Monoclonal antibodies reactive with human myeloid leukaemia cells.

Hybridomas producing monoclonal antibodies that bind to determinants on myelogenous leukaemia blast cells were developed using fresh leukaemia blasts as immunogens. These monoclonal antibodies were used to quantitate the amount of a variety of antigens on both leukaemia and normal blood cells in a radioimmunoassay. The ability of these antibodies to mediate complement-dependent lysis of leukaemia cells and normal blood cells was evaluated. Significant quantitative differences in the expression of a variety of cell surface antigens were observed among different myeloid leukaemia cell samples, normal cells, and other forms of leukaemia. However, none of the monoclonal antibodies studied were found to be specific for myeloblasts. Despite the fact that these antibodies bound to both leukaemia and normal cells, several of them mediated complement-dependent cytotoxicity which was restricted to leukaemic myeloblasts (AML-1-211, AML-2-30, CML-18, CML-75, CML-115, and CML-150). None of these clones, alone or in any combination, were capable of lysing normal lymphocytes or monocytes. Others were specifically cytotoxic to leukaemia cell lines (AML-1-99), B cells and some leukaemia samples (AML-2-9), and myelomonocytic leukaemia cell samples and normal monocytes (AML-2-23). Several of the monoclonal antibodies from this panel appear to be promising for future clinical applications.     

Monoclonal antibodies which react with lymphocyte-lysed target cells and which cross-react with complement-lysed ghosts.

Monoclonal antibodies were raised against a human natural killing system and screened against targets lysed either by human lymphocytes in antibody-dependent cellular cytotoxicity (ADCC), or by human complement. Two monoclonals were identified which bound specifically to both types of killed targets. More detailed studies with one antibody showed that it inhibited ADCC, both as intact antibody and as the F(ab')2 fragment. Intact antibody enhanced natural (NK) killing, although the F(ab')2 fragment inhibited NK killing. The data support the hypothesis that lymphocyte-mediated killing involves a complex analogous in nature to the complement membrane attack complex. In addition, the antibodies provide evidence to suggest that this complex has antigenic determinants in common with the complement membrane attack complex, and indicate the possibility that the two systems are derived from a common ancestor.     

Monoclonal anti-tuberculosis antibodies react with DNA, and monoclonal anti-DNA autoantibodies react with Mycobacterium tuberculosis.

Classical models of experimental autoimmune diseases, such as adjuvant arthritis entail the use of mycobacteria. Furthermore, BCG immunotherapy may be followed by arthritic symptoms. To test the infection-autoimmunity relationship of mycobacteria, we used monoclonal antibodies raised against M. tuberculosis and against DNA. Murine monoclonal anti-TB antibodies were found to react with ssDNA, dsDNA and other polynucleotides. Monoclonal anti-DNA autoantibodies derived from patients and mice with SLE bound to three glycolipids shared among all mycobacteria and derived from mycobacterial cell wall. Prior incubation of the antibodies with ssDNA and other polynucleotides or with glycolipid antigens inhibited binding. These results indicate that infecting mycobacteria share antigens with human tissue, thus accounting in part for the production of autoantibodies in mycobacterial infections.     

Carbohydrate-specific human heterophile antibodies in normal human sera that react with xenogeneic cells

Human heterophile antibodies (HHA) that are present in normal human sera (NHS) play an important role in hyperacute xenograft rejection. The aim of this study was to analyze the occurrence, mode of action and molecular specificity of HHA in NHS that are directed against xenogeneic lymphocytes (isolated from mouse, rat, guinea pig, rabbit, cattle and pig) and isolated rat pancreatic islets. All sera contained variable amounts of HHA that killed the target cells via the classical complement pathway. The cytotoxic activity of these HHA was specifically inhibited by certain carbohydrates (alpha-D-melibiose, beta-lactose, beta-gentiobiose, beta-cellobiose, D-mannose, N-acetyl-beta-D-mannosamine and alpha-D-rhamnose) and by rat IgM. By means of affinity chromatography with immobilized inhibitors we obtained an antibody preparation of mainly IgG type from NHS (up to 3.5 mg/10 ml serum) that reacted strongly with rat lymphocytes and isolated rat pancreatic islets. Though thus far residual xenospecific antibody activity has remained in the sera even after multiple affinity chromatography, these data suggest that specific elimination of HHA is feasible and that it may be thus possible to overcome a major obstacle to xenotransplantation.     

Monoclonal antibodies reactive with normal and neoplastic T cells in paraffin sections

Without fresh or frozen tissue, it previously has been impossible to confirm the T-cell nature of reactive or neoplastic lymphoid cells. The availability of antibodies reactive with T cells in paraffin sections now allows retrospective analysis of a large number of cases. Two commercially available monoclonal antibodies, MT1 and MT2, were tested for their reactivities with T cells in a wide range of formalin-fixed, paraffin-embedded tissues, including 130 cases of immunologically characterized lymphoma. In reactive lymph nodes, MT1 stained the T-cell areas, whereas MT2 stained both the T-cell areas and mantle-zone B lymphocytes. MT1 stained 38 of 55 T-cell lymphomas (69.1%; 94.7% of cases from one hospital that used a shorter fixation time, and 55.6% of cases from another hospital that used a longer fixation time). MT2 stained only 6 (10.9%) of the T-cell lymphomas. Among the 74 cases of B-cell lymphoma, 3 (4.0%) were stained by MT1 and 30 (40.5%) by MT2.MT1 was also reactive with 3 of 4 cases of granulocytic sarcoma, as expected from its reactivity with normal granulocytes. Neither MT1 nor MT2 stained Reed-Sternberg cells or their variants in HodgKin's disease. We conclude that MT1 is a valuable marker for T cells, particularly when used with a panel of antibodies reactive with B cells in paraffin sections. MT2 is of limited value because of its cross-reactivity with many B-cell lymphomas.     

Monoclonal antibodies recognizing normal and retrovirus-transformed chicken hematopoietic cells.

The avian hematopoietic system has long been an invaluable model to study the mechanisms of cell growth and differentiation. We have developed six MAbs against either chicken embryonic hematopoietic precursor cells or retrovirus-transformed cells. MAbs Mo1, Mo2, and Mo3 recognized transformation-associated markers expressed in AMV-transformed nonproducer cell line-BM2. Not only were these markers expressed 7 to 10 folds higher on BM2 than on normal monocytic cells, but their expression was drastically reduced when BM2 cells were induced to differentiate into macrophages by PMA. The control of marker expression is associated with v-myb-transforming cascade, since another monocytic lineage-specific oncogene, v-myc, did not enhance the expression of these markers. MAb Em1 detected a marker that is normally present in 20% of the cells from the 30/50% interface of a discontinuous percoll gradient of normal 4-day-embryo yolk sac. Its expression is also found in AEV-transformed cells and MSB1 cells. The epitope for Em1 was exposed after neuraminidase treatment on erythroleukemia cell line 6C2, which suggested that sialylation and/or glycosylation is pivotal in regulating the expression of specific markers in differentiation pathways during embryogenesis and tumorigenesis. MAb Em2 recognized proliferating hematopoietic cells after the fourth day of embryogenesis. MAb Em3, on the other hand, is presumed to be specific for an oncofetal antigen expressed in various transformed cells but only in 10% of the cells from 30/50% interface of a discontinuous percoll gradient of normal 4-day-embryo yolk sac. These MAbs will be useful for dissecting the expression of differentiation markers within normal versus abnormal differentiation pathways in molecular terms.     

Monoclonal antibodies distinguish macrophages and epithelioid cells in sarcoidosis and leprosy.

Existing anti-macrophage monoclonal antibodies are unable to differentiate between macrophages and epithelioid cells. In search of more precise reagents, we have applied recently developed antibodies to lesions of sarcoidosis and leprosy. UCHM1 and Leu-M3 stained both granulomas and surrounding histiocytes. However, in lesions with epithelioid granulomas there was a clear distinction between cells identified by RFD9 (epithelioid and giant cells) and RFD7 (macrophages in the surrounding mantle and normal tissue), whereas macrophages in the non-hypersensitivity granulomas of lepromatous leprosy were labelled by both the latter antibodies. In lung biopsies, alveolar macrophages were also labelled by both RFD7 and RFD9. These reagents may be useful for studying pathogenic mechanisms in granuloma formation.     

Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes.

Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function.     

Monoclonal antibodies against nucleophosmin mutants: potentials for the detection of acute myeloid leukemia

Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.     

Monoclonal antibodies which react with bovine T-lymphocyte antigens and induce blastogenesis: tissue distribution and functional characteristics of the target antigens.

In this study we report on the tissue distribution and functional characteristics of bovine T-cell differentiation antigens recognized by the monoclonal antibodies (mAbs) IL-A26, IL-A27, and IL-A28. All three mAbs are able to stimulate proliferation of peripheral blood mononuclear cells (PBM) and inhibit proliferation of responder cells in mixed leucocyte cultures (MLC). MAbs IL-A27 and IL-A28 are believed to react with the same molecule, which is different to that recognized by IL-A26 as determined by a number of criteria. MAbs IL-A27 and IL-A28 inhibit binding of one another, but not of IL-A26. MAbs IL-A27 and IL-A28 react with 25% of thymocytes confined to the medulla, whereas IL-A26 reacts with approximately 80% of thymocytes, including medullary and cortical populations. MAbs IL-A27 and IL-A28 react with thymocytes which express BoT4 or BoT8 singularly, whereas IL-A26 reacts with all cells which express BoT4 or BoT8, either singularly or dually, in addition to all thymocytes which react with IL-A27/28. Only IL-A26 inhibits spontaneous sheep erythrocyte (E)-rosette formation by bovine T cells. Based on tissue distribution and functional characteristics, IL-A26 is believed to recognize the bovine homologue of CD2, designated BoT2, whereas IL-A27/28 reacts with a mature T-cell antigen. Cells reactive with the mAbs constitute approximately 60% of bovine PBM. Using these mAbs in dual immunofluorescence analyses, at least three populations of bovine T cells are demonstrable in PBM. The majority of T cells are BoT4+ or BoT8+ and also react with IL-A26/27/28. A second small population of PBM is negative for BoT4 and BoT8 but is IL-A26/27/28+. A third population (less than 5%) is BoT4-/BoT8-/ILA27/28- but reacts with IL-A26.     

Expression of myeloid differentiation antigens on normal and chronic granulocytic leukemia erythroid progenitor cells identified by monoclonal antibodies

The antigenic phenotype of erythroid progenitor cells (BFU-E and CFU-E) in bone marrow and peripheral blood from normal individuals and patients with chronic granulocytic leukaemia (CGL) was studied using 14 myeloid monoclonal antibodies (Mc Abs) in a complement dependent cytotoxicity assay followed by culture in methylcellulose. Mc Abs which reacted with normal CFU-GEMM and CFU-GM antigens usually reacted strongly with normal and CGL-BFU-E. In contrast, the majority of myeloid Mc Abs used in the study reacted poorly or did not recognize the antigens on normal and CGL CFU-E. HLA-DR antigens recognized by L243 Mc Ab were expressed on the majority of BFU-E from blood and bone marrow of normal individuals and CGL patients. On the other hand, those antigens were not expressed on normal and leukaemic CFU-E. Two Mc Abs, R1.B19 and WGHS29.1, which recognised the antigens on "late" CFU-GM and not on "early" CFU-GM reacted with a higher proportion of BFU-E from the marrow and blood of CGL patients than of normal subjects. These results indicate that BFU-E in CGL are more differentiated than their normal counterparts. The Mc Ab 54/39, frequently expressed on platelets, recognized a higher percentage of BFU-E and CFU-E from CGL patients than from normal individuals. This may suggest that in CGL there exists a tendency for the expression of megakaryocytic markers on erythroid progenitor cells.     

T-prolymphocytic leukemia with circulating carrot-like cells

A case of T-prolymphocytic leukemia leading to rapid demise of a 67-year-old man is reported. He presented with multiple skin lesions and splenomegaly. A unique feature was that a proportion of circulating leukemic cells assumed bizarre shapes, resembling carrots. The leukemic cells expressed the T-cell markers T11, T8 and Dako-T2, and the natural killer cell markers NKH1, Leu7 and Leu 11b.     

Characterization by ultrastructural cytochemistry of normal and leukemic myeloid cells reacting with monoclonal antibodies

Six monoclonal antibodies (McAb) of the FMC series with specificity for granulocytic or monocytic cells were investigated by means of the immunogold method in combination with the myeloperoxidase reaction at ultrastructural level. This method allowed a precise characterization of the level of myeloid maturation at which the antigens detected by the McAb were expressed and confirmed their specificity for the two myeloid lineages. In addition, it facilitated the identification of granulocytic and monocytic cell components in cases of acute myeloid and chronic myelomonocytic leukemia. Blast cells did not react with any of the McAb used, and the reactivity in more mature cells was parallel to the development of cytoplasmic granules. The pattern of reactivity was similar in normal and leukemic cells, thus suggesting that these McAb could be applied to studies of normal myelopoiesis and to the classification of leukemia.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

Monoclonal antibodies against differentiation antigens on human macrophages

Human macrophages matured in vitro from blood monocytes without additional exogenous activation were used to produce monoclonal antibodies (mAbs). Ten of them were selected for high avidity and the best discrimination between mature macrophages and a panel of other cells including freshly prepared monocytes, B cells, T cells, granulocytes and thrombocytes. Five mAbs (MAX. 1, MAX. 2, MAX. 3, MAX. 11, MAX. 24) were found to detect macrophage differentiation antigens. One mAb (MAX. 26) detects an antigen which is shared by mature macrophages and T cells.     

Monoclonal antibodies to rat Kupffer cells. Anti-KCA-1 distinguishes Kupffer cells from other macrophages.

Two monoclonal antibodies, anti-KCA-1 and anti-KCA-2, directed against rat Kupffer cells (hepatic sinusoidal macrophages) were developed. Immunohistologic studies of the liver and analysis of isolated hepatic cells by immunofluorescence and flow cytometry showed that the reactivity of these antibodies was restricted to macrophages. Both KCA-1+ and KCA-2+ cells were located predominantly in the periportal region; in contrast, Ia+ sinusoidal cells were located primarily in the centrilobular region. Macrophagelike cells within the portal tracts expressed KCA-2 but not KCA-1. These findings indicate the presence of heterogeneity within the macrophage population of the liver. Anti-KCA-1 reactivity appeared to be almost entirely restricted to Kupffer cells; only a few macrophages in the thymus and a small number of cells in the bone marrow expressed KCA-1. In contrast, KCA-2 was more widely distributed; splenic, lymph node, and intestinal macrophages were intensely stained with anti-KCA-2. These studies indicate that KCA-1 is a marker of Kupffer cells.