Kawakami Lab – Live imaging of autoimmunity

One of the key pathological features of Multiple Sclerosis, a human autoimmune disease, is mononuclear cell infiltration within the central nervous system (CNS). Among those cells, autoantigen-specific CD4+ T helper cells play a central role in initiation of CNS inflammation. The aim of our work is to visualize and to understand the cellular and molecular mechanisms of T cell infiltration into the CNS beyond the blood-brain barrier and induction of CNS inflammation.

We use intravital two-photon microscopy to visualize genetically labelled autoreactive T cells in Experimental Autoimmune Encephalomyelitis (EAE), an animal model for Multiple Sclerosis. For T cell labelling, conventional Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) are used. Additionally, we perform functional imaging by using calcium sensing proteins.. Flow cytometry, immune fluorescence confocal imaging and quantitative PCR are used to support findings from intravital imaging.

The encephalitogenicity of T cells depends on TCR specificity and host. We have shown previously that the encephalitogenic potential is also related to T cell activation in the CNS. Highly encephalitogenic T cells were activated in the CNS, whereas weakly encephalitogenic T cells were not. However, it is largely unknown what factors contribute to different degrees of T cell activation. We visualized and compared the behaviour of highly and weakly encephalitogenic T cells in the animal. We are especially interested in differences in antigen presentation within the CNS.

The encephalitogenic T cells migrate into the CNS through the Blood-Brain barrier, a tight membrane which separates CNS and periphery. Although integrina4, which is the target of Natalizumab, one of the most effective treatments for MS patients, is known to be involved, the detailed molecular mechanism for this step is still largely unknown. We are exploring the molecule which can be used as therapeutic target.

Varying calcium concentrations within migrating T cells were detected using a FRET-based calcium biosensor. Images were acquired by intravital two-photon microscopy. Encephalitogenic T cells are activated in the CNS; however, the percentage of activated T cells goes only up to 40%. By conventional immunological analysis, it is hard to answer where and how T cells are activated within the target organ. In contrast, by using a recently developed calcium sensing protein, we can visualize T cell activation in vivo at single cell level. To this end, we express Fluorescence Resonance Energy Transfer (FRET) based calcium sensing protein in T cells and use them for intravital two-photon imaging.

Encephalitogenic T cells exist in healthy individuals although they do not induce inflammation in the majority of cases. Recent findings suggest that gut microbiota trigger the first step of inflammation by stimulating T cells to initiate CNS infiltration. By using a transgenic mouse line with high numbers of encephalitogenic T cells in combination with activation sensors and intravital imaging, we try to visualize the stimulation in the gut-associated lymphoid tissues and unveil the mechanism of the process. In addition, we study the phenotypical changes of those encephalitogenic T cells following stimulation.