Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.

In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.

2-photon imaging of phagocyte-mediated T cell activation in the CNS.

Autoreactive T cells can infiltrate the CNS to cause disorders such as multiple sclerosis. In order to visualize T cell activation in the CNS, we introduced a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) as a real-time T cell activation indicator. In experimental autoimmune encephalomyelitis, a rat model of multiple sclerosis, we tracked T cells interacting with structures of the vascular blood-brain barrier (BBB). 2-photon imaging documented the cytoplasmic-nuclear translocation of fluorescent NFAT, indicative of calcium-dependent activation of the T cells in the perivascular space, but not within the vascular lumen. The activation was related to contacts with the local antigen-presenting phagocytes and was noted only in T cells with a high pathogenic potential. T cell activation implied the presentation of an autoantigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon instillation of exogenous autoantigen. Activation did not cogently signal long-lasting arrest, as individual T cells were able to sequentially contact fresh APCs. We propose that the presentation of local autoantigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination, enabling them to attack the target tissue.

Intravital imaging of autoreactive T cells in living animals.

Developing technologies now allow observing cellular motility and functions in the living animal. Here, we introduce the method of intravital imaging by using two-photon microscopy. To acquire images with good quality, a highly stablilized tissue is required. Additionally, physiological parameters of the animal need to be controlled during the entire period of intravital imaging. We image rat autoreactive T cells within the spinal cord leptomeninges. Those autoantigen specific T cells were labeled in vitro by using fluorescent protein coding retroviral vectors. Adoptively transferred T cells migrate into the spinal cord with highly reproducible time kinetic. Intravital imaging was performed within the deeply anesthetized animals. Although two-photon microscopy is a powerful tool, the penetration depth in certain tissues, like the spinal cord parenchyma, is still limited. To overcome this issue, imaging of explanted acute spinal cord slices was performed under nearly physiological conditions. Results obtained from intravital imaging will strengthen the "snap shots" analysis such as FACS and quantitative PCR, and can provide new insight into cellular mechanisms in vivo.