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Free keywords:
antigen presentation; central nervous system;
intravital imaging; T cell activation
Abstract:
To 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.
