A human B cell differentiation antigen selectively expressed on mature B cells identified by a monoclonal antibody (HB-2)

A monoclonal IgM antibody (HB-2), produced against a membrane antigen on the Raji, B cell line, reacted by indirect immunofluorescence with 2 to 40% of lymphoblasts from the B cell lines, Raji, Daudi, SN-1036, and SB but not with other types of cell lines, including pre-B, myeloid, melanoma, or T cells. HB-2 antibody reacted with 10 ± 3% of normal blood mononuclear cells, and was unreactive with monocytes, granulocytes, platelets, or erythrocytes. Two-color immunofluorescence revealed that HB-2 antigen expression was confined to cells bearing surface Ig. An interesting finding was the fact that 25% of plasmablasts induced by pokeweed mitogen also expressed the HB-2 antigen. However, pre-B and plasma cells from normal bone marrow did not express the HB-2 antigen either on their membrane surface or in their cytoplasm. Analysis of 85 leukemias revealed that the HB-2 antigen was expressed on acute and chronic B cell leukemias and Burkitt's lymphomas, but not on malignacies of pre-B, T, myelocytic, or plasma cells. The results suggest that expression of the HB-2 antigen is confined to mature B cells.     

IL-2 receptor {alpha} chain expression during early B lymphocyte differentiation

The IL-2/IL-2 receptor (IL-2R) has been studied intensivelybecause of its potential function in the development and regulationof the immune system. The IL-2R chain has been shown to be expressedon CD4^–CD8minus; thymocytes and activated T and B cells.In this report, we show that IL-2R is also expressed on precursorB cells in the bone marrow. Its expression is initiated by functionalrearrangement and expression of Ig µ heavy chain geneand is down-regulated when immature B cells mature and expressIgD. Its potential function in early B cell differentiationis discussed in comparison with its role in thymocyte differentiation.     

Evidence that FMC7 is a human B cell differentiation antigen.

FMC7 is a 105-kDa B cell restricted antigen which is expressed on about 50% of adult human peripheral blood B cells. Seven to ten days following booster immunization with tetanus toxoid, peripheral blood contains a small population of B cell blasts with an increased density of FMC7. The majority of anti-tetanus toxoid antibody secreting cells (both IgM and IgG) are however found in FMC7- B cells. These data indicate that upon in vivo B cell activation FMC7 expression initially increases. B cells involved in antibody secretion have lost the FMC7 determinant.     

Augmentation of B7 expression by herpes simplex virus antigen

B7 costimulation plays a major role in herpes simplex virus (HSV) immunity. Effects of immunoregulatory cytokines (interferon-gamma [IFN-gamma] and interleukin-10 [IL-10]) and HSV antigens on B7-1 (CD80) and B7-2 (CD86) expression on monocytes during HSV-2 infection were investigated using flow cytometry. Our results demonstrate that HSV-2 infection effects the expression of B7 isoforms (B7-1 and B7-2) on monocytes in two ways, with opposing outcomes. It abrogates the IFN-gamma upregulation of B7-1 and B7-2 and also augments expression of B7-1 and B7-2 on monocytes through an IFN-gamma independent mechanism. The clinical significance of these opposing effects may be related to the pathogenesis of recurrent HSV disease in immunocompetent hosts.     

Terminal defects of B lymphocyte differentiation

In humans several abnormalities can occur during terminal B cell differentiation, leading to primary humoral immunodeficiencies. A recent study provided evidence of a qualitative defect of the affinity antibody maturation in some patients affected with common variable immunodeficiency syndrome, the molecular basis of which remains unknown. Several genetic defects in class switch recombination leading to a hyper-IgM syndrome have recently been delineated. Besides the well-known role of CD40-CD40 ligand interaction, they definitively demonstrate the requirement of CD40-mediated nuclear factor kappa B activation and the essential role of a newly described molecule, the activation-induced cytidine deaminase, in B cell terminal differentiation.     

Evidence for membrane antigen(s) specific for human B lymphoblasts

IgM antibodies against surface antigen(s) of a subpopulation of PHA-induced lymphoblasts have been found in the sera (20%) of infectious mononucleosis patients by indirect immunofluorescent staining. Maximum staining was obtained at 3°C. Evidence is presented that this subpopulation is derived from B lymphocytes. Common antigenic determinants were also present on four human lymphoid cell lines.     

Interaction of hepatitis B surface antigen (Australia antigen) with membrane vesicles of Pseudomonas aeruginosa.

A lysogenic strain of Pseudomonas aeruginosa was cultured from the dialysis fluid of a patient on chronic hemodialysis treatment whose blood contained hepatitis B surface antigen (HB8Ag). When this bacterium was incubated for 4 to 7 days with serum containing HB8Ag or with purified HB8Ag, a loss of the HB8Ag-specific immunological reactivity was observed. Bacteriophages can be induced from the isolated P. aeruginosa with mitomycin C; the phages, after purification on CsCl gradients, also lyse P. aeruginosa strain 25102 (ATCC). Subsequent to gradient centrifugation of the lysate, a fraction was found with a density around 1.40 g/ml that inactivated HB8Ag after a 4-h incubation at 37 C as determined by counterelectrophoresis and hemagglutination inhibition. The activity was not found in appreciable amounts in other gradient fractions. The electron microscope shows that the active fraction contains envelope vesicles of 45 to 60 nm in diameter. In spite of their loss in HB8Ag activity, the HB8Ag particles (22nm) appeared morphologically intact. These findings suggest that an enzyme(s) is present in the vesicle fraction which inactivates antigenic determinants on HB8Ag particles. Thus, the presence of these bacteria in environments such as feces, dialysis tanks, and contaminated drinking water may prevent the detection of HB8Ag.     

Expression of binding sites for peanut agglutinin during murine B lymphocyte differentiation.

Haemopoietic cells from several murine organs were examined for the presence of peanut agglutinin (PNA) receptors. Foetal liver and adult bone marrow contained a number of cells, which were PNA+ but did not stain with conventional T and B markers. Some of the PNA+ cells, however, were pre-B cells, as shown by the presence of cytoplasmic IgM in the absence of cell surface Ig. Cells in the T lineage retained their PNA receptors during maturation, although these became masked by sialic acid on mature peripheral blood T cells. Cells of the B lineage, in contrast, gradually lost their PNA receptors as they matured from pre-B to mature surface immunoglobulin positive B lymphocytes.     

Expression of the Epstein-Barr virus (EBV)-encoded membrane antigen (LMP) increases the stimulatory capacity of EBV-negative B lymphoma lines in allogeneic mixed lymphocyte cultures

Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) lines are poor stimulators in allogeneic mixed lymphocyte cultures compared to EBV-transformed lymphoblastoid cell lines derived from the same individuals. We have previously shown that the stimulatory capacity of the tumor cells is increased after EBV conversion (Avila-Carino et al., Int. J. Cancer 1987. 40: 691). As a first step towards the identification of the viral gene product responsible for this change we have studied the influence of the EBV latent membrane protein (LMP) on the stimulatory capacity of the EBV-negative BL lines BL41 and DG75 and the B lymphoma line BJAB. Four LMP-transfected sublines of BL41, four DG75 LMP transfectants and one LMP-transfected subline of BJAB showed a significantly stronger stimulatory capacity than the original line. The effect was directly proportional to the amount of LMP detected in each transfectant but was not due to reactivation of LMP-specific memory cells since lymphocytes from EBV-seropositive and -seronegative individuals responded equally. In order to define the relation between LMP expression and induction of stimulatory capacity, DG75 was transfected with constructs containing the LMP gene under the control of an heat-shock promoter. The peak of LMP expression in heat shock-treated cells preceded the appearance of stimulatory capacity by 6-12 h suggesting that critical amounts of the protein may be required to induce the phenotypic change recognized by the T cells. LMP influenced in a dose-dependent manner the expression of the adhesion molecules LFA-1, LFA-3 and ICAM-1 and B cell activation markers CD23 and CD39 in transfected sublines of BL41, but did not affect the expression of these markers in the DG75 and BJAB cell line. All LMP-expressing transfectants showed an increased capacity to form conjugates with unprimed allogeneic lymphocytes.     

Effect fixation on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue

The identification of lymphoid surface membrane antigens in tissue sections using immunohistochemical techniques is becoming increasingly important for the diagnosis and classification of lymphoproliferative disorders. Many of the lymphocyte specific monoclonal antibodies used, however, can only be applied to frozen tissue sections. In this paper we report the successful application of a number of these antibodies to paraffin processed tissue utilizing alternative fixatives and the highly sensitive immunogold-silver staining method. The best fixatives for this purpose were formol dichromate, periodate-lysine-paraformaldehyde (PLP) and a novel fixative formed from the addition of a dichromate solution to PLP.     

Direct helper T cell-induced B cell differentiation involves interaction between T cell antigen CD28 and B cell activation antigen B7.

Cognate interactions between major histocompatibility complex class II antigen (Ag)-reactive CD4+ T helper (Th) and Ag-presenting B cells induce first the activation of B cells and their subsequent differentiation into Ig-secreting cells (IgSC). The Th cell-associated homodimeric glycoprotein CD28 has been implicated as an important regulator of Th activation. Recently, B cell-associated early activation Ag B7 has been identified as a ligand for the CD28 molecule. In this study, we have examined using monoclonal antibodies (mAb) the roles of CD28 and B7 molecules during the Th-B cell cognate interactions leading to the differentiation of B7+ B cells. Anti-CD28 mAb 9.3 specifically inhibited proliferative responses of CD4+ T cells to both allogeneic B cells and soluble Ag-presenting autologous non-T cells. In addition, anti-CD28 mAb 9.3 inhibited Th-induced differentiation of alloantigen-presenting B cells into ISC. Similar inhibition of both Ag-induced Th activation and B cell differentiation into ISC was observed using mAb BB1 which recognizes a B cell-associated molecule B7. In contrast, non-cognate Th-independent exogenous interleukin 6-induced differentiation of B7+ B cells into ISC was not inhibited by mAb to either molecule. These results clearly demonstrate the involvement of CD28 on Th and its ligand B7 on B cells during cognate Th-B interactions leading to the differentiation of B cells. Furthermore, these results also suggest the development of new mAb-based therapeutic approaches for exaggerated B cell activation associated with certain autoimmune diseases such as systemic lupus erythematosus.     

Prostate-specific membrane antigen (PSMA) expression in breast cancer and its metastases

The present study was undertaken to investigate the expression of prostate-specific membrane antigen (PSMA) in normal breast tissues, in cancerous breast tissues and in distant metastases from patients with breast cancer. Immunohistochemical analysis was performed to determine PSMA expression and angiogenic activity using anti-PSMA mAb and anti-CD31 mAb respectively. Immunofluorescence staining was applied to confirm the exact co-localization of PSMA and CD31. We observed different patterns of PSMA expression between normal and cancerous tissues. Normal breast tissues showed PSMA expression only in normal glandular cells. However, primary breast tumors and distant metastases showed PSMA expression in tumor cells and in tumor-associated neovasculature. PSMA score group status in primary breast tumors was significantly associated with histologic type and tumor grade (p?=?0.026 and p?=?0.004 respectively). Distant metastases showed higher PSMA expression in tumor-associated neovasculature comparing with primary tumors. Moreover, brain tumor-associated neovasculture had significantly higher expression of PSMA comparing with bone tumor-associated neovasculture. The localized binding of PSMA mAb to the neovasculature endothelium was confirmed with the double Immunofluorescence staining. ⁶⁸Ga-PSAM imaging of a patient with metastatic breast cancer showed strong tracer uptake in all known skeletal metastases. To the best of our knowledge, this study is the second one that has assessed PSMA expression in a large number of breast cancer patients. Our findings showed that PSMA is particularly expressed in tumor-associated neovasculature of breast tumors and its distant metastases, thus enhancing the evidence on the potential usefulness of PSMA as a therapeutic vascular target.     

Identity of PB76 differentiation antigen and lymphocyte alkaline phosphatase

Alkaline phosphatases (APases, EC 3.1.3.1) are ecto-enzymes bound to cell membranes by a phosphatidyl-inositol anchor. We have previously shown that APase is present on activated murine B cells and its expression correlates with the process of B cell differentiation into immunoglobulin secretion. Recently, a monoclonal antibody (mAb), G-5-2, that recognizes a 76-kDa molecule preferentially expressed on the surface of pre-B and plasma cells (PB76) was described. Some features shared by APase and PB76 differentiation antigen suggest that the G-5-2 mAb might be specific for lymphocyte APase. Here, we have analyzed this possibility and found an absolute correlation between PB76 expression in cells and their APase activity. Although PB76 has been described as a B cell-restricted marker, PB76 is also expressed on some T cells, such as the YAC-1 T cell lymphoma, that are known to bear APase. Treatment of YAC-1 cells with phosphatidylinositol-specific phospholipase C resulted in a quantitatively correlated removal of both APase and PB76 antigens. Moreover, we demonstrate that PB76 antigen has APase activity using an enzyme-antigen immunoassay with the G-5-2 mAb. We conclude that PB76 and lymphocyte APase are one and the same antigen.     

Dual pathway of B lymphocyte differentiation in vitro

Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and Θ surface markers and autoradiography after [³H] thymidine uptake. The changes observed were related to biochemical parameters such as incorporation of [³H] thymidine into DNA, Ig biosynthesis and secretion. Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig. Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with [³H] thymidine. We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.     

Markers of B lymphocyte differentiation in the chicken

A study was made of the ontogeny and tissue distribution of seven antigen systems associated with B lymphocyte development. A panel of seven monoclonal antibodies (MAbs) directed against avian bursal and peripheral B lymphocytes was developed. The spectra of cellular reactivity and size of respective antigens indicate that these MAbs appear to react with determinants of chicken B lymphocytes that had not been previously described. Immunofluorescence analysis of cell surface antigens using this panel indicated that dynamic changes in antigen expression are associated with early ontogeny and maturation of the B cell lineage. The yolk sac contained subpopulations of hematopoietic cells reactive with three MAbs (CB10, CLA3 and Hy86b5) which possibly mark the pre-bursal stem cell population. At or near the time of surface IgM expression in the embryonic bursa, B cells expressed antigens detected by the CB7, CB8, CB9 and CB11. In the juvenile chicken, CB7 reacted with immature bursal lymphocytes, while CB8, CB9 and CB10 reacted with bursal lymphocytes and a subpopulation of peripheral B lymphocytes. The antigens defined by Hy86b5 and CB11 were expressed on mature B lymphocytes but on only a subpopulation of bursal lymphocytes. The MAb CLA3 detected an antigen expressed on all leukocytes but not erythroid cells. Expression of some of these antigens by cells of the myelomonocytic lineages suggests a possible functional or ontogenetic relationship between the B lymphocyte and myeloid lineages.     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

Structural similarity between a primitive chordate membrane heterodimer and lymphocyte antigen receptors

Botryllus schlosseri is a colonial tunicate that shared a common ancestor with the lineage leading to mammals about 450 million years ago, and flourishes today along the California coast. Prior studies of Botryllus populations have demonstrated the presence of a co-dominantly expressed, highly polymorphic histocompatibility locus (Fu/HC) controlling the acceptance (fusion) or rejection of new individuals into a parabiotic colony. Intercolonial blood cell contact, and recognition of self/not self, precedes both fusion and rejection reactions. Efforts to understand the evolution of the immune system necessitate study of cell surface molecules involved in cell-cell recognition events in primitive species. In mammals, birds, amphibians, and fishes clonally distributed lymphocyte surface molecules that are responsible for antigen recognition (B cell immunoglobulins and T cell receptors) can be distinguished by the disulfide linkage that pairs two or more polypeptides containing constant and variable regions. We have identified a disulfide-linked, heterodimeric (alpha beta) cell surface molecule in Botryllus with biochemical resemblance to mammalian lymphocyte antigen receptors. Observed charge variants of constituent chains of the tunicate protein described here do not correlate with Fu/HC allelic diversity. Both chains of this heterodimer can be resolved into several isoforms which are not based upon post-translational carbohydrate or phosphate additions. Comparisons of iodinated tryptic peptides from two beta chain isomorphs reveal one distinct and several common peptides.     

Specific binding of poly(I)-poly(C) to the membrane of murine B lymphocyte subsets.

Indirect immunofluorescence revealed that 13% of BALB/c and 33% of CBA spleen cells of B type carry specific binding sites at their surface for double-stranded poly(I).poly(C). Pretreatment of BALB/c spleen cells with anti-mouse immunoglobulin serum increased the number of B cells capable of binding poly(I).poly(C) indicating the existence of a second B lymphocyte subpopulation carrying masked poly(I).poly(C)-binding sites. Pretreatment of the cells with mitogenic doses of either lipopolysaccharide (LPS) or single-stranded polynucleotides, e.g. poly(I) or double-stranded poly(A).poly(U), failed to affect binding of poly(I).poly(C) to the cells. Poly(I).poly(C) converts small poly(I).poly(C)-binding lymphocytes into lymphoblasts carrying poly(I).poly(C)-binding sites. Lymphoblasts derived from LPS-stimulated cells do not carry poly(I).poly(C)-binding sites. Thymocytes or splenic T cells failed to bind poly(I).poly(C). As measured by thymidine uptake, CBA mice containing a higher percentage of poly(I).poly(C)-binding cells, are high responder mice to poly(I).poly(C), compared with low responder BALB/c mice.     

Differential expression of the HECA-452 antigen (cutaneous lymphocyte associated antigen, CLA) in cutaneous and non-cutaneous T-cell lymphomas

The monoclonal antibody HECA-452 identifies an antigen that is primarily expressed on high endothelial venules, the preferred site of lymphocyte extravasation in lymphoid tissues, and also on a subpopulation of myelomonocytic cells and some T-cells. We investigated the expression of the HECA-452 antigen, also called the cutaneous lymphocyte associated antigen, in primary cutaneous and primary non-cutaneous T-cell non-Hodgkin's lymphomas. The tumour cells of cutaneous T-cell non-Hodgkin's lymphomas were positive in 53% of cases, while only 5% of the non-cutaneous lymphomas were positive. These differences were also present in morphologically identical tumours. Thus, the tumour cells in six out of 10 primary cutaneous anaplastic large cell T-cell lymphomas were positive, while they were positive in none of 24 primary non-cutaneous anaplastic large cell T-cell lymphomas. In general, primary cutaneous and primary nasal T-cell non-Hodgkin's lymphomas were devoid of HECA-452 positive high endothelial venules, whereas most nodal T-cell non-Hodgkin's lymphomas contained HECA-452 positive high endothelial venules. These observations suggest that the HECA-452 antigen might be related to a skin-associated type of lymphoid tissue and to lymphomas originating in the skin. However, the results of HECA-452 expression in secondary sites, and the clinical data of the primary cutaneous large cell lymphomas did not support the concept that HECA-452 is functionally involved in homing to the skin, or that loss of the HECA-452 antigen is related to tumour progression of primary cutaneous T-cell lymphomas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sialylation patterns of lymphocyte function-associated antigen 1 (LFA-1) differ between T and B lymphocytes

Lymphocyte function-associated antigen 1 (LFA-1) was immunoprecipitated from various types of surface-radioiodinated murine lymphocytes, and analyzed by two-dimensional polyacrylamide gel electrophoresis. LFA-1 alpha and beta chains from splenic B lymphocytes had the same apparent molecular weights as, but distinct isoelectrofocusing patterns from, their counterparts from thymocytes or splenic T lymphocytes. The splenic B lymphocytes lacked a basically charged population of alpha chain, while the thymocytes and the splenic T lymphocytes showed both the acidic and the basic portions. Furthermore, the beta chain of the former migrated more towards the acidic end than that of the latter. No difference was found between LFA-1 molecules of the same lineage of cells from several strains of mice whose H-2 haplotypes were different from one another. When murine lymphocyte lines were examined, LFA-1 with various isoelectrofocusing patterns were recognized. The charge difference again reflected the difference in lymphocyte lineage, but in a more exaggerated manner than that seen with cells from mice. The average acidity of both chains of LFA-1 decreased in the order of B cell lines, pre-B cell lines and T cell lines. The lineage-dependent charge difference of either chain disappeared after neuraminidase treatment of LFA-1, indicating that lymphocyte differentiation was accompanied by changes in LFA-1 sialylation.