Multiplex dynamic reporters enable single cell temporal analysis of costimulatory and cytokine receptor engagement effects on TCR signaling

Authors

• W Chan
• M Cao
• M Smelkinson
• J Kabat
• C Bradfield
• JL Hor
• RN Germain

Abstract

Intracellular signal dynamics are key to unraveling the complex decision-making processes cells use to enable appropriate responses to a wide range of extracellular stimuli. T cell antigen receptor signaling pathways have been an intense area of investigations for decades; however, very few studies had probed the dynamics of multiple downstream signaling pathways in individual live lymphocytes with high spatial-temporal resolution. Utilizing an optimized split-GFP complementation detection system (CDS) with GFP11-tagged endogenous RelA, c-Rel or NFAT2, combined with the ERK, JNK or p38 kinase translocation reporter (KTR), we developed novel live cell dual reporter systems that allow for high-resolution single-cell quantification of the MAPK, NFAT and NF-kappaB activation dynamics via confocal time-lapse microscopy. We probe signaling dynamics in response to anti-CD3, anti-CD28, the combination and anti-CD3 plus TNF as a model of early feedback regulation of T cell activation. In addition to revealing differences between TCR and CD28 signaling in these pathways, our studies identify a unique second-wave of RelA translocations that is specifically induced by the combination of TNF and TCR-CD3 signaling, regardless of CD28 engagement, without influencing ERK activation dynamics. Unlike the initial response to TNF or TCR-CD28 stimulation, these sustained secondary RelA translocation events are dependent on induced cytokine secretion or dynamic membrane receptor expression. These data illustrate how the cellular environment can specifically influence the process of immune cell signaling and compensate for the need of a core costimulatory signal that is commonly provided through cell-cell interaction to promote optimal cell survival and proliferation.

Scientific Focus Area: Systems Biology

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