EBV receptor)

inm nology letters ELSEVIER

Immunology Letters 54 (1996) 201-204

Membrane and soluble forms of CD21 (the C3dg/EBV receptor) V6ronique Fr6meaux-Bacchi, Elizabeth Fischer, Michel D. Kazatchkine* I N S E R M U430 and Universit~ Pierre et Marie Curie, H6pital Broussais, 96 rue Didot, 75014 Paris. France

1. Introduction Human CD21 (complement receptor type 2 (CR2)) is a single chain 145 kD glycoprotein that binds the surface-fixed cleavage fragments of C3, iC3b/C3dg/C3d and serves as the receptor for Epstein-Barr virus (EBV) on B-lymphocytes [1-3]. CD21 was also shown to bind an additional ligand, CD23, which exists as a membrane receptor on hematopoietic cells and in a soluble form in serum [4]. A soluble CD21 molecule retaining the ligand binding properties of the membrane form of the receptor has been recently characterized [5]. The capacity of membrane and soluble CD21 to interact with its counter-receptor CD23 opens the prospect of extended immunoregulatory functions for the CD21 molecule.

2. Structural properties of CD21 The,CD21 coding sequence spans 35 kb within the RCA gene cluster located on chromosome 1 [3]. A functional CD21 promoter region containing sequences with significant similarity to TATA, SP-I, AP-2, AP-1like sites has been cloned. Four regions of homology have been described between the CD21 promoter sequences and those of the human CD23 promoter [6]. CD21 is comprised of a 954 aa extracellular domain, a 24 aa transmembrane domain and a 34 aa cytoplasmic domain. The extracellular domain is entirely composed of 15 or 16 tandemly repeated SCR sequences which are homologous to those described in other C3/C4binding proteins. Each SCR of CD21 contains invariant residues involved in a triple-loop conformation. The presence of 11 Asn-X-Ser/Thr sites for potential glycosylation was determined within the extracellular domain * Corresponding author. 0165-2478/96/$12.00 © 1996 Elsevier Science B.V. All rights reserved PII S01 6 5 - 2 4 7 8 ( 9 6 ) 0 2 6 7 3 - 9

of CD21 and biosynthetic studies have characterized a non-glycosylated precursor of CD21 of M r 111 kD and a mature glycosylated form of Mr 145 kD. The cytoplasmic domain of CD21 contains the sequence T S Q K that constitutes a potential protein kinase C substrate and the EAREVY sequence, a potential substrate for tyrosine kinase. Phosphorylation of CD21 has been observed following stimulation of tonsillar B-cells and of B-lymphoblastoid cells with P M A and following activation of peripheral Blymphocytes with anti-/t antibodies. CD21 has initially been described as the receptor for the C3dg fragment of C3. The binding site of C3 to CD21 has been mapped to residues 1205 1214 in the C3d region of the molecule. The binding site is less accessible in the native C3 molecule and in the C3b fragment so that the latter molecules exhibit a lower affinity for CD21 than C3bi, C3dg and C3d [7]. CD21 also serves as the receptor for EBV which binds to CD21 through its envelope glycoprotein gp350/220. By analysis of CD21 deletion mutants and CR1-CR2 chimeric genes transfected in COS cells, the two NH2terminal SCRs have been shown to be necessary and sufficient for the binding of both gp350/220 and C3dg [8]. Recent observations demonstrate that human T-cell lines and human thymocytes may be infected with EBV in vitro, which relates to the expression of CD21 by these cells [9]. An additional ligand for CD21 is CD23, a type II transmembrane protein expressed on a variety of hemopoietic cell types that serves as a low-affinity receptor for IgE. Fluorescent liposomes carrying CD23 were shown to interact specifically with CD21 on Bcells, some T-cells, follicular dendritic cells and with hamster kidney cells transfected with CD21 cDNA [4]. Binding of CD23 involves a lectin-sugar interaction with SCR5-8 and a protein-protein interaction with SCRI-2 of CD21 [10]. Human CD21 is expressed by B-lymphocytes and B-lymphoblastoid cell lines, human

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thymocytes [11,12] a fraction of human T-lymphocytes [13], certain leukemia T-cell lines, the pharyngeal and cervical epithelium and by follicular dendritic cells [1]. The level of expression of CD21 is developmentally regulated, it is highest on mature B-lymphocytes and on a subpopulation of immature blastic thymocytes [12]. Expression of CD21 on B- and T-lymphocytes is upregulated by infection of these cells with the lymphocytotropic viruses EBV and HTLV-1 [14].

3. Functions of membrane CD21

CD21 is involved in human B-cell activation and proliferation. Thus, EBV has been shown to induce proliferation of B-lymphocytes independently of T-cells and accessory cells. Co-ligation of CD21 with the B-cell antigen receptor amplify the capacity of mIgM to trigger proliferation and to induce the release of Ca 2+ from intracellular stores in B lymphocytes [15]. On the surface of B-lymphocytes, CD21 was found to form a complex with the membrane proteins CD19 [16] TAPA1 and Leu-13 [17]. CD19 behaves as the signal transducing molecule of the complex and upon cross-linking with the B-cell receptor is coupled via PTK to PLC and P13'kinase [18]. Co-ligation of the CD21/CD 19 complex with the antigen-receptor decreases the requirement for the number of mIgM molecules that need to be ligated to trigger B-cell activation. This may occur under physiological conditions if B-cells encounter C3d-opsonized antigens or CD23-expressing antigen presenting cells. Recently, secondary cellular responses mediated through interaction of CD21 with membrane and soluble CD23 have been evidenced. Thus, subset of antiCD21 monoclonal antibodies has been shown to behave in a similar fashion to soluble CD23 in decreasing the occurrence of apoptosis in germinal center B-cells [19]. The effect of soluble CD23 on IL-4-induced IgE production by blood mononuclear cells may be reproduced with anti-CD21 antibodies [4]. CD21/CD23 pairing also occurred between membrane forms of the receptors and were shown to be involved in homotypic adhesion of B-cells [20]. There is evidence that CD23CD21 interactions between T- and B-cells are required for presentation of soluble antigens to specific CD4 + T-clones and IgE production [21]. Although the function of CD21 on T-cells has not been fully established, we have shown that CD21 is partially associated to CR1 in the T-cell membrane and capable of inducing a Ca + + signal in the HPB-ALL T-cell line [22]. No report evidences that CD21 is a part of a preformed complex in T-cell although two proteins of 105/55 Mr were reported to be co-immunoprecipitated with CD21 using the anti-CD21 OKB7 mAb from T-cell lines lysates, suggesting that the complex associates following ligation of CD21 on the cell membrane [23].

4. Soluble CD21

Several surface proteins of lymphocytes are released in the extracellular environment and plasma by enzymatic cleavage. The presence of CD21 antigen has been reported in culture supernatants of lymphocytic cell lines, by using hemagglutination and solid-phase immunoassay [24,25]. Two forms of soluble CD21 of Mr 72 and 130 kDa have been described following affinitypurification of culture supernatants of biosyntheticallylabeled Raji cells and of a lymphoblastoid cell line, respectively [26,24]. We have recently characterized a soluble form of CD21 that is spontaneously released by B- and T-lymphocytes. Immunoprecipitation with antiCD21 mAbs of culture supernatants of surface-and biosynthetically-labeled B- and T-cell lines revealed a single band of apparent M r 135 kDa. The molecule exhibited an Mr that was 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) from B- and T-cells was time-dependent and correlated with a parallel decrease in the expression of the membrane associated molecule, indicating that sCD21 is generated by cleavage and shedding of the membrane receptor. The CD21 protein was also found in culture supernatants of tonsillar B-cells and normal human thymocytes [5]. Epitopic analysis using combinations of anti-CD21 mAbs indicated that sCD21 and membrane CD21 were similarly recognized by mAbs directed against SCR 1-2, SCR 4-5 and SCR 9-11 [5]. Soluble CD21 retained the ability to bind the ligands of the membrane form. Thus, we have observed that affinitypurified sCD21 bound purified human iC3b and human recombinant CD23 using an ELISA and using the BIAcore technology. In the latter experiments purified sCD21 exhibiting a single band of Mr 135 kDa upon SDS PAGE analysis and silver staining was immobilized indirectly to the sensor ship using anti-CD21 mAb. Fig. 1 depicts the kinetics of binding of CD23 and iC3b to and dissociation from sCD21-anti-CD21 complexes. The calculated Kd of CD23 and iC3b for sCD21 coupled to F97 anti-CD21 mAb at apparent equilibrium was 392 and 29 nM respectively. Consistent with data reported by others [24,25], we observed the presence of a soluble form of CD21 in normal human serum indicating that the shedding process occurs in vivo. The soluble form of CD21 in human serum exhibited a similar Mr to that of the molecule shed by B- and T-cells in culture and was recognized by all anti-CD21 mAbs that we tested. It is noteworthy that increased amounts of both CD21 and CD23 have been reported in sera in B-cell chronic lymphocytic leukaemia [27]. Significantly increased levels of CD21 were also found in EBV-related infection and malignancies that may be related to the ability of EBNA-2 to enhance membrane expression and shedding of soluble CD21 following transfection of the gene in B- and T-cell lines [28].

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Soluble forms of membrane receptors may exhibit immunoregulatory functions, such as the inhibition of interactions between membrane receptors and their ligands or ligand transporters. The infusion of soluble recombinant CD21 has been shown to suppress responses to T-cell dependent antigens in mice, suggesting a role for sCD21 in modulation of immune responses in vivo [29]. The ability of soluble CD21 to bind iC3b represents a potential mechanism by which the soluble receptor could participate in the transport of opsonized immune complexes. Upon gel filtration of normal human serum, we have detected sCD21 in two different elution peaks suggesting that the molecule is partially associated with other proteins in serum. Blotting experiments of sCD21 that had been affinity-purified from serum revealed the presence of CD23, C3, IgG and IgE, indicating that sCD21 circulates in serum through two different complexes. Soluble CD21 may be released in a complex with a molecule with which it is associated on the cell surface; alternatively, it may form new complexes with other proteins released from the cells or in serum.

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Time (seo) Fig. 1. BiacoreTM measurement of the binding of CD23 (A) and iC3b (B) to immobilized sCD21/anti-CD21-mAb complexes. The refractive index resulting from the binding of CD23 and iC3b to the immobilized complexes was expressed as resonance units (RU). The figure shows overlay plots of the sensorgrams obtained after the injection of CD23 (50 pg/ml) or iC3b (50/~g/ml) to sCD21 immobilized on F97 and BU32 anti-CD21-mAbs. No binding was observed when recombinant renine was injected using the same experimental conditions.

2113

Acknowledgements This work was supported by Institut National de la Sant6 et de la Recherche M6dicale (INSERM), Association de la recherche contre le Cancer (ARC), France.

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