DEVELOPMENTAL REGULATION OF TCR-CD3-DEPENDENT [CA2-DEFICIENT T-LYMPHOCYTES(](I) RESPONSES OF INDIVIDUAL NORMAL AND PP59(FYN))

The aim of this study was to ascertain whether different types of T-ce ll receptor (TCR)-mediated [Ca2+](i) signals could begin to explain th e different cellular responses of mature and immature T cells to ligat ion of the TCR-CD3 complex. Using a digital fluorescence imaging syste m, we measured and compared [Ca2+](i) of individual cells from immatur e and mature murine T-cell populations following application of CD3-ep silon monoclonal antibody (mAb). Our approach revealed distinctions am ong developmental subsets which were not seen by previous measurements of [Ca2+](i) in bulk cell populations. The CD3-mediated [Ca2+](i) res ponses of individual thymocytes were very complex. Latencies to peak [ Ca2+](i) varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+](i ) responses may be an important informative parameter for TCR-CD3 sign alling. In addition, among cells responding to CD3 mAb, higher peak [C a2+](i) responses correlated with maturity (CD4(+)CD8(+) thymocytes < single-positive thymocytes < splenic T cells). Examination of cells fr om pp59(fyn) deficient mice showed that pp59(fyn) deficiency affects t he amplitude and probability, but not the latency or synchrony, of CD3 -mediated [Ca2+](i) responses of CD4(+)CD8(+) and CD4(+)CD8(-) thymocy tes. All subsets showed equivalent receptor-independent mobilization o f [Ca2+](i). These developmentally distinct [Ca2+](i) features most pr obably reflect meaningful developmental changes in how the TCR-CD3 com plex couples to intracellular signalling machinery including pp59(fyn) . By clearly showing how [Ca2+](i) responses change during development , these results support the hypothesis that distinctive types of [Ca2](i) responses drive thymocyte differentiation.