Expression of B-cell-specific markers in different Burkitt lymphoma subgroups

Forty-three Burkitt lymphoma (BL) lines were examined for the expression of 5 monoclonal antibody (MAb)-identified B-cell-specific markers and immunoglobulin production. All (13) EBV-negative BL lines were CALLA+ LB-1-, whereas 30 EBV-carrying lines showed a more heterogeneous pattern. In the EBV-negative lines, the follicle mantle zone markers BA-1 and 35.1C5 were expressed concordantly, at a different level in each line. This coordination was disrupted in EBV-carrying lines. In the EBV-negative lines, there was also an inverted correlation between the expression of 35.1C5 and the germinal center marker BLA, suggesting that some etiologically important event, perhaps the translocation, had fixed the cells at different stages of their transition from one zone to the other. This inverted relationship was also disrupted in the EBV-carrying lines, suggesting that EBV can interfere with the maturation program of the BL cell. This conclusion was also supported by a comparison between 5 EBV-negative BL lines and their EBV-converted sublines. All converted lines have undergone marker changes, but the degree and nature of these changes was different for each EBV-BL line. Both the coordinated expression of BA-1 and 35.1C5 and the inverted relationship between CALLA and LB-1 were disrupted in several other convertants. We have reexamined our previous finding (Ehlin-Henriksson and Klein, 1984) that the majority of the variant translocation-carrying BL lines were CALLA- LB-1+, in contrast to the majority of the typical translocation carriers that were mostly CALLA+ LB-1-. All II EBV-negative lines were CALLA+ LB-1-, irrespective of the type of translocation. Among the EBV-carrying lines, 4 of 17 typical (8;14) translocation carriers were CALLA- LB-1+, whereas 7 of the 12 variant translocation-carrying lines were CALLA- LB-1+. The remaining two expressed both antigens to some extent. The difference is statistically significant at the 0.03 level.     

Immunophenotypic characterization of follicle-center-cell-derived non-Hodgkin's lymphomas

The distribution of the BLA, CALLA (CD 10), AC-2 (CD 39), MHM-6 (CD 23), LB-I, and 351C5 (CD 45R) antigens in 40 non-Hodgkin's lymphomas was demonstrated by immunohistochemical staining of frozen tissue sections. Nine out of 10 centroblastic and centrocytic follicular and diffuse type of lymphomas (CB/CC F/D) and all 10 cases of CB/CC follicular lymphomas were BLA+ and CALLA+. A few cases also showed weak expression of activation antigens (AC-2, MHM-6 and LB-I) and 351C5. In contrast, 3 CC and 3 lymphoblastic (non-Burkitt) lymphomas showed a heterogeneous pattern of distribution with dominating activation antigen expression. A single case of lymphoblastic lymphoma of Burkitt-like type expressed BLA and CALLA but not activation antigens. In reactive follicular center and FCC lymphomas different cell populations appeared to express BLA and activation antigens, respectively. Assessment of staining intensity and proportion of the stained cells indicated that almost all BLA+ cells are CALLA+. CALLA+ BLA- cells were regularly present, in addition. The co-expression of BLA and CALLA in the same cell was confirmed by double immuno-enzymatic staining. By the same technique, BLA+ and CALLA+ cells were shown to be activation-antigen negative.     

Lymphokine-activated killer (LAK) cells discriminate between Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells

We have generated in vitro lymphokine-activated killer (LAK) cells from healthy donors by stimulating their mononuclear leukocytes with recombinant interleukin-2 (rIL-2) (100 U/ml). After 6 days in culture, the lytic properties of the LAK cells were analyzed in the 51Cr-release assay by utilizing a target panel of 6 paired lines consisting of an Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line and an EBV-transformed lymphoblastoid cell line (LCL) from the same donor, the Raji BL line and the natural killer (NK) cell-sensitive K562 line. The patterns of lysis showed that the LAK cells discriminated between two categories of BL cell lines. Group I/II BL tumor cells which expressed the common acute lymphoblastic leukemia antigen (CALLA), the BL-associated glycolipid antigen (BLA) and phenotypically resembled biopsy cells were strongly lysed whereas group III BL cells which had assumed an LCL-like phenotype during culture and lacked the CALLA and BLA surface markers were only poorly lysed. The LCL targets were generally resistant to lysis but the K562 cell line was particularly sensitive. The outcome of cell depletion and monoclonal antibody (MAb) studies indicated that the LAK cell populations were phenotypically and functionally heterogeneous and consisted of at least 2 subpopulations of effector cells; a tumor-specific component and an NK-cell-mediated component.     

Endemic Burkitt's lymphoma: phenotypic analysis of tumor biopsy cells and of derived tumor cell lines

Tumor cells from 10 patients with Epstein-Barr virus-positive endemic Burkitt's lymphoma (BL) have been examined for cell surface phenotype, both at the biopsy stage and during BL cell line outgrowth in vitro, the cultures being followed for up to 150 passages. In all 10 cases, the biopsy cells showed coexpression of the common acute lymphoblastic leukemia antigen (CALLA) and of the BL-associated glycolipid antigen (BLA) with no accompanying expression of several "lymphoblastoid" cell surface markers defined by selected monoclonal antibodies. During cell line establishment and in vitro passage, the individual BL cell lines showed different degrees of progression toward a more "lymphoblastoid" cell surface phenotype, some even losing CALLA and BLA expression while retaining the chromosomal translocations indicative of their malignant origin. This differential capacity for phenotypic progression in vitro explains much, if not all, of the heterogeneity of the BL cell phenotype apparent from many previous studies with panels of long-established lines. Such heterogeneity in vitro belies the true homogeneity of the tumor cell phenotype in vivo.     

T cell receptor gene rearrangements in B-precursor acute lymphoblastic leukemia correlate with age and the stage of B cell differentiation

Children with ALL diagnosed at less than 2 years of age have a poor prognosis when compared with older children. In an effort to identify biologic features of ALL in children less than 2 that might explain this difference, we performed extensive immunophenotypic and molecular genetic analyses on a series of patients. For comparison purposes patients were divided into four groups: CALLA- (CD10-) infants less than 2 years of age at diagnosis (n = 10), CALLA- children greater than 2 years of age at diagnosis (n = 10), CALLA+ infants (less than 2 years, n = 21) and CALLA+ children (older than 2 years, n = 21). No immunophenotyping differences in CALLA- or CALLA+ subgroups were identified when cases less than 2 were compared with cases greater than 2 years of age at diagnosis. The most interesting results were in the CALLA- group where 94% of the samples expressed the B cell antigen CD19 but 27% co-expressed CD7. Double labeling experiments confirmed leukemic blast cells co-expressed CD19 and CD7. The double-labeled cells represent either leukemic conversion of a precursor cell which has not yet committed to B or T cell lineage or aberrant expression of these antigens. Molecular genetic studies demonstrated that all samples, regardless of the patients' age or immunophenotype, had rearrangement of the Ig heavy chain gene. The most striking molecular results were in CALLA- patients; in patients less than 2 at diagnosis neither the beta- nor the gamma-chain gene of the T cell receptor (TCR) was rearranged, whereas DNA from 5 of 10 patients over the age of 2 demonstrated beta- or gamma-chain TCR gene rearrangements. The percentage of CALLA+ cases under the age of 2 years with rearrangements in TCR genes is less than that found in CALLA+ cases over the age of 2 years. The finding of no TCR rearrangements in CALLA- ALL and a decreased number of gamma-TCR rearrangements in CALLA+ cases under the age of 2 suggest that age may affect TCR gene rearrangements in lymphoblasts. The molecular differences in TCR gene rearrangements do not appear to correlate with the response to therapy.     

SH2D1A expression in Burkitt lymphoma cells is restricted to EBV positive group I lines and is downregulated in parallel with immunoblastic transformation

The SH2 domain containing SH2D1A protein has been characterized in relation to the X-linked lymphoproliferative disease (XLP), a primary immunodeficiency that leads to serious clinical conditions after Epstein-Barr virus (EBV) infection. The SH2D1A gene is mutated in the majority of XLP patients. We previously detected SH2D1A in activated T and NK cells, but not in B lymphocytes. We have found SH2D1A protein in Burkitt lymphoma (BL) lines, but only in those that carried EBV and had a Group I (germinal center) phenotype. All the EBV-carrying Group III (immunoblastic) and the EBV-negative BL lines tested were SH2D1A-negative. Motivated by these differences, we studied the impact of EBV and the cellular phenotype on SH2D1A expression. We approached the former question with BL sublines after both the loss of the virus and subsequent reinfection. We also tested original EBV-negative BL lines carrying transfected EBV genes, such as EBNA1, EBNA2, EBNA6, EBER1, 2 and LMP1, respectively. In our experiments, no direct relationship could be seen between EBV and SH2D1A expression. We modified the phenotype of the Group I BL cells by LMP1 transfection or CD40 ligation. The phenotypic changes, indicated by expression of immunoblastic markers, e.g., SLAM, were accompanied by downregulation of SH2D1A. It seems, therefore, that the presence of EBV and the phenotype of the cell together regulate SH2D1A expression in the BL cells. It is possible that SH2D1A is expressed in a narrow window of B cell development represented by germinal center cells.     

Induction of cell differentiation in Burkitt lymphoma lines. BLA: a glycolipid marker of B-cell differentiation

We have previously described an MAb referred to as 38.13 which reacts with a glycolipid membrane antigen (named BLA) on Burkitt lymphoma (BL) lines. The BLA antigen and other B-cell differentiation markers have been studied on BL lines treated with sodium butyrate, agents from the phorbol-diester series (with or without differentiating properties) and teleocidin. With all differentiation inducers tested, expression of BLA as well as of surface IgM decreased on the induced cells whereas that of BI increased and that of HLA DR remained stable. No BL cells studied reacted with anti-IgD, with B2 or with LBI MAbs, either before or after induction. An EBV-negative line (BJAB) was compared to its Epstein-Barr virus (EBV)-converted subline (BJAB/B95). Both EBV-positive and EBV-negative lines gave comparable results. These data demonstrate that BL cells could be moved along their differentiation pathway by chemical inducers and suggest that BLA represents a new glycolipid marker of early B-cell differentiation.     

Cell phenotype dependent expression of MHC class I antigens in Burkitt's lymphoma cell lines.

Burkitt's lymphoma (BL) is a highly malignant B cell tumor characterized by three types of chromosomal translocation which constitutively activate the c-myc oncogene by juxtaposing it to Ig coding sequences. Epstein-Barr virus (EBV) infection, hyperendemic malaria and HIV-caused immunosuppression are thought to contribute to the pathogenesis of the tumor. Cell lines derived from EBV carrying and EBV negative BLs often show altered MHC class I antigen expression. The defects include a lower expression of all HLA class I antigens compared to EBV transformed normal B-blasts, and selective down-regulation of certain HLA-A and HLA-C alleles. As a consequence BL cells are often resistant to cytotoxic T lymphocyte (CTL) mediated destruction. Alleles selective down-regulations are found only in cell lines that maintain the tumor cell phenotype while shift towards a more activated 'B-blast like' phenotype is accompanied by HLA class I up-regulation. A similar pattern of HLA class I expression can be found in a subpopulation of germinal center B cells which express a 'BL like' phenotype. Our findings suggest that the HLA class I expression of BL cells reflects the characteristics of the normal B cell precursor and is probably not the result of immune selection.     

Karyotypic patterns in acute mixed lineage leukemia

We performed cytogenetic and immunologic studies of blast cells from 13 children with acute mixed lineage leukemia (AMLL) to discern patterns of chromosome alteration and antigen expression that would assist in classification of this disease entity. Six patients with 11q23 translocations--including four with the t(11;19), one with the t(9;11), and one with the t(1;11)--were characterized by a young age and hyperleukocytosis. A B cell-associated antigen (CD19) and HLA-DR antigens were expressed by blast cells from all patients; only one case was positive for the common acute lymphocytic leukemia antigen (CALLA, CD10). A myeloid-associated antigen (CD13) was expressed by blast cells from one patient at diagnosis and from another at relapse; it was also expressed by cells from the remaining four patients after brief in vitro culture without addition of differentiating agents. Four patients with t(9;22)(q34;q11) were characterized by an older age and hyperleukocytosis. Each of these cases was positive for CD13, CD19, and HLA-DR, and three were positive for CALLA. The 11q23 translocation was associated with CALLA- ALL marked by a myeloid phenotype, whereas the t(9;22) occurred in cases of acute myeloid leukemia with a CALLA+ lymphoid phenotype. One case had a 7q35-q36 translocation, which involves the region of the T cell receptor beta-chain gene. Our results suggest that karyotypic alterations can be used to refine the classification of AMLL.     

A monoclonal antibody with anti-Burkitt lymphoma specificity. I. Analysis of human haematopoietic and lymphoid cell lines

38-13 is a hybridoma-produced monoclonal rat IgM which appears to define a Burkitt's lymphoma-associated antigen (BLA). In this paper, we described the reactivity of 38-13 with a panel of human haematopoietic and lymphoid cell lines. In indirect immunofluorescence (IF) assays, 15 of 26 Burkitt's lymphoma (BL) lines studied were clearly stained with 38-13 (from 13 to 100% positive cells) by microscope, with varying numbers of heavily labelled cells. In these positive cell lines, fluorescence-activated cell-sorter (FACS) analysis demonstrated that BLA was actually present on all the cells. Positive BL included Epstein-Barr virus (EBV) genome-carrying lines and EBV-negative ones; thus, BLA is not related to the presence of EBV. Most of the 15 BL cells that reacted with 38-13 contained a typical t(8;14) translocation, but had variant translocations such as t(2;8) and t(8;22). The cells were derived from BL patients of different geographical origins and clinical features. Four BL lines were poorly stained and seven were negative with 38-13 in IF assays. The 32 EBV-positive lymphoblastoid cell-lines (LCL) studied were negative. In three line pairs, consisting of a tumor line and an LCL from the same patient, only the BL line was demonstrated to react with 38-13. A series of non-BL cells, including haematopoietic, lymphoid and solid tumor lines, all failed to react with 38-13. Various attempts to modulate the expression of BLA on BL cells were unsuccessful. However, it cannot be ruled out that BLA is actually a transient B-cell differentiation marker.     

The role of natural-killer-cells in the pathogenesis of epstein-barr virus-associated burkitt-lymphoma in a scid mouse model

Epstein-Barr virus (EBV) is associated with both benign and malignant lymphoproliferative processes. Recently, mice with severe combined immunodeficiency (SCID) have been described that develop EBV-induced lymphomas when inoculated with peripheral blood lymphocytes from EBV-seropositive individuals. To investigate the pathogenesis of EBV-associated Burkitt lymphomas, we intraperitoneally inoculated SCID mice with cells from EBV-infected Burkitt lymphoma (BL) cell lines. In general, cells from BL lines developed into BL-like tumors. Certain BL cell lines, however, were not particularly tumorigenic in these animals. Antibody capable of depleting mice of natural killer cells (anti-asialo GM1) favored the development of these Burkitt lymphomas. The pathogenetic implications of this animal model for human disease is discussed.     

末梢血中EB病毒潜伏感染的静息B细胞模型的建立

目的 应用EB病毒(EBV)感染末梢血CD5⁺B细胞,建立体内储存EB病毒的静息B细胞模型。方法 应用磁珠技术分离脐胎血中的CD5⁺B细胞;从新酶素耐性的Atada-EBV细胞中提取EB病毒对CD5⁺B细胞进行转染制备CD5⁺B/EBV细胞;流式细胞仪、Western blot法检测CD5⁺B/EBV细胞的病毒免疫表型和基因表型;免疫荧光双重染色BrdU、Ki67和EBNA2检测CD5⁺B/EBV细胞增殖活性。结果 流式细胞仪检测显示CD5⁺B/EBV细胞病毒免疫表型CD20、CD80、CD38和CD86表达增强,CD23没有表达;CD5⁺B/EBV细胞EBNA2和LMP1病毒相关潜伏感染基因显著增高;免疫荧光染色检测EBNA2阳性的CD5+B/EBV细胞可见BrdU阴性细胞。经因子激活CD5⁺B/EBV后可见Brdu阴性细胞中有Ki67阳性细胞存在,而在EBNA2阳性细胞中可见Ki67阴性细胞,验证CD5⁺B/EBV细胞中静息B细胞的存在。结论 本研究建立的CD5⁺B/EBV细胞具有体内EBV潜伏感染的静息B细胞的特征。     

EBNA promoter usage in EBV-negative Burkitt lymphoma cell lines converted with a neomycin-resistant EBV strain

Latent Epstein-Barr virus (EBV) uses two alternative strategies to express the Epstein-Barr nuclear antigens (EBNAs). Resting normal B cells harboring latent virus and Burkitt's lymphoma (BL) cells use monocistronic messages generated from the Q promoter (restricted strategy). EBV-transformed immunoblasts express all EBNAs by using giant messages generated from the W/C promoter (full program). Whether the virus establishes the restricted program on primary infection of a BL cell (or its progenitor) or, alternatively, whether such cells are generated by phenotypic down-regulation from the immunoblast is unclear. We found previously that conversion of EBV-negative BL lines to EBV-positive sublines required repeated exposure to large virus doses. The converted sublines used the full program. However, the possibility that cells with a full program had a selective advantage during the long period of in vitro passage could not be excluded. We therefore infected EBV-negative BL lines with recombinant EBV carrying a neomycin resistance marker. Most convertants of the 12 lines tested were positive for YUK splicing, indicative of the full program, but some were also positive for the restricted QUK splice program. One convertant DG75 line showing both YUK and QUK was cloned and gave rise to stable QUK users. We conclude that EBV infection of established BL lines can give rise to subclones with either the full or the restricted program. The fact that all EBVs carrying BL lines use the restricted program in vitro may be a consequence of immunoselection.     

Expression of EGFR and follicular dendritic markers in lymphoid follicles from patients with Castleman's disease

This study investigated the useful morphologic and immunophenotypic findings for the diagnosis of Castleman's disease (CD). We focused on the distribution and expression of follicular dendritic cells (FDC) in lymphoid follicles from patients with CD. Eleven CD cases of the hyaline vascular (HV) variant and six cases of the plasma cell (PC) variant were studied using tissue microarray and paraffin resistant monoclonal antibodies CD21, CD35, and EGFR, a new novel marker of FDC, as well as an antibody against human herpes virus 8 (HHV8). Epstein-Barr virus (EBV) was detected by means of in situ hybridization with a fluorescein isothiocyanate-labeled EBV-encoded RNA (EBER) specific oligonucleotide. The FDC network of the PC variant (n=4) was similar to that seen in normal or reactive germinal centers. In contrast, all HV variants and 2 cases of the PC variant were either expanded, disrupted, or exhibited multiple tight collections of FDC both in germinal centers and in mantle zone lymphocytes. The expanded mantle zone lymphocytes were CD20+, Bcl2+, PAX5+, and MUM1- with less number of CD3+ T cells admixed. Other features of the HV variant included follicular regression and vascular ingrowth of the germinal centers, whereas features of the PC variant were follicular hyperplasia and interfollicular plasmacytosis. In addition, EBV infection was positive in three CD cases, and one case had co-expression of HHV8 and EBV infection. Taken together, we found immunophenotypic differences of mantle zone lymphocytes and FDC network patterns of lymphoid follicles in CD. Thus, we conclude that these differences are relevant for the differential diagnosis of the two histopathologic variants of CD.     

Subgroup analysis of BL-cell lines with polyclonal BL-specific antibodies

A Burkitt's lymphoma (BL)-specific antibody (anti-GP70), previously described, was used to analyse 22 different BL-cell lines. The results indicated specificity of antibodies to lines that contain both surface membrane Ig (SmIg) and cytoplasmic Ig (CyIg). BL-cell lines derived from more immature B cells that do not have SmIg but rather have only CyIg were negative. A comparative study with antibody to common ALL antigen (CALLA) and to another BL-specific antigen (BLA) revealed coexpression of GP70 with those two antigens, but no identity between them.     

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.     

EBV-negative and -positive Burkitt cell lines variably express receptors for B-cell activation and differentiation

The expression of receptors for proliferation and differentiation factors was analyzed by indirect immunofluorescence on 29 Burkitt lymphoma (BL) cell lines previously classified into 3 groups on the basis of their reactivity with 8 monoclonal antibodies (MAbs), including anti-CALLA, BL13 and TU1. BL13 and HB5 antibodies recognize different epitopes of the EBV/CR2 receptors. The determinant recognized by BL13 has been previously shown to be expressed only on cell lines of the first two groups, supposed to derive from the germinal center and to be negative on a third group of lines of putative BM origin and established from sporadic cases of BL. In contrast, and as expected from its reactivity on normal B cells in the BM or in the lymph nodes, HB5 antibody reacts with all BL lines except one. The receptor for transferrin is expressed on the 29 lines. Two new MAbs, Bac-1 and B1H5, could recognize respectively receptors for BCGF1 and BCGF2. Bac-1 reacts with 15 of 17 BL lines belonging to the first two groups and 7 of 12 BL lines of the third group; 14 of 15 EBV + lines express Bac-1. No BL line expresses B1H5. The IL2 receptor is weakly expressed on 5 EBV + cell lines and one EBV (-) line. All delta are BCGF1-positive. The almost constant expression of BCGF1 receptor on EBV + cell lines is the only strict relation between the expression of receptors for growth factors and their characteristics (i.e. EBV association, translocation, ethnic origin and clinical presentation). The maturation stage or the origin of BL cell lines in relation to the expression of growth factor receptors and the functional significance of these receptors will be discussed.