CBL561 Sigma-AldrichAnti-CD24 Antibody, clone SN3

The fetal mouse omentum has been shown to be a source of precursors that exclusively reconstitutes Ly1+ B cells and the closely related Ly1- sister population, but not conventional B cells or T cells. We have extended these studies to compare B cell development in the human fetal omentum, liver, and spleen, and to demonstrate that the pro/pre-B cell compartment (CD24+, sIgM-) is detected in the omentum and liver but not spleen as early as 8 wk of gestation. From 8 to 12 wk of gestation, the proportions of IgM+ cells that were pre-B cells (cIgM+/sIgM-) in the omentum and liver were 53 +/- 15% and 45 +/- 13%, respectively, and IgM+ cells were not detectable in the spleen. After 12 wk, the percentage of pre-B cells was unchanged in the fetal liver (41 +/- 10%) but decreased significantly in the omentum (25 +/- 14%); pre-B cells were now detected in the spleen but at much lower percentages (2 +/- 3%) than either the omentum or liver. The nuclear enzyme, Tdt, was detected in approximately 25% of the CD24+ cells in the omentum and liver during the 8-12-wk time period, however, Tdt+ cells were not detected in the spleen. Approximately 40% of the mature B cells found in the omentum and spleen were CD5+ compared with only 20% in the liver. These results demonstrate that the fetal omentum, like the fetal liver and bone marrow, is a primary site of B cell development.

The CD24 Ag is present on human B cells from the earliest stages of B lineage development and is lost on terminal B cell maturation to plasma cells. This Ag is also expressed on mature forms of granulocytes. We studied the effects of CD24 mAb on the function of both lymphocytic and myeloid cell types. We found a clear-cut increase in free cytoplasmic calcium when tonsil B cells or mononuclear cells from B cell chronic lymphatic leukemia patients were preincubated with CD24 mAb and further stimulated with goat F(ab')2 anti-mouse Ig antibodies. The same experimental setting with granulocytes instead of B lymphocytes led to the triggering of hydrogen peroxide production in these cells. CD24 Ag is expressed at higher levels on activated granulocytes and is anchored to the plasma membrane by a phosphoinositol linkage. In conclusion we provide evidence that the CD24 Ag are functional molecules in cells of different lineage, playing a signal transducing role in cell function.

The CD24 human B cell-associated antigen was originally characterized in this laboratory with the use of monoclonal antibody BA-1 combined with a standard radioimmuneprecipitation technique, and was reported to be a three chain glycoprotein complex of 45, 55, and 65 kilodaltons. We have compared our standard radioimmuneprecipitation technique (BA-1 ascites followed by rabbit anti-mouse IgM-coated protein A-Sepharose) to BA-1 conjugated directly to CNBr-Sepharose (BA-1-Sepharose) and report striking differences in the electrophoretic profile of CD24 immuneprecipitates. The CD24 immuneprecipitate obtained with BA-1-Sepharose showed a single broad band with a relative mobility of 42 kilodaltons. A series of experiments performed with the two immuneprecipitation techniques, reducing or nonreducing electrophoretic conditions, and addition of preformed mock BA-1 immuneprecipitate to BA-1-Sepharose immuneprecipitates convincingly demonstrated that the previously described 55 and 65 kilodalton components were artifacts caused by co-migration of CD24 with IgG and IgM heavy chains, respectively. Because of the consistent association and co-migration of CD24 with IgG heavy chains, we investigated the possibility that CD24 might be related to the 45 kilodalton Fc-gamma receptor expressed on B cells and eosinophils. We found, however, no evidence for such a relationship by cross adsorption analysis with BA-1-Sepharose and IgG-Sepharose.

We have generated and characterized three new monoclonal antibodies (mAbs), termed SN3, SN3a, and SN3b, which are directed to sialic acid of a glycoprotein(s) on human non-T leukemia cells. These mAbs were generated by immunizing mice with an antigen preparation isolated from cell-membrane glycoconjugates of NALM-1, a pre-B leukemia cell line. The initial characterization of the mAbs consisted of a sensitive cellular radioimmunoassay against various cultured human leukemia-lymphoma (HLL) and nonmalignant cell lines. They strongly reacted with all four (all three in the case of SN3a) non-T/non-B HLL cell lines tested and both pre-B HLL cell lines tested. However, they reacted with only one of three B HLL cell lines tested. In addition, these mAbs did not react with other cell lines, which include T- and myelomonocytic HLL cell lines and nonmalignant B-cell lines. Normal peripheral blood cells were also tested; the mAbs reacted with B cells and granulocytes but not with T cells, monocytes, erythrocytes, or platelets. In a test using SN3 and SN3b with uncultured cell specimens derived from various cancer patients, the mAbs primarily reacted with non-T/non-B and B HLL specimens, as well as with chronic myelocytic leukemia specimens. The biochemical nature of antigenic determinants defined by the three mAbs was studied by treating the non-T leukemia cells with sialidase and proteases. The results show that the antigenic determinants defined by these mAbs all contain a sialic acid residue(s) that is attached to the cells via a protein backbone(s). Competitive binding experiments show that binding of SN3 to the leukemia cells was blocked almost completely by SN3a and SN3b, as well as by BA-1. Both SN3 and SN3a are IgG1 antibodies, whereas SN3b is an IgM antibody; SN3b showed a strong complement-mediated cytotoxic activity against non-T leukemia cells.

CLL B cells may be induced to form B-cell colonies in vitro. Colonies formed are monoclonal and appear to reflect the circulating malignant B-cell clone in vitro. Using hybridoma-produced monoclonal antibodies (MAB) and an in vitro B-cell colony assay, we have provided a characterization of the antigenic phenotype of the clonogenic CLL B cell. B-cell colony growth in both patients and normals was not altered by prior incubation with either MAB or complement (C') alone. CLL B-cell colony formation was markedly reduced after treatment with T101 and C', while normal colonies were unaffected (8 +/- 2 versus 107 +/- 10). None of the residual CLL B-cell colonies after T-101 and C' treatment reacted with T-101. However, BA-1 and la reactivity were still seen in residual CLL B-cell colonies following T-101 treatment. In contrast, a similar percentage reduction of B-cell colony growth was seen for both normals and CLL patients following treatment with BA-1 (76% versus 81%) and Q5/13 (89% versus 92%). These studies suggest that the CLL progenitor cell is characterized by the phenotype la+, BA-1+, T-101+. Better definition of the CLL progenitor cell has potential implications with regards to clinical utilization of MAB in the treatment of CLL.

Lymphoblasts from 59 children with non-T, non-B acute lymphoblastic leukaemia were studied with monoclonal antibodies to four cell-surface proteins. 87% of the children had lymphoblasts positive for HLA-DR, 82% for p30, 75% for p24, and 72% for CALLA. The commonest composite phenotype was HLA-DR+ p30+ CALLA+ p24+. Significant correlations were seen between expression of HLA-DR, p30, and CALLA, but not p24. p30- and CALLA phenotypes were found in patients with high white-blood-cell counts (WBC) and splenomegaly. With standard chemotherapy, disease-free survival from time of remission was shorter in p30- and CALLA- patients than in others. Splenomegaly was associated with poor disease-free survival and provided prognostic information independent of phenotype. High WBC was less significant than phenotype in predicting outcome and was not independent of phenotype.