Multiple Sclerosis (MS) is an autoimmune disease, which features a highly complex pathogenic cascade and involves the infiltration of mononuclear cells into the brain and spinal cord. Although many essential steps in the development of the disease remain ambiguous, experimental and clinical studies indicate that autoreactive CD4+ helper T cells are crucial for induction of inflammation in the central nervous system (CNS). By using Experimental Autoimmune Encephalomyelitis (EAE) as an animal model for MS, it was previously shown that encephalitogenic T cells mature in peripheral organs. Next, T cells migrate to the CNS, become activated, and initiate inflammation. During their sojourn, the T cells perceive stimuli and respond to their microenvironment through signal transduction mechanisms.
To portray the serial signaling in transfer EAE (tEAE), two activation reporters, a FRET-based calcium biosensor and a fluorescent NFAT activation marker were combined with in situ two-photon microscopy. The Twitch calcium sensor can detect weak signals which are accumulated within the cells and lead to T cell activation. On the other hand, NFAT sensor can reliably detect T cell activation induced by antigen recognition in vivo. During T cell maturation in the spleen, both myelin basic protein (MBP) specific encephalitogenic and OVA specific control T cells displayed similarly low frequent and short-lasting calcium signaling. This process was driven by chemokine and MHC class II-dependent signals. Next, arrived at leptomeningeal blood vessels, the portal to the spinal cord, intravascular T cells presented minimal calcium activity. Short-lasting calcium signaling was detected only during rolling-crawling transitions. After extravasation, in spinal cord leptomeningeal space and parenchyma, the T cells responded with high, sustained calcium plateaus, and NFAT translocation. T cells presented longer-lasting elevated calcium levels (>2 min) after contacting local antigen presenting (APC) cells, whereas OVA specific T cells presented only short calcium spikes. Each APC displayed different potential to stimulate T cells likely due to the limited availability of immunogenic myelin proteins. This T cell reaction was most pronounced in the prodromal phase, and followed a ‘first come – first served’ rule for antigen recognition. When MHC class II was blocked by intrathecal injection of blocking antibody, MBP specific T cells presented only short-lasting calcium signaling similar to those in the spleen. Accordingly the treatment reduced the infiltration of T cells and clinical severity.
To directly correlate the activation of encephalitogenic T cells with their calcium signaling, a new combined sensor was generated. The co-expression of Twitch and ∆NFAT protein required a set of fluorescent proteins (FP) that would have minimal bleed through in their emission channels. Five FPs were evaluated, and mRuby2 was selected as the red analogous counterpart of ∆NFAT-GFP. In combination with Twitch, ∆NFAT-mRuby2 displayed similar translocation kinetics compared to ∆NFAT-GFP and presented adequate two-photon absorption. Nonetheless, more detailed studies need to be performed regarding the dual sensors‘ transduction efficiency.