Multiphoton intravital microscopy analysis of signaling in T cells reveals persistent NFAT activation during dynamic APC scanning. (121.21)

Abstract

Abstract The Ca2+ dependent activation and nuclear shuttling of the cytoplasmic transcription factor NFAT is a hallmark of productive T cell receptor (TCR) triggering. However, the kinetics of NFAT activation and the relationship to the Ca2+ dependent migratory stop signal have not been explored in vivo. We used Multiphoton Intravital Microscopy (MP-IVM) to visualize the subcellular dynamics of a fluorescent reporter of NFAT signaling (NFAT-eGFP) in cytotoxic T cells during their antigen-dependent interactions with various APC in lymph nodes (LNs) and in tumor tissue in vivo. During interactions with peptide-loaded B cells representing optimal APC in LNs, t1/2 of NFAT nuclear translocation was 1 minute. Relocation to the cytoplasm upon contact cessation occurred with a slower t1/2 of ~20 minutes. During its delayed nuclear export, NFAT continued to drive transcription of IFN-gamma. We therefore hypothesized that NFAT may facilitate the integration of TCR signals received through serial APC contacts. To test this in vivo, we performed MP-IVM to visualize contacts between cytotoxic T cells and tumor cells, representing suboptimal APCs. We observed that among T cells with continuously nuclear NFAT, ~30% remained motile while engaging in transient, serial contacts with multiple tumor cells. Hence, NFAT slow off-kinetics may constitute a mechanism of short-term biochemical memory enabling the integration of intermittent TCR signaling into a continuous transcriptional response.