Activation of B lymphocytes by Epstein-Barr virus/CR2 receptor interaction

Epstein-Barr virus (EBV) infects and transforms human B lymphocytes. The virus receptor was shown to be identical to the complement receptor CR2. The consequences of EBV/CR2 receptor interaction on B lymphocyte activation were analyzed by infection of B cells with the transforming (B95-8) and the nontransforming (P3HR1) virus strains and with UV-inactivated B95-8 virus. Similar to mitogens and antibodies against surface IgM and CR2 receptor, transforming and nontransforming EBV induced the release of leukocyte migration inhibitory factor (LIF). LIF production occurred early after infection and was not affected by irradiation of the B95-8 virus with doses of UV-light which prevented the expression of viral functions. B lymphocytes infected with UV-inactivated virus responded to T cell-derived B cell growth factors. The results demonstrate that binding of EBV to the CR2 receptor activates the B cells. Progression of the infected cells in the activation pathway required helper cell-derived factors or signals provided by the intact viral genome. The nontransforming P3HR1 virus induced a low level of RNA synthesis and increase of cell size and major histocompatibility complex class I antigen expression. Only the transforming virus induced expression of EBV nuclear antigens and cellular DNA synthesis.     

Structural and functional analysis of CR2/EBV receptor by means of monoclonal antibodies and limited tryptic digestion.

The receptor for the C3d fragment of the third component of complement, CR2, has recently been shown also to act as the receptor for the Epstein-Barr virus (EBV) and to be involved in the control of B-cell proliferation. In order to define functionally important domains on this molecule, we produced monoclonal antibodies to several distinct epitopes. CR2 was purified from a NP-40 lysate of human tonsils by a new method involving sequential chromatography on lentil lectin Sepharose 4B and DEAE-Sephadex and used to immunize mice. After fusion we obtained four stable hybridoma lines producing antibody to CR2. Specificity of these antibodies for CR2 was ascertained by immunofluorescence analysis on a panel of various cells known to possess CR2, by their reactivity in a recently described ELISA for C3 receptors, by Western blotting with purified CR2 and immunoprecipitation from 125I-labelled Raji cells. These four antibodies were found to recognize three distinct epitopes localized on the same fragments of 95,000, 72,000, 50,000, 32,000 and 28,000 MW obtained after mild tryptic digestion of CR2. The 72,000 MW fragment contains the binding site for C3d. Two monoclonal antibodies recognizing the same epitope did not inhibit the binding of C3d-coated sheep erythrocytes to Raji cells, whereas the other two antibodies against distinct epitopes did inhibit in the presence of a second antibody. All four monoclonal antibodies stimulated the proliferation of human peripheral blood B cells.     

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.     

Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping

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.     

Cell cycle control of a Burkitt lymphoma cell line: responsiveness to growth signals engaging the C3D/EBV receptor.

CR2, the receptor for the C3d fragment of the third complement component and for Epstein-Barr virus (EBV) has been shown, on mouse B cells, to be involved in the control of B-cell proliferation by acting as a receptor for macrophage-derived growth factors. We examined whether the growth of a Burkitt lymphoma cell line, RAJI, could be influenced by ligands of human CR2. In serum-free culture, purified human C3d, as well as three monoclonal antibodies to distinct epitopes on human CR2, were capable of enhancing the growth rate of RAJI cells two to five-fold. This effect could not be observed if even trace amounts of serum were present in the culture medium. Simultaneous addition of pairs of antibodies did not enhance the growth rate, suggesting that a particular engagement of CR2 may be critical in order to induce a stimulatory effect. These results indicate that in a homologous serum-free human B-cell system human C3d as well as monoclonal antibodies to human CR2 can induce B-cell proliferation and that CR2-mediated triggering of B cells can be induced via epitopes other than the C3d-binding site. In addition we conclude that--unlike normal human B cells--at least some human B-lymphoma cells respond to CR2-mediated stimuli in the absence of any T-cell derived factors. Therefore the control mechanisms exerted through CR2 must still be intact on these autonomously growing cells.     

Expression of CRl and CR2 Complement Receptors following Epstein-Barr Virus Infection of Burkitt''s Lymphoma Cell Lines

Epstein-Barr virus (EBV) infection of human B lymphocytes involves a specific receptor closely associated with, or identical to, the C3d complement receptor, CR2. Thus, 25 out of 29 EBV-positive Burkitt's lymphoma (BL) cell lines but none of 15 EBV-negative BL lines were found to express C3 receptors. Furthermore, in vitro infection with EBV of six EBV-negative cell lines resulted in the expression of C3 receptors in association with that of EBV-determined nuclear antigen (EBNA). Rosette assays using erythrocytes coated with human C3b, C3bi, and C3d, inhibition of rosette formation with anti-receptor antibodies, and flow cytometry analysis of stained cells demonstrated that EBV-converted lines expressed C3b and C3d receptors, CR1 and CR2. Anti-receptor antibodies recognized an average of 40,700 anti-CR1 and 140,000 anti-CR2 binding sites on an EBV-converted line (BL41/B95), whereas no specific binding occurred on the corresponding EBV-negative (BL41) cells. Because CR1 and CR2 are involved in B-cell proliferation and/or differentiation, enhanced expression of C3 receptors following the interaction between EBV and B cells and/or subsequent infection of the cells by EBV may provide a basis for positive control of B lymphocyte proliferation by EBV.     

B cell activation by the nontransforming P3HR-1 substrain of the Epstein-Barr virus (EBV)

The P3HR-1 substrain of Epstein-Barr virus does not transform B cells. This defect is known to be determined by the loss of the coding sequence for the nuclear antigen EBNA-2. The virus can attach to and enter resting B cells. The initial events after EBV infection are reminiscent of those induced by polyclonal B cell activators. Similar to the effect of these, P3HR-1 virus lowers membrane IgD expression on B cells and abrogates the transient elevation of activation markers BB-1 and LB-1 induced by the culture conditions. An important event of B cell activation is the acquisition of competence to respond to specific growth factors produced by T cells. This was induced by the P3HR-1 virus. The infected B cells had elevated [3H]thymidine incorporation when exposed to the supernatant of PHA-treated T cells. The EBV receptor is identical with the complement receptor CR2. Ligand binding to CR2 has been shown both with mouse and human B cells to deliver certain activation signals. Therefore, it is possible that the early step of activation by EBV is initiated through the binding to the receptor and is thus a cell surface event.     

Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies

The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage.     

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.     

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.     

Antibody-independent activation of the classical pathway of complement by Epstein-Barr virus

A purified preparation of Epstein-Barr virus (EBV) has been shown to activate the classical complement pathway by direct interaction with the first component of complement, C1, without the intervention of antibody. No evidence was found for activation of the alternative pathway. Following classical pathway activation the specific affinity of EBV for B cells can be presumed to be lost since the virus will become opsonized for clearance by phagocytic cells bearing complement receptors, CR1 and CR3. This activation is further evidence that complement plays a role in defence mechanisms independently of antibody activity.     

Patterned entry and egress by Epstein-Barr virus in polarized CR2-positive epithelial cells

In polarized epithelium direction of viral entry and release correlates with proclivity of a virus to establish local versus systemic infection. The Epstein-Barr virus (EBV), whose principal tissue reservoir is B lymphocytes, also has disease manifestations in epithelium, suggesting intertissue spread potentially influenced by epithelial cell polarity. We stably transfected the B lymphocyte EBV receptor (CR2/CD21) into Madin-Darby canine kidney (MDCK) epithelial cells used extensively to study effects of cell polarity on infection by both DNA and RNA viruses. CR2/CD21 was detected on both apical and basolateral surfaces of polarized MDCK cells, with predominant expression basolaterally. However, infectivity was up to four-fold greater apically, suggesting that endogenous cell surface molecules, sorted asymmetrically onto polarized plasma membranes, may be involved in EBV entry into MDCK cells. EBV gp350/220, a replicative cycle glycoprotein added to the virus envelope on egress through the cell membrane, was immunolocalized by confocal microscopy to basolateral cell surfaces only. Apical entry of EBV with subsequent basolateral release of newly replicated virus favors systemic infection by viral dissemination to underlying lymphocytic aggregations. Under conditions of long-term culture, latent EBV was not stably maintained in these cells, suggesting that the epithelial phase of acute EBV infection may be transient.     

Characterization of murine complement receptor type 2 and its immunological cross-reactivity with type 1 receptor.

We have previously demonstrated that some mAbs prepared against mouse complement receptor type 1 (CR1) bind a 150,000 Mr protein in addition to the 190,000 Mr CR1 protein. We now identify the 150,000 Mr murine protein as complement receptor type 2 (CR2), since: (i) one of the monoclonal antibodies that bind this protein inhibits rosette formation between mouse B cells and C3d-bearing sheep erythrocytes; (ii) as is known for human CR2, this protein is present on B lymphocytes but not T lymphocytes; and (iii) this protein must have affinity for C3b, since it has weak factor I cofactor activity. In addition, this protein resembles the 145,000 Mr human CR2 molecule in size. Since four of the five mAbs that were produced by immunization with CR1 also bound CR2, and they bind to different CR1 epitopes, it seems that murine CR1 and CR2 share multiple epitopes. Injection of mice with one of the CR1-CR2 cross-reactive mAbs almost eliminated both CR1 and CR2 expression, but did not decrease B cell numbers or the expression of B cell IgM, Ia, or B220 antigens. In contrast, injection of mice with a non-cross-reactive anti-CR1 antibody only modulated CR1 expression. These antibodies should thus provide useful tools for the study of the in vivo roles of B cell complement receptors.     

Differences in Epstein-Barr virus (EBV) receptors expression on various human lymphoid targets and their significance to EBV-cell interaction

This study was aimed at quantitating, by means of fluorescence-activated cell sorter (FACS), EBV binding to different types of target cells, and at learning about a possible relation between EBV receptor density and the fate of cell-surface bound virus. We used fluoresceinated virus preparations of two strains of EBV (B95-8: lymphocyte transforming strain; P3HR-1: non-transforming strain) to analyze quantitatively the expression and density of EBV receptors on different human lymphoid cell lines and on B lymphocytes from both EBV-seropositive and -seronegative donors. FACS analysis was also used as a tool to approximate the cell surface area of the different lymphoid cells examined. Our results indicate that: (a) after accounting for the difference in cell surface dimensios, the fluorescence intensity of EBV-bound Raji (a B line) cells was three to four times higher per unit area than that of EBV-bound fresh B lymphocytes from an EBV-seropositive donor; (b) Molt-4 (a T line) cells bound about 21-fold less P3HR-1 EBV and 6-fold less B95-8 EBV than Raji cells per unit area; (c) B lymphocytes from EBV-seronegative adult donors bound only about one third as much virus as B cells from seropositive individuals; (d) two B lymphocyte sub-populations can be identified in the peripheral blood in regard to their ability to bind EBV, regardless of the EBV antibody status of the donor; (e) the EBV receptor on Molt-4 cells appears structurally different from the one found on Raji cells since EBV binding to Molt-4 cells was not blocked by a monoclonal antibody (OKB7) specific to the complement receptor (CR2). Further, in contrast to Raji cells, Molt-4 expressed a differential binding activity for each of the two EBV strains used. Taken together, the important differences observed in regard to EBV attachment to various targets also appear to relate to the fate of cell-surface bound virus: i.e., virus penetration might be determined, at least in part, by the density of EBV receptors on the target cell surface; thus the receptor density may play a major role in viral infection.     

Peripheral T-cell lymphoma complicated by a proliferation of large B cells

We studied 14 cases that showed a morphologic appearance of peripheral T-cell lymphoma and contained substantial numbers of CD20+ large B cells. In all but 2 cases, the CD20+ large cells showed a mix of kappa and lambda light chain expression. Two cases showed a focal predominance of kappa expression. In situ hybridization using the EBER1 probe for detection of Epstein-Barr virus (EBV) RNA was performed on every case. EBV RNA was present in 10 cases. Of 8 cases with EBV RNA stained by immunohistochemistry for the latent membrane protein of EBV, 6 were positive. Double-labeling immunohistochemistry and in situ hybridization confirmed that EBV was present in the large B cells. Polymerase chain reaction (PCR) analysis showed a clonal rearrangement of the T-cell receptor (TCR)-gamma chain gene in 12 of 13 cases tested. One additional case showed a clonal rearrangement of the TCR-beta chain gene by Southern blot hybridization. PCR analysis showed a clonal immunoglobulin gene rearrangement in 5 cases, a suggestion of a clonal rearrangement in 1, an oligoclonal pattern in 4, and a polyclonal pattern in 4. The finding of large B and T cells may result in a misdiagnosis of a reactive process or of T-cell-rich B-cell lymphoma. The presence of EBV in some cases could cause further confusion with the reactive T- and B-immunoblastic proliferation of infectious mononucleosis.     

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.