Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant.
The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.
D000818:Animals; D000911:Antibodies, Monoclonal; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods; D003114:Colony-Forming Units Assay; D000939:Epitopes / Q000032:analysis*; D006183:H-2 Antigens / Q000032:analysis*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008815:Mice, Inbred Strains; D013154:Spleen / Q000276:immunology; D013950:Thymus Gland / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006183:H-2 Antigens; C024755:H-2K(K) antigen