Intracellular calcium dependence of gene expression in single T lymphocytes

In T lymphocytes, intracellular Ca²⁺ concentration ([Ca²⁺](i)) rises within seconds of T-cell antigen-receptor stimulation and initiates the synthesis and secretion of interleukin 2, a cytokine essential for T-cell proliferation and the immune response. Using video-imaging techniques, we tracked [Ca²⁺](i) signals in individual T cells and measured subsequent expression of a β-galactosidase reporter gene (lacZ) controlled by the NF- AT element of the interleukin 2 enhancer. [Ca²⁺](i) spikes elicited by monoclonal antibody binding to the CD3ε subunit of the T-cell receptor were positively correlated with gene expression, but varied widely between individual cells and were therefore difficult to relate quantitatively to lacZ expression. The [Ca²⁺](i) dependence of NF-AT-regulated gene expression was determined by elevating [Ca²⁺](i) with either thapsigargin or ionomycin and then 'clamping' [Ca²⁺](i) to various, stable levels by altering either extracellular [Ca²⁺] or extracellular [K⁺]. Raising [Ca²⁺](i) from resting levels of 70 nM to between 200 nM and 1.6 μM increased the fraction of cells expressing lacZ, with K(d) ≃ 1 μM. Activation of protein kinase C enhanced the [Ca²⁺](i) sensitivity of gene expression (K(d) = 210 nM), whereas stimulation of protein kinase A inhibited [Ca²⁺](i)-dependent gene expression. The experiments described here provide single-cell measurements linking a second messenger to gene expression in individual cells.