B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes

The characterization of a new B cell-specific antigen (B4) is described in this report. With the use of a monoclonal antibody to B4, it was shown that B4 is present on B cells isolated from peripheral blood and lymphoid organs, on cell lines derived from normal and malignant B cells, and on tumor cells isolated from patients with B cell-derived neoplasms. B4, in contrast, was not detected on normal, activated, or malignant cells of T or myeloid origin. The B4 antigen is distinct from known B cell antigens, including sIg, Ia, B1, B2, Fc, and C3. Examination of mitogen-stimulated B lymphocytes suggests that the B4 antigen initially increases with B cell activation and then is lost at the terminal stage of B cell differentiation. Moreover, the observation that B4 is expressed on almost all early B cell tumors suggests that it may precede B1, CALLA, cytoplasmic mu, and B2 in early B cell ontogeny.     

Characterization of a human B cell-specific antigen (B2) distinct from B1

A human B lymphocyte-specific antigen (B2) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence and quantitative absorption, B2 was shown to be expressed exclusively on Ig+ B cells isolated from peripheral blood and lymphoid tissues. In contrast, B2 was not found on monocytes, resting and activated T cells, Null cells, or granulocytes, nor was it found on cell lines or tumor cells of T cell or myeloid origin. Functional studies demonstrated that only B2 antigen-positive splenocytes could be induced to differentiate into plasma cells under the stimulus of pokeweed mitogen, further confirming the B cell specificity of B2. It was then demonstrated that the B2 antigen was distinct from the previously described B cell-surface determinants including surface immunoglobulin, Ia-like antigens, and Fc and C3 receptors. More importantly, the B2 antigen has been clearly shown to be distinct from the previously described B cell-specific antigen, B1, by its m.w. and expression on normal and malignant B lymphocytes. The distinct distribution of B2 on normal and malignant lymphocytes supports the notion of B cell heterogeneity and provides further evidence for existence of subpopulations of human B lymphocytes.     

A new human T-cell differentiation antigen: Unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.PJM is a Junior Faculty Clinical Fellow of the American Cancer Society.     

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.     

A novel human T cell antigen preferentially expressed on mature T cells and shared by both well and poorly differentiated B cell leukemias and lymphomas

A new lymphocyte differentiation antigen shared by all normal T cells and some malignant B cells was defined by a monoclonal antibody designated 12.1. This antibody reacted with all peripheral blood T cells but not with normal B cells and B cell lines. Analysis with a fluorescence activated cell sorter showed that the expression of 12.1 antigen changes during T cell maturation. Most thymocytes, blasts of acute T cell leukemia, and cells from established leukemic T cell lines bear a small amount of 12.1 antigen. In contrast the majority of peripheral blood T cells, activated T cells, and leukemic T cells of the Sezary syndrome bear a large amount of 12.1 antigen. Unexpectedly, antibody 12.1 reacted with leukemic cells from most patients with B-type chronic lymphocytic leukemia (CLL) and some patients with lymphosarcoma cell leukemia (LSCL). Among these leukemias, expression of the 12.1 antigen was not correlated with the stage of B cell maturation, with the amount of surface immunoglobulin on the cells, or with the presence or absence of monoclonal gammapathy. In a comparative serologic analysis the antigen defined by antibody 12.1 was distinct from the p67 T cell antigen (defined by monoclonal antibody 10.2) that is also known to be expressed by B-type CLL cells.     

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.     

A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside

Monoclonal antibody D1.1 originally prepared against the B49 cell line derived from a rat brain tumor was shown to react with a ganglioside present in fetal rat brain. We have found that this antigen is also present in human malignant melanoma tumors as well as many melanoma cell lines. The ganglioside from human melanoma cell lines migrates between GM1 and GM2 on one-dimensional thin layer chromatography. Analysis by two-dimensional thin layer chromatography with intermediate ammonia treatment suggests that the ganglioside contains one or more base-labile O-acyl esters. Mild base hydrolysis under conditions known to remove O-acyl esters results in complete loss of antigenic reactivity. Thus, the alkali-labile moiety is a critical component of the epitope recognized by the antibody. Analysis of the sialic acids of total gangliosides from [6-3H]glucosamine-labeled melanoma cells showed that approximately 10% of these molecules are O-acylated. Similar analysis of the purified ganglioside showed that greater than 30% of the sialic acids comigrated with authentic 9-O-acetyl-N-acetylneuraminic acid. The antibody did not cross-react with normal human skin melanocytes nor with any of a large number of normal human adult and fetal tissues. The antibody also did not react with numerous other malignant cell lines studied. These findings suggest that the antigenic epitope defined by antibody D1.1 contains an O-acylated sialic acid and may arise from aberrant O-acetylation occurring in human malignant melanoma cells.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

A human monoclonal antibody directed to blood group i antigen: heterohybridoma between human lymphocytes from regional lymph nodes of a lung cancer patient and mouse myeloma

A fusion of human lymphocytes released from regional lymph nodes of papillary adenocarcinoma of lung cancer with mouse myeloma P3-X63-Ag8-U1 cells resulted in a stable hybridoma-secreting human IgM antibody (NCC-1004) that reacts with a large proportion of squamous cell carcinomas of lung and esophagus as well as carcinoma of thyroid glands. However, the antibody also reacts with normal red blood cells, B lymphocytes, and a few other limited loci in normal tissues such as the basal cells of bronchial epithelium and the basal cell layer of stratified squamous epithelium, as well as endothelium and alveolar lining epithelium. The antigen defined by NCC-1004 has been characterized as blood group i antigen on the basis of the following results. The antibody preferentially agglutinates cord erythrocytes in contrast to adult erythrocytes. The agglutination was obvious at 4 degrees C, but diminished greatly at 37 degrees C, and was enhanced after sialidase treatment. The antibody specifically reacts with lacto-norhexaosylceramide (nLc6) and sialosyllacto-norhexaosylceramide (IV3NeuAcnLc6), but does not react with lacto-neotetraosylceramide (nLc4), sialosyllacto-neotetraosylceramide (IV3NeuAcnLc4), lacto-isooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6; I antigen), and other standard glycolipids so far tested. The properties of the antibody and its antigen are identical to those previously described for the i blood group system. Inasmuch as the hybridoma was established by hybridization of lymphocytes derived from regional lymph nodes of lung cancer, and the antigen was found in the patient's lung cancer tissue, the i antigen in lung cancer is probably recognized as a tumor-associated antigen by the host's immune cell system.     

Cytochemical examination of acid phosphatase and beta-glucuronidase enzymes in low-grade B cell non-Hodgkin's lymphomas

The differential diagnostic significance of acid phosphatase and beta-glucuronidase were studied in 77 cases of low-grade B cell non-Hodgkin's lymphomas. In most cases the results of cytochemical enzyme studies performed on malignant cells of the bone marrow were evaluated. B cell chronic lymphocytic leukaemia, centrocytic and centroblastic/centrocytic lymphomas were characterized by a weak or a negative acid phosphatase and beta-glucuronidase activity. Stronger positivity was observed in immunocytoma and in Waldenstr?m's macroglobulinaemia, while the highest activity was found in multiple myeloma. Hairy cell leukaemia of B cell origin showed intensive tartrate-resistant acid phosphatase activity. The cytochemical examination of these lysosomal enzymes may be useful in the diagnosis of low-grade malignant lymphomas of B cell origin by completing other methods.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

BLAST-2 [EBVCS], an early cell surface marker of human B cell activation, is superinduced by Epstein Barr virus

Activation of human B cells by pokeweed mitogen (PWM), protein A, anti-IgM, or EBV infection results in the expression of a new surface antigen, termed BLAST-2 [EBVCS]. This marker appears before the cells undergo blast transformation as assessed by the initiation of DNA synthesis and expression of the BLAST-1 antigen. Thus, the BLAST-2 [EBVCS] antigen is expressed on both activated and lymphoblastoid cells. The antigen is, in addition, restricted to B cells, as it is not found on cells of T or myeloid lineage derived from peripheral blood, cell lines, or neoplastic cells. However, it is readily detected on chronic lymphocytic leukemia cells of B cell origin and in the germinal centers of tonsils and lymph nodes. Like the BLAST-1 antigen, BLAST-2 [EBVCS] is expressed at a high level only on EBV-transformed B lymphoblasts and has a m.w. close to 45,000. Immunoprecipitation experiments show, however, that the two antigens are expressed on distinct populations of molecules.     

Flow cytometric characterization of chronic lymphocyte leukaemias using orthogonal light scattering and quantitative immunofluorescence

Light scattering properties and antigen distribution of lymphocytes labeled with the monoclonal antibodies CD 5 and CD 20 were determined for 19 patients with a chronic B-cell derived leukaemia. The density of the antigen detected by the monoclonal antibody CD 5 appeared to be considerably lower on malignant B-lymphocytes of the patients as compared with T lymphocytes. A large variation was observed in the amount of receptors for the monoclonal antibodies CD 5 and CD 20 on the malignant cells of the different patients. B-cell chronic lymphocytic leukaemia (B-CLL) patients were clearly distinguishable from leukaemic follicular non Hodgkin lymphoma patients (LF-NHL, formerly lymphosarcoma cell leukaemia) and from a patient with a prolymphocytoid transformation (PLT) of the B-CLL according to the amount of the antigens for CD 5 and CD 20. Within the B-CLL patient population, no relation of progression of the disease with distribution of these antigens could be observed. In one patient the extraordinary phenotype CD 20+, CD 11+, leu 8+, CD 5- of the malignant lymphocytes was observed. An experimentally simple method to differentiate between the various chronic lymphocytic leukaemias (CLL) appeared to be the determination of orthogonal light scattering properties of lymphocytes. In healthy donors one can always distinguish two populations of lymphocytes in the orthogonal light scatter histograms. Lymphocytes of B-CLL patients show one uniform population with a relatively small orthogonal light scattering signal, lymphocytes of our patients with PLT of B-CLL or with LF-NHL show one uniform population with a relatively large orthogonal light scattering signal.     

B5, a new B cell-restricted activation antigen

The characterization of a new human B cell-restricted activation antigen (B5) is described in this report. With the use of a monoclonal antibody to B5, we show that B5 can be detected on peripheral blood or splenic B cells after 1 day of stimulation with either anti-immunoglobulin, protein A, Epstein Barr virus, or pokeweed mitogen. In contrast, B5 was not expressed on resting B, T, or myeloid cells. More important, B5 could not be detected on activated T cells or monocytes. The B5 antigen was expressed on some lymphoblastoid B cell lines and B cell neoplasms but was not expressed on leukemias or lymphomas of T or myeloid origin. The B5 antigen is distinct from previously reported B cell activation antigens by its m.w. and pattern of cellular expression. These studies suggest that B5 is a novel B cell-restricted activation antigen, which may be useful to study the events of early human B cell activation.     

A monoclonal antibody defining a lymphoma-associated antigen in man

A monoclonal antibody (Ab 89) was developed against a lymphoma-associated antigen on the tumor cells of a patient (N.B.) with a B cell, poorly differentiated lymphocytic lymphoma (D-PDL). By indirect immunofluorescence, Ab 89 was shown to react only with N.B. lymphoma cells and was unreactive with normal N.B. lymphoid cells, normal fractionated peripheral blood cells, other normal lymphoid tissues, fetal tissues, and non-lymphoid malignant cells. In addition, Ab 89 was unreactive with conventional immunoglobulins, private and public HLA antigens, or Ia-like antigens. More importantly, Ab 89 was reactive with some B cell lymphoid malignancies. Of the 57 B cell lymphomas tested, it was found that Ab 89 reacted with approximately 10% of B cell D-PDL and B cell chronic lymphocytic leukemia (CLL). Of interest was the finding that N.B. serum contained a circulating antigen which could specifically block the reactivity of Ab 89. The data obtained suggests that Ab 89 defines a tumor-associated antigen shared by a unique subgroup of B cell lymphomas. The group of patients reactive with Ab 89 did not fall into any histopathologic classification system. These data support the notion that there is greater heterogeneity of B cell lymphomas than may have been previously recognized.     

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.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.