A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity

Abstract

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.

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].…”

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

“…In brief, BMMfs and BMDCs derived from WT and NOX2-deficient mice were allowed to endocytose unprocessed whole Ag or preprocessed antigenic peptides for a defined period. The activation of CD4 + T cells isolated from OTII and 2D2 mice and coincubated with the APCs were assessed by measuring surface expression of the early activation markers CD69 and CD25 as previously described (15,25,31,33,35,53,54).…”

“…Further phagocytosis of myelin debris by APCs (particularly macrophages) increases the presentation of myelin-derived Ags to effector T cells, creating a positive feedback loop that amplifies the inflammatory process to a level that is clinically apparent. Hence, the local and recruited macrophages act as the central "gatekeepers" of T cell reactivation and ultimately the initiation and propagation of clinical EAE (11,(13)(14)(15). Thus, EAE presents a good model for studying the generation of MHC-II-restricted autoepitopes, from both exogenous and endogenous sources, by different APCs in different tissues.…”

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

“…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).…”