CR2/CD21) As a Receptor for IFN-Α: a Potential Role in Systemic Lupus Erythematosus This Information Is Current As of September 26, 2021

Characterization of Human Complement Receptor Type 2 (CR2/CD21) as a Receptor for IFN-α: A Potential Role in Systemic Lupus Erythematosus This information is current as of September 26, 2021. Rengasamy Asokan, Jing Hua, Kendra A. Young, Hannah J. Gould, Jonathan P. Hannan, Damian M. Kraus, Gerda Szakonyi, Gabrielle J. Grundy, Xiaojiang S. Chen, Mary K. Crow and V. Michael Holers

J Immunol 2006; 177:383-394; ; Downloaded from doi: 10.4049/jimmunol.177.1.383 http://www.jimmunol.org/content/177/1/383 http://www.jimmunol.org/ References This article cites 69 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/177/1/383.full#ref-list-1

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of Human Complement Receptor Type 2 (CR2/CD21) as a Receptor for IFN-␣: A Potential Role in Systemic Lupus Erythematosus1

Rengasamy Asokan,* Jing Hua,† Kendra A. Young,* Hannah J. Gould,‡ Jonathan P. Hannan,* Damian M. Kraus,* Gerda Szakonyi,§ Gabrielle J. Grundy,¶ Xiaojiang S. Chen,ʈ Mary K. Crow,† and V. Michael Holers2*

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-␣. IFN-␣, a multifunctional cytokine important in the innate immune system, has recently been Downloaded from proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-␣ in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-␣ interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-␣-responsive genes. Thus, IFN-␣ represents a fourth class of extracellular ligands for CR2 and interacts with the same http://www.jimmunol.org/ domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-␣ receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction. The Journal of Immunology, 2006, 177: 383–394.

uman complement receptor (CR)3 type 2 (CR2/CD21) is sights into the structure and biophysical characteristics of the a multifunctional receptor for three well-described ligand recognition mechanism of CR2 were provided by the solu- H classes of ligands. These ligands include complement tion of the x-ray crystallographic structure of the short consensus

C3 fragments iC3b, C3d,g, and C3d (1), which are covalently at- repeat (SCR)1-2 domain of CR2 with C3d (6) as well as without by guest on September 26, 2021 tached to target Ags, the gp350/220 viral coat protein of the EBV the C3d ligand (7). Subsequent in silico modeling (8), analytical (2), and the immunoregulatory protein CD23 (3, 4). CR2 is ex- ultracentrifugation, x-ray and neutron scattering (9), as well as pressed on B cells, follicular dendritic cells, and a subset of pe- mutagenesis (10) analyses have resulted in the current understand- ripheral and thymic T cells (reviewed in Ref. 5). Substantial in- ing that the SCR1-2 domain of CR2 interacts with C3d by both charge-dependent and charge-independent means. CR2 has been primarily viewed as a B cell coreceptor whose *Department of Medicine and Department of Immunology, University of Colorado biologic role is to amplify Ag receptor-mediated signal transduc- Health Sciences Center, Denver, CO 80262; †Mary Kirkland Center for Lupus Re- search, Hospital for Special Surgery, New York, NY 10021; ‡Randall Centre for tion. In support of this conclusion, coligation of CR2 with the Molecular Mechanisms of Cell Function, King’s College London, New Hunt’s House, membrane BCR results in enhanced intracellular calcium release, Guy’s Campus, London, United Kingdom; §Department of Biochemistry and Molec- proliferation, and/or activation of MAPK, as well as up-regulation ular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262; ʈDepartment of Molecular and Computational Biology, University of Southern Cal- of B7 molecules (11–15). Enhancement of BCR-dependent cell ifornia, Los Angeles, CA 90089; and ¶Laboratory of Molecular Biology, National activation by CR2 is due to the association of CR2 with CD19 and Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 CD81 in a B cell-specific signal transduction complex (16, 17), where coligation of BCR with CR2/CD19 amplifies signals with- Received for publication October 24, 2005. Accepted for publication April 13, 2006. out efficient engagement of inhibitory phosphatases (14, 15). 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 In addition to enhancing BCR-dependent signals, ligation of with 18 U.S.C. Section 1734 solely to indicate this fact. CR2 alone has been reported to result in several other phenotypes, 1 This work was supported by the Lupus Research Institute (to V.M.H., M.K.C., and including the induction of homotypic adhesion (18), NF-␬B acti- R.A.), National Institutes of Health Grants R0-1 CA53615 (to V.M.H.) and R0-1 vation (19), IL-6 generation (20), Ag uptake and presentation to T AR050829 (to M.K.C.), and the Alliance for Lupus Research (to M.K.C.). cells (21, 22), and actin polymerization (23). 2 Address correspondence and reprint requests to Dr. V. Michael Holers, Division of Rheumatology, Department of Medicine, Box B-115, University of Colorado Health CR2 has been shown to be one of several receptors for human Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: CD23 (3, 4). CD23 is an immunoregulatory protein found both on [email protected] cell membranes and as a soluble protein. CD23 interacts with CR2 to 3 Abbreviations used in this paper: CR, complement receptor; SCR, short consensus increase production of IgE in the presence of IL-4 (3), rescue germi- repeat; SLE, systemic lupus erythematosus; IFNAR, IFN-␣ receptor; BCCP, biotin carboxyl carrier protein; BSP, biotinylation signal peptide; IPTG, isopropyl ␤-D-thio- nal-center B cells from apoptosis (24), provide T cell-activating sig- galactoside; SA, streptavidin; rh, recombinant human; FC, flow cell; HPRT, hypo- nals by B cell APCs (25), and promote T-B cell adhesion (26). xanthine guanine phosphoribosyltransferase; IFIG, IFN-␣-inducible gene; MX1, myxovirus (influenza virus) resistance 1; PRKR, dsRNA-dependant protein kinase; In mice, CR2 is encoded along with the larger receptor CR1 by RE, relative expression. the Cr2 gene, which produces both proteins through alternative

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 384 CR2/CD21 AS AN IFNAR splicing of a common mRNA (27). Support for a critical role of CCG GGC CAT TTC TTG TGG CTC TCC TCC GCC-3Ј and 5Ј-GAA CR2 in the immune response has been provided by results in TGC GGC CGC ACT ACT AAA AAT TTC TTC ACA TAC TGG CAT Ϫ/Ϫ TTT GG-3Ј) that also generate Sfi and NotI restriction endonuclease en- Cr2 mice that are deficient in CR2 (as well as mouse CR1). Ј Ј Ϫ/Ϫ zyme sites at the 5 and 3 ends, respectively. A clone containing aas Cr2 mice demonstrate substantial defects in Ag-specific, T 70–156 of the biotin carboxyl carrier protein (BCCP) was provided by Dr. cell-dependent and T cell-independent humoral immune responses J. Lambris (Department of Pathology and Laboratory Medicine, University (28–30) that is due to a lack of receptor on both B cells and fol- of Pennsylvania, Philadelphia, PA), as was a clone encoding the biotin licular dendritic cells (31). Cr2Ϫ/Ϫ mice also demonstrate defects holoenzyme synthetase protein Bir A, which catalyzes the incorporation of biotin into BCCP (46). A Bir A enzyme kit was also purchased from Avid- in B cell memory (32, 33) and the development of the natural Ab ity. Sequences encoding CR2 SCR1-4 alone, or with BCCP, were then repertoire (34, 35). cloned into the pSecTag2/Hygro vector (Invitrogen Life Technologies) In addition to having an important role in the immune response such that soluble SCR1-4 would be expressed with and without C-terminal to foreign Ags, recent studies have strongly suggested that CR2 BCCP (Fig. 1). Before transfection, each cDNA underwent nucleotide se- plays a key role in maintaining tolerance to self-Ags such as quence analysis (University of Colorado Cancer Center) to assure the presence of the anticipated DNA sequence. ssDNA, dsDNA, chromatin, and histones, as well as the development of experimental systemic lupus erythematosus (SLE) (13, 36, Transfection, expression, and purification of CR2 SCR1-4 and 37). The molecular explanation for this phenotype is incompletely CR2 SCR1-4-BCCP understood. One hypothesis suggests that enhanced autoimmunity The cDNAs encoding the recombinant proteins SCR1-4 and SCR1-4- to nuclear Ags in the relative or complete absence of CR2 and/or BCCP were stably expressed in the NSO and HEK 293 mammalian cell CR1 function may be due to the requirement for complement and lines using electroporation and Lipofectamine 2000 (Invitrogen Life Tech-

CR2/CR1 in Ag capture in the bone marrow (36, 38). Another nologies), respectively. For NSO cell line expression, the CR2 SCR1-4 Downloaded from cDNA was linearized and then electroporated at 100 microfarads/300 V in hypothesis is that, in the absence of CR2/CR1, there is ineffective 0.5 ml of a PBS solution containing 50 ␮g of herring sperm DNA and 6 ␮g deletion of autoreactive B cells by C3/C4-bound Ags (13, 36). of SCR1-4 cDNA. Cells were then serially diluted in medium under se- Although the exact role of CR2 in maintaining tolerance to DNA, lection with 800 ␮/ml hygromycin. For HEK 293 cell expression, cells histones, and chromatin is unclear, markedly lower expression of were recovered by 0.1% trypsin treatment and release from a flat-bottom ϫ 5 ␮ human CR2 in patients with SLE (39) and mouse models of SLE flask, counted, and then seeded at 2.5 10 in 500 l growth medium containing no serum and no antibiotics. For transfection of the CR2 SCR1- before the onset of detectable humoral autoimmunity (40) have 4-BCCP cDNA, 1 ␮g of linearized plasmid and 3 ␮l of Lipofectamine http://www.jimmunol.org/ suggested that alteration of this function of CR2 is likely to be 2000 in 50 ␮l of Opti-MEM (Invitrogen Life Technologies)-reduced serum pathophysiologically important in the development of human SLE. medium was mixed and incubated at 37°C for 30 min, followed by incu- IFN-␣ has recently been proposed as a molecule that is also bation in tissue culture wells for 24–48 h. Following this incubation, cells were diluted serially and grown in the presence of 800 ␮g/ml hygromycin. centrally implicated in the pathogenesis of SLE, a hypothesis that In both expression systems, after 2–3 wk, individual clones were recovered is supported by several lines of investigation. For example, a sub- and screened for CR2 protein expression by ELISA. The secreted protein set of patients with SLE demonstrate a peripheral blood lympho- obtained from both of these cell line supernatants was concentrated and cyte mRNA profile consistent with IFN-␣ stimulation (41). In ad- stored at Ϫ70°C until sufficient volume was ready for purification. dition, lupus-prone New Zealand Black mice whose expression of CR2 SCR1-4 and CR2 SCR1-4-BCCP were both purified in an identical fashion. Supernatant was concentrated and dialyzed in 50 mM PBS over- ␣ by guest on September 26, 2021 the type I IFN- receptor (IFNAR) has been eliminated by gene night at 4°C. Dialyzed supernatant was loaded on a previously equilibrated targeting demonstrate amelioration of autoimmune disease (42). HB-5-Sepharose 4B anti-CR2 mAb (which specifically recognizes an One previous series of experiments has shown that IFN-␣ epitope in CR2 SCR3-4) affinity column at 4°C. Following washing of blocks EBV binding to, and capping of, CR2 on B cells (43, 44). unbound protein, bound protein was eluted using a buffer previously de- ␣ scribed (47) to maintain CR2 activity that contains 0.2 M acetic acid, 150 In addition, IFN- was reported to contain a peptide sequence mM NaCl, 1 mM PMSF, 1 ␮M leupeptin, and 1 ␮M pepstatin (pH 2.5). similar to one from C3d that was proposed to be at the C3d-CR2 The eluate was immediately neutralized with 1.0 M Tris buffer (pH 7.4). binding interface (43). That particular IFN-␣-derived peptide was The eluted protein was pooled, concentrated, and dialyzed in 50 mM PBS reported to bind to purified CR2 in vitro and moderately blocked overnight at 4°C. All CR2 proteins purified through this method were then the interaction of iC3b/C3d-coated zymosan particles with B cells. finally passed through a protein G-Sepharose column to remove a trace ␣ amount of anti-CR2 mAb HB-5 that leaches from the column during this Although these data provided some evidence that IFN- could be purification method. an authentic ligand for CR2, the conclusions from this study were challenged by the finding that the C3d-derived peptide sequence CR2 SCR1-2 and CR2 SCR1-15 expression and purification used as the basis for the IFN-␣ comparison was found to not be CR2 SCR1-2 produced in Pichia pastoris and CR2 SCR1-15 produced in near the CR2 binding site for C3d in the C3d-CR2 cocrystal struc- baculoviral Sf9 cells were produced as described by Guthridge et al. (48). ture (6). Briefly, the human CR2 SCR1-2 domain was cloned into the expression plasmid pPICZ␣ (obtained from Invitrogen Life Technologies) and used to For this reason, in addition to the emerging interests related to transfect the X33 wild-type strain of P. pastoris. A highly producing trans- the potential roles of both IFN-␣ and CR2 in the development of fected clone was grown by fermentation. To purify CR2 SCR1-2, the cell SLE, we have re-examined the interactions of this ligand-receptor supernatant was buffered to pH 4.0, and the supernatant was passed over a pair. To provide a relative context for this ligand-receptor study, SP-Sepharose column and eluted using 10 mM formate at pH 4.0 with a ␣ 0–0.5 M linear NaCl salt gradient. The eluted CR2 SCR1-2 material was we have also compared the affinity and kinetics of IFN- binding pooled, concentrated, and deglycosylated overnight at 37°C with 33,000 U to CR2 with three other well-characterized ligands for this recep- EndoH/ml (New England Biolabs). The deglycosylated sample was passed tor. We find, using surface plasmon resonance, ELISA, and IFN- over SP-Sepharose, and a 0.0–0.5 M NaCl gradient was used to elute the ␣-responsive gene analysis, that IFN-␣ exhibits readily detectable, deglycosylated CR2 SCR1-2. Positive fractions were pooled and concen- high-affinity interactions with CR2. These data suggest that the trated. The protein size and purity was assured by both nonreducing and reducing SDS-PAGE analysis using NuPAGE 10% Bis-Tris gels and MES roles of these two proteins in the development of autoimmunity NuPAGE running buffer (Invitrogen Life Technologies). Aminoterminal may be mechanistically linked in the pathogenesis of SLE. amino acid sequencing and MALDI-TOF mass spectroscopy analysis was also performed to confirm the exact molecular mass. This same material Materials and Methods was also used in the cocrystalization studies with C3d (6). Expression vectors Human CR2 SCR1-15 was expressed in Sf9 insect cells as a recombinant soluble protein in the baculovirus expression system. The cDNA cod- Human CR2 SCR1-4 was amplified by PCR from a previously used CR2- ing for the first 15 SCR domains plus the native human Kozac consensus encoding expression vector (45) using the primers (5Ј-CCG GGC CAG sequence and signal peptide as a soluble protein was cloned into the The Journal of Immunology 385 Downloaded from http://www.jimmunol.org/

FIGURE 1. Forms of human CR2 used in studies. Soluble CR2 SCR1-2 (A), CR2 SCR1-4 (B), full-length soluble CR2 SCR1-15 (C), and biotinylated CR2 SCR1-4-BCCP (D). pVL1393 baculoviral expression vector (BD Pharmingen). After infection column in 20 mM Tris (pH 7.1) and a MonoS HR5 column in 50 mM MES of the insect cells with the recombinant baculovirus, spinner cultures were (pH 7.0), where in all three cases C3d was eluted using a linear salt gra- grown for 5 days at 27°C. The supernatants were removed, filtered, and dient up to 1 M NaCl. The protein purity was confirmed using SDS-PAGE. concentrated, and the concentrate was dialyzed overnight into 50 mM PBS by guest on September 26, 2021 at 4°C. To purify CR2 SCR1-15, the dialyzed supernatant was loaded on a EBV gp350 expression and purification previously equilibrated HB-5 Sepharose mAb column, and protein was purified as noted above for CR2 SCR1-4. As previously described (50), a 70-kDa fragment of the EBV gp350/220 coat protein was cloned into the pVI-Bac transfer vector (BD Pharmingen). Human C3dg-biotin and C3d expression and purification This contained a melittin signal sequence and a recombinant protein with a C-terminal polyhistidine tag. This fragment of gp350/220 was produced Human C3dg-biotin (49) and C3d (48) were grown and purified as de- by infecting Sf9 insect cells with the gp350/220-packaged baculovirus par- scribed previously. Briefly, C3dg-biotin was cloned into the pET11b vector ticles. The cultures were grown, and the culture supernatant was processed (Novagen). This construct consisted of a bicistronic bacterial expression as described above for CR2 SCR1-15. The 70-kDa fragment of gp350/220 vector with a T7 promoter, an N-terminal polyhistidine, and a T7 epitope was then purified using a Con A Sepharose column. The concentrated tag, followed by the human C3dg sequence (C3 residues 953 to 1303, in supernatant was loaded on a previously equilibrated column. The unbound 1010 which Cys at its thioester site was mutated to Ala: Swiss-Prot code protein was washed off with 20 mM Tris-HCl (pH 7.4) containing 0.5 M P01024), a GGGSGGGS linker, and a C-terminal biotinylation signal pep- NaCl. The bound protein was eluted with increasing concentration of ␣-D- tide (BSP; LGGIFEAMKELRD) for birA-catalyzed biotinylation. A sec- methylmannoside or ␣-D-methylglucoside. The eluted protein was concen- ond ribosomal binding site allows for translation of the birA gene in vivo trated, dialyzed into PBS, and stored at Ϫ20°C. Protein purity was assessed in cells that also express the recombinant C3dg-BSP protein. C3dg-biotin by SDS-PAGE. was produced in the Escherichia coli BL21 pLysS Codon Plus strain (Strat- agene) transfected with the C3dg bicistronic vector. The cultures were CD23 expression and purification induced with 25 mM isopropyl ␤-D-thiogalactoside (IPTG) at 28°C and shaken overnight. The harvested pellets were resuspended in PBS at pH 8.0 Using the numbering from Swiss-Prot accession no. P06734, derCD23 with Complete EDTA-free protease inhibitor tablets (Roche-Boehringer comprises the amino acids Ser156 to Glu298. The human derCD23 construct Mannheim) and lysed using four freeze thaw cycles. DNase (4,000 U) and was subcloned from CD23 cDNA by PCR into pET5a. Overexpression of RNase (50 ␮l of 0.5 mg/ml stock) were added, and the lysate was clarified CD23 was induced with 0.4 mM IPTG in the E. coli host strain, BL21 by sonication and centrifugation at 8,000 ϫ g, followed by mixing for 30 (DE3) (pLysS) (Invitrogen Life Technologies) cultured in M9 minimal min with 10 ml of Talon (Co2ϩ-immobilized metal affinity chromatogra- medium. The cells were incubated for a further3hat37°C then harvested phy) resin. Unbound material was washed off using PBS at pH 8.0 and 10 and stored at Ϫ20°C. The recombinant protein was present as inclusion mM imidazole, and the C3dg-biotin was eluted by a linear gradient to 100 bodies and extracted as reported (51), including 100 U of DNase I in the mM imidazole and further purified using a Superdex-75 gel filtration col- first solution, and replacing Nonidet P-40 with Igepal (Sigma-Aldrich). The umn (Amersham Biosciences). Biotinylation was confirmed by Western final inclusion body preparation in 6 M guanidine was refolded via a glu- blots using streptavidin (SA)-HRP, which detected bands that also reacted tathione intermediate (52). CD23 was purified by hydrophobic interaction with a rabbit anti-C3dg Ab. chromatography using Phenyl Sepharose 6 (high substituted) Matrix (Am- Human C3d was expressed by transfection of the pET15b-C3d plasmid ersham Biosciences) equilibrated with 25 mM Tris-HCl (pH 7.5), contain- into BL21 pLysS Codon Plus E. coli (Stratagene). Cultures were grown as ing 1.5 M ammonium sulfate. The refolded protein was adjusted to 1.5 M described for C3dg-biotin above, the bacterial pellet was harvested in ammonium sulfate and filtered through a cellulose acetate filter (0.8-␮m DEAE starting buffer (20 mM Tris; pH 7.1) plus Complete EDTA-free pore; Sattorius). Purified CD23 was eluted by a decreasing ammonium protease inhibitor tablets, and lysis was performed as described above. C3d sulfate concentration gradient. Fractions containing CD23 were pooled, was purified using a DEAE HiPrep column, followed by a MonoQ HR5 concentrated by ultrafiltration (10-kDa cutoff YM10 cellulose membranes; 386 CR2/CD21 AS AN IFNAR

Millipore), and dialyzed into 25 mM Tris-HCl (pH 7.5), 137 mM NaCl, CR2 SCR1-15 bound to IFN-␣ was detected using anti-CR2 mAb HB-5,

and 2 mM CaCl2. which is specific for SCR3-4, followed by anti-goat mouse IgG (Jackson ImmunoResearch Laboratories) at 1/1000. Color was developed using IFN-␣ source for biophysical studies ABTS, and the OD was measured at an absorbance of 405 nm. Human recombinant human (rh)IFN-␣ in phosphate buffer was obtained Measurement by ELISA of the interaction of CR2 with the from PBL Biomedical Laboratories. For surface plasmon analysis, ex- ligands human IFN-␣, CD23, C3d, and gp350 change of IFN-␣ into the working buffer was accomplished using a Cen- tricon (Millipore). Dose-dependent binding of CR2 to its ligands was studied using the ELISA method in which 5 ␮g/ml rhIFN-␣, CD23, C3d, and gp350 were immo- Bir A enzyme expression and purification bilized on 96-well ELISA plates (Costar) in bicarbonate buffer (pH 9.0) overnight at 4°C. Plates were then washed and blocked with PBS Tween 20 The biotin holoenzyme synthetase protein, Bir A, catalyzes the incorpora- containing 1% BSA. Serial dilutions of CR2 SCR1-15 were then added to tion of biotin into BCCP. This protein was expressed in E. coli using a the wells in PBS containing 50 mM NaCl (pH 7.4) and allowed to incubate clone that was provided by Dr. J. Lambris (Department of Pathology and for 1 h binding at 37°C. Following washing, the amount of CR2 SCR1-15 Laboratory Medicine, University of Pennsylvania, Philadelphia, PA) and bound to the wells was measured using anti-CR2 mAb HB-5 as described purified according to previously described method (46). Following growth above. at 37°C in YT medium containing 50 ␮g/ml ampicillin, 0.25 mM IPTG was added to induce protein expression. The expressed protein in the cell Measurement by surface plasmon resonance of the interaction of lysate was purified by Ni-NTA affinity column. Briefly, the cell lysate was CR2 with IFN-␣, CD23, C3d, and gp350 dialyzed in 10 mM Tris, 300 mM NaCl, and 10 mM imidazole (pH 8.0) overnight at 4°C. The dialyzed supernatant was incubated with 5 ml of CR2 binding to its ligands human IFN-␣, CD23, C3d, and gp350 was Ni-NTA resin for 60 min at 4°C and then packed into a column. The studied using BIAcore 3000 (BIAcore) with SA-coupled and carboxym- unbound protein was washed with 10 mM Tris with 20 mM imidazole pH ethyl-dextran (CM5) chips. All experiments were performed in 10 mM Downloaded from 8.0. The bound protein was eluted with increasing concentrations of imi- HEPES, 50 mM NaCl, 1 mg/ml carboxymethyl-dextran, and 0.05% sur- dazole (0.01–0.25 M) in 0.02 M PBS (pH 7.5) containing 0.5 M NaCl. The factant P-20 (pH 7.4) at 25°C. The chip regeneration buffer used 1 M NaCl eluted protein was dialyzed against 50 mM Tris, 200 mM KCl, and 5% in place of 50 mM NaCl. For kinetic analysis, biotinylated CR2 SCR1-4- glycerol (pH 7.5) at 4°C and stored at Ϫ70°C. BCCP (1268 RU) or C3dg-biotin (1221 RU) were immobilized on a SA chip. IFN-␣ (789 RU), CD23 (1228 RU), and gp350 (1466 RU) were Site-specific biotinylation of CR2 SR1-4-BCCP using the Bir A immobilized on a CM5 chip using amine-coupling chemistry per manu- facturer’s instructions. Immobilization was performed on either flow cell

enzyme http://www.jimmunol.org/ (FC)2 or FC4, and FC1 and FC3 were used as control cells. Binding was Site-specific biotinylation was performed following the method of Sarrias measured at 30 ␮l/min to avoid mass transfer effects. At this flow the initial et al. (46). To accomplish this, CR2 SCR1-4-BCCP protein was dialyzed on-rate was maximum. Flow was allowed for several seconds to establish against 40 mM Tris containing 5.5 mM MgCl2 and 100 mM KCL (pH 8.0) baseline, and then various concentrations of analyte were injected. The overnight at 4°C. Following this, 30 ␮g of SCR1-4-BCCP protein was association was allowed for 120 s, and the dissociation of the complex was incubated with 365 ␮g of purified Bir A protein, 24 ␮g of d-biotin, and 20 monitored for 120 s. For IFN-␣ instead of 120 s, 150 s was allowed for mMATPfor1hat37°C. Excess biotin was removed by a desalting pD-10 both association and dissociation phase to allow optimal binding. BIAe- column followed by extensive dialysis against PBS. Protein was stored at valuation 3.1 (BIAcore) software was used to analyze the binding data, Ϫ70°C in aliquots until use. using global fitting where best fit was indicated by a low residual and ␹ (2) values Ͻ10. This analysis yields a K ,K, and K for protein:protein SDS-PAGE and Western blot analysis of recombinant proteins a d D interactions. by guest on September 26, 2021 Protein size and purity was determined using nonreducing SDS-PAGE and Inhibition of CR2-ligand interaction by mAbs recognizing the Western blot analysis. Purified CR2 SCR1-4 and CR2 SCR1-4-BCCP were first two SCRs of CR2 electrophoresed on a NuPAGE Novex 10% Bis-Tris gel with MOPS Nu- PAGE as the running buffer. Proteins were then transferred to a nitrocel- To determine whether the first two SCR domains were directly involved in lulose membrane and incubated sequentially with anti-CR2 mAb HB-5 and CR2 binding to IFN-␣, CR2 SCR1-15 was preincubated for1hat37°C peroxidase-conjugated goat anti-mouse IgG secondary Ab (Jackson Immu- with IgG1 anti-CR2 mAbs (171, 1048, 994, 629) directed to the SCR1-2 noResearch Laboratories). In another blot, the same proteins were trans- domain (53) or a control IgG1 anti-human factor B mAb (a gift from Dr. ferred to a nitrocellulose membrane to evaluate for biotinylation efficiency. J. Thurman, Division of Nephrology and Hypertension, University of Col- In both experiments, nonspecific binding was blocked with PBS 0.05% orado Health Sciences Center, Denver CO). Preincubated CR2 SCR1-15 Tween 20 containing 10% milk. The incorporation of biotin was assessed samples were then added for1hat37°C to an ELISA plate that had been by reactivity with peroxidase-conjugated SA (BD Pharmingen) at 1 ␮g/ml. precoated as described above with IFN-␣, CD23, C3d, and gp350. The Proteins were detected using the ECL kit (Amersham Biosciences). In ad- amount of CR2 SCR1-15 bound to the wells was detected using the anti- dition, N-terminal amino acid sequence analysis was performed for the CR2 mAb HB-5 followed by goat anti-mouse IgG (Jackson ImmunoRe- CR2 SCR1-4 and CR2 SCR1-4-BCCP proteins to confirm the correct pro- search Laboratories) at a 1/1000 dilution. Color was developed using tein identity. To accomplish this, a 12% denaturing minigel was prepared ABTS, and OD measured at an absorbance of 405 nm. Percentage of in- and run with SDS-PAGE running buffer containing 100 mM reduced glu- hibition was calculated by the following: 1 Ϫ [(average experimental tathione at 37°C and stored overnight at 4°C before loading the sample. OD Ϫ background OD)/(maximum average OD Ϫ background OD)] ϫ Next, CR2 SCR1-4 and CR2 SCR1-4-BCCP proteins were electrophoresed 100. in running buffer prepared by substituting glutathione with 2-ME. Bands were transferred to polyvinylidene difluoride membrane, and the mem- Cross-competition analysis using CD23, C3d, and gp350 brane was stained with 0.1% Coomassie blue in 50% methanol for 5 min. To determine the relationships between the IFN-␣ binding site and other Following slight destaining, the transferred protein bands were excised CR2-ligand interaction sites, 2 ␮g/ml rhIFN-␣ (IFN-␣; BioSource Inter- with a razor blade, and N-terminal sequence analysis was then performed national) was coated on 96-well ELISA plates (Costar) in bicarbonate in the Protein Sequencing Core (National Jewish Medical and Research buffer (pH 9.0) overnight at 4°C. Plates were then washed and blocked with Center, Denver, CO). PBS Tween 20 containing 1% BSA. Serial dilutions of CD23, C3d, and ␮ Characterization by ELISA of pH and NaCl dependence of CR2 gp350 were made on a diluter plate. CR2 SCR1-15 at 2 g/ml was then ␣ added to the serially diluted wells, as well as to control wells with buffer SCR1-15 binding to IFN- alone, and incubated for1hatroom temperature. These samples were then ␣ CR2 SCR1-15 was dialyzed overnight at 4°C against 50 mM PBS with added to IFN- -bound ELISA plates and incubated for 1 h. Following differing pH (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) and in a separate experiment washing, the amount of CR2 SCR1-15 bound to the wells was measured 50 mM PBS containing differing NaCl concentrations (25, 50, 75, 100, using anti-CR2 mAb HB-5 as described above. 6 125, and 150 mM). Then, 5 ␮g/ml human rhIFN-␣ (5 ϫ 10 U/ml) (PBL B cell culture and stimulation Biomedical Laboratories) was immobilized on 96-well ELISA plates (Costar) in bicarbonate buffer (pH 9.0) overnight at 4°C. Following wash- Human B cells were isolated from healthy donor blood using the Ro- ing of the plate, soluble CR2 SCR1-15 dialyzed against differing pH and setteSep Human B cell Enrichment Cocktail (StemCell Technologies). Pu- NaCl buffers was added and incubated for 60 min at 37°C. After washing, rified B cells were cultured at a density of 2 ϫ 105/0.1 ml in 96-well The Journal of Immunology 387

flat-bottom plates in RPMI 1640 supplemented with L-glutamine (2 mM), HEPES (20 mM), penicillin (100 U/ml), streptomycin (100 ␮g/ml), and o ␣ 10% FBS at 37 C, 5% CO2. B cells were cultured with medium, rhIFN- (IFN-␣; BioSource International) or IFN-␣ plus anti-CR2 mAb 171 or its isotype control mouse IgG2A (R&D Systems). The B cells were preincubated with Abs for 1 h before adding IFN-␣. After6hofculture, B cells were lysed and stored at Ϫ70oC. RNA extraction and quantitative real-time RT-PCR RNA was extracted from each lysate using the RNeasy Mini Kit (Qiagen). A total of 0.4 ␮g of this RNA was reverse-transcribed to cDNA in a 20-␮l reaction using SuperScript III RNase H- Reverse Transcriptase (Invitrogen Life Technologies). cDNA obtained from each sample was diluted 1/40, and 10 ␮l was amplified in a 25-␮l real-time PCR using 0.4 ␮M sense and antisense primers and the 2ϫ iQ SYBR Green Supermix (Bio-Rad). Hypoxanthine guanine phosphoribosyltransferase (HPRT)1 was used as a housekeeping gene. Primer sequences for IFN-␣-inducible genes (IFIG) and HPRT1 were as follows: myxovirus (influenza virus) resistance 1 (MX1) forward, 5Ј-TACCAGGACTACGAGATTG-3Ј; MX1 reverse, 5Ј-TGCCAGGAAGGTCTATTAG-3Ј; dsRNA-dependant protein kinase (PRKR) forward, 5Ј-CTTCCATCTGACTCAGGTTT-3Ј; PRKR reverse, 5Ј-TGCTTCTGACGGTATGTATTA-3Ј; HPRT1 for- Ј Ј Ј Downloaded from ward, 5 - TTGGTCAGGCAGTATAATCC-3 ; HPRT1 reverse, 5 - FIGURE 2. SDS-PAGE and Western blot analysis under nonreducing Ј GGGCATATCCTACAACAAAC-3 . conditions of CR2 SCR1-4 and CR2 SCR1-4-BCCP. Lane 1, m.w. marker; B cells cultured with medium alone were included in each assay to lane 2, BSA; lane 3, CR2 SCR1-4; and lane 4, CR2 SCR1-4-BCCP. Anti- provide a basis for normalization across experiments. Results for each culture condition are expressed as relative expression (RE) compared with B CR2 mAb HB-5 was used to detect CR2. cells cultured with medium. Details of the real-time PCR method have been described in detail (54). Briefly, gene amplifications for each sample were

performed in triplicate using the iCycler Real-Time Detection Systems http://www.jimmunol.org/ (Bio-Rad). Standard curves for MX1 and PRKR genes were generated using cDNA to determine efficiency. Melting curve analysis was performed we believe that this minor difference is due to differential glyco- for all PCR products to assure specific amplification. To calculate RE in a sylation, because there are two glycosylation sites predicted in this B cell lysate sample of MX1 and PRKR, each sample was amplified with domain, and the differences in apparent m.w. are not present fol- primers for both the target gene and housekeeping gene in separate wells. lowing treatment of the proteins with PN-glycanase (data not Values for target genes MX1 and PRKR and the housekeeping gene HPRT1 were subtracted from the corresponding reference sample. The shown). In addition, aminoterminal protein sequence analysis differences were then used as exponents with the base equal to 1 plus the demonstrated the identical and expected sequence for each of the value of the efficiency of that PCR. The MX1 and PRKR values were four forms (data not shown). divided by the HPRT1 value for each sample, and the result was the rel-

We used CR2 SCR1-4-BCCP because that would allow us to by guest on September 26, 2021 ative gene expression of each unknown sample. Results are expressed as percentage of inhibition compared with expression induced by rhIFN- ␣. avoid the usual method of coupling to chips in a random fashion using conventional amine coupling chemistry methods. Rather, we Protein determination have used a more standardized method by generating a form of

The absorption coefficient for each protein calculated at A280 was used to CR2 SCR1-4 that, following addition of biotin using purified Bir determine protein concentrations. A enzyme to the C-terminal BCCP protein (46), could be properly Statistical analyses oriented on SA-coated chips. The ligands C3d and C3dg-biotin were expressed in E. coli and Analyses were performed using Student’s t test. purified as described previously (6, 48). EBV-gp350 was also ex- Results pressed and purified using an established method (50). Expression Generation and characterization of recombinant proteins and purification of the lectin-like domain of CD23 is detailed in Binding sites for the ligands C3d, gp350, and CD23 exist within Materials and Methods. the SCR1-2 domain of CR2. To study these CR2-ligand interactions in comparison to IFN-␣, we used two previously described Identification of optimal pH and NaCl concentration for ligand- forms of soluble recombinant CR2 containing SCRs 1-2 and 1-15 binding experiments (Fig. 1), each of which interacts with C3d and gp350 at high affinity (48). In addition, for this current study, we have created a We first sought to determine whether CR2 would bind to IFN-␣ by new form of soluble recombinant CR2 containing SCR1-4 (Fig. 1), ELISA and to then characterize the optimal binding conditions for both with (CR2 SCR1-4-BCCP) and without (CR2 SCR1-4) a bi- further studies. This strategy follows prior experiments in which otinylation site at the C terminus. We reasoned that the addition of ELISA has been used to characterize the binding of CR2 to C3d the SCR3-4 domain would allow us to detect binding of this pro- and gp350, and also the finding that optimal binding to C3d occurs tein to ligands with the nonligand-blocking mAb HB-5, which re- at neutral pH and a 50 mM NaCl concentration (46, 48). Fig. 3 acts with the SCR3-4 domain. In addition, the incorporation of a demonstrates the results of these analyses. First, CR2 SCR1-15 biotinylation site at the C terminus would allow us to reproducibly does clearly bind to IFN-␣ by ELISA (see also Fig. 4A for BSA orient the ligand binding domain of CR2 to a SA chip, as described specificity control). In addition, optimal binding conditions ap- previously (46). peared to be very similar to those for C3d, with a decrease in We used the mammalian cells NSO and HEK 293 to express apparent affinity below a pH of 6.5 and above 50 mM NaCl. Be- CR2 SCR1-4 and CR2 SCR1-4-BCCP, respectively, and proteins cause of that, and previous studies in which comparisons were were purified using mAb HB-5-Sepharose affinity column. Fig. 2 done under neutral pH and low salt conditions (46, 48), all addi- demonstrates these proteins. Although there are two bands present, tional experiments were performed in PBS (pH 7.4) and 50

34/35 Kd for CR2 SCR1-4 and 40/41 Kd for CR2 SCR1-4-BCCP, mM NaCl. 388 CR2/CD21 AS AN IFNAR

FIGURE 3. pH (left) and NaCl (right) dependent binding of soluble CR2 SCR1-15 to plate-bound IFN-␣ by ELISA. Binding was measured using anti-CR2 mAb HB-5 that will bind to CR2 SCR3-4. Mean Ϯ SEM for three experiments is shown.

Specific and dose-dependent binding of CR2 to IFN-␣, CD23, In the first experiments, we examined the interaction of hu- C3d, and gp350 man IFN-␣ as a solution phase analyte to the immobilized CR2 Fig. 4 demonstrates the results of experiments in which each of the SCR1-4-BCCP on a SA chip. We found that the binding reac- four ligands, or BSA as a control, is first bound to an ELISA plate. tion was dose-dependent but not saturable at the highest tested Subsequently, decreasing doses of CR2 SCR1-15 are added, fol- concentration of 2000 nM. Similarly, using the same chip, we studied CD23, C3d, and gp350 binding. We found that CD23 lowed by washing and detection of bound CR2 by anti-CR2 mAb Downloaded from HB-5. These results demonstrate a remarkable consistency in binding was dose dependent but, similarly to IFN-␣, not satu- which highly specific interactions and dose-dependent interactions rable at the highest tested concentration of 4000 nM. C3d and are readily detected with each of the four ligands, including IFN-␣. gp350 demonstrated dose-dependent binding that was saturable at the highest tested concentration of 125 nM. All ligands dem- Kinetic analysis of the interaction of CR2 SCR1-4 with human onstrated a comparable affinity for CR2 SCR1-4-BCCP, be- IFN-␣, CD23, C3d, and gp350 by surface plasmon resonance tween 263 nM and 467 nM. http://www.jimmunol.org/ In this study, we have examined the interaction of CR2 with hu- We used BIAevaluation software version 3.1 to evaluate man IFN-␣, CD23, C3d, and gp350 by surface plasmon resonance whether these ligand-receptor pairs exhibited simple 1:1 bind- technology using a BIAcore 3000 to compare the binding kinetics ing or whether more than one binding site could be detected. of human IFN-␣, C3d, gp350, and CD23 for surface-attached CR2 The data analysis revealed that the binding reaction of the ex- SCR1-4. In these experiments, C-terminal biotin-tagged CR2 perimental curve (gray color) does not fit well to a simple 1:1 SCR1-4-BCCP on a SA chip was studied with the ligands human Langmuir binding model but rather fits to a two-site binding IFN-␣, CD23, C3d, and gp350 as solution phase analytes. For each model (solid lines). These analyses suggest the presence of one of the ligands, the Ka,Kd, and KD were measured and calculated high-affinity binding site and a second much lower (2–3 logs or (Fig. 5, A–D, and Table I). greater difference) affinity binding site in the CR2 SCR1-4 by guest on September 26, 2021

FIGURE 4. Dose-dependent binding of CR2 SCR1-15 to its plate-bound ligands IFN-␣ (A), CD23 (B), C3d (C), and gp350 (D) by ELISA. Binding was measured using anti-CR2 mAb HB-5 that will bind to CR2 SCR3-4. Mean Ϯ SEM for three experiments is shown. The Journal of Immunology 389 Downloaded from http://www.jimmunol.org/

FIGURE 5. Surface plasmon resonance sensograms demonstrating the association and dissociation of IFN-␣ (A), CD23 (B), C3d (C), and gp350 (D)to by guest on September 26, 2021 the immobilized CR2 SCR1-4-BCCP. Nanomolar concentrations of injected analyte are indicated at the right hand side of each sensogram. Solid lines are the result of global ﬁtting analysis, and the gray lines are the normalized experimental sensogram kinetic data.

domain. For this interaction, we report only the higher affinity was injected as the solution phase analyte, dose-dependent and ␹2 binding site KD values (Table I). values are the goodness of saturable binding was observed. Similar dose-dependent binding fit parameter describing how precisely the experimental curve interactions were seen with both CR2 SCR1-4 and CR2 SCR1-15 fits to the proposed binding model. The ␹2 values range from for each of the immobilized ligands, including IFN-␣. However,

0.46 to 3.0 (Table I) and show that a two-site binding model fits some differences are present in the Ka and Kd when the ligand is well to the experimental data. bound by amine coupling chemistry to the CM5 chip as compared To determine what domain of CR2 is capable of binding to with when CR2 SCR1-4-BCCP-biotin is bound to the SA-coated IFN-␣ as well as confirm that the interaction occurs in the reverse chip. Notably, in this analysis C3d, gp350, and IFN-␣ were found orientation during surface plasmon resonance analysis, we used to bind to the solution phase analyte CR2 SCR1-2 at higher affin- soluble CR2 SCR1-2, SCR1-4, and SCR1-15 with chip-bound ities when compared with CR2 SCR1-4 and CR2 SCR1-15. In ␣ IFN- , again in direct comparison to other ligands. Fig. 6 and general, in the case of CR2 SCR1-4 and CR2 SCR1-15, the KD Table II demonstrate the results of these studies using each of the decreases as compared with SCR1-2. In contrast, however, CD23 four chip-bound ligands. When the CR2 SCR1-2 domain that con- demonstrates a comparatively lower affinity when binding with tains known binding sites for the three well-characterized ligands CR2 SCR1-2 and CR2 SCR1-4 as compared with CR2 SCR1-15.

Table I. Kinetic values for the interaction of CR2 with its ligands IFN-␣, CD23, C3d, and EBV-gp350

a b c ␹2d Ligand Analyte kd1(1/s)/ka1(1/M s) KD1 CR2 SCR1–4 biotin IFN-␣ 8.9 ϫ 10Ϫ3/2.1 ϫ 104 0.423 ␮M 0.46 CR2 SCR1–4 biotin CD23 7.9 ϫ 10Ϫ2/3.0 ϫ 104 0.263 ␮M 0.12 CR2 SCR1–4 biotin C3d 6.6 ϫ 10Ϫ2/2.1 ϫ 105 0.314 ␮M 3.0 CR2 SCR1–4 biotin EBV-gp350 3.6 ϫ 10Ϫ2/7.7 ϫ 104 0.467 ␮M 2.7

a Experiments performed at 50 mM NaCl (average values represents from 3–4 determinations). b Ka, on rate; kd, off rate. c Equilibrium dissociation constant (kd/ka). d ␹2 goodness-of-ﬁt parameter. 390 CR2/CD21 AS AN IFNAR Downloaded from http://www.jimmunol.org/

FIGURE 6. Surface plasmon resonance sensograms demonstrating the association and dissociation of CR2 SCR1-2 (A–D), SCR1-4 (E–H), and SCR1-15 (I–L) to immobilized IFN- ␣ (A, E, and I), CD23 (B, F, and J), C3d (C, G, and K), and gp350 (D, H, and L). Nanomolar concentrations of injected analyte by guest on September 26, 2021 are indicated at the right hand side of each sensogram. Solid lines are the result of global ﬁtting analysis, and the gray lines are the normalized experimental sensogram kinetic data.

This is likely due to the presence of an additional carbohydrate- SCR1-4, and SCR1-15, respectively. For this interaction (Table depending binding site in CR2 SCR5-8 (4). II), we report only the higher affinity binding site KD values. Similarly to the results obtained using CR2 SCR1-4-BCCP ␣ coupled to the SA chip, analysis of binding data for all three Mapping of IFN- ligand interactions using informative anti- CR2 proteins to the immobilized ligands did not fit into simple CR2 mAbs 1:1 Langmuir binding model but rather fit better to a two-site Previous studies using inhibitory mAbs as well as cross-competi- model. ␹2 values for this proposed binding model fit well, with tion of ligands have demonstrated that the binding sites for C3d, a range of 0.33–4.0, 0.15–10.0, and 0.25–3.5 for CR2 SCR1-2, gp350, and CD23 overlap within the SCR1-2 domain (4, 53, 55).

Table II. Kinetic values for the interaction of CR2 SCR1–2, SCR1–4, and SCR1–15 with its ligands IFN-␣, CD23, C3d, and EBV-gp350

a b c ␹2d Ligand Analyte kd1(1/s)/ka1(1/M s) KD1 IFN-␣ CR2 SCR1–2 1.2 ϫ 10Ϫ1/2.9 ϫ 105 0.042 ␮M 0.33 CD23 CR2 SCR1–2 3.3 ϫ 10Ϫ2/2.1 ϫ 103 15.71 ␮M 4.0 C3d-biotin CR2 SCR1–2 6.1 ϫ 10Ϫ2/3.4 ϫ 105 0.179 ␮M 1.9 EBV-gp350 CR2 SCR1–2 0.171/2.2 ϫ 105 0.077 ␮M 0.21 IFN-␣ CR2 SCR1–4 0.3/3.6 ϫ 105 0.833 ␮M 0.15 CD23 CR2 SCR1–4 3.6 ϫ 10Ϫ3/2.6 ϫ 104 0.138 ␮M 1.8 C3d-biotin CR2 SCR1–4 5.0 ϫ 10Ϫ2/8.1 ϫ 104 0.617 ␮M 10.0 EBV-gp350 CR2 SCR1–4 0.462/3.1 ϫ 106 0.149 ␮M 0.31 IFN-␣ CR2 SCR1–15 0.179/4.4 ϫ 103 41.6 ␮M 0.25 CD23 CR2 SCR1–15 3.3 ϫ 10Ϫ3/4.3 ϫ 104 0.076 ␮M 3.5 C3d-biotin CR2 SCR1–15 3.5 ϫ 10Ϫ1/1.6 ϫ 105 2.2 ␮M 3.1 EBV-gp350 CR2 SCR1–15 0.155/1.4 ϫ 104 11.1 ␮M 2.75

a Experiments performed at 50 mM NaCl (average values represents from 3–4 determinations). b Ka, on rate; kd, off rate. c Equilibrium dissociation constant (kd/ka). d ␹2 goodness-of-ﬁt parameter. The Journal of Immunology 391 Downloaded from http://www.jimmunol.org/

FIGURE 7. Competition ELISA measuring relative ability of anti-CR2 mAbs to block soluble CR2 SCR1-15 binding to plate-bound ligands IFN-␣ (A), by guest on September 26, 2021 CD23 (B), C3d (C), and gp350 (D). Binding was measured using anti-CR2 mAb HB-5 that will bind to CR2 SCR3-4 and not compete with any of these four mAbs (52). Results demonstrate that mAbs 171, 1048, 994, and 629 are inhibitory in the same relative rank order for each ligand. E, Cross-competition ELISA measuring relative ability of CD23, C3d, and gp350 to block CR2 SCR1-15 interactions with plate bound IFN-␣. Mean of three representative experiments.

In the next set of experiments, we determined whether a set of of relative inhibition. These results suggest that each ligand binds anti-CR2 mAbs generated to and reactive with the SCR1-2 domain to a closely related site on CR2 SCR1-2. that we had previously created (53) would inhibit the binding of IFN-␣ to CR2. As shown in Fig. 7, a very similar pattern of inhibition is found when comparing the relative ability of mAbs to Cross-competition analysis block IFN-␣ binding to CR2 as found when comparing them to Previous studies have shown that IFN-␣ may serve to directly C3d (mAb 171Ͼ1048Ͼ994Ͼ629). Importantly, the relative level block EBV infection of B cells through receptor competition. Our of inhibition for these four mAbs is identical with that we have results have suggested that IFN-␣ may as well directly block the previously reported for C3d and gp350 (53). We now show that the binding of other ligands to CR2. To determine whether IFN-␣ other two ligands, CD23 and IFN-␣, demonstrate a similar pattern directly blocks CR2-ligand interaction, cross-competition binding

FIGURE 8. IFIG response is partially inhibited by monoclonal anti-CR2 Ab. Varying concentrations (0.1 ␮g/ml, 1 ␮g/ml, 5 ␮g/ml) of anti-CR2 mAb 171 or mouse isotype control IgG2a were preincubated with human B cells for 1 h, then rhIFN-␣ (500 U/ml) was added to the cells. After6hofculture, RE of two IFIGs (MX1 and PRKR) was determined. Results are expressed as percentage of inhibition compared with expression induced by rhIFN-␣. .p Ͻ 0.05 ,ء .Mean Ϯ SEM for three experiments is shown 392 CR2/CD21 AS AN IFNAR analyses were performed using IFN-␣-coated wells and determin- domain in solution, because the binding of C3d and gp350 to ing the effects on the binding of CR2 by adding increasing amounts SCR1-2 (48), as well as CD23 to this domain (4), have been di- of CD23, C3d, and gp350 in solution. Fig. 7E demonstrates the rectly shown to be independent of CR2 SCR1-2 glycosylation. results of this analysis and reveals a rank order of competition of Previous studies using inhibitory mAbs as well as cross-com- gp350ϾC3dϾCD23. This likely reflects the relationships between petition of ligands have demonstrated that the binding sites for ligand binding sites, although we cannot rule out a contribution to C3d, gp350, and CD23 overlap within the SCR1-2 domain (4, 53, the results by the relative differences in affinities. Nevertheless, the 55). In this study, we have evaluated whether a set of anti-CR2 results are consistent with the presence of closely related binding mAbs generated to and reactive with the SCR1-2 domain that we sites for each ligand. had previously created (53) would inhibit the binding of IFN-␣ to CR2. As shown in Fig. 7, a very similar pattern of inhibition is Identification of IFIG partially inhibited by anti-CR2 mAb 171 found when comparing the relative ability of mAbs to block IFN-␣ Previous studies have reported inhibition of EBV binding to B binding to CR2 as found when comparing them to C3d (mAb cells by IFN-␣, and we have demonstrated in vitro binding in 171Ͼ1048Ͼ994Ͼ629). There is also direct inhibition of the bind- studies shown above. To determine whether there were biologic ing of IFN-␣ to CR2 shown by the other ligands. These results, in consequences of the interaction with CR2 on IFIG expression, we conjunction with the observation that CR2 SCR1-2 alone binds used purified B cells and determined using real-time PCR analysis IFN-␣ in surface plasmon resonance studies, are most consistent the effects of anti-CR2 mAb 171 on expression of IFIG MX1 and with the binding site for IFN-␣ lying within SCR1-2. However, PRKR. We found that both were partially inhibited in a dose- additional sites of interaction are not ruled out by our studies. dependent manner by anti-CR2 mAb in comparison to control Notably, Delcayre et al. (43) reported that mAb HB-5, which in- Downloaded from IgG2a mAb (Fig. 8). Thus, a component of the IFIG response of B teracts with SCRs 3-4, but not SCR1-2-reactive mAb OKB7 cells to IFN-␣ is due to the interaction of this cytokine with CR2. blocked IFN-␣ binding to B cells. Therefore, we cannot rule out an important contribution to IFN-␣ binding by regions of CR2 outside Discussion of SCR1-2. In this study, we have shown that IFN-␣ binds to CR2 with an IFN-␣ is a member of the type I family of IFNs that are secreted

affinity that is comparable to three other well-characterized ligands by fibroblasts and lymphocytes, as well as in large quantities by http://www.jimmunol.org/ for this receptor studied in parallel and that B cells respond to this immature plasmacytoid dendritic cells (reviewed in Ref. 56). The by increases in IFIG expression. Each of the four ligands studied type I family consists of structurally homologous cytokines that interacts with CR2 when either in the fluid phase or when chip- are involved in the control of viral pathogenesis and cancer, and bound. Based on the ability of SCR1-2 to bind IFN-␣ in surface exhibit both antiviral and antiproliferative effects on cells. Cur- plasmon resonance analysis as well as inhibition experiments with rently, IFN-␣ is also used for treatment of several diseases such as informative mAbs using the full-length CR2 SCR1-15 molecule, it hepatitis C and various types of lymphoid cancers (57). appears that IFN-␣ interacts with CR2 within the same SCR1-2 One well-described complication of the therapeutic administra- domain as the other three ligands. As part of these studies, we have tion of IFN-␣ is the development of autoimmunity, especially lu- also created a novel form of CR2, CR2 SCR1-4-BCCP, that can be pus-like autoantibodies (58). SLE is a chronic autoimmune disease by guest on September 26, 2021 readily biotinylated, linked to SA-coated chips, and used in surface characterized by autoantibodies, the formation of immune com- plasmon resonance studies in an orientation that mimics the in vivo plexes, and target organ injury, as well as increased levels of situation. IFN-␣ (59) and an IFN-␣ signature in peripheral blood mRNA (41, We found that human IFN-␣ binds in a dose-dependent manner 54). Plasmacytoid dendritic cells through the secretion of IFN-␣ to surface-attached CR2 SCR1-4-BCCP as well as to CR2 can promote the differentiation of B cells into plasma cells in an SCR1-2, SCR1-4, and SCR1-15 in solution when IFN- ␣ is chip- IL-6-dependent manner (60). In addition, IFN-␣ itself can lead to bound. A slow Ka as well as Kd was observed as compared with the activation of B cells in vitro in a mixed PBMC population (61). C3d and gp350, and the association and dissociation characteristics IFN-␣ can also both promote the development of B cells in the were observed to be more similar to CD23. Identically to C3d, bone marrow as well as enhance BCR-dependent activation events gp350, and CD23, analysis of the binding data did not fit well to such as calcium flux, proliferation, and induction of activation a simple 1:1 Langmuir binding model but rather are more consis- markers (62). tent with a two-site binding interaction. However, the high-affinity Inappropriate expression of IFN-␣ from plasmacytoid dendritic site is several logs greater than the lower and thus provides the cells may play a key role in breaking tolerance in SLE (56, 58). In greatest impact on the receptor-ligand interaction. contrast, whereas the majority of studies suggest that IFN-␣ pro- One interesting but unanticipated aspect of the results is the motes the development of lupus-like autoimmunity, some caution relative decrease in the apparent affinity of CR2 for IFN-␣ as well is necessary because MRL/lpr mice are protected from the devel- as C3d and gp350 as the length of the molecule is increased from opment of autoimmunity and target organ injury by type I IFNs (63). SCR1-2 to SCR1-4 and then SCR1-15. This is in contrast to the The structure of IFN-␣ family members and their receptor com- effects on the apparent affinity for CD23 as the length is increased. ponents has been determined by x-ray crystallography and NMR ␣ We believe this result is due to the presence of a flexible proximal spectroscopy (reviewed in Ref. 64). For example, human IFN- 2b SCR domain structure in solution, a situation that allows the two consists of an ␣ helical structure with a high degree of similarity dispersed binding sites on CD23 in SCR1-2 and SCR5-8 (4) to to other type I IFNs (65). Type I IFNs including IFN-␣ shared a come together, but one that also partially obstructs SCR1-2 inter- common receptor that consists of two subunits, IFNAR1 and IF- actions with the ligands that do not use these proximal SCRs (data NAR2, which associate with each other following ligand binding not shown). This is the subject of ongoing analyses. An alternate (66). IFNAR2 exhibits high-affinity binding of type I IFNs with a ϳ ϳ explanation we have considered is that the effect is due to differ- KD of 3 nM, which increases 20-fold following association of ences in glycosylation of the CR2 proteins, because SCR1-2 is IFNAR2 into a ternary complex with ligand, whereas the affinity of Ͼ deglycosylated before use and CR2 SCR1-4 and CR2 SCR1-15 are IFNAR1 alone is lower ( 100 nM KD) (66). not. However, we are unaware of any known effects of differences A reasonable question that is raised by our studies is what is the in glycosylation on direct binding of ligands to the CR2 SCR1-2 biologic role of this IFN-␣ interaction with CR2. There are several The Journal of Immunology 393 possibilities. First, based on our results demonstrating an effect of 3. Aubry, J. P., S. Pochon, P. Graber, K. U. Jansen, and J. Y. Bonnefoy. 1992. CD21 anti-CR2 mAb on the IFIG response, at least a portion of this is a ligand for CD23 and regulates IgE production. Nature 358: 505–507. 4. Aubry, J. P., S. Pochon, J. F. Gauchat, A. Nueda-Marin, V. M. Holers, P. Graber, response is due to CR2, and it is possible that some genes are C. Siegfried, and J. Y. Bonnefoy. 1994. CD23 interacts with a new functional induced in B cells by IFN-␣ solely or preferentially through CR2. extracytoplasmic domain involving N-linked oligosaccharides on CD21. J. Im- It is relevant to point out that several IFN-␣-dependent activation munol. 152: 5806–5813. 5. Holers, V. M. 2001. Complement. In Principles and Practices of Clinical Im- events in primary human B cells have not been completely inhib- munology. R. Rich, ed. Mosby, St. Louis, pp. 21.1–21.8. ited when cells are treated with an inhibitory anti-IFNAR-1 mAb 6. Szakonyi, G., J. M. Guthridge, D. Li, K. Young, V. M. Holers, and X. S. Chen. ␣ 2001. Structure of complement receptor 2 in complex with its C3d ligand. Science (61), which is consistent with our results. Second, IFN- binding 292: 1725–1728. may impart a BCR-independent signal through CR2 in a similar 7. Prota, A. E., D. R. Sage, T. Stehle, and J. D. Fingeroth. 2002. The crystal struc- fashion as previously described for other ligands, where homotypic ture of human CD21: implications for Epstein-Barr virus and C3d binding. Proc. ␬ Natl. Acad. Sci. USA 99: 10641–10646. adhesion (18), NF- B activation (19), IL-6 generation (20), and 8. Morikis, D., and J. D. Lambris. 2004. The electrostatic nature of the C3d-com- actin polymerization (23) have been described as potential out- plement receptor 2 association. J. Immunol. 172: 7537–7547. comes. Third, IFN-␣ may block the effects of CD23 as it binds to 9. Gilbert, H. E., J. T. Eaton, J. P. Hannan, V. M. Holers, and S. J. Perkins. 2005. Solution structure of the complex between CR2 SCR1–2 and C3d of human CR2 on B cells and promotes isotype switching to IgE (3). Fourth, complement: an X-ray scattering and sedimentation modeling study. J. Mol. Biol. as previously suggested, IFN-␣ may serve to directly block EBV 346: 859–873. infection of B cells through receptor competition (43), or may 10. Hannan, J. P., K. A. Young, J. M. Guthridge, R. Asokan, G. Szakonyi, X. S. Chen, and V. M. Holers. 2005. Mutational analysis of the complement block the binding of other ligands to CR2. Consistent with this receptor type 2 (CR2/CD21)-C3d interactions reveals a putative charged SCR1 possibility, experiments using ELISA have shown that IFN-␣ can binding site for C3d. J. Mol. Biol. 346: 845–858. block other ligands from interacting with CR2. And finally, al- 11. Carter, R. H., M. O. Spycher, Y. C. Ng, R. Hoffman, and D. T. Fearon. 1988. Downloaded from ␣ Synergistic interaction between complement receptor type 2 and membrane IgM though it is unlikely that IFN- would coligate CR2 with the BCR on B lymphocytes. J. Immunol. 141: 457–463. similarly to C3d-bound Ags, down-modulation of BCR-dependent 12. Kozono, Y., R. Abe, H. Kozono, R. G. Kelly, T. Azuma, and V. M. Holers. 1998. signals through sequestration of the CR2-associated molecule Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells. J. Immunol. 160: 1565–1572. CD19 (67) may occur and interfere with CD19-dependent BCR 13. Boackle, S. A., V. M. Holers, X. Chen, G. Szakonyi, D. R. Karp, E. K. Wakeland, signal amplification (68). Each of these possibilities is a testable and L. Morel. 2001. Cr2, a candidate gene in the murine Sle1c lupus suscepti- bility locus, encodes a dysfunctional protein. Immunity 15: 775–785. hypothesis for future experiments. http://www.jimmunol.org/ 14. Poe, J. C., M. Hagesawa, and T. F. Tedder. 2001. CD19, CD21, and CD22: An additional important question for further analysis is whether multifaceted response regulators of B lymphocyte signal transduction. Int. Rev. IFN-␣ binds to mouse CR2. This is relevant because, whereas Immunol. 20: 739–762. C3-derived ligands bind mouse and human CR2 comparably, 15. Lyubchenko, T., J. dal Porto, J. C. Cambier, and V. M. Holers. 2005. Co-ligation of the B cell receptor with complement receptor type 2 (CR2/CD21) using its gp350 does not bind mouse CR2 (54, 69), and mouse CD23 has natural ligand C3dg: activation without engagement of an inhibitory signaling never been convincingly shown to bind mouse CR2. Thus, whether pathway. J. Immunol. 174: 3264–3272. IFN-␣ is similar to the former or latter situation is important to 16. Fearon, D. T., and R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13: resolve. 127–149. Finally, these data again point to intriguing relationships be- 17. Tedder, T. F., L. J. Zhou, and P. Engel. 1994. The CD19/CD21 signal transduc- tween CR2 and SLE. As noted above, decreased levels of CR2 tion complex of B lymphocytes. Immunol. Today 15: 437–442. by guest on September 26, 2021 18. Bradbury, L. E., G. S. Kansas, S. Levy, R. L. Evans, and T. F. Tedder. 1992. The appear to promote loss of tolerance in this disease. In addition, CD19/CD21 signal transducing complex of human B lymphocytes includes the prior infection with EBV, another CR2 ligand, has been closely target of antiproliferative antibody-1 and Leu-13 molecules. J. Immunol. 149: 2841–2850. linked with the development of SLE (70), and increased serum 19. Sugano, N., W. Chen, M. L. Roberts, and N. R. Cooper. 1997. Epstein-Barr virus levels of EBV have been identified in patients with SLE (71). We binding to CD21 activates the initial viral promoter via NF-kB induction. J. Exp. now confirm in this study that IFN-␣, also associated with SLE, is Med. 186: 731–737. 20. D’Addario, M., T. A. Libermann, J. Xu, A. Ahmad, and J. Menezes. 2001. Ep- another ligand for CR2 and that a portion of the IFIG response that stein-Barr virus and its glycoprotein-350 upregulate IL-6 in human B-lympho- is linked to SLE is due to CR2 interactions. In toto, these obser- cytes via CD21, involving activation of NF-␬B and different signaling pathways. vations continue to support the hypothesis that CR2 is a key con- J. Mol. Biol. 308: 501–514. 21. Boackle, S. A., M. A. Morris, V. M. Holers, and D. R. Karp. 1998. Complement tributor to the pathogenesis of SLE, potentially through several opsonization is required for presentation of immune complexes by resting pe- molecular and immune mechanisms. ripheral blood B cells. J. Immunol. 161: 6537–6543. 22. Cherukuru, A., P. C. Cheng, and S. K. Pierce. 2001. The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens. Acknowledgments J. Immunol. 167: 163–172. We thank Dr. John Lambris (Department of Pathology and Laboratory 23. Melamed, I., L. Stein, and C. M. Roifman. 1994. Epstein-Barr virus induces actin Medicine, University of Pennsylvania, Philadelphia, PA) for providing the polymerization in human B cells. J. Immunol. 158: 1998–2003. Bir A-expressing clone. We thank Dr. Paul J. Cachia (Manager, Biophysics 24. Bonnefoy, J. Y., S. Henchoz, D. Hardie, M. J. Holder, and J. Gordon. 1993. A Core Facility, University of Colorado Health Sciences Center (UCHSC), subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis. Eur. J. Immunol. 23: 969–972. Denver CO) for suggestions during initial BIAcore data analysis; Dr. 25. Grosjean, I., A. Lachaux, C. Bella, J. P. Aubry, J. Y. Bonnefoy, and D. Kaiserlian. Joshua M. Thurman (Division of Nephrology and Hypertension, UCHSC) 1994. CD23/CD21 interaction is required for presentation of soluble protein an- for providing control IgG1 anti-factor B mAb; and Dr. Liudmila Kulik tigen by lymphoblastoid B cell lines to specific CD4ϩ T cell clones. Eur. J. Im- (Division of Rheumatology, UCHSC) for providing C3dg-biotin. CR2 and munol. 24: 2982–2986. 26. Bjork, P., C. Elenstrom-Magnusson, A. Rosen, E. Severinson, and S. Paulie. gp350 baculoviral clones were grown in the Tissue Culture Core of the 1993. CD23 and CD21 function as adhesion molecules in homotypic aggregation UCHSC Cancer Center. of human B lymphocytes. Eur. J. Immunol. 23: 1771–1775. 27. Molina, H., T. Kinoshita, K. Inoue, J.-C. Carel, and V. M. Holers. 1990. A Disclosures molecular and immunochemical characterization of mouse CR2: evidence for a The authors have no financial conflict of interest. single gene model of mouse complement receptors 1 and 2. J. Immunol. 145: 2974–2983. 28. Ahearn, J. M., M. B. Fischer, D. A. Croix, S. Georg, M. Ma, J. Xia, X. Zhou, References R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the Cr2 1. Iida, K., L. Nadler, and V. Nussenzweig. 1983. 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