Differentiation of thymocytes from CD3-CD4-CD8- through CD3-CD4-CD8+ into more mature stages induced by a thymic stromal cell clone.

We have investigated the capacity of our established thymic stromal cell clone (MRL104.8a) or its derived factor(s) to induce the differentiation of immature thymocytes. Culture of purified adult murine double-negative (CD4-CD8-, indicated here as CD4-8-) thymocytes on the MRL104.8a thymic stromal cell monolayer for 1 day resulted in the induction of an appreciable percentage of CD4-8+ thymocytes. A bone marrow-derived stromal cell monolayer or a L929 fibroblast monolayer failed to generate CD4-8+ cells. This differentiation could also be induced by a semipurified sample of the MRL104.8a culture supernatant, which contained a thymic stroma-derived T-cell growth factor capable of contributing to the growth of double-negative immature thymocytes. CD4-8+ thymocytes generated 1 day after coculture with the MRL104.8a cells or the sample containing thymic stroma-derived T-cell growth factor were found to be CD3- and J11d+, excluding the possibility of expansion of mature (CD3+4-8+) thymocytes present in the thymus. More importantly, when the culture period was extended to 2 or 3 days, an appreciable number of CD4+8+ and single-positive (CD4+) cells were generated on the MRL104.8a monolayer. Thus, these results provide the direct demonstration that CD3-4-8- immature thymocytes are promoted to differentiate through a rapidly cycling intermediate (CD3-4-8+) into double- and single-positive cells by a specialized thymic stromal component.