SC3 monoclonal antibody defines a novel specific human B-cell surface antigen differentially expressed on B-cell leukaemias and lymphomas and involved in the proliferation of normal and malignant B lymphocytes

The monoclonal antibody SC3 was raised against the NK leukaemia cell line YTindi. It detected a 98-kDa surface antigen with weak expression on a restricted number of leukaemia cell lines under reducing conditions. SC3 mAb labelled 5-10% of normal peripheral blood lymphocytes corresponding almost exclusively to B lymphocytes, and 60-70% of tonsillar B cells. It did not react with erythrocytes, platelets or monocytes whereas it stained granulocytes. The aim of the present study was to examine the expression and functional effects of SC3 mAb reactive epitope on normal and malignant B cells. Most SC3+ B cells from healthy donors were CD23+, some co-expressed CD5 and CD27 and a few were CD38+. SC3 epitope was expressed exclusively by B-lineage malignant proliferations, including B-lineage ALL. Practically, all B-CLL studied expressed SC3 mAb reactive epitope although with variable intensity, while MCL and PLL were negative. Other low grade and high grade B-NHL were variably stained. SC3 mAb alone triggered the proliferation of CD2-depleted PBL and significantly increased the proliferation induced by suboptimal concentrations of LPS. This effect was much weaker with B-CLL cells but was increased after cross-linking with an anti-IgM antibody. The restricted expression pattern combined with molecular weight and functional data indicate that SC3 mAb may detect a novel B-cell antigen mostly expressed by early and naive B cells. Although its expression in B-cell malignancies was not limited to a single differentiation stage, it might confer specific functional characteristics to the positive malignant cells.     

In vitro production of interleukin 1 by normal and malignant human B lymphocytes

In this study, the capacity of normal and neoplastic B lymphocytes to release interleukin 1 (IL 1) has been investigated. Peripheral blood B cells from normal donors were isolated by depletion of E rosetting cells and by positive selection of cells expressing surface immunoglobulin (sIg) or the B1 marker. Peripheral blood B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) were purified by removal of E rosetting cells followed by complement-mediated cytotoxicity with selected monoclonal antibodies. All of the normal B cell suspensions and the large majority of the B-CLL cells produced in culture high amounts of IL 1 in the absence of any apparent stimulus. Control experiments ruled out that small numbers of monocytes in the B cell suspensions could represent the source of IL 1. These data support the contention that B cells participate to the immune response as accessory cells for T cell activation not only by physically presenting antigen, but also by releasing IL 1.     

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig^P) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig^M), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig^P and hCTLA4Ig^M had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig^P and hCTLA4Ig^M had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ, but did not suppress the expression of macrophage-derived cytokines, including TNF-α and IL-1β, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig^P is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig^M.     

Activation of human B lymphocytes by a particulate antigen

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.     

Phosphorylation of the B1 (CD20) molecule by normal and malignant human B lymphocytes

The B1 molecule (CD20) is a phosphoprotein found only on B lymphocytes. Multiple isoforms of the B1 molecule are expressed with Mr of 33,000, B1(33) and Mr of 34,500-36,000, B1(35). In this study it was found that nonproliferating B cells did not incorporate 32PO4 into B1 although phosphorylated class I histocompatibility molecules were easily detected. In contrast B1 isolated from proliferating or malignant B cells or B cell lines was heavily phosphorylated. Cross-linking B1 on the cell surface by antibody resulted in enhanced phosphorylation of B1 as did exposure to phorbol esters, and the membrane permeable diacylglycerol analog 1,2,-dioctanoylglyceron. B1(33) and B1(35) produced identical peptide maps following limited proteinase digestion. However, B1(35) contained both phosphoserine and phosphothreonine, while B1(33) only contained phosphoserine. In addition alkaline phosphatase was able to remove the phosphate residue(s) that resulted in generation of the B1(35) form of B1 but was unable to remove the phosphorylation of B1(33). These results suggest that phosphorylation of B1 molecules is associated with proliferation and that the different Mr forms of B1 result from the phosphorylation of B1 at different sites. Also, the finding that antibody binding to B1 generated a transmembrane signal may explain why antibody binding to B1 alters B cell function.     

Expression of neurotrophin receptors in normal and malignant B lymphocytes

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).     

Qc-1, a novel Q-region-controlled CTL determinant expressed on B lymphocytes

Using cloned cytotoxic T lymphocytes (CTL), we have identified a Q region controlled determinant with a unique strain and tissue distribution. Several strains that express the classically defined Qa-2 determinant and other Q region controlled determinants do not express the CTL determinant. In addition, strain BALB/cByJ, which does not normally express any Q region controlled cell surface determinant, expresses this new determinant. Cross-reactivity between the Q region controlled CTL determinant and a Kk region controlled class I product (probably H-2Kk) was observed. Finally, among lymphocytes, the CTL determinant is expressed preferentially (if not exclusively) on B cells, thus distinguishing it from all previously described Q region controlled determinants, which are expressed predominantly on T cells. We provisionally designate this novel Q region controlled CTL determinant Qc-1. The possibility that Qc-1 is recognized together with a self antigen is discussed.     

Leukotriene B4 enhances activation, proliferation, and differentiation of human B lymphocytes

Highly purified human tonsillar B lymphocytes at different stages of activation were incubated with leukotriene B4 (LTB4). As a key marker for activation, we used the CD23 Ag. LTB4 enhanced the CD23 expression on resting B cells in synergy with B cell-stimulating factors from 4% to 50%. Maximal effect of LTB4 was observed at 10(-10) M to 10(-12) M. LTB4 also augmented the S and M phase entries as well as Ig secretion in synergy with IL-2 and IL-4. In contrast, 5S,12S-dihydroxyeicosatetraenoic acid, an isomer of LTB4, and leukotriene C4 lacked these effects. The results indicate that LTB4 amplifies lymphokine-driven activation, replication, and differentiation of human B lymphocytes.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.     

Distinctive expression of the T4 antigen in normal and stimulated lymphocytes

Correlated light scatter and fluorescence flow cytometric analysis of human peripheral blood lymphocytes showed that the expression of the T4 antigen was higher in the larger lymphocytes than in the smaller lymphocytes. A similar expression pattern was observed for HLA Class I antigens but not for T3 and T8, whose expression was independent of cell size. Results with lymphocytes from spleen, lymph node, and tonsil were comparable to those of peripheral blood. Thymocytes, however, were smaller and expressed less T4 and T8 than peripheral lymphocytes. In studies of lymphocytes stimulated in vitro with allogeneic cells or pokeweed mitogen, two populations of T4-positive cells were observed: one of large cells expressing high amounts of T4 and one of small cells expressing low amounts of T4. Similar patterns were seen with T8, although less consistently. In contrast, the expression of T3 was the same in both large and small cells. The large cells expressing high amounts of T4 were not restricted to cells engaged in DNA synthesis or mitosis. This was established by selectively analyzing cells in the G0G1 phases of the cell cycle and by studying stimulated lymphocytes no longer undergoing proliferation. Taken together, these results suggest that immature T lymphocytes are small and express low amounts of T4 and T8. We postulate that as they differentiate, cell size and T4 expression increase proportionally, both parameters increasing even further after antigenic or mitogenic stimulation. The quantitative expression of T4, and probably of T8 but not of T3, is therefore intimately related to maturation and activation of lymphocytes, a fact that may conceivably be related to a functional role of these surface molecules.     

Expression of CD9 antigen on normal activated human B cells

The expression of the CD9 pre-B acute lymphoblastic leukemia (ALL)-associated antigen was studied. CD9-positive B cells were enriched in the in vivo-activated buoyant B cell population isolated from tonsils. Small tonsil B cells activated in vitro with either PWM, phorbol 12-myristate 13-acetate (TPA), or anti-Ig plus low Mr B cell growth factor (BCGF) also demonstrated increased CD9 expression. The peak of CD9 expression (30-40% positive cells) occurred after 4-6 days of activation. The kinetics of increased CD9 expression was similar to that of the 4F2 activation antigen. CD9 antigen expression on tonsillar B cells as well as on pre-B leukemic cell lines was associated with protein kinase C activation. Two phorbols that activate protein kinase C (TPA; phorbol 12,13-dibutyrate) induced expression of the CD9 antigen whereas a phorbol analogue that does not activate C kinase (4-alpha-phorbol 12,13-didecanoate) and an analogue that is a very weak agonist (phorbol 12-myristate 13-acetate-4-0-methyl ether) were unable to induce CD9 expression on tonsil B cells or on the cell lines. The effect of the anti-CD9 monoclonal antibody, DU-ALL-1, on B cell mitogenesis was studied. Dense or buoyant tonsillar B cells were cultured in the presence or absence of DU-ALL-1 antibody plus PWM, anti-Ig, and BCGF, DU-ALL-1 antibody did not inhibit or augment the mitogenic response of resting or activated B cells. These results indicate that the CD9 pre-B ALL antigen is present on a population of normal activated tonsillar B cells and that its induction of expression is associated with protein kinase C activation.     

Signal transduction through the B cell antigen receptor is normal in ataxia-telangiectasia B lymphocytes.

The rare human genetic disorder ataxia-telangiectasia (A-T) has multiple consequences including a variable degree of immunodeficiency. Khanna and co-workers (Khanna, K. K., Yan, J., Watters, D., Hobson, K., Beamish, H., Spring, K., Shiloh, Y., Gatti, R. A., and Lavin, M. F. (1997) J. Biol. Chem. 272, 9489-9495) evaluated signaling in Epstein-Barr virus (EBV) immortalized A-T lymphoblastoid cell lines (LCLs), derived from the B cells of A-T patients. They showed that A-T lymphoblastoid cells lack signaling through the B cell antigen receptor and concluded that the fault in A-T encompasses intracellular signaling in B cells. However, it is established that EBV latent membrane protein 2A (LMP2A) blocks signaling in EBV-bearing cells by interaction with cellular tyrosine kinases. To test whether the reported fault in A-T B cells was not inherent in A-T but the result of influence of wild-type EBV, we derived A-T LCLs with wild-type or LMP2A-deleted EBV and studied signaling in these cells in response to cross-linking the B cell antigen receptor. We report that intracellular calcium mobilization and tyrosine phosphorylation in LMP2A-depleted LCLs derived from A-T patients is indistinguishable from that in LMP2A-depleted LCLs derived from normal controls. Further, signaling is blocked similarly in A-T and normal lymphoblastoid cells bearing wild-type EBV. In conclusion there is no evidence of any defect in B cell receptor signal transduction in A-T B cells.     

Direct activation of human B lymphocytes by Candida albicans-derived mannan antigen

We have studied the activation of human resting B cells by a carbohydrate antigen, mannan, with a polymannose branched repetitive structure. Mannan has been extracted from the cell wall of the Candida albicans yeast. For this purpose, dense G0 B lymphocytes were purified from tonsils. Mannan antigen was shown to trigger B cell activation, since an increase of cell volume and RNA synthesis occurred. B cell proliferation was observed following addition of recombinant interleukin 2, but not following addition of recombinant interleukin 4 or low-molecular-weight BCGF. The B cell activation appears to be mannan-specific since B cells obtained from mannan-sensitized subjects but not from unsensitized subjects were responsive. The observation that mannan antigen can directly activate specific dense B lymphocytes can be related to the previous observation that the in vitro anti-mannan antibody production does not require a cognate T-B cell interaction.     

Phorbol ester induces tyrosine phosphorylation in normal and abnormal human B lymphocytes

Tumor-promoting phorbol esters have been found to bind and activate phospholipid/Ca2+-dependent or C-kinase, and several of their effects, including proliferative responses in lymphocytes, have been assumed to be related to activity of this enzyme. However, phorbol esters have also recently been found to stimulate tyrosine phosphorylation in certain other cell types, and we therefore studied tyrosine kinase activity in normal and chronic lymphocytic leukemia (CLL) peripheral blood B lymphocytes stimulated with phorbol ester. High levels of tyrosine labeling were observed in unstimulated cells with major endogenous substrates of 75K, 66K, 43K, and 28K in Triton-soluble material, and of 56K to 61K in Triton-insoluble material; this profile was essentially similar in normal and CLL B cells. Treatment with phorbol ester for time periods varying from 20 min to 48 hr led to qualitative increases in tyrosine labeling of these phosphoproteins, as measured both in vitro and in intact cells "in vivo." Although the relative abundance of tyrosine phosphorylation as a percentage of total labeling was variable due to concomitant enhancement of serine and threonine phosphorylation, exogenous peptide substrate assays confirmed the increased tyrosine kinase activity quantitatively. Enhanced tyrosine phosphorylation was succeeded or accompanied in both normal and abnormal B cells by cellular activation, as judged by increased [3H]thymidine uptake, and terminal differentiation of CLL cells. These findings provide further evidence implicating tyrosine kinases in B lymphocyte activation.     

Granulocyte-macrophage colony-stimulating factor as an autocrine survival factor for mature normal and malignant B lymphocytes

The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.     

A new B cell differentiation antigen (BDA) on normal and leukemic human B lymphocytes that is distinct from known DR (Ia-like) antigens.

A newly defined human B cell differentiation antigen, designated as BDA, has been defined and partially characterized. BDA is expressed on normal human B cells and lymphocytic leukemia cells at all stages of known differentiation (pre-B cells to plasma cells). It is distinct from DR (Ia-like) determinants and other known B cell surface constituents.     

Effects of a placental hormone, human chorionic somatomammotropin on normal and malignant cells

Studies were made on the effects of human chorionic somatomammotropin (HCS) on normal and malignant cells and tissues in Swiss mice. HCS was found to produce a significant increase in the fresh weight of normal liver, kidney, spleen, testis, ovary and uterus. Total cell counts of leukocytes and erythrocytes were elevated. The percentage of granulocytes in blood was found to be increased and the percentage of lymphocytes was decreased following HCS treatment. HCS stimulated the growth of ascitic Ehrlich's carcinoma and Sarcoma 180, and nucleic acid synthesis by these tumor cells. A depression in the mitogen induced blastogenesis of lymphocytes was also noted following HCS treatment.