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

Abstract

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.

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

“…These reactions led to T-cell activation, as previously documented by the NFAT-GFP sensor (8). Similar to EAE in the mouse (12), the velocity of the T cells was inversely correlated to shape and amplitude of calcium signaling (Fig.…”

“…This indicator allows the documentation of changes in the intracellular calcium levels, although the limited scanning speed of our intravital two-photon setup (∼1 s per frame) does not resolve calcium oscillations of <1 Hz. Elevated and sustained calcium signaling may induce NFAT translocation from the cytosol to the nucleus, a process that we followed using a recently described NFAT-GFP fusion protein (8).…”

“…In the lymph nodes, lung, and spleen, freshly activated T cells acquire a functional phenotype that enables them to successfully overcome the cerebrovascular BBB (5,6). Beyond the vascular barrier, the T cells are confronted with phagocytes that, by presenting myelin autoantigen, usher them into the compact CNS white matter (7)(8)(9). This itinerary implies that the autoimmune T cell drifts sequentially through different milieus where it interacts with diverse local stroma structures through intercellular membrane contacts and/or soluble signals in the presence or absence of autoantigens.…”

“…To correlate calcium changes with the activation events downstream of calcium signaling, we followed the calcium-dependent nuclear translocation of a truncated Nuclear Factor of Activated T Cells (NFAT)-GFP fusion protein (8,14,15).…”

Molecular anatomy and functions of the choroidal blood-cerebrospinal fluid barrier in health and disease

“…On the CSF side of the arachnoid the subarachnoid space is home to numerous tissue resident MHC class II expressing macrophages, some of them even in tight contact with the arachnoid barrier and perforating the arachnoid lining at the CSF side with their processes [13]. Subarachnoid macrophages exert scavenger functions and present CNS antigens to T cells entering this CSF space as shown by elegant in vivo live cell imaging studies [8,84,107]. Combined parabiosis and fate-mapping studies in transgenic mice provided recent evidence that subarachnoid macrophages arise from hematopoietic precursors during embryonic development and establish a stable long-lived population of innate immune cells in this compartment [52].…”