A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.
The CD31 antigen, a member of the immunoglobulin superfamily with a possible cell adhesion function, is expressed on approximately 50% of peripheral blood lymphoid cells at relatively low intensity (10-20% of the level on monocytes). In the accompanying paper we showed that a mAb, 5A2.G5, which identifies a glycosylation-dependent epitope of the CD31 antigen, bound to fewer lymphocytes than two other CD31 mAb, B2B1 and 2BD4, although the 3 antibodies bound equally well to monocytes. We have now analyzed the pattern of expression of epitopes of the CD31 antigen on lymphoid cell subpopulations using two-color immunofluorescence and flow cytometry. Large granular lymphocytes (CD16+), CD8-positive T cells and B cells (SMIg+) were mostly CD31-positive as indicated by the binding of mAb B2B1 and 2BD4. Single populations displaying some overlap with the negative control were obtained in each case. In contrast, CD4-positive T cells fell into two discrete populations with respect to CD31 antigen expression. mAb 5A2.G5 displayed weaker binding to all lymphoid cell types, indicating that the pattern of glycosylation of the CD31 antigen differs between lymphocytes (of all types) and cells of the myeloid lineages. The heterogeneity of CD31 antigen expression by CD4-positive cells was further examined by dual-labelling of purified CD4 cells with mAb B2B1 and CD45RA or CD29 mAb which identify naive and memory T cells respectively. The CD31 antigen was found to be preferentially expressed by the CD45RA-positive, naive cell population.
Three monoclonal antibodies (MAbs) to the human c-kit receptor tyrosine kinase (P145c-kit), derived in independent laboratories, have been extensively used in studies of c-kit expression and the role of its ligand, steel factor (SLF), in hemopoiesis and mast cell differentiation and function. In this study, the relationship between the epitopes they identify, and their effects on SLF binding, receptor internalization, and signal transduction are compared. Epitope mapping studies carried out on the high P145c-kit-expressing cell line HEL-DR showed that SR-1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacting epitopes. SR-1 potently blocked the binding of SLF to P145c-kit on these cells and also on cells of the factor-dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conversely, SLF partially blocked the binding of SR-1 and YB5.B8 to cells, while binding of 17F11 was actually enhanced by SLf on some target cells. Preincubation of HEL-DR and MO7e cells with MAbs prior to exposure to SLF revealed that 17F11 itself brought about partial down-regulation of P145c-kit and did not inhibit SLF-mediated down-regulation. SR-1 caused minimal down-regulation and inhibited SLF-mediated receptor internalization. YB5.B8 had minimal effects on either cell line in this assay. To determine whether the antibodies had any agonist activity, they were compared with SLF for their ability to bring about receptor phosphorylation in intact MO7e cells. All three antibodies induced detectable tyrosine phosphorylation with 17F11 being the most effective, while YB5.B8 was the least effective. Finally, the ability of the antibodies to influence the proliferation of the MO7e cells was examined. As expected, SR-1 potently inhibited the proliferative response to SLF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but reproducible agonist activity.
3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.
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