Developmental regulation of TCR-CD3-dependent [Ca²⁺](i) responses of individual normal and pp59(fyn)-deficient T lymphocytes

The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca²⁺](i) signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca²⁺](i) of individual cells from immature and mature murine T-cell populations following application of CD3-ε monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca²⁺](i) in bulk cell populations. The CD3-mediated [Ca²⁺](i) responses 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 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca²⁺](i) responses correlated with maturity (CD4⁺ CD8⁺ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59(fyn)-deficient mice showed that pp59(fyn) deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca²⁺](i) responses of CD4⁺ CD8⁺ and CD4⁺ CD8^- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca²⁺](i). These developmentally distinct [Ca²⁺](i) features most probably reflect meaningful developmental changes in how the TCR-CD3 complex 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.