Stimulus dependence of the action of small-molecule inhibitors in the CD3/CD28 signalling network.

Abstract

Cells in the body are exposed simultaneously to a multitude of various signals. Inside a cell, molecular signalling networks integrate this information into a physiologically meaningful response. Interestingly, in the cellular testing of drug candidates, this complexity is largely ignored. Compounds are tested for cells that are challenged with one stimulus only. The activation of T lymphocytes through engagement of the T cell receptor (TCR)-CD3 complex and CD28 coreceptor is a prominent example for a cellular response that depends on the integration of signals. We investigated the cellular response characteristics of this network at different strengths of receptor and coreceptor activation. A novel cellular microarray-based approach, in which various combinations of antibodies directed against the CD3 complex and CD28 were spotted, was employed for analysing the stimulus dependence of activation of the transcription factor NFAT and actin reorganisation. For both responses, quantitative differences in inhibitor activity were observed. Remarkably, for IL-2 expression, which was detected by standard ELISA, low doses of the Src-family kinase inhibitor PP2 strongly potentiated IL-2 expression at high-level, but not at low-level, CD28 co-engagement. Therefore, for a physiologically highly relevant signalling network, the cellular response might vary qualitatively with only quantitative variations of a stimulus. This level of complexity should be considered in early cellular drug testing.