Heterogeneity of immature (Lyt-2-/L3T4-) thymocytes. Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements.
Thymocytes that bear neither Lyt-2 nor L3T4 differentiation Ag (2-4- thymocytes) contain the precursors of mature Lyt-2+ and L3T4+ T cells. In the present study, we have identified four major subpopulations of 2-4- cells in adult C57BL/6 mice that differ in surface phenotype and in situ proliferative status. Two-color immunofluorescence analysis with RL-73 (a mAb recognizing an as yet unidentified activation Ag) and PC-61 (an anti-IL-2R mAb) revealed three distinct subsets of 2-4-thymocytes: RL-73+ IL-2R- (30%), RL-73+/-IL-2R+ (45%), and RL-73- IL-2R- (25%). The RL-73+ IL-2R- subset had the highest percentage of large blasts and cycling cells, whereas the RL-73+/- IL-2R+ and RL-73- IL-2R- subsets had intermediate and low percentages, respectively, indicating that in situ proliferation correlated better with RL-73 intensity than with IL-2R expression. An additional marker, heat-stable Ag (HSA), was found to further subdivide the RL-73- population into RL-73- HSA- (10% of total 2-4-) and RL-73- HSA+ (15%) fractions. The two latter (RL-73-) subsets appeared to be more "mature" than the former since they expressed high levels of Lyt-1 and appeared later during fetal thymus ontogeny. In parallel with the phenotypic analysis, we compared the in vitro activation requirements of each of the four purified 2-4- subsets. All four populations proliferated well to the combination of phorbol ester (PMA), ionomycin, and IL-2. In response to PMA and ionomycin (without added IL-2), only RL-73- HSA-cells proliferated and this proliferation was correlated with IL-2 production. However, if IL-1 was included with PMA and ionomycin then all four populations responded. Finally, a proliferative response to Con A or mitogenic anti-Thy-1 mAb was observed only for RL-73- HSA+ and (to a lesser extent) RL-73- HSA-cells. These data indicate that each of the four phenotypically distinct subpopulations of immature thymocytes can also be distinguished on the basis of their in vitro activation requirements.
Howe
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RC
Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland.
MacDonald
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HR
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D000950:Antigens, Ly / Q000032:analysis*; D002453:Cell Cycle; D004987:Ethers / Q000494:pharmacology; D005260:Female; D007375:Interleukin-1 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015759:Ionomycin; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010641:Phenotype; D013601:T-Lymphocytes / Q000145:classification* / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013950:Thymus Gland / Q000196:embryology
D000945:Antigens, Differentiation, T-Lymphocyte; D000950:Antigens, Ly; D004987:Ethers; D007375:Interleukin-1; D007376:Interleukin-2; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate