Characterization and differentiation of CD4-, CD8- thymocytes sorted with the Ly-24 marker.
Heterogeneity within the CD4-, CD8- thymocyte population was explored by flow microfluorometry on thymocytes from 6-wk-old female C57BL/6 mice. Double negative cells were obtained by twice killing thymocytes with anti-CD4 and anti-CD8 antibodies. The resultant population lacked CD4, CD8, and Ig on cell surfaces; it contained cells bearing Ly-24 (27%), Ly-6C (6%), and Ly-5 (B220) detected by 6B2 (6%). These markers are the same as those characteristic of lpr/lpr T cells; they also are found on bone marrow cells. In order to investigate the maturational pathway of CD4-, CD8- thymocytes, such cells were cultured in vitro for 6 days with phorbol myristate acetate + calcium ionophore. There was a marked increase in cells bearing Ly-24 with time; by 6 days essentially all bore Ly-24. A lesser increase (to 15%) in 6B2 + Thy-1 positive cells was observed. Small numbers of cells bearing CD4 and/or CD8 also were found after 6 days in vitro. In additional studies, CD4-, CD8- cells were first sorted with respect to Ly-24 and then cultured with phorbol myristate acetate + calcium ionophore. Ly-24+ cells proliferated vigorously and formed clusters whereas Ly-24- cells did not. The former gave rise to large numbers of CD4+, CD8+ cells; the latter exhibited little differentiation. These studies demonstrate substantial heterogeneity within the CD4-, CD8- thymocyte population. Use of the markers Ly-24, Ly6C, and 6B2 allows a subdivision of such progenitor thymocytes. Different stages of maturation as well as possible lineages of cells may be investigated by combining such hemopoietic cell surface markers with in vitro culture.
Gause
W C
WC
Cellular Immunology Section, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD 20892.
Mountz
J D
JD
Steinberg
A D
AD
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D002454:Cell Differentiation; D002478:Cells, Cultured; D005434:Flow Cytometry; D051379:Mice; D008810:Mice, Inbred C57BL; D013601:T-Lymphocytes / Q000145:classification / Q000276:immunology*; D013950:Thymus Gland / Q000166:cytology* / Q000276:immunology
D000945:Antigens, Differentiation, T-Lymphocyte