T cell activation induced by cross-linking CD3 and CD28 leads to silencing of Epstein-Barr virus/C3d receptor (CR2/CD21) gene and protein expression

Complement receptor II (CR2) also known as CD21 is the receptor for C3d on immune complexes. In humans it serves as a receptor for the Epstein-Barr virus (EBV). CR2 is expressed on B cells and in low density in the T cell lineage. EBV can infect T cells and EBV-positive T lymphomas have been described. Although CR2 mRNA is readily detectable in T cells, the function of CR2 in human T lymphocytes remains elusive. Here we have analyzed the expression of CR2 in normal and activated T cells. PCR analyses and immunofluorescence/confocal microscopy of peripheral blood T cells and of activated T cells shows considerable reduction in CR2 mRNA and protein expression upon activation. The downregulation of CR2 expression may modulate life span or immunological reactivity of T cells and the susceptibility of cells to infection by lymphotropic viruses.     

Epstein-Barr virus/C3d receptor (CR2, CD21) activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways

We previously demonstrated that human C3d or pep16, a 16-amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2-positive cells depleted in CR2 molecules by absorption with anti-CR2 antibodies or CR2-negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein-Barr virus capsid protein or OKB7, an anti-CR2 monoclonal antibody (mAb). HB5, another anti-CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB-5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non-activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2-CD19 complexes. Both pathways were TAPA-1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA-1.     

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor,CR2) that binds iC3b and CD23

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short concensus repeats (SCR) 1–2, SCR 4–5 and SCR 9–11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore^TM technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1–2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.     

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase,respectively

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.     

Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.     

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.     

gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4)

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.     

Complement receptor type two (CR2,CR21)

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.     

Expression of Epstein-Barr virus nuclear antigen-2 (EBNA2) induces CD21/CR2 on B and T cell lines and shedding of soluble CD21

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) expressed as a fusion protein with the hormone-binding domain of the estrogen receptor was used to study expression of CD21 and other surface markers in different cell lines. Special emphasis was placed on cell lines with a normally low expression of CD21, especially on T cell lines. After induction of EBNA2, a substantial increase in CD21 mRNA was observed, as well as increased production of membrane CD21. This was found not only in cell lines of B cell origin, but also in the T cell line Jurkat. The amount of CD21 was quantitated by means of a fluorescence immunoassay, and found to correlate with the presence of EBNA2 protein. A decrease in EBNA2 abundance was associated with complete loss of cell-associated CD21. As we could also detect large amounts of soluble CD21 (sCD21) in the supernatant of the transfected cell lines, which exceeded the total amount contained in the respective cell lysates, this indicates considerable shedding of the newly synthesized receptor molecules induced by EBNA2, comparable to the situation described for CD23. It further provides an explanation of the recent findings of increased sCD21 levels in sera of patients with EBV-associated disease, and suggests a possible additional function of EBNA2 in vivo.     

Activation of human platelets through gp140, the C3d/EBV receptor (CR2)

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab')2 fragments.     

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein-Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as-yet-unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon-11-encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21-pool. CD21 shedding is induced by stimulation with PMA plus Ca(2+) ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Immunopathology associated with Epstein-Barr virus (EBV) infection: Evidence for interactions with T-lymphocyte EBV receptor CD21

Epstein-Barr virus (EBV), also termed Human Herpes Virus 4, is the causative agent of infectious mononucleosis and may be a cofactor in some human cancers. The virus has also been suggested to play a role in human autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. X-linked lymphoproliferative syndrome caused by the deficiency of the Sarc homology 2 domain protein 1A, also termed signaling lymphocyte activation marker-associated protein, can result in immune dysfunction and death after EBV infection. The EBV-related immunopathology in X-linked lymphoproliferative syndrome and prototypical autoimmune syndromes is summarized in this review. A novel model of viral interaction with complement receptor CD21, which is also the receptor for EBV, is proposed to account for both the immunological abnormalities of X-linked lymphoproliferative syndrome and autoimmune diseases associated with EBV infection. The pathogenesis of both X-linked lymphoproliferative syndrome and EBV-associated immune diseases is proposed to result from increased direct infection of T cells by EBV through the T-cell complement receptor CD21 expressed on T cells. A prediction of this model is that therapy designed to decrease CD21-mediated EBV infection of T lymphocytes could also be beneficial in the treatment of some autoimmune diseases.     

The structural basis for complement receptor type 2 (CR2, CD21)-mediated alternative pathway activation of complement: studies with CR2 deletion mutants and vaccinia virus complement-control protein-CR2 chimeras

The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.     

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and immune response. Our recent analysis of this phenotype suggested that signaling through hCR2 and presumably mouse CD19 on the B cell surface, during bone marrow development, could result in the observed changes in B cell function in these mice. To test this hypothesis, we back crossed hCR2(high) transgenic mice onto the CD19(-/-) background. CD19(-/-)hCR2(high) mice were found to possess even fewer mature B cells than their CD19(+/+)hCR2(high) littermates, demonstrating that loss of CD19 exacerbated the effects elicited through hCR2. This data suggests that CD19 provides a survival signal during B cell development in this model. Next, we examined if the removal of the main ligand for CR2, namely C3d, through back-crossing onto the C3(-/-) background could restore normal B cell development. However, we found only minor recovery in peripheral B cell numbers and no obvious change in function. This was despite a three-fold increase in the level of hCR2 expression on B cells isolated from the spleen or bone marrow of C3(-/-)hCR2(high) mice when compared with C3 sufficient littermates. These data demonstrate that hCR2 is integrated in mouse B cell signaling and that the downstream effects of hCR2 expression during early B cell development are partially but not completely due to interaction with C3 fragments and signaling through CD19 in the bone marrow environment.     

CR1(CD35) and CR2(CD21) complement C3 receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein-Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1⁺ and CD1^? cells in the thymus. Two subsets of CR2⁺ thymocytes were defined expressing low and high density of the receptor. The CR2⁺⁺ subset represented 20% of CR2⁺ thymocytes and co-expressed the CR1 receptor. CR2⁺⁺ thymocytes expressed an immature CD1^dull, CD3^?, CD4^dull, CD8^?, CD7⁺⁺ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement-opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.     

Structure and functions of gp 140, the C3d/EBV receptor (CR2) of human B lymphocytes

Analysis of the interaction of human C3 fragments with human B lymphoma cell line, led us to isolate gp 140, the C3 receptor of Raji cells. Rabbit anti-gp 140 was prepared against this highly purified receptor. Using these polyclonal antibodies, it was found that: gp 140 is the C3d receptor (CR2) which reacts with the C3d site expressed on C3d, C3dg, C3bi and at a less extent on C3b. Gp 140 is a specific marker of human B lymphocytes; gp 140 is also the Epstein-Barr virus receptor (EBVR); CR2 is a membrane site involved in B-cell regulation; and the C3d/C3dg receptor (CR2) of human B lymphocytes is distinct to the C3dg receptor (CR4) of human neutrophils.     

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin''s disease is frequently associated with CR2 (EBV receptor) expression

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.