B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

Differential expression of the major histocompatibility antigen complex (MHC) on a series of Burkitt's lymphoma lines

We compared the expressions of class I and class II major histocompatibility antigen complex (MHC) on the surface of Jijoye and P3HR-1 cells of Burkitt's lymphoma sublines. Jijoye cells had a large amount of class I and class II MHC antigens, whereas these antigens were less expressed on P3HR-1 cells. On a subline of P3HR-1 K cells the expression of class I antigen markedly diminished and class II antigen was undetectable. On the other hand, Jijoye, P3HR-1, and P3HR-1 K cell lines were confirmed to be Epstein-Barr virus (EBV) nonproducer, low producer, and high producer, respectively. The chemical activation of EBV genome by treating P3HR-1 cells with 12-O-tetradecanoyl phorbol-13 acetate (TPA) and n-butyrate resulted in inhibition of the expression of class I and II antigens, while the addition of retinoic acid, an inhibitor of virus replication, blocked the decrease in the MHC antigen expression. These findings suggested that there might be an inverse correlation between the virus production and the expression of class I and II MHC antigens.     

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.     

Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.     

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.     

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.     

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.     

Activation of protein kinase C modulates the expression of the T3/T cell antigen receptor complex on human T lymphocytes

Activators of protein kinase C induced a rapid decrease (within 15 min) in the surface expression of the T3 antigen and T-lymphocyte antigen receptor (Ti) on HPB-ALL cells, and a concomitant phosphorylation of the T3 gamma and delta polypeptides; the gamma chain was more extensively phosphorylated than the delta chain. No phosphorylation of the T3 epsilon chain and the Ti alpha and beta polypeptides was detected. Evidence was obtained that the T3 gamma chain is phosphorylated only on serine residues.     

Expression of CR2 (the C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes.

The expression of CR2 (the C3dg/EBV receptor, CD21) on normal human T lymphocytes was investigated using purified peripheral blood T cells and indirect immunofluorescence with biotinylated anti-CR2 mAb and streptavidin-phycoerythrin. Thirty to 40% of normal peripheral blood T lymphocytes expressed CR2 Ag. The cells expressed three nonoverlapping epitopes of CR2. The specificity of the staining for CR2 epitopes was demonstrated by the ability of unlabeled anti-CR2 mAb but not of anti-CR1 mAb of the same isotype to compete for the binding of biotinylated anti-CR2 mAb to T cells. The intensity of staining of T lymphocytes with anti-CR2 mAb was approximately 10-fold lower than that of peripheral blood B cells. CR2 was immunoprecipitated from purified T lymphocytes as a single protein of apparent Mr 145,000. The presence of CR2 on normal human T lymphocytes suggests that the receptor may modulate the function of T cells in the immune response and the susceptibility of the cells to infection by lymphocytotropic viruses.     

Monoclonal and anti-idiotypic anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on glycoprotein 140, the EBV/C3dR, expressed on human B lymphocytes

Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Comparative structure and evolution of murine CR2. The homolog of the human C3d/EBV receptor (CD21)

The complete nucleotide sequence of murine complement receptor type 2 (CR2) was determined from two overlapping cDNA clones derived from a lambda gt11 library of late pre-B cell origin. Comparison of the predicted sequence of the 1014 amino acid murine homolog with that of human CR2 revealed marked evolutionary conservation. The murine molecule was 65% identical to human CR2 overall, lacking a single repetitive sequence variably present in man. The 15 approximately 60-75 amino acid short consensus repeats (SCR) that constitute the entire extracellular domain of murine CR2 were 53 to 81% identical to and could be directly aligned with the human protein. As reported, the cytoplasmic tail shared 79% amino acid identity with human CR2, whereas that of the transmembrane was only 33%. Murine CR2 contained 16 potential N-linked glycosylation sites of which 6 were conserved, 4 altered, and 6 lost during human evolution. The hydropathicity profile of the two molecules was nearly colinear with some variation in the N-terminal region of the first repeat, as well as within the sixth and twelfth repeats. RNA blot analysis revealed a approximately 4.0 to 5.0 kb message in murine B lymphocytes, which was absent in T lymphocytes (thymus and spleen), liver, brain, lung, kidney, and heart. A method was devised to more precisely compare the repeat structures. An identity matrix analysis suggests that human ancestral CR2 evolved before divergence of the rodent and primate branches of the evolutionary tree through a series of predictable gene duplications, possibly giving rise to the precursor of human CR1 and murine CRY. The marked structural similarity between the human and murine receptors suggests functional conservation as well.     

Monoclonal antibodies to the human C3b/C4b receptor (CR1) enhance specific B cell differentiation

The addition of monoclonal antibodies against the human C3b/C4b receptor (CR1) to cultures of peripheral blood lymphocytes in the presence of suboptimal amounts of TNP bound to polyacrylamide beads enhanced by 150 to 400% the specific anti-TNP response, as measured by a plaque-forming cell assay on day 7. Anti-CR1 antibodies similarly enhanced the anti-fluorescein antibody response. Enhancement only occurred in cultures performed in the presence of the relevant antigen. No enhancing effect on the anti-TNP response was observed on addition to cultures of monoclonal antibodies directed against other surface antigens of B cells or an anti-T cell antibody of the same subclass as that of anti-CR1 antibodies. Anti-CR1 antibodies alone did not induce nonspecific B cell proliferation and did not provide B cells with a first signal for proliferation in the presence of a source of B cell growth factors. Anti-CR1 antibodies did not enhance the nonspecific proliferative response of B cells to growth factors derived from PHA-stimulated T cells, semi-purified BCGF 20 KD, BCGF 50 KD, or recombinant IL 2 in the presence of anti-mu. In this respect, the effect of anti-CR1 antibodies differs from that of anti-CR2 antibodies which interact with early stages of B cell activation. In contrast, anti-CR1 antibodies enhanced specific differentiation of antigen-activated B cells in the absence of T cells when soluble T cell factors were provided. Similar results were obtained by using either of two sources of differentiation factors, the MLA-144 supernatant or a 30 to 15 KD fraction from PHA-stimulated T cells. These results indicate that triggering of CR1 on B cells positively regulates the specific antibody response to low doses of antigen by enhancing B cell differentiation whether T cell help is provided by intact T cells or by T cell-derived differentiation factors.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.     

Combined treatment with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha up-regulates the expression of HLA class I determinants in Burkitt lymphoma lines

Cell lines derived from Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt lymphoma (BL) have a low or defective expression of polymorphic HLA class I determinants compared to EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin and are resistant to lysis by cytotoxic T lymphocytes (CTL) specific for the corresponding determinants (M. G. Masucci, S. Torsteinsdottir, J. Colombani, C. Brautbar, E. Klein, and G. Klein, Proc. Natl. Acad. Sci. USA 84, 4567, 1987; S. Torsteinsdottir, C. Brautbar, E. Klein, G. Klein, and M. G. Masucci, Int. J. Cancer, 41, 913, 1988). In order to investigate whether this phenotypic trait of the tumor cells can be modulated by agents known to enhance HLA class I antigen expression, pairs of LCL and BL lines were cultured in the presence of recombinant human interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Three low HLA A11 expressor EBV-negative BL lines, DG 75, BL 28, and BL 41, reacted significantly stronger with the anti-HLA A11 monoclonal antibody (Mab) AUF 5.13 after combined treatment with 500 U/ml IFN-gamma and 500 U/ml TNF-alpha. Reactivity with the AUF 5.13 and with other anti-polymorphic class I Mab's was up-regulated also in in vitro EBV-converted sublines of BL 28 and BL 41. The increment of antigen expression depended on the baseline expression in untreated cells. It was largest for the low expressor lines and decreased proportionally to the level of up-regulation induced by EBV conversion. Up-regulation of HLA A11 was accompanied by induction of sensitivity to HLA A11-specific CTLs in BL 28 and its converted subline E95A BL28 while BL 41 and E95A BL 41 remained resistant. The treatment did not affect significantly HLA A11 expression of two EBV-carrying, low HLA A11 expressor BL lines, WW-1-BL and WW-2-BL, and of the EBV-carrying BL 72 line that had a high spontaneous expression. The results suggest that the down-regulation of class I antigen expression is reversible in some but not all BL lines.