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Epstein-Barr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis.

The type 2 complement receptor, CR2, a B lymphocyte surface glycoprotein, is known to be a component of the EBV receptor. We now demonstrate that the major EBV outer membrane glycoprotein, gp350/220, is a highly specific ligand for CR2. EBV or beads coated with purified recombinant gp350/220 adsorb to normal B lymphocytes, cap with CR2, become endocytosed into vesicles, and are released into the cytoplasm. This is the first demonstration of herpesvirus glycoprotein-cell glycoprotein receptor interaction in viral adsorption and penetration. The capping of CR2 in response to virus, gp350/220-coated beads, or anti-CR2 monoclonal antibodies is associated with cocapping of surface immunoglobulin. Interaction between CR2 and surface immunoglobulin may be important in modulating the B cell activation that normally follows EBV infection or exposure to antigen.

Adsorption↗

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.

Amino Acid Sequence↗

Catalogue of Epstein-Barr virus (EBV) receptors on human malignant and non-malignant hematopoietic cell lines.

Epstein-Barr virus (EBV) can induce a broad spectrum of hematological diseases, especially in immune deficient patients. We assayed for receptor for EBV (EBVR) using fluoresceinated viral particles on 44 human hematopoietic cell lines derived from patients with T, B, and non-T, non-B acute lymphocytic leukemia (ALL), non-lymphoid leukemia, Burkitt lymphoma, myeloma and several unique lines we and others have recently developed. All 31 EBV nuclear-associated antigen (EBNA) negative cell lines were of neoplastic origin. Seven of 13 EBNA-positive cell lines were of normal cell origin. Four of 25 non-B (surface immunoglobulin negative) EBNA-negative neoplastic cell lines were EBVR-positive. Three of six EBNA-negative B-cell (surface immunoglobulin positive) lines were EBVR-positive. Nine of 13 EBNA-positive Burkitt and non-Burkitt cell lines strongly expressed EBVR. Four EBNA-positive Burkitt lymphoma cell lines exhibited EBVR only to a limited degree. Studies of the cell lines for EBVR, complement receptors (CR) and surface immunoglobulin (SIg) revealed that presence of SIg does not obligate the presence of EBVR. Functional EBVR accompanied SIg among EBNA-negative cell lines. SIg-negative cell lines can possess EBVR. Fourteen of 16 EBVR-positive lines were also positive for CR. The EBVR assay is a useful tool for assessing the potential role of EBV in the induction of hematopoietic disorders.

Antigens, Surface↗

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.

B-Lymphocytes↗

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.

Antigens, Differentiation, B-Lymphocyte↗

Binding sites of the Epstein-Barr virus and C3d receptor (CR2, CD21) for its three intracellular ligands, the p53 anti-oncoprotein, the p68 calcium binding protein and the nuclear p120 ribonucleoprotein.

Epstein-Barr virus/C3d receptor (CR2, CD21) interacts with three intracellular proteins: the p53 anti-oncoprotein expressed in human B lymphoma cells, the p68 calcium binding protein expressed in normal B lymphocytes and the nuclear p120 ribonucleoprotein (RNP). We previously demonstrated that p53 and p68 interacted with the intracytoplasmic carboxy-terminal domain of CR2. To analyse the amino acid sequence of CR2 binding sites for p53 and p68, we synthesized different peptides whose sequences were derived from this carboxy-terminal domain. Thus, CR2 bound to p53 and p68 through two distinct binding sites localized on the N-terminal and on the central part of its carboxy-terminal domain, characterized by the amino acid sequences of KHRERNYYTD and KEAFHLEARE, respectively. CR2 site reacting with the nuclear p120RNP was determined using either anti-CR2 mAb directed against its extracellular domain or pep34, pep14/SCR3 and pep14/SCR4, synthetic peptides whose sequences corresponded to the intracellular 34 amino acid domain or to sites of the extracellular domain of CR2, respectively. Data support that CR2 interacts with p120RNP through the DEGYRLQGPPSSRC amino acid sequence of its extracellular SCR4 domain. Furthermore, phosphorylation of CR2 inhibits its interaction with the nuclear p120RNP. Binding of CR2, through its intracellular and extracellular domains, with the p53 oncoprotein and p120RNP, respectively, and the co-localization of these three proteins on nuclear interchromatin fibrils, suggest that CR2 could act as a crosslinker between these two nuclear proteins to regulate their functions.

Amino Acid Sequence↗

Pep34, a synthetic peptide whose sequence corresponds to the intracytoplasmic domain of the Epstein-Barr virus receptor (CR2, CD21), regulates human B lymphocyte proliferation triggered through CR2.

CR2 is involved in regulation of human B lymphocyte proliferation by interacting, through distinct domains, with extracellular, cell surface or intracellular components. Contribution of CR2 intracytoplasmic domain in CR2 regulatory functions remains unclear. Thus, we used pep34, a 34 amino acid synthetic peptide whose sequence corresponds to CR2 intracytoplasmic domain. Pep34 was incorporated into B lymphocytes which were then activated by EBV or C3d through CR2. Our data demonstrate that pep34 inhibits 100% B lymphocyte proliferation triggered by EBV or C3d. Irrelevant peptide had no effect. When B lymphocyte proliferation was triggered by a multipotent B cell activator as SAC, pep34 did not exert any inhibitory effect. Our data demonstrate that pep34 inhibits B lymphocyte proliferation only when lymphocytes are triggered through CR2. Thus, this strongly supports that despite its short length. CR2 intracytoplasmic domain participates to regulatory functions of this receptor.

Amino Acid Sequence↗

Soluble forms of CD21 and CD23 antigens in the serum in B cell chronic lymphocytic leukaemia.

By using pairs of monoclonal antibodies (MAbs) to different epitopes on CD21 and CD23 antigens, it has been shown that both antigens are readily detectable in cell-free supernates of cultures of B cells expressing these antigens on the cell surface. The antigens remained in the soluble fraction after high speed centrifugation. Sera from normal individuals contained significant amounts of CD21 antigen, whereas little CD23 antigen was detectable. By contrast CD23 but not CD21 antigen was present in urine. Sera from patients with B cell chronic lymphocytic leukaemia (B-CLL) contained increased amounts of both antigens. The levels were related to the surface expression of antigen on the leukaemic cells and the number of cells in the blood. The possible functional role of soluble forms of B cell antigens and the diagnostic potential of their detection in body fluids are discussed.

Antibodies, Monoclonal↗

Expression of EBV/C3d receptors on T cells: biological significance.

There is overwhelming evidence that a single polypeptide serves as a receptor for both the Epstein-Barr Virus (EBV), and for certain enzymatic fragments of C3. This receptor, termed CR2 (CD21), is known to be expressed on the surfaces of B cells, and a large body of evidence suggests that CR2, or related structures, are also expressed on cells of the T lineage. Here, Constantine Tsoukas and John Lambris review the studies of CR2 expression in T cells and offer some speculation on its possible biological significance.

Animals↗

A new pair of surface molecules involved in human IgE regulation.

The molecules controlling IgE production are the subject of intense study in the effort to find new ways to treat allergic diseases. One candidate, the CD23 molecule, a low affinity receptor for IgE, was recently identified to interact with another molecule, named CD21, in the regulation of IgE production.

B-Lymphocytes↗

FDC-specific functions of p55TNFR and IKK2 in the development of FDC networks and of antibody responses.

The FDC-specific molecular signals required in the formation of FDC networks, B cell follicles, and germinal centers (GCs) have remained poorly understood. We used FDC-specific gene targeting to investigate the function of p55TNFR and IKK2 in lymphoid organ structure and function. Here we show that FDC-specific expression of p55TNFR is necessary and sufficient to promote FDC network and B cell follicle formation, restore the expression of CXCL13 and VCAM-1/ICAM-1 in FDCs, and lead to productive GCs. Notably, FDC-specific disruption of IKK2 does not affect formation of FDC networks. Yet, after antigen engagement or immune complex (IC) deposition, FDCs lacking IKK2 fail to upregulate VCAM-1 and ICAM-1, and GCs remain sterile. These findings demonstrate that IKK2-independent function of p55TNFR on FDCs is sufficient to support the development of FDC networks and GCs, while FDC-specific IKK2 is indispensable for the generation of efficient humoral immune responses.

Animals↗

Analysis of the regulatory role of BAFF in controlling the expression of CD21 and CD23.

The TNF family member BAFF serves to promote the survival and differentiation of maturing splenic B cells. The major receptor for BAFF (BAFF-R) is expressed by the transition 2, marginal zone and follicular, mature conventional B-2 cell populations; functional BAFF/BAFF-R signaling is required for T1 to T2 cell B cell maturation. Induced expression of CD23 and CD21 is also coincident with the T1 to T2 maturation stage. A key question we address in this report is if BAFF signaling directly induces CD21 and CD23 gene transcription and expression at this B cell transition point, or if their expression is simply coincident with B cell maturation and differentiation. We present data that supports the contention that BAFF does not preferentially induce the expression of CD23 or CD21 at the T1 to T2 transition, nor does exogenous BAFF lead to preferential increased expression of these proteins/genes in mature B cell populations. The analysis of LPS-induced splenic B cells from BAFF-R defective (A/WySnJ) mice did not show the preferential induction of expression of CD21 or CD23 that might have been expected if NF-kappaB-p52 protein was lacking due to insufficient BAFF-R signaling in cells bearing this mutation. Indeed, chromatin immunoprecipitation analysis demonstrated stable NF-kappaB-p52 complexes on CD21 and CD23 genes obtained from both wild type and A/WySnJ B cells. FACS analysis of splenic B cells from 1-, 2-, 3- and 6-week-old A/WySnJ mice demonstrated a block in differentiation (thus reducing overall B cell numbers) resulting in a failure of such cells to express CD21 but allowing for the expression level of CD23 per cell to reach levels approaching wild type. We have dubbed this CD23(HI)CD21(LO) subset as the T1b transition B cell. These data support the recognized role of BAFF as promoting the survival and differentiation of splenic B cells but do not support a model of BAFF signaling directly inducing the expression of the CD21 and CD23 proteins via translocation of NF-kappaB-p52 species.

Aging↗

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life.

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.

Age Factors↗

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4.

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.

Adolescent↗

Epstein-Barr virus receptors on human pharyngeal epithelia.

The apparently strict tropism of Epstein-Barr virus (EBV) for B lymphocytes has been attributed to the existence of a B-lineage-specific surface molecule, the C3d receptor, which also functions as a receptor for EBV. Two monoclonal antibodies against different determinants on the EBV/C3d receptor of B cells were shown to react with pharyngeal epithelia in a cell differentiation-dependent manner. These findings, which raise the possibility of direct virus entry into a naturally exposed epithelium, strengthen the evidence in favour of an epithelial reservoir of EBV infection in vivo and identify a means whereby the virus/epithelium interactions leading to nasopharyngeal carcinoma might be initiated.

Antibodies, Monoclonal↗