Barbro Ehlin-Henriksson

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

SummaryWe have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BackgroundThe developmental stage from which stems the malignant B cell population in Burkitt’s lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rearranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents.Materials and MethodsRearranged Vκ region genes from 10 κ-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three κ-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined.ResultsAll BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged Vκ genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines).ConclusionsThe detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for μ chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.

Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposis sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitts lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

Host cell phenotype-dependent methylation patterns of Epstein-Barr virus DNA

We have shown previously that the EBNA 1 and latent membrane protein encoding regions of the Epstein-Barr virus (EBV) genome are highly methylated at CCGG sequences in the Burkitts lymphoma (BL)-derived cell line Rael, but are unmethylated in a lymphoblastoid cell line (LCL) harbouring the same virus. To examine whether this is a regular phenomenon, we compared the methylation patterns of selected regions (BamHI C, W, H, M, E, K and N fragments) of EBV DNA in representative EBV-carrying cell types of normal and neoplastic origin. Analysis of HpaII and MspI cleavage patterns showed that all probed regions were highly methylated in all six BL biopsy samples, but hypomethylated in the four LCLs immortalized by the virus. EBV DNA was also highly methylated in the nude mouse-passaged C15 nasopharyngeal carcinoma strain and partially methylated in the C18 strain. Eight BL lines propagated in vitro, ranging from a typical BL group I to a more LCL-like group III phenotype, showed heterogeneous levels of methylation. Rael, the only stable group I cell line, carried highly methylated viral genomes. The other cell lines, which have drifted to an LCL-like blastic phenotype to various degrees, showed more moderate or low viral DNA methylation. Two sublines of the BL cell line Jijoye, which could be classified as groups II and III, respectively, showed a corresponding difference in EBV DNA methylation.(ABSTRACT TRUNCATED AT 250 WORDS)

B‐lymphocyte subpopulations are equally susceptible to Epstein–Barr virus infection, irrespective of immunoglobulin isotype expression

While Epstein–Barr virus (EBV) is known to establish latency in the memory B‐cell compartment, there is controversy as to whether the memory or the naïve B cell is the initial target for infection. Here we have explored the infectability of the B‐cell subsets contained in peripheral blood and tonsils, as distinguished by their surface expression of the immunoglobulin isotypes that help to define naïve and memory pools. First we show that both CD21 and major histocompatibility complex (MHC) class II molecules – respectively, the major receptor and co‐receptor for EBV on B cells – are expressed at similar levels on blood and tonsillar B cells, irrespective of surface immunoglobulin class, indicating that each of the subsets demonstrate an equal potential, at least for infection. Then, following in vitro infection of total tonsillar B cells, we found that the relative frequencies of immunoglobulin (Ig)M‐, IgG‐ and IgA‐positive cells containing EBV‐encoded Epstein–Barr virus nuclear antigen 5 (EBNA5) protein at 48 hr were similar to those of the starting population. However, IgD expression was uniformly decreased, probably as a consequence of cellular activation. These data indicate that recirculating B cells have both the potential for, and susceptibility to, initial infection by EBV, irrespective of the immunoglobulin isotype expressed.

EBV-encoded EBNA-5 associates with P14ARF in extranucleolar inclusions and prolongs the survival of P14ARF-expressing cells.

Epstein‐Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus‐encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2‐hybrid system, we have shown that EBNA‐5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull‐down assay. Moreover, expression of EBNA‐5 increased the survival of p14ARF‐transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA‐5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.

CD40 ligation downregulates EBNA‐2 and LMP‐1 expression in EBV‐transformed lymphoblastoid cell lines

Epstein‐Barr virus (EBV) drives the proliferation of human B cells in vitro and during primary infection in vivo. The transformed immunoblasts express nuclear proteins EBNA1–6, transcribed from the Cp/Wp promoter, and the membrane proteins LMP‐1, ‐2A and ‐2B (lymphoblastoid type of latency). EBV persists through life in resting memory B cells with a restricted type of latency in the absence of the Cp/Wp promoter activity. Since CD40 crosslinking can reportedly inhibit the growth of EBV‐transformed lymphoblastoid cell lines (LCLs), we have examined the effect of CD40 ligation on the expression of EBNAs and LMP‐1 and on Cp EBV promoter activity together with several phenotypic markers. CD40 crosslinking led to a partial downregulation of EBNA‐2, EBNA3–6 and LMP‐1 in LCLs, paralleled by downregulation of Cp promoter activity. It also induced upregulation of the germinal center marker CD77 on the LCL cells. Our findings suggest that the encounter of proliferating EBV‐transformed immunoblasts with CD40L, as would occur when normal B cells generate memory cells in germinal centers, may switch the viral transcription program from the full lymphoblastoid to a more restricted latency program in a proportion of cells. This would permit virus persistence in the B‐cell memory compartment.

Changes in chemokines and chemokine receptor expression on tonsillar B cells upon Epstein–Barr virus infection

Chemokines and chemokine receptors are likely to play important roles in the pathogenesis of Epstein–Barr virus (EBV) ‐associated disease. The primary EBV infection occurs in the oropharynx where the virus infects mainly tonsillar B cells. We have previously shown that CXCR4 expression on tonsillar B cells is modulated by EBV. Here, CXCR5 and CCR7 expression, which is important for migration into lymphoid tissue, was followed for 14 days after EBV infection of tonsillar B cells. Early after infection (2 days) there were only minor changes in CXCR5 and CCR7 expression. However, at day 7 the expression of CXCR5, as well as of CCR7, was decreased and by day 14 these molecules were no longer present at the cell surface. Furthermore, EBV infection affects the chemotactic response to CXCL13 and CCL21 (the ligands for CXCR5 and CCR7, respectively) with a reduction of ligand‐induced migration at day 2. Using gene expression profiling, we identified an additional set of chemokines and chemokine receptors that were changed upon EBV infection in comparison with non‐infected tonsillar B cells. In particular, messenger RNA expression for CCR9 and the complement receptor C5AR1 was increased. Both receptors mediate homing to mucosal tissue. The alterations of the expression of these molecules may lead to retention of EBV‐infected tonsillar B cells in the interfollicular region of the tonsil.

Epstein–Barr virus infection negatively impacts the CXCR4-dependent migration of tonsillar B cells

The primary Epstein–Barr virus (EBV) infection occurs in the oropharynx, where the virus infects B cells and subsequently establishes latency in the memory B‐cell compartment. EBV has previously been shown to induce changes in the cell surface expression of several chemokine receptors in cell lines and the transfection of EBNA2 or LMP1 into a B‐cell‐lymphoma‐derived cell line decreased the expression of CXCR4. We show that in vitro EBV infection reduces the expression of CXCR4 on primary tonsil B cells already 43 hr after infection. Furthermore, EBV infection affects the chemotactic response to stromal cell‐derived factor (SDF‐1)α/CXCL12, the ligand for CXCR4, with a reduction of SDF‐1α‐induced migration. To clarify whether this reduced migration is EBV‐specific or a consequence of cell activation, tonsillar B cells were either infected with EBV, activated with anti‐CD40 and interleukin‐4 (IL‐4) or kept in medium. Activation by anti‐CD40 and IL‐4 decreased the CXCR4 expression but the CD40 + IL‐4‐stimulated cells showed no reduction of chemotactic efficacy. Our finding suggests that changing the SDF‐1α response of the EBV‐infected B cells may serve the viral strategy by directing the infected cells into the extrafollicular areas, rather than retaining them in the lymphoepithelium.

EPSTEIN-BARR VIRUS GENOMES ARE FOUND PREDOMINANTLY IN IgA-POSITIVE B CELLS IN THE BLOOD OF HEALTHY CARRIERS

B lymphocytes have been identified as the main reservoir of latent Epstein‐Barr virus (EBV) in healthy virus carriers. We have established a semi‐quantitative PCR method to estimate the EBV genome load in the blood B‐cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM‐, IgG‐ and IgA‐positive B cells. Between 80% and 90% of the viral DNA was found in the IgA‐positive compared with the IgA‐negative fraction. Int. J. Cancer 83:50–54, 1999.