Characterization by Flow Cytometry and Fluorescence Microscopy of a Novel p34 kD Proliferation-Associated Antigen on the Surface Membrane of Normal and Leukemic Human Leukocytes
Monoclonal antibodies (MoAb) that recognize different surface membrane antigens have proven useful in classifying human leukocytes as different subsets. A recently developed murine MoAb 53.6 (IgG2a), raised against the HEL human erythroleukemia cell line has been shown to identify a p34 kDa surface membrane antigen that is associated with cell proliferation. The p34 kDa antigen is a non-glycosylated acid polypeptide whose structural gene is encoded on human chromosome 11. Moreover, the p34 kDa antigen is expressed on different human hematopoietic cell lines that represent leukemic leukocyte subsets of T cells, B cells, and myelomonocytic cells that are in different stages of maturation and differentation. Reported herein are studies that were undertaken to evaluate the utility of MoAb 53.6 for identifying proliferating leukocytes in the blood of patients with leukemia. It is anticipated that this MoAb 53.6 will prove to be a useful addition to panels of MoAb that are used currently for phenotypic analyses. Moreover, it is postulated that MoAb 53.6 will serve as a suitable substitute for the cumbersome DNA assay procedures employing propidium iodide. The immediate research objectives were to: (a) define by flow cytometry and fluorescence microscopy the expression of p34 kDa on peripheral blood mononuclear cells (PBMC) of healthy subjects; (b) determine the expression of the p34 kDa antigen on PBMC that have been activated in vitro with a combination of MoAb anti-CD3 and human recombinant interleukin 2 (hrIL-2) to generate lymphokine-activated killer (LAK) T cells; (c) compare the binding of MoAb 53.6 to that of other MoAb (e.g. CD3, CD4, and CD8); and (e) correlate the expression of the p34 kDa antigen using both PBMC and whole blood assay procedures. PBMC were isolated using Ficoll-Hypaque. The thoroughly washed PBMC were phenotyped using an indirect procedure incorporating a primary MoAb (usually MoAb 53.6, or its isotypic control) and a secondary heteroantibody (i.e., goat antimouse IgG-(Fab’)2 -FITC. In the whole blood assay method, the cells were labeled as described for the PBMC, and then exposed to ammonium chloride erythrocyte-lysing agent; the white blood cells (WBC; total leukocyte population) were then analyzed. Quantitative binding of the antibody was defined by flow cytometry and histograms were created that defined the cell relative number versus relative fluorescence intensity. These studies have shown: (a) MoAb 53.6 was expressed, but at very low levels on the majority (75%) of normal PBMC; (b) MoAb 53.6 binding to normal PBMC was specific and was not inhibited by IgG-Fc blocking agents; (c) p34 kDa antigen was upregulated on PBMC that had been activated with anti-CD3 + hrIL-2; (d) MoAb 53.6 binding (i.e., relative fluorescence intensity) to LAK cells was comparable to that observed with CD3, CD4, and CD8; and (f) similar levels of p34 kDa antigen was expressed using PBMC and whole blood assay method. These studies have identified the p34 kDa antigen on freshly isolated normal human PBMC, and have shown that this peptide is upregulated on PBMC that have been activated in vitro. Blast cells of patients with leukemia were also shown to display the p34 kDa antigen. In the future, studies will be conducted to characterize the expression of the p34 kDa antigen on the blood and bone marrow leukocytes of patients with different types of leukemia and who are in different phases of their disease.