Clonogenic potential of murine CD4+8+ thymocytes. Direct demonstration using a V beta 6-specific proliferative stimulus in Mlsa mice

Although considerable indirect evidence supports the hypothesis that CD4+8+ thymocytes are developmental intermediates in the generation of mature (CD4+8- or CD4-8+) T cells, the ability of these cells to proliferate in vitro has been highly controversial. We demonstrate here that a fraction of purified murine CD4+8+ thymocytes can be induced to proliferate in response to immobilized anti-TCR mAb. To exclude possible proliferation by trace mature T cell contaminants, we have exploited our recent finding that in Mlsa mice mature V beta 6-bearing thymic T cells are virtually absent (less than or equal to 0.5%) due to clonal deletion, whereas V beta 6 +CD4+8+ thymocytes are present in much higher numbers (approximately 3%). Proliferation of sorted CD4+8+ thymocytes from Mlsa mice was therefore induced at limiting dilution with immobilized anti-V beta 6 mAb to select against any contaminating mature T cells. Under optimal culture conditions, the frequency of CD4+8+ thymocytes proliferating specifically to anti-V beta 6 mAb (1/1000) was higher than those obtained for purified CD4-8+ (1/2000) or CD4+8- (1/5000) subsets, thus demonstrating directly that a proportion (in this case 3%) of CD4+8+ thymocytes are potentially clonable. During culture, V beta 6 +CD4+8+ thymocytes gave rise to a mixture of phenotypically "immature" (CD4-8-) and "mature" (CD4-8+) T cells. This system should be valuable for further analysis of the elusive CD4+8+ thymocyte subset.