In vitro differentiation and commitment of CD4+ CD8+ thymocytes to the CD4 lineage, without TCR engagement

Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells. Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells. The signals, however, still remain to be identified. Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect. Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and TCR transgenic mice. We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4+ CD8lo T cells that have mostly committed to the CD4 lineage. The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation. The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.