Real-time in vivo analysis of T cell migration and activation in the periphery and in the central nervous system using a genetically encoded calcium indicator (P4103)

Abstract

Abstract Interaction of T cells with cells of the surrounding milieu may result in activation of one or both partners. In particular, T cell activation following recognition of antigen presented by antigen presenting cells (APCs) triggers a swift rise of intracellular calcium levels within the T cell. Synthetic calcium indicators have been successfully used to monitor the activation status of T cells in vitro. However, these chemical dyes do not persist intracellularly for a sustained period which creates a major challenge for calcium imaging in the living animal. We now present a newly developed fluorescence resonance energy transfer (FRET)-based, genetically encoded calcium indicator (GECI) expressed in (auto)antigen-specific CD4+ T cells to study T cell activation in vivo at real time. Using two-photon microscopy, we explored the responses of retrovirally transduced, GECI-expressing T cells to antigen. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around APCs concomitant with an instant rise of cytosolic calcium levels. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium levels while still meandering through the space. This approach enables us for the first time to follow the migration and activation patterns of T cells in vivo during the course of the disease.