Search

In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1.2), suggesting a role in locomotion. On dividing multinegative thymocytes, FMC46 was found almost exclusively along the cleavage furrow, implicating it in detachment processes. We conclude that the properties of the LAM-1 molecule recognized by FMC46 are consistent with a role in detachment phases of motility and of cell interactions.

A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

The technique of somatic cell hybridization has been used to produce a monoclonal antibody (designated FMC7) against the human B lymphoblastoid cell line HRIK. By indirect immunofluorescence FMC7 reacted with a total of 3 out of 10 B cell lines, but failed to bind T and Null cell lines. On normal peripheral blood, FMC7 bound between 3 and 6% of mononuclear cells. When normal peripheral blood mononuclear cells were fractionated, FMC7 reacted with only the B-enriched fraction, staining some but not all cells expressing SMIg. Double marker analysis indicated that the antigen detected by FMC7 is not identical with any of the conventional B cell markers, including surface membrane IgM, C3 receptors, Fc receptors, and the binding site for mouse erythrocytes. Another monoclonal antibody, FMC1, which reacts specifically with B lymphocytes, binds a separate antigenic determinant. Thus, FMC7 appears to recognize a previously undetected marker present specifically on a subpopulation of human B lymphocytes. FMC7 reacts with some but not all B cell leukemias and may have diagnostic applications in leukemia.

A somatic cell hybrid line which secreted antibody reacting selectively with a proportion of the white cells in human blood was prepared. The hybridoma appeared to be monoclonal, and the antibody secreted stained 67% of the lymphocyte population in blood. It reacted less well with granulocytes and monocytes. The lymphocytes stained comprised 80% of the T cells and 50% of the B cells. The antibody showed no recognizable pattern in its reactivity with cell lines and leukaemic cells, although B cells tended to react less well than T cells, null cells, or myeloid leukaemic cells. The expression of the antigenic determinant is discussed in relation to the classification of leucocytes. This determinant and certain other markers exhibited differential expression on closely related cells, and yet were shared by more distantly related cells.

A hybridoma has been isolated from the products of fusion of a myeloma cell line with spleen cells from mice immunized with a human B cell line. After cloning, the hybridoma secretes antibody with the following properties: (i) Human B-lymphohlastoid cell lines react with the antibody while T and null cell lines do not. (ii) The antibody reacts with the majority of leucocytes in the blood of patients with CLL, but with a minority of cells in the blood of patients with A ML or ALL of the null or T type, (iii) The antibody reacts with 9-21% of mononuclear cells in normal peripheral blood. The reacting cells are not T cells and overlap extensively with cells identified as B cells by other markers. The antigen identified by this antibody appears to be distinct from known B cell markers, and is put forward as a new B cell marker with diagnostic potential. [ABSTRACT FROM AUTHOR]