Abstract

AbstractTo visualize the entire process of encephalitogenic T cell infiltration into the target organ, we performed intravital imaging by using two‐photon microscopy in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Intravital imaging documented that T cells first appear in the leptomeningeal blood vessels where they crawl in an integrin‐dependent manner and scan the intraluminal surface for extravasation sites. After diapedesis, the T cells continue to crawl on the abluminal surface, where they meet local antigen presenting cells (APC) that can provide stimuli to the T cells for the subsequent infiltration into the central nervous system (CNS) parenchyma. Although flow cytometric analysis documented that the infiltrated T cells upregulated their activation markers in the CNS meninges, it was unclear at which scanning step the activation occurred. We recently introduced two genetically encoded fluorescent T cell activation sensors for intravital imaging. The first is a fluorescent resonance energy transfer‐based Ca2+ sensor for the quantification of the intracellular Ca2+ concentration, a major step in T cell receptor signaling. The second sensor is a truncated nuclear factor of activated T cells fused to green fluorescent protein, which subcellular localization corresponds to the T cell activation state. Introducing these sensors into the lymphocytes enables the visualization of the interactions of encephalitogenic T cells with different blood–brain barrier structures, and allows us to assess the functional aspect of these interactions directly in vivo. This model system can be further used to evaluate therapeutic compounds and to better understand the activities of T cells in vivo.

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