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PubMed1993

Epstein-Barr virus (EBV) nuclear-antigen-2-induced up-regulation of CD21 and CD23 molecules is dependent on a permissive cellular context.

The Epstein-Barr virus (EBV) induces unlimited growth of B lymphocytes in vitro, a phenomenon known as immortalization. The elucidation of the mechanisms by which EBV de-regulates B-cell proliferation in vitro will permit an understanding of how the virus contributes in vivo to the genesis of Burkitt's lymphoma (BL) and of lymphoproliferations in immunosuppressed patients. At present, no single EBV immortalizing gene has been identified, and the hypothesis has been made that many viral genes cooperate in establishing an autocrine loop of secretion leading to immortalization. Constitutive expression of B-cell surface molecules such as CD21 and CD23, specifically implicated in the control of B-cell proliferation, is indeed induced at the surface of immortalized B lymphocytes. The expression of the viral nuclear antigen 2 (EBNA2) has been shown to be in part responsible for CD21 and CD23 up-regulation, and EBNA2 is suspected to be a transactivator of cellular genes, although this point remains to be demonstrated. The role of EBNA2 gene, independently of other viral genes, has been investigated by transfection into B-lymphoma lines, but conflicting results have been reported. To further investigate its role in the regulation of CD21 and CD23 molecules, we have compared the effects of EBNA2 expression in 2 sets of B-lymphoma lines infected with P3HR1 EBV strain, and/or transfected with EBNA2 gene. We report here that: (i) EBNA2 expression is not a sufficient condition to induce CD21 and CD23 upregulation, EBNA2's effects are highly dependent on the cellular context, and moreover can be modified by infection with P3HR1 virus; (ii) EBNA2 induces activation of CD23 expression in a very particular way, namely, an increased quantity of CD23 steady-state RNA coding for the form A of the protein, which is not detectable at the cell surface but directly secreted.

Antigens, Viral↗
PubMed1991

Epstein Barr virus/complement C3d receptor is an interferon alpha receptor.

Interferon alpha contains a sequence motif similar to the complement receptor type two (CR2/CD21) binding site on complement fragment C3d. Antibodies against a peptide with the CR2 binding sequence on C3d react with a peptide carrying the IFN alpha CR2 binding motif (residues 92-99) and with recombinant IFN alpha. The IFN alpha-derived peptide, as well as recombinant IFN alpha, inhibits C3bi/C3d interaction with CR2 on the Burkitt lymphoma Raji. The direct interaction of IFN alpha and CR2 is inhibited by polyclonal anti-IFN alpha, anti-CR2 and anti-C3d peptide antibodies as well as by C3bi/C3d, EBV coat protein gp350/220 and IFN but not by IFN gamma. [125I]IFN alpha binding to Raji cells is inhibited by polyclonal anti-IFN alpha and anti-CR2 antibodies, by peptides with the CR2 binding motif and partially by C3bi/C3d. Monoclonal anti-CR2 antibody HB5, but not OKB-7, blocks IFN alpha binding to Raji cells. CR2 or CR2-like molecules may therefore be the major IFN alpha receptors on B lymphocytes.

Amino Acid Sequence↗
PubMed1996

Structure-based modeling of the ligand binding domain of the human cell surface receptor CD23 and comparison of two independently derived molecular models.

CD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C-type lectin domain: immunoglobulin E (IgE), CD21, and the beta 2-integrins CD11b and CD11c. CD23 specifically interacts in a calcium-dependent manner, "lectin-like" with carbohydrate moieties expressed on CD21 and CD11b/c, but also "lectin-unlike" with protein epitopes on IgE. As a first step in analyzing the multiple binding specificities associated with CD23 in more detail, we report a detailed molecular model of the lectin-like domain of human CD23 (hCD23). The model was built based on information provided by X-ray structures of mannose binding protein (MBP) and E-selectin, both of which are members of the calcium-dependent (C-type) lectin superfamily. Sequence-structure comparisons suggest that hCD23 is structurally more similar to MBP than to E-selectin. The hCD23 model is compared to an independently derived model. Although the CD23-carbohydrate and CD23-protein interactions are both calcium dependent, analysis of the model suggests the presence of distinct binding sites for these ligands.

Amino Acid Sequence↗
PubMed1993

EBV membrane receptor (CR2) is phosphorylated by protein kinase C (PKC) in the early stages of virus entry into lymphoblastoid cells line (Raji).

Labeling the EBV membrane with octadecylrhodamine-b-chloride (R18) we were able to monitor spectrofluorometrically the early events of EBV fusion, under conditions in which we could affect PKC activity. Binding of EBV to Raji cells induces PKC translocation from the cytosol to the plasma membrane and 32P incorporation into its cellular receptor CR2. CR2 phosphorylation is completely inhibited when cells are preincubated with the PKC inhibitor calphostin c. This treatment also generates a strong inhibition of EBV fusion. Taken together this result suggests a key role of CR2 phosphorylation in the EBV entry into Raji cells.

Adenosine Triphosphate↗
PubMed1993

Elevated secretory IgA antibodies to Epstein-Barr virus (EBV) and presence of EBV DNA and EBV receptors in patients with cervical carcinoma.

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.

Analysis of Variance↗
PubMed1995

Interaction of Fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

3T3 Cells↗
PubMed1995

Epstein-Barr virus infection of CR2-transfected epithelial cells reveals the presence of MHC class II on the virion.

Epithelial cell lines transfected with the Epstein-Barr virus (EBV) receptor CR2 are susceptible to infection by EBV. Following infection with certain EBV strains we found that these cells became positive for MHC class II. The class II was confirmed as being of viral and not target cell origin by immunostaining with HLA-specific monoclonal antibodies. Electron microscopic immunogold staining confirmed the presence of MHC class II on the surface of the virion. While some MHC class I was also found on the EB virion, other cell surface molecules were absent. Dual color immunofluorescence and confocal microscopy analysis demonstrated colocalization of class II with EBV-encoded structural proteins (MA and VCA) in infected epithelial cells. However, preincubation of EBV with antibodies against either MHC class I or MHC class II failed to affect either EBV binding or EBV infection. The presence of MHC on the surface of the EB virion may be a consequence of the intracellular pathways through which productive virus exits from the cell and may influence the target cell tropism of EBV.

Animals↗
PubMed1997

Epstein-Barr virus in synergy with tumor-promoter-induced malignant transformation of immortalized human epithelial cells.

It is difficult to study how Epstein-Barr virus (EBV) causes transformation of human epithelial cells. The major difficulty is that cultured human epithelial cells do not express EBV receptor (complement receptor 2, CR2), hence EBV cannot infect such epithelial cells directly. In order to investigate the role of EBV in the transformation of human epithelial cells, pSG-CR2-Hyg carrier was transfected into immortalized human epithelial cells (293 cells) to express EBV receptor. EBV could infect these CR2-positive cells directly, and expressed EBV antigens. EBV-infected epithelial cells grew in piles with multiple cellular layers and lost contact inhibition in vitro. In soft-agar culture containing 12-0-tetradecanoylphorbol 13-acetate (TPA), EBV-infected 293 cells formed more and larger colonies. When EBV-infected 293 cells were transplanted subcutaneously into nude mice, and treated with TPA, poorly differentiated carcinoma was induced. These results suggest that EBV could induce the malignant transformation of immortalized human epithelial cells in synergy with TPA.

Animals↗
PubMed1984

Human monoclonal antibodies: methods of production and some aspects of their application in oncology.

The currently available methods for the production of human monoclonal antibodies (Mabs) are: mouse-human hybrids, human-human hybrids, EBV transformation, and fusion of EBV-transformed specific B-lymphocytes with a malignant cell line. Each method has its advantages and drawbacks, but they are all inferior to an optimal functioning mouse hybridoma system. A significant obstacle is the lack of a reproducible method to obtain high amounts of antigen-specific B-lymphocytes. However, it seems that newly developed in vitro antigen-priming methods may be a significant aid to the solution of this problem. Human Mabs have already been obtained against some types of human cancer, and the reactivity pattern has provided some biological information that seems not to be obtainable with non-human Mabs. It furthermore seems that the technology for production of human Mabs now has progressed to the stage where it permits a realistic hope that they soon can be applied in the diagnosis and treatment of human cancer.

Animals↗
PubMed1989

Enhancement of Epstein-Barr virus/C3d receptor (EBV/C3dR or CR2) and nuclear p120 ribonucleoprotein phosphorylation by specific EBV/C3dR ligands in subcellular fractions of the human B lymphoma cell line, Raji.

We present herein the first evidence that interaction of specific EBV/C3dR ligands, as human C3bi/C3d and anti-EBV/C3dR MoAb, with EBV/C3dR enhanced significantly, in a dose dependent process, phosphorylation of EBV/C3dR and p120 RNP present in subcellular fractions, as purified plasma membranes and nuclei, of the human B lymphoma cell line, Raji. The use of kinase effectors allowed to detect some of the kinases involved in these phosphorylations. Pp60src-like phosphotyrosine kinase and protein kinase C were involved in the phosphorylation of plasma membrane or nuclear EBV/C3dR. An additional calcium/calmodulin-dependent kinase was also involved in nuclear EBV/C3dR phosphorylation. P120 RNP phosphorylation was under the control of protein kinase C and of CaCl2/Calmodulin-dependent kinase but also of casein kinase II.

Burkitt Lymphoma↗
PubMed1986

gp 140, the C3d/EBV receptor (CR2), is phosphorylated upon in vitro activation of human peripheral B lymphocytes.

gp 140, the C3d/EBV receptor (CR2), is a specific marker of human B lymphocytes. Very recent data suggest that CR2 is a membrane site involved in early B cell activation. These properties of CR2 led us to analyze the molecular events associated with gp 140. We analyzed whether in some conditions of B lymphocyte activation, CR2 could be phosphorylated. We have found that when highly enriched peripheral B cells were cultured for 48 h with anti-mu Ab and/or SAC, in order to provide an optimal activating signal, phosphorylation of the CR2 was induced.

B-Lymphocytes↗
PubMed1986

Purification of the B lymphocyte receptor for the C3d fragment of complement and the Epstein-Barr virus by monoclonal antibody affinity chromatography, and assessment of its functional capacities.

The human C3d receptor (complement receptor type 2, CR2), that also serves as the B lymphocyte receptor for the Epstein-Barr virus, was purified from detergent lysates from the B lymphoblastoid cell lines, SB and Raji, by monoclonal antibody affinity chromatography using the anti-CR2 monoclonal antibody, HB-5. Relative to the concentration of cellular protein and receptor that was initially solubilized by detergent, the procedure provided a 37,000-fold purification with a 40-50% recovery of CR2. The purified receptor presented a single Coomassie blue-stained band when analyzed by SDS-PAGE, and it retained its function of binding to C3-Sepharose. The N-terminus of CR2 was blocked. The amino acid composition was significantly similar to that of the C3b/C4b receptor, factor H and C4 binding protein, suggesting that CR2 may be a member of this newly defined protein family. However, CR2 did not exhibit the regulatory functions of these proteins, namely, the decay dissociation of the classical or alternative pathway C3 convertases and serving as a cofactor for the cleavage of C3b.

Amino Acids↗
PubMed1992

A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

Antigens, CD↗
PubMed1993

A multiparameter flow cytometric method to study surface molecules involved in interactions between subpopulations of cells.

The interactions between T and B lymphocytes are mediated by several antigen-independent adhesion molecules including LFA-1/ICAM-1 and CD2/LFA-3. Recently new pairs of adhesion molecules involved in T and B interactions have been described: CD28/B7, CD5/CD72 and CD45RO/CD22. In order to study these heterotypic adhesion events, the phenotypes of the subpopulations as well as new potential adhesion molecules involved in conjugate formation, we have developed a flow cytometric method which analyses conjugate formation between T and B cells. The two types of cells were loaded with two vital intracellular dyes: human T lymphocytes purified from blood or tonsils were labelled with BCECF-AM (green fluorescence) and the B lymphoblastoid cell line, RPMI 8866 was labelled with Indo-1-AM (blue fluorescence). The two labelled cell populations were mixed, gently centrifuged for 5 min and then incubated at 37 degrees C in a waterbath for 5 min. The cells were then gently resuspended by inversion and analysed with a double laser flow cytometer. This method permitted us to discover new molecular interactions since preincubation of the two populations with monoclonal antibodies directed against some surface molecules inhibited conjugate formation. As an example, using this technique we found that the low affinity IgE receptor, CD23 and the CR2/EBV receptor are involved in T cell/B cell adhesion and can therefore be considered as a new pair of adhesion molecules. This method also seems to be applicable to recombinant cells bearing a single adhesion molecule such as LFA-1 and ICAM-1. A particular advantage of the two intracellular dyes we used is that they are compatible with the dyes commonly used for classical simultaneous triple colour immunofluorescence (phycoerythrin and Cy-Chrome). We were thus able to determine the subpopulations involved in forming conjugates and we found that T-B conjugates were preferentially formed by CD4, CD45RO positive T cells, which are believed to be the memory T lymphocytes.

Antigens, Surface↗