be �2 � 105

M�1s�1, and from the koff, the half-life was estimated to be 0.1 s.In comparison, measurements made at the same time show that theaffinity for human SIRP�-CD47 interaction was Kd � 2 �M (datanot shown) and the koff � 1.6 s�1 (0.4 s half-life; Fig. 3). The koff

is independent of concentration and is the same as that determinedpreviously (13). However, the Kd value for human SIRP�-CD47was lower (8 �M) than that reported in this study (2 �M), probablydue to a higher proportion of active CD47 in this preparation. Thecomparison shown above is valid and is the best estimate, becausethe same CD47 preparation was used for both SIRP� and SIRP�;thus, SIRP� has �10-fold lower affinity for CD47 than SIRP�.

Expression of SIRP� on human peripheral blood leukocytes

Recombinant soluble SIRP� � CD4 protein was used to raise mAbin mice. The resulting mAb were screened against the three knownSIRP proteins by ELISA. Four mAb (mAb OX116–OX119) werepicked for further study by flow cytometry using transfected cells.None of the mAb stained untransfected 293T cells (data notshown). From the mAb obtained, only one, mAb OX119, appeared

FIGURE 2. SIRP gene predictionand genomic localization. A, Dia-gram showing a map of the genes andapproximate locations of the SIRPcluster on human chromosome20p13. B, Diagramatic representationof the five members of the SIRP genefamily in humans, showing predictedstructural features (50–52). Alsoshown are the two possible splicevariants of SIRP� predicted fromcDNA.

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to be largely specific for SIRP� (Fig. 4), although some marginalbinding to SIRP� transfectants was observed. The specificity ofOX119 was confirmed by immunoprecipitation, because thisshowed a single immunoprecipitated band of �55 kDa (Fig. 4).The positive control mAb SE5A5 bound with a band of the ex-pected size for SIRP� and also a particularly prominent band cor-responding to SIRP�. Studies with transfectants confirmed thatthis mAb binds SIRP� and SIRP�, but not SIRP� (Table II), al-though the prominence of the SIRP� band was unexpected. Dataproduced from transfected cells with some of the mAb may notreflect the specificity of staining on ex vivo cells, because althoughmAb OX116 apparently stained SIRP�-transfected 293T cells, itdid not cross-react with SIRP� by ELISA and did not stain theU937 cell line (that expresses SIRP� and SIRP�) or PBLs. It maybe that alternative glycosylation by some cell lines (e.g., 293T) canalter the specificity of some mAb. Of the other mAb, OX117 cross-reacted with SIRP� (SIRP�/DAP12-transfected RBL cells weregifts from E. Tomasello and E. Vivier, Centre d’Immunologie deMarseille-Luminy, Marseille, France), and OX118 cross-reactedwith SIRP� (Fig. 4). A summary and comparison of SIRP mAbspecificity is shown in Table II (other mAb were SE5A5 and B1D5(32) and ILA24 (3)). The expression of SIRP� was equally ex-pressed in the presence or the absence of DAP12, in marked con-trast to SIRP�, which requires coexpression of DAP12 for surfaceexpression (Fig. 4).

The OX119 mAb stained the majority (70%) of human PBMC(Fig. 4), and this was repeated in several different unrelated indi-viduals (data not shown). Staining on peripheral blood myeloidcells was generally negative/very low (Fig. 4). The marginal levelof staining seen in some cases (data not shown) was assumed to becross-reactivity due to high levels of SIRP� on these cells, whichwere negative for SIRP� cDNA by PCR (data not shown). SIRP�/SIRP� staining (mAb SE5A5) is shown for comparison. Withinthe lymphocyte population, the majority of T cells expressedSIRP� (85% of CD3 cells), with 92% of CD4 and 71% of CD8high

T cells (excluding CD8low NK cells) staining. The expression levelof SIRP� on CD25� T cells is equivalent to that on CD25� Tcells. There was no change in relative SIRP� proportions based onCD45Ra/Rb expression (results not shown). There was, however,a reduction in the overall percentage of SIRP�-expressing cellsafter 2-day activation of PBMC with Con A, although the expres-sion levels remained approximately the same on the remainingpositive cells (Fig. 4). Approximately 10–20% of CD19 B cellswere labeled, with some variation between individuals (Fig. 4).

Both SIRP� and SIRP� can induce a functional interactionthrough CD47

To show that SIRP� can influence the behavior of CD47 on Tcells, an apoptosis assay was used in a similar manner to that

FIGURE 3. Affinity and dissociation rate of soluble human CD47 bind-ing to human SIRP�. A,- BIAcore trace showing CD47 � CD4 binding toSIRP� � CD4 and SIRP� � CD4, but not SIRP� � CD4 or CD4 control,which were immobilized onto streptavidin-coated flow cells at 1000 RU. B,For affinity measurements at 37°C, biotinylated SIRP� � CD4 (1600 RU) or

CD4 control (1400 RU) were immobilized onto streptavidin-coated flowcells. The indicated concentrations of CD47 � CD4 were injected overSIRP� � CD4 (solid line) or CD4 (dotted line). C, The difference betweenthe response at equilibrium in the SIRP� � CD4 and the control flow cellwas plotted against the CD47 � CD4 concentration. A Kd of 23 �M wascalculated by nonlinear curve fitting of the Langmuir isotherm (line) to data(F) from B with the negative control subtracted. The inset shows Scatchardplots of binding data for both SIRP� (circles) and SIRP� (squares), wherea linear fit indicates monomeric binding. D, The dissociation of solubleCD47 � CD4 from SIRP� � CD4 (squares; 1600 RU), SIRP� � CD4 (dia-monds; 1000 RU) or control CD4 (triangles; 1600 RU). The response in thecontrol flow cell was subtracted from that in the SIRP� cells for calculationof Koff values. The data were then normalized; thus, 100% represents thestart of the dissociation phase, and first-order exponential decay curveswere fitted to the data.

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described by Manna and Frazier (40). To overcome the low affinityof the SIRP-CD47 interaction and achieve binding of recombinantproteins to cells, a high avidity system of biotinylated SIRP� fu-sion protein bound to avidin-Sphero beads was used. These, ormAb (also bound to beads as a control), were incubated with Jurkator U937 cells in tissue culture medium for 2–3 h at 37°C beforelevels of apoptosis were measured with annexin. As expected, theCD47 mAb (clone 1796; Cymbus Biotechnology) induced highlevels of apoptosis. The results show that SIRP� and SIRP� in-duced apoptosis at much higher levels than with control beads inboth Jurkat and U937 cells (Fig. 5a). The slight reduction in ap-optosis induction by SIRP� vs SIRP� is consistent with loweraffinity binding to CD47. Levels of apoptosis induction were com-parable to the CD47 mAb itself (Fig. 5, b and c). The CD47 mAbMCA911-coated beads show that not all CD47 mAb are capable ofinducing apoptosis as has been previously described (40). Abs thatcross-linked the CD47-associated integrin CD51/61 had no effecton apoptosis, indicating a lack of direct involvement by CD47-associating integrins. The effect could also be specifically blockedby either excess soluble CD47 protein (CD4 chimera) or theOX119 mAb (Fig. 5c).

Activation of T cells modulates CD47 expression and, hence,SIRP�/SIRP� binding

Human PBMC were stimulated with Con A for up to 4 days. Toensure consistency in the results, cells were frozen at each timepoint, and all staining was conducted at the same time and with thesame preparations of reagents. To measure binding of SIRP pro-teins with CD47, the high avidity system of avidin-coated, fluo-rescently labeled beads saturated with biotinylated SIRP� � CD4,SIRP� � CD4, or CD4 control was used. SIRP� binding to PBMCwas mediated solely by CD47, as a CD47 mAb blocked binding ofthe beads to the cells (Fig. 6a). The results presented in Fig. 6 showthat PBMC at time zero had weak binding to SIRP� or SIRP�

fluorescent beads despite expression of CD47. Upon Con A acti-vation, PBMC expression of CD47 increased �2-fold until day 2before falling to lower levels by day 4. Despite this modest in-crease in CD47 expression, binding with both SIRP� and SIRP�

beads increased dramatically. However, SIRP� binding decreasedmore quickly once CD47 levels started falling. Thus, the reductionin affinity between SIRP� and SIRP� does not alter the overallamount bound at optimum levels of CD47, but, instead, changesthe threshold of binding at intermediate concentrations of CD47.

FIGURE 4. Specificity of SIRP�mAb and staining on human periph-eral blood leukocytes. A, Flow cyto-metric analysis of mAb OX116,OX117, OX118, and OX119 staining(f) or an isotype-matched controlmAb (�) on SIRP�- and SIRP�-transfected 293T cells, SIRP�/DAP12-transfected RBL cells andthe U937 cell line. B, Immunopre-cipitation of SIRP from humanPBMC with mAb as indicated. C,Staining with the SIRP�/SIRP�-re-acting mAb OX117 on 293T cellstransfected with either SIRP� orSIRP� alone or transfected togetherwith DAP12. D, For flow cytometryof human PBMC, granulocytes,monocytes, and lymphocytes weregated on the basis of forward and sidescatter. Human peripheral bloodgranulocytes and monocytes fromtwo individuals (i and ii) were stainedwith mAb OX119 or SE5A5 (f) oran isotype-matched control mAb(�). Two-color analysis of lympho-cyte gated cells are represented bydot plots with the indicated mAb.Quadrants were set with isotype-matched control mAb, and the per-centage of events within the quadrantis indicated, apart from CD8, whereonly CD8high T cells were gated toavoid CD8low NK cells. E, Expres-sion of SIRP� on Con A-activatedPBMC. Histograms show SIRP�staining on CD25�-gated cells.

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Thus, a threshold of CD47 expression is required before SIRP�beads can remain bound. Around the threshold concentration,small changes in CD47 lead to dramatic differences in SIRP� bind-ing, as shown in Fig. 6. The higher affinity SIRP� interaction al-lows a lower threshold, leading to smaller changes in binding rel-ative to the initial binding seen on day 0. These findings are inagreement with other low affinity cell surface molecular interac-tions, such as that between CD2 and CD48 (36).

DiscussionThese results show evidence for the expression and function of anovel member of the SIRP gene family in humans. The increasednumber of SIRP genes expressed at the cell surface and the finding

that most mAb cross-react on different SIRP gene products meansthat previous mAb binding data need to be reassessed to determinewhether functional effects were unique to a particular SIRP gene.A summary of the current known specificity of various SIRP mAbis shown in Table II. We have shown that the SIRP gene familycontains three highly related genes, all of which can be expressed.There were two additional genes with some sequence identity tothe SIRPs, although these have not been proven to exist at theprotein level.

Of the known expressed SIRP genes, all have very differentintracellular signaling potential. SIRP� seems to signal via inter-action of phosphotyrosines in cytoplasmic ITIM motifs with theShp-1 and Shp-2 protein tyrosine phosphatases (2), whereasSIRP� can impart signals via interaction with the DAP12 adapter(41), possibly through subsequent activation of Src family typetyrosine kinases. SIRP� is unlikely to transmit a signal to the cy-toplasm, because it has a negligible cytoplasmic tail with no ty-rosines capable of being phosphorylated and no charged trans-membrane amino acids capable of interacting with othertransmembrane signaling proteins, such as DAP12.

SIRP� was originally described as SIRP-b2 (33), which sug-gests that it is a subtype of the SIRP� gene. We have termed itSIRP�, because it is a separate gene product distinct from SIRP�or SIRP�. Analysis of the sequence from the mature expressedprotein shows the same level of sequence identity between thegenes, although the signal sequence of SIRP� is more similar tothat of SIRP�. As SIRP� is well conserved between mice andhumans, it seems likely that duplication of an ancestral SIRP�gene gave rise to a common CD47 binding precursor of SIRP� andSIRP�, followed by further duplication and divergence of SIRP�

FIGURE 5. SIRP� ligation of CD47 caninduce apoptosis in Jurkat and U937 cells. a,Two-color flow cytometric analysis of cellsstained with 7-AAD and annexin-PE after in-cubation with the indicated coated Spherobeads. b, Histograms show annexin-PE bind-ing on Jurkat cells after incubation withSphero beads coated with the indicated fu-sion protein (CD4, SIRP� � CD4, orSIRP� � CD4) or mAb (CD3, CD47 (1796and MCA911), or CD51/61). The cells weregated to remove 7-AAD-stained dead cells.c, Assay to show specificity of effects ofbead binding on Jurkat cells, using either sol-uble CD47 protein (CD4 as a control) orOX119 mAb (isotype-matched control mAbas control).

Table II. A comparison of SIRP mAb specificity between differenthuman SIRP proteins based on staining of transfected 293T cells(SIRP� and SIRP�) or SIRP�/DAP12-transfected RBL

SIRP� SIRP� SIRP�

OX116 �a � �OX117 � � �OX118 � � �OX119 �a,b �a,b �SE5A5 � � �ILA24 � � �BID5 � � �c

a Negative on U937 cells.b Marginal staining apparent on SIRP� and SIRP� transfectants due to very high

expression levels in these cells.c Positive by ELISA. Note, mAb OX116, OX117, and OX119 were all SIRP�

negative by ELISA.

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into a non-CD47-binding protein, possibly driven by a viral patho-gen. However, the low identity (�57%) of the putative SIRP�orthologue in mice (EMBL accession no. XM_283831) may indi-cate that it duplicated independently in this species, and the ap-parent lack of an SIRP� gene indicates that mouse SIRP� evolveddirectly from SIRP�. The identity of a SIRP� ligand remains un-known, but it is possible that it binds a pathogen, from the analogywith Ly49 in mice. Most mouse strains have an inhibitory Ly49form, but some, in addition, have a closely related activatory formthat can bind a CMV protein and confer resistance to thisvirus (42, 43).

The finding of two related proteins that bind the same ligandwith differing affinities has also been described for other cell sur-face receptors. CD80 binds to both CD28 and CTLA-4 with Kd of4 and 0.4 �M, respectively, and CD80 favors CTLA-4 engagementover CD28 (44, 45). It is also notable that the higher affinity in-teraction (CD80-CTLA4) is inhibitory, and this parallels the higheraffinity SIRP�-CD47 inhibitory interaction. The threshold effectobserved with multivalent binding of SIRP� to CD47 on activation

of T cells implies that in vivo, resting T cells would not signal toeach other via CD47 due to the low affinity/avidity of SIRP�, evenif cell-cell contact were taking place. However, activation, leadingto increased CD47 expression, would allow for the SIRP�-CD47interaction to take place. This would be transient, and the interac-tion would be lost on chronically activated T cells when theirCD47 levels decrease. This means that freshly activated CD47high

T cells may be on a “knife edge,” where any extra signals throughCD47 could lead to their destruction and uptake by neighboringmyeloid cells (46, 47). Indeed, ligation of CD47 has been associ-ated with apoptosis and uptake of cells in some experimental sys-tems (48). This functional consequence of CD47 ligation was re-produced in this study using SIRP� fusion protein on Jurkat cells.The existence of SIRP� on T cells means that they have anothermechanism for inducing signaling through CD47 with possiblemultiple effects on integrin function and T cell behavior. Thus, theability of T cells to directly send signals via CD47 has importantimplications for T cell biology.

AcknowledgmentsWe are grateful to Michael J. Puklavec and Steve Simmonds for help withmAb production, to H. J. Buhring for providing mAb SE5A5 and B1D5, toE. Tomasello and E. Vivier for SIRP�/DAP12 transfectants and M. Col-onna for SIRP� cDNA, and to J. Sedgwick for DAP12 vector.

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