Structural requirements for C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral infection.

The structure of CR2, the human C3d,g/EBV receptor (CR2/CD21) consists of 15 or 16 60-70 amino acid repeats called short consensus repeats (SCRs) followed by a transmembrane and a 34-amino acid intracytoplasmic domain. Functions of CR2 include binding the human complement component C3d,g when it is covalently attached to targets or cross-linked in the fluid phase. In addition, CR2 binds the Epstein-Barr virus (EBV) and mediates internalization of EBV and subsequent infection of cells. In order to explore functional roles of the repetitive extracytoplasmic SCR structure and the intracytoplasmic domain of CR2, we have created truncated CR2 (rCR2) mutants bearing serial deletions of extracytoplasmic SCRs and also the intracytoplasmic tail. We then stably transfected these rCR2 mutants into two cell lines, murine fibroblast L cells and human erythroleukemic K562 cells. Phenotypic analysis of these expressed mutants revealed that 1) The C3d,g- and EBV-binding sites are found in the two amino-terminal SCRs of CR2, 2) expression of SCRs 3 and 4 is further required for high affinity binding to soluble cross-linked C3d,g, 3) the intracytoplasmic domain of CR2 is not required for binding C3d,g or EBV but is necessary for internalization of cross-linked C3d,g as well as for EBV infection of cells, 4) monoclonal anti-CR2 antibodies with similar activities react with single widely separated epitopes, and 5) no functional roles can yet be clearly assigned to SCRs 5-15, as rCR2 mutants not containing these SCRs show no major differences from wild-type rCR2 in binding or internalizing cross-linked C3d,g or mediating EBV binding and infection.     

Analysis of epitope expression and the functional repertoire of recombinant complement receptor 2 (CR2/CD21) in mouse and human cells

We transfected human complement receptor 2 (CR2/CD21) cDNA containing eukaryotic expression constructs into CR2-negative mouse L cells and human K562 erythroleukemia cells. We subsequently selected stably transformed cells that expressed human CR2, as assessed by flow microfluorimetry analysis and immunoprecipitation of 125I-labeled surface membranes using the monoclonal anti-CR2 antibody, HB5. Utilizing flow microfluorimetry analysis, epitopes recognized by anti-CR2 mAb HB5, OKB7, B2, and four other anti-CR2 antibodies were detected on CR2 expressing transfectants but not parental cells. In addition, CR2 expressing transfected cells efficiently formed rosettes with sheep erythrocyte intermediates bearing human C3bi and C3d, but not C4b or C3b, consistent with the known ligand specificity of CR2. CR2 containing transfectants were also demonstrated to specifically bind EBV. Infection with EBV of CR2 expressing L cells and K562 cells resulted in mean expression of Epstein-Barr nuclear Ag (EBNA) at 48 h in 0.35% of CR2 expressing L cells and 3.7% of CR2 expressing K562 cells. Parental L cells and K562 cells did not express EBNA after EBV infection. These results indicate that CR2 alone is sufficient to transfer both C and EBV receptor functions to heterologous cells. In addition, expression of EBNA was found to be significantly higher in human K562 than mouse L cells, both expressing the same recombinant receptor. These results suggest that mechanisms other than CR2 binding lead to inefficient EBV infection and/or EBNA synthesis in mouse fibroblasts.     

Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.     

OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2

OKB7, an IgG2 mouse monoclonal antibody, binds to the C3d receptor (CR2) of human B lymphocytes in or near the active site of the receptor. OKB7 precipitates the same molecule as monoclonal antibodies B2 and HB-5 which have previously been reported to react with the C3d receptor at epitopes distant from the active site. The epitope recognized by OKB7 is distinct from those bound by B2 and HB-5 as shown in competitive binding experiments and is near the complement binding site since OKB7 blocks EAC3d rosetting at concentrations as low as 1 microgram/ml in the absence of cross-linking antibodies.     

Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2).

In pursuing studies on the early events in the infection of human B cells by Epstein-Barr virus (EBV), we examined the host cell attachment phase with a panel of B-cell-specific monoclonal antibodies. One of the monoclonal antibodies, OKB7, directly blocked the attachment of purified EBV to B lymphocytes in the absence of a second anti-immunoglobulin antibody and thereby prevented EBV infection of tonsil and peripheral blood B cells. Although earlier studies have shown a close association of the EBV and complement receptor (CR2), an anti-CR2 monoclonal antibody, anti-B2, did not directly block the binding of EBV to B cells. A comparison of the structures recognized by these monoclonal antibodies on various cell types and their functional and physiochemical properties was undertaken. Flow cytometric analysis revealed that the molecules detected by OKB7 and anti-B2 were coexpressed to the same extent on B cells but were not expressed on T-cell lines. OKB7 and anti-B2 both immunoprecipitated a 145,000-molecular-weight membrane protein with an isoelectric point of 8.2 from membrane extracts of Raji lymphoblastoid cells. OKB7 and, to a lesser extent, anti-B2 directly blocked the attachment of C3d,g-coated fluorescent microspheres and sheep erythrocytes bearing C3d to B cells, indicating that these antibodies also react with CR2. These studies indicate that the EBV-CR2 receptor is a single membrane glycoprotein which possesses multiple antigenic and functional epitopes.     

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.     

CR2 ligands modulate human B cell activation

A considerable body of evidence from this and other laboratories indicates that complement receptor type 2 (CR2) modulates B cell activation and growth. In the present studies we have examined the effects of three different types of CR2 ligands, i.e., monomeric, aggregated, and latex-bound C3dg; mAb to different CR2 epitopes; and UV-inactivated, non-transforming EBV (EBVUV) for their actions on highly purified, high density resting tonsil B cells. Although none of these ligands induced B cells to enter the cell cycle or synergized with either anti-mu or low m.w. B cell growth factor in triggering B cell mitogenesis, aggregated C3dg, latex-bound C3dg, the OKB7 anti-CR2 mAb, and EBVUV-enhanced thymidine incorporation by phorbol ester-activated tonsil B cells. Such enhancement was not T cell or monocyte dependent. The major action of the CR2 ligands thus seems to be to enhance the transition of B cells activated by certain stimuli from the G1 to the S phase of the cell cycle. In contrast to the action of aggregated and latex-bound C3dg, monomeric C3dg was inhibitory for phorbol ester and aggregated C3dg-induced B cell activation. The HB-5 anti-CR2 mAb, which reacts with a different epitope on CR2 from that of OKB7, did not synergize with PMA in B cell activation. These data provide additional evidence for a role for the CR2 in the control of B cell growth and provide a useful model for studying the CR2-mediated signals that affect the growth of B cells.     

Genomic analysis reveals a duplication of eight rather than seven short consensus repeats in primate CR1 and CR1L: evidence for an additional set shared between CR1 and CR2

We report the discovery of previously unrecognised short consensus repeats (SCRs) within human and chimpanzee CR1 and CR1L. Analysis of available genomic, protein and expression databases suggests that these are actually genomic remnants of SCRs previously reported in other complement control proteins (CCPs). Comparison with the nucleotide motifs of the 11 defined subfamilies of SCRs justifies the designation g-like because of the close similarity to the g subfamily found in CR2 and MCP. To date, we have identified five such SCRs in human and chimpanzee CR1, one in human and chimpanzee CR1L, but none in either rat or mouse Crry in keeping with the number of internal duplications of the long homologous repeat (LHR) found in CR1 and CR1L. In fact, at the genomic level, the ancestral LHR must have contained eight SCRs rather than seven as previously thought. Since g-like SCRs are found immediately downstream of d SCRs, we suggest that there must have been a functional dg set which has been retained by CR2 and MCP but which is degenerate in CR1 or CR1L. Interestingly, dg is also present in the CR2 component of mouse CR1. The degeneration of the g SCR must have occurred prior to the formation of primate CR1L and prior to the duplication events which resulted in primate CR1. In this context, the apparent conservation of g-like SCRs may be surprising and may suggest the existence of mechanisms unrelated to protein coding. These results provide examples of the many processes which have contributed to the evolution of the extensive repertoire of CCPs.     

Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.     

C3 synthetic peptides support growth of human CR2-positive lymphoblastoid B cells

The nature of CR type 2 (CR2)-ligand interactions which leads to the activation of human B cells was analyzed by using synthetic peptides and CR2-positive cell lines. The third component of C (C3) supported the growth of human lymphoblastoid B cells in serum-free medium containing human transferrin. This effect was inhibited by an antibody to C3d (mAb 130) which specifically inhibits C3d binding to CR2, but not by other anti-C3 mAb. Synthetic peptides corresponding to the CR2-binding site on C3d, P28 (residues 1187-1214) or multivalent P13 [1202-1214)4-template), supported the proliferative response of CR2-positive human lymphoblastoid lines in a similar way as C3 and this response could be inhibited by the anti-CR2 mAb OKB7. The proliferative response to C3 or peptides was dose dependent and a 60-fold higher concentration of P28 peptide was required to induce the same level of proliferation as C3. This stimulation of growth was observed only on CR2 expressing cell lines Raji and Daudi, and not on the CR2-negative Burkitt lymphoma cell line Rael and the monocytic cell line U937. In contrast to the stimulatory effect of P28 and P13-template, monomeric P14 (1201-1214) was not able to support the growth of these cell lines. This peptide, however, inhibited the proliferative response of the CR2-positive lines to C3, P28, and multivalent-P13, thus indicating that cross-linking of the CR2 receptor is necessary for B cell proliferation. Another peptide, E12 (from glycoprotein (GP)350, the major EBV outer membrane GP) which shows a high degree of similarity with P14, also inhibited the proliferative response of Raji cells, suggesting that this segment on GP350 is involved in the interaction of EBV with CR2. The possibility of using the above peptides as well as other peptides with "tailor-made" structure in studying the multifunctional role of C3 is discussed.     

Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions

The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.     

Genomic organization and polymorphisms of the human C3d/Epstein-Barr virus receptor

The human C3d/Epstein-Barr virus receptor (CR2/CD21) is a 145-kDa protein primarily expressed on mature B lymphocytes. CR2 is a member of the regulators of complement activation (RCA) gene family found on band q32 of chromosome 1. The RCA proteins are characterized by the presence of 60-70 amino acid short consensus repeats (SCR). A full length CR2 cDNA was cloned and used to identify overlapping cosmid genomic clones. Analysis of CR2 exon-intron junctions revealed the presence of three types of exons in the short consensus repeat region of CR2. First, four exons each of which encodes two SCR are present. Five exons encode a single SCR. Six exons encode SCRs which are split in identical positions. The order of these types of exons is in a repeated array of four SCRs, indicating that the contemporary CR2 gene likely evolved from a more primitive gene containing four SCRs. The CR2 full length cDNA clone was used to find restriction fragment length polymorphisms (RFLPs). Restriction enzyme TaqI generated 2.55- and 2.10-kilobase (kb) polymorphic bands. This RFLP was mapped near the exon containing the first two SCRs. HaeIII digestion generated polymorphic bands of 1.45, 1.55, and 1.75 kb. The HaeIII 1.45-kb RFLP band maps near the exon containing the 15th SCR. The TaqI and HaeIII RFLPs will provide tools for the genetic analysis of CR2. The organization of the CR2 gene provides insights into the evolution of human CR2 and the RCA gene family.     

Inhibition of Epstein-Barr virus infection in vitro and in vivo by soluble CR2 (CD21) containing two short consensus repeats.

The extracellular domain of CR2, the Epstein-Barr virus (EBV)/C3d receptor of B lymphocytes, contains 15 or 16 tandemly arranged short consensus repeat elements (SCR). Recombinant CR2 proteins containing SCR 1 and 2 fused to Staphylococcus aureus protein A (PA-CR2) and to murine complement factor H SCR 20 (CR2FH) were expressed in Escherichia coli and in insect cells, respectively. These recombinant CR2 molecules retained functional activity as indicated by their ability to bind to C3dg in an enzyme-linked immunosorbent assay and to inhibit EBV gp350/220 binding to B cells. PA-CR2 and CR2FH were as efficient in blocking EBV gp350/220 binding as the full-length CR2 extracellular domain, indicating that the first two SCR of CR2 contain the majority of the ligand binding activity of the receptor. PA-CR2 and CR2FH inhibited EBV-induced B-cell proliferation in vitro and blocked the development of EBV-induced lymphoproliferative disease in severe combined immunodeficient mice reconstituted with human lymphocytes. These studies indicate that soluble forms of truncated CR2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication.     

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

采用原位杂交及电镜检测CR2转染小鼠细胞的EB病毒感染

EB病毒不能感染小鼠是因为小鼠CR2受体构像与人的不同,通过对小鼠CR2受体进行定点空变,然后将野生型和突变型小鼠CR2／1（MCR2／1）及人CR2（hCR2）用基因转移技术导入小鼠鼻咽上皮细胞系（TMNE）进行表达,观察转染阳性细胞是否具有结合EB病毒的能力,EBER－1杂交结果显示,只有转染hCR2和空变型MCR2（mtMCR2)的TMNE细胞可以感染EB病毒,但是前感染EB病毒的阳性率比后高的高得多。电镜结果也进一步证实EB病毒可以感染这两种细胞,这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。     

Evolutionary relationships among proteins encoded by the regulator of complement activation gene cluster

Evolutionary relationships among members of the regulator of complement activation (RCA) gene cluster were analyzed using neighbor-joining and parsimony methods of phylogenetic tree inference. We investigated the structural and functional similarities among short consensus repeats (SCRs) of the following human proteins: the alpha chain of the C4b-binding protein (C4bpalpha), factor H (FH), factor H-related proteins (FHR-1 through FHR-4), complement receptors type 1 (CR1) and type 2 (CR2), the CR1-like protein (CR1L), membrane cofactor protein (MCP), decay accelerating factor (DAF), and the sand bass proteins, the cofactor protein (SBP1) and its homolog, the cofactor-related protein (SBCRP-1). Also included are the beta chain of the human C4b-binding protein (C4bpbeta) and the b subunit of human blood-clotting factor XIII (FXIIIb). Our results indicate that the human plasma complement regulators, FH and C4bpalpha, fall into two distinct groups on the basis of their sequence divergence. Homology among RCA proteins is in agreement with their chromosomal location, with the exception of C4bpbeta. The evolutionary relationships among individual short consensus repeats are confirmed by the exon/intron structure of the RCA members. Structural similarities among repeats of the RCA proteins correlate with their functional activities and demonstrate the importance of the N-terminal SCRs.     

Role of CR2 in the human adult and neonatal in vitro antibody response to type 4 pneumococcal polysaccharide.

A number of studies have indicated that the complement receptor type 2 (CR2), which is the receptor for C3d, a degradation fragment of the complement component C3, regulates B lymphocyte activation and growth. Early reports have described that C3 regulates T cell-dependent (TD) antibody responses. The involvement of CR2 in the antibody response to T cell-independent type 2(TI-2) antigens was investigated because neonatal B cells, which are unresponsive to TI-2 antigens both in vivo and in vitro, express a significantly decreased level of CR2 as compared to B cells of adult donors. We utilized type 4 pneumococcal polysaccharide (PS4) as a model TI-2 antigen. In order to study the relationship between CR2 and the response to PS4, B cells were costimulated with PS4 and monoclonal antibodies (MAb) to CR2. HB5 and OKB7 anti-CR2 monoclonal antibodies enhanced the in vitro response of adult B cells to PS4, as measured in a PS4-specific spot-forming cell assay. Neonatal B cells could only be induced to respond to PS4 using high concentrations of OKB7 anti-CR2 MAb. The 8-mercaptoguanosine (8MGuo), an agent that can overcome the in vitro unresponsiveness to PS4 of neonatal B cells, increased CR2 expression on adult and neonatal B cells. Furthermore, 8MGuo synergizes strongly with anti-CR2 antibodies in augmenting the anti-PS4 antibody response. Data presented in this report provide evidence of CR2 involvement in the antibody response to PS4 and that the neonatal B cell unresponsiveness to TI-2 antigens may be due to the decreased expression of CR2.