Immunocytometric characteristics of a monoclonal antibody (Bra55) recognizing the leukocyte common antigen (LCA)

The immunofluorescence cytofluorometric reactivity pattern of monoclonal antibody Bra55 (IgG1) elicited with a non-T, non-B ALL cell line (REH), with a panel of human neoplastic hemopoietic cell lines (including non-T, non-B, T and myeloid leukemia cell lines) and with isolated peripheral blood lymphocytes, monocytes and granulocytes from healthy donors corresponded to the previously described microscopic immunofluorescence, ELISA, immunoprecipitation and immunocytochemic data indicating that this monoclonal antibody recognizes a 170-220 kDa cell surface glycoprotein (leukocyte common antigen) expressed selectively on hemopoietic cells. The purified, FITC-conjugated Bra55 monoclonal antibody was effectively inhibited in its binding to the surface of LCA-positive cells by reference anti-LCA monoclonal antibodies; no inhibition of this activity by LCA-unrelated monoclonal antibodies (such as anti-MHC class I and class II antibodies) was observed. These data confirm the previously reported hemopoietic cell specificity (anti-LCA, CD45) of the Bra55 monoclonal antibody.     

Monoclonal antibodies to two adhesive cell surface antigens (CD43 and CD59) with different distribution on hematopoietic and non-hematopoietic tumor cell lines.

Two new murine monoclonal antibodies were prepared by hybridoma technique after immunization with the immature pluripotent leukemia cell line K562. The monoclonal antibody Bra10G (IgG2b) reacted in a non-lineage pattern with all examined hematopoietic neoplastic cell lines and peripheral blood cells (granulocytes, lymphocytes, erythrocytes) of healthy donors, with the exception of monoblastoid cell line U-937 and B lymphoma cell line Daudi. This monoclonal antibody immunoprecipitated an 18-20 kDa cell surface protein expressed also on the cell surface of examined non-hematopoietic (malignant glioma, melanoma and breast carcinoma) cell lines. These properties and the efficient inhibition of Bra10G binding to the cell surface of K562 cells by the reference CD59 monoclonal antibody (MEM-43) indicated that Bra10G belongs to the CD59 cluster of monoclonal antibodies which identify the human protectin molecule. The monoclonal antibody Bra7G (IgM) reacted with a 95 kDa cell surface protein expressed on hematopoietic cells (with the exception of erythrocytes) and was absent on the examined non-hematopoietic neoplastic cell lines. These data together with a partial inhibition of Bra7G binding by the reference CD-43 monoclonal antibody suggested the CD43 (leukosialin, sialophorin) specificity of this monoclonal antibody.     

Supplementary characteristics of anti-MHC class II monoclonal antibodies elicited by an ALL cell line: immunofluorescence cytofluorometry, C-dependent cytotoxicity, two-dimensional analysis of antigen

Monoclonal antibodies directed to MHC class II antigen(s), elicited by a non-T, non-B ALL cell line, were characterized by immunofluorescence flow cytofluorometry and ELISA immunofiltration measurements of their immunoreactivity with selected neoplastic hemopoietic cell lines, determination of their complement-dependent cytotoxic activity against isolated peripheral blood B and T lymphocytes and by two-dimensional electrophoretic analysis (isoelectric focusing, SDS-PAGE) of radiolabeled, immunoprecipitated by these antibodies cell surface antigens. Patterns of these immunological reactivities, as well as two-dimensional radioimmunoprecipitation patterns (acidic heavy chain p35 and basic light chain p30) of antigens recognized by these antibodies confirm their anti-MHC class II specificity. One of these antibodies (braFB6; IgG2b) displayed identical pattern of expression on cell lines and cell types as the typical anti-MHC class II antibodies, but immunoprecipitated only a single chain p30 radioiodinated cell surface protein (with two-dimensional pattern close to the beta-chain of MHC class II DR antigen). These properties indicate the ability of braFB6 monoclonal antibody to recognize a nonpolymorphic determinant of DP-MHC class II antigen.     

Brb, a Platelet Alloantigen Involved in Neonatal Alloimmune Thrombocytopenia

Serum from a pregnant woman with the May-Hegglin anomaly contained a platelet-specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mother's own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb- for the mother, PLA1+, Baka+, Bra-/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet-GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May-Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet-specific antibody was present in the serum of the 7 unrelated May-Hegglin subjects. The antigen recognized by this platelet-specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.     

Hemopoietic cell line distribution and immunoprecipitation of cell surface proteins recognized by two newly prepared monoclonal antibodies elicited by a human non-T, non-B leukemia cell line

Two newly prepared monoclonal antibodies elicited by a human non-T, non-B acute lymphoblastic leukemia cell line REH recognized distinct antigenic specificities characterized by the pattern of their immunofluorescence reactivities with a panel of hemopoietic cell lines and by immunoprecipitation of 125I-lactoperoxidase radioiodinated cell surface proteins, as well as periodate/tritiated borohydride radiolabeled cell surface sialoglycoproteins. Monoclonal antibody anti-p30 (BraFB6; IgG2b) recognized an antigen similar in its distribution to MHC class II antigens and immunoprecipitated a p30 cell surface protein, radiolabeled by lactoperoxidase catalyzed radioiodination. Monoclonal antibody anti-gp95 (BraEA10; IgG3) reacted in immunofluorescence intensively with non-T, non-B, T-leukemia and myeloid leukemia cell lines, less intensively with lymphoblastoid and lymphoma cell lines of B-phenotype and no reactivity was observed with examined non-hemopoietic human tumor cell lines. This antibody immunoprecipitated a lactoperoxidase radioiodinated and periodate/NaB3H4 tritium-radiolabeled cell surface sialoglycoprotein of approximately 95k (gp95) with variability in its apparent molecular weight, related to the origin of cells utilized for radiolabeling and immunoprecipitation.     

Detection of an early activation-dependent cell surface antigen on human T-lymphocytes

The binding properties of a monoclonal antibody to an early activation marker of human lymphocytes are described. The monoclonal antibody BL-Ac/p26 binds to an antigen which is expressed on activated lymphocytes. This antigen is not detectable on resting lymphocytes or other blood cells. The surface radioiodinated antigen of activated T cells isolated by the monoclonal antibody BL-Ac/p26 is separated by SDS-PAGE in a major 26k Da component and a minor band (32 kDa) under reducing conditions. These polypeptide chains form disulphide linked dimers on the cell surface (Mr 55-77 kDa). This 26 kDa antigen is a very early activation marker of T lymphocytes. Human T lymphocytes stimulated by mitogens (PHA, ConA or PWM), anti-TCR/CD3 monoclonal antibodies or allogeneic leucocytes express this 26 kDa antigen after 2-4 hours.     

HLA class I antigen display on hepatocyte membrane in chronic hepatitis B virus infection: its role in the pathogenesis of chronic type B hepatitis

It has been suggested that cytotoxic T cells are involved in the recognition and lysis of the infected hepatocytes in chronic hepatitis B virus infection, and that the target antigen is probably HBcAg which is displayed on the hepatocyte membrane during active viral replication. However, studies in other viral infection have demonstrated that cytotoxic T cells recognize viral antigen on the infected cells only in the context of HLA class I antigens. To test whether this mechanism is also operative in chronic hepatitis B virus infection, we studied the expression of HLA class I antigens in livers from 35 patients with chronic hepatitis B virus infection by indirect immunofluorescence using monoclonal antibody against HLA class I antigens. The blocking effect of monoclonal antibody against HLA class I antigens on the in vitro T cell cytotoxicity to autologous hepatocytes was also studied. The results revealed that HLA class I antigen was undetectable on the hepatocyte membrane in all of 10 HBeAg-positive carriers with minor hepatitic activity, whereas it was demonstrated in 15 (88%) of the 17 HbeAg-positive patients with chronic active liver disease and in 7 (87%) of the 8 anti-HBe-positive "normal" carriers. The in vitro T cell cytotoxicity to autologous hepatocytes in six HBeAg-positive patients with chronic active liver disease was significantly inhibited by preincubation of hepatocytes with monoclonal antibody (10 to 40 micrograms per ml) against HLA class I antigen, but not by monoclonal antibody against HLA class II antigens and non-HLA-associated surface molecules (Leu 11).(ABSTRACT TRUNCATED AT 250 WORDS)     

Human neoplastic cell line distribution, immunoprecipitation and immunohistopathological study of a gp200 cell surface glycoprotein (LCA) detected by a monoclonal antibody elicited with an ALL cell line

The antigen recognized by a newly produced monoclonal antibody (bra55; IgG1) elicited by the non-T, non-B acute lymphoblastic leukemia cell line REH 6, was expressed on all examined hemopoietic neoplastic cell lines (including non-T, non-B, T, B and myeloid leukemia cell lines), but not on examined nonhemopoietic human tumor cell lines (such as carcinoma, sarcoma, melanoma and neuroblastoma cell lines), as demonstrated by indirect immunofluorescence and enzyme-linked immunoassay. Specific immunoprecipitation of 125I-lacto-peroxidase radioiodinated cell surface proteins and sodium metaperiodate/tritiated sodium borohydride 3H-radiolabeled cell surface sialoglycoproteins followed by electrophoretic analysis (SDS-PAGE) demonstrated that the immunoprecipitated antigen is a cell surface 200 kDa sialoglycoprotein (on the non-T, non-B ALL cell line REH 6), with variation in its electrophoretic mobility (in the Mr range of 170,000-210,000) on different examined cell lines. These properties are characteristic for the leukocyte common antigen (LCA, T200). Immunoperoxidase staining of several normal and malignant tissues, as well as some nonhemopoietic tumor tissues confirmed the type of antigen tissue distribution pattern characteristic for LCA.     

A monoclonal antibody detecting a novel antigen expressed in the HTLV-I- infected cells

A monoclonal antibody, FTF 148, was prepared by hybridizing murine myelomal cells (NS-1) and spleen cells of BALB/c mice immunized with cultured cells derived from an adult T cell leukemia (ATL) patient (KUT- 2 cells). This monoclonal antibody reacted with all of the human T cell leukemia virus I (HTLV-I)-infected cell lines tested but did not react with other T cell lines derived from acute lymphocytic leukemia, Epstein-Barr virus-transformed B cell lines, or an erythroleukemic cell line. This monoclonal antibody was not directed to viral antigens because it reacted equally well with almost all KUT-2 and MT-1 cells, only 1% to 3% of which were ATL-associated antigen-positive. In contrast to interleukin 2 receptors expressed on both ATL cells and normal phytohemagglutinin-stimulated blasts, this antigen was not expressed on the latter cells. The antigen, mainly expressed on the cell membrane, was analyzed by metabolic labeling with 3H-leucine and surface labeling with 125I followed by cell lysis and immunoprecipitation with the FTF 148 antibody. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that p50 and p74 proteins were specifically precipitated and the antigen was also different from the product of the Xs gene of HTLV-I.     

Major histocompatibility complex class I molecule serves as a ligand for presentation of the superantigen staphylococcal enterotoxin B to T cells.

Superantigens, such as staphylococcal enterotoxin B (SEB), elicit a strong proliferative response in T cells when presented in the context of major histocompatibility complex (MHC) class II molecules. We observed a similar T-cell response, when MHC class II-negative epidermal cell lines were employed as antigen-presenting cells. Immunoprecipitation studies indicated that the ligand to which SEB bound had a molecular mass of 46 kDa. Radiolabeled SEB could be immunoprecipitated from isolated membrane proteins on the SCC13 epidermal cell line with a monoclonal antibody directed against the MHC class I molecule, and transfection of the K-562 cell line with MHC class I molecules showed a 75% increased SEB-binding capacity compared with the nontransfected MHC class I- and class II-negative counterpart. In functional studies, antibodies to the MHC class I molecule inhibited T-cell proliferation by at least 50%. From these studies, we conclude that MHC class I molecules on malignant squamous cell carcinomas serve as ligands for SEB, which, given the appropriate costimulatory signals, is sufficient to allow for superantigen-induced T-cell proliferation.     

Monoclonal antibodies (series Bra-) of broad (non-lineage) or restricted (myelomonocytic) reactivities elicited with granulocytes or K 562 cells

A series of monoclonal antibodies was obtained by hybridoma technology after immunization with granulocytes from healthy donors or K 562 immature erythroid-myeloid leukemia cells. Three different types of reactivities with examined hematopoietic-, nonhematopoietic cells and cell lines were observed by microscopic immunofluorescence, immunocytofluorometry and enzyme-linked immunoassay (ELISA), as follows: (i) broad, non-lineage type of two monoclonal antibodies (Bra10G and Bra7F1) with examined hematopoietic and nonhematopoietic human neoplastic cell lines, (ii) non-lineage type of reactivity restricted to hematopoietic cell lines, and (iii) restricted (myelomonocytic) pattern of binding to myeloid cell lines and healthy donors' granulocytes (monoclonal antibodies Bra4F1, BraC8 and Bra1F2). Monoclonal antibodies Bra10G and BraC6 were shown to immunoprecipitate specifically a heterodimeric two-chain cell surface protein p200,95 from cell lysates of lactoperoxidase radioiodinated U 937 cells with recognized epitope localized on the heavy chain (as shown by immunoblotting experiments). Antibodies with restricted myelomonocytic type of reactivity exhibited minor quantitative differences in their microscopic immunofluorescence and immunocytofluorometric patterns of reactivities with examined myeloid leukemia cell lines (K 562, HL 60, U 937) and healthy donors' granulocytes. The monoclonal antibody Bra4F1 was defined by the 4th International Workshop on Leukocyte Differentiation Antigens as CD15 (with typical selective reactivity towards myelomonocytic leukemia cells and cell lines as well as healthy donors' granulocytes) recognizing the X-hapten carbohydrate antigenic determinant.     

Luteinizing hormone triggers a molecular association between its receptor and the major histocompatibility complex class I antigen to produce cell activation

We studied the involvement of major histocompatibility (MHC) class I antigens on the mechanism of LH/hCG receptor activation. For this purpose we investigated the effects of anti-MHC class I antibodies on hormone-receptor interaction, signal transduction, and MHC class I antigen-receptor interaction. Monoclonal antibodies against MHC class I antigen were able to stimulate testosterone production in mouse Leydig cells with the same potency as LH. This biological effect depends on the concentration of antibody used and could be abolished by a LH antagonist. There is a perfect parallelism, for each monoclonal antibody, between the specificity for a particular haplotype and the response of the target cells from the strains carrying such a haplotype. The same antibodies were able to precipitate the soluble LH/hCG receptors, as both a hormone-receptor complex and a free receptor. The results suggest that bound hormone triggers an association of the MHC class I antigen with the LH/hCG receptor, resulting in activation of the target cell.     

Studies on the distribution of the antigens detected by some newly prepared monoclonal antibodies in normal hemopoietic and leukemic cells

A great number of hybridoma clones has been developed in two independent fusions after immunization with cells of a human non-T, non-B REH leukemic cell line, recognizing different differentiation antigens on leukocytes. Only four of these new murine monoclonal hybridoma-derived antibodies (Bra20, Bra30, BraFB6 and BraEA10) have been used in our study for determination of their reactivity spectrum in normal blood cells and fresh lymphoid and non-lymphoid leukemic cells. The resulting reactivity spectra of these four monoclonal antibodies in normal hemopoietic and leukemic cells in indirect immunofluorescence assay are in agreement with supposed Ia-like reactivity for Bra20, Bra30 and Bra FB6 monoclonal antibodies and "common" leukocyte specificity for BraEA10 monoclonal antibody.     

Biochemical identification of the antigen recognized by the monoclonal pan-B cell antibody Y29/55

To examine the biochemical structure of the antigen recognized by the monoclonal pan-B cell antibody, Y29/55, the Daudi- and Jurkat-cell lines were labeled by two different methods and immunoprecipitation experiments were carried out. After surface labeling with iodine, two bands with molecular weights of about 38 and 42 kD were observed. The same two proteins were precipitated after biosynthetic labeling with (35S)-methionine from B cells and, to a lesser extent, from T cells. Therefore, it seems that the same proteins, or proteins with similar molecular weight, exist intracellularly in T cells as exist on the surface of, and possibly intracellularly, in B cells. It was confirmed that 80-90% of normal blood-derived B cells were stained with Y29/55 by indirect immunofluorescence. Double-labeling experiments with the pan-B cell antibodies Leu 16 (CD 20) and Leu 12 (CD 19) showed a B-cell population which could be stained with both antibodies (Y29/55 and Leu 16 or Y29/55 and Leu 12). A minor cell population was stained with the antibody Y29/55 alone. Our findings indicate that the antibody Y29/55 recognizes a B-cell antigen, which has not been described previously.     

Fluorescent double labeling of normal and malignant hematopoietic cells by monoclonal antibodies (FITC) and anthracycline cytostatic drug (Daunomycin): a cytometric technique for analysis of drug uptake in hematopoietic cell subpopulations

A technique of simultaneous double labeling of normal and neoplastic hematopoietic cells with FITC-conjugated monoclonal antibodies directed to selectively expressed hematopoietic cell surface antigens (green fluorescence) and the anthracycline cytostatic drug (Daunomycin, red fluorescence) was described. Flow cytometric analysis of double labeled cells permitted anthracycline cell content determination in peripheral blood lymphocytes, granulocytes, monocytes from healthy donors, T- (MOLT-4), non-T, non-B (REH) and myelomonocytic (U-937) leukemic cell lines. After mixing peripheral blood lymphocytes from healthy individuals with cultured leukemic cells labeled on a restrictively expressed hematopoietic cell differentiation antigen (CALLA-CD10-, MHC class II-DR-antigen, a myelomonocytic differentiation antigen) detected by corresponding monoclonal antibodies (DGH-10-1-A9,Bra30, BraC8), the described technique allowed separate measurements of anthracycline content in leukemic cells vs. peripheral blood lymphocytes from healthy donors. Potential diagnostic aspects and research utilization of this technique are discussed.     

Synergistic enhancement of class I major histocompatibility complex antigen expression in K562 cells induced by recombinant human interferon-gamma and tumor necrosis factor in combination

The effects of recombinant preparations of human interferon-gamma (rIFN-gamma) and tumor necrosis factor (rTNF), alone or in combination, on class I or class II major histocompatibility complex (MHC) antigen induction were studied using K562, a multipotent hematopoietic precursor cell line. Class I antigens were weakly induced by rIFN-gamma; however, rTNF at any concentration examined (1-1000 U/ml) showed no effect on the induction of class I or class II antigens in the cells. rIFN-gamma (600 U/ml) induced approximately 20% of the cells to express class I antigens after 72-h exposure, whereas 81% of the cells demonstrated class I antigens on their cell surfaces when the cells were simultaneously exposed to 600 U/ml of rIFN-gamma and 1000 U/ml of rTNF. The class II MHC antigens were not induced by the treatments with rIFN-gamma or rTNF, alone or in combination. A synergistic increase of mRNA for class I MHC molecules was demonstrated by treatments of the cells with rIFN-gamma and rTNF in combination. rTNF, but not rIFN-gamma, weakly induced granulocyte-monocyte antigens on the cell surface; however, no synergism was observed on the induction of these antigens by the combined treatments with rIFN-gamma and rTNF. These results indicate that class I MHC antigen expression on K562 cells can be induced by IFN-gamma in cooperation with TNF in a manner different from myeloid antigen expression.     

Distribution of cell surface glycoprotein CD9 (P24) antigen on megakaryocyte lineage leukemias and cell lines

We herein describe CD9 (p24) antigen as existing on the cell surface of megakaryocyte lineage leukemias as well as megakaryocytic leukemia cell line, MEG-01 and HEL, by means of fluorescence-activated cell sorter (FACS IV) with a panel of monoclonal antibodies (Mabs). We found CD9 antigen expression on the cell surface of megakaryoblastic leukemias as well as MEG-01 and HEL cells. Furthermore, CD9 antigen expression increased while culturing these cells with phorbol esters, and was also found in the cytoplasm by means of indirect immunofluorescence test. These findings clearly showed that CD9 antigen exists on the cell surface of megakaryocytic cells which are capable of synthesizing the CD9.     

Identification of a unique tumor-specific antigen as a novel class I major histocompatibility molecule.

Cancers induced by physical or chemical carcinogens express tumor-specific antigens that are uniquely specific for any given tumor; therefore, there is a seemingly endless variety of these unique antigens. We have studied a UV-induced fibrosarcoma, designated 1591, to elucidate the obscure molecular nature and genetic origins of unique tumor-specific antigens. A monoclonal antibody raised against syngeneic 1591 tumor cells has unique tumor specificity. This tumor-specific monoclonal antibody precipitated from the tumor a 45-kDa molecule associated with a 12-kDa molecule having the pI of beta2-microglobulin. This and other evidence indicated that the 1591 tumor expresses a novel class I molecule. A 1591 variant selected for the absence of binding to the monoclonal antibody lacked the novel class I MHC molecule as well as reactivity with cytotoxic T lymphocytes specific for the 1591 tumor. Furthermore, tumor cells bearing the antigen are rejected while variants that have lost the antigen grow progressively. Fourteen of 14 host-selected progressor tumor variants lost reactivity with the monoclonal antibody and provided further evidence that this novel class I molecule is a transplantation antigen on the parental 1591 tumor required for immune rejection. The identification of a unique tumor-specific antigen as a novel class I major histocompatibility complex gene product allows us to search for the possible genetic mechanisms involved and to explore further the role such molecules play in tumor immunity and malignancy.     

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens.

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.