Multiple marker analysis of resting and activated murine thymocytes.

The expression of differentiation antigens and terminal deoxynucleotidyl transferase (TdT) were studied with cultured murine thymocytes using the immunofluorescent technique. Four different culture conditions were used: i) medium alone; ii) addition of Con A; iii) addition of Con A stimulated splenocyte supernatant (CSS); iv) addition of both Con A and CSS. The percentages of viable cells and their absolute numbers were evaluated. TL expression was strongly decreased in all four cases, with the almost total disappearance of TL+ cells in Con A + CSS-stimulated cultures. In contrast, TdT+ cells remained well represented. The distribution of cells expressing or not expressing Lyt antigens varied according to the different culture conditions. The only cell type to exceed its initial number was the TdT+ Lyt- one, in cultures performed in the presence of Con A + CSS. The absolute number of Qa-2+ cells was initially reduced by 50% after 2 h in culture, and was then maintained for 3 days. This number was increased only in the presence of Con A + CSS. Therefore, Qa-2+ cells appear much more resistant than the other thymocyte subpopulations in culture, and even proliferate under stimulating conditions. The proliferating Qa-2+ cells also expressed TdT, as detected by the immunofluorescent technique, but enzymatic detection of the enzyme gave unsignificant results. Proliferating Qa-2+ cells presented either Lyt1 or both Lyt1 and Lyt2 antigens. The expression of H-2K antigen was also strongly increased in stimulated cultures, but Qa-4 and Qa-5 antigens could not be detected. These results suggest that Con A and CSS stimulated both mature cells and precursor cells (as shown by TdT expression). The possible relevance of these findings for the theories of thymocyte ontogeny is discussed.