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2-photon imaging of phagocyte-mediated T cell activation in the CNS
Marija Pesic, … , Hartmut Wekerle, Naoto Kawakami
Marija Pesic, … , Hartmut Wekerle, Naoto Kawakami
Published February 1, 2013
Citation Information: J Clin Invest. 2013;123(3):1192-1201. https://doi.org/10.1172/JCI67233.
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Technical Advance Immunology

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

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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.

Authors

Marija Pesic, Ingo Bartholomäus, Nikolaos I. Kyratsous, Vigo Heissmeyer, Hartmut Wekerle, Naoto Kawakami

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Figure 1

ΔNFAT-GFP as an activation marker of T cells.

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ΔNFAT-GFP as an activation marker of T cells.
(A) Native and truncated v...
(A) Native and truncated versions of NFAT. aa numbers are indicated. TAD, transcription activation domain. (B) Confocal images of resting in vitro TMBP-NFAT-GFP cells stained with membrane dye PKH26 and DAPI, before and after application of ionomycin. Scale bars: 2 μm. (C) Time kinetics of cytoplasmic-nuclear (c-n) ΔNFAT-GFP translocation upon ionomycin stimulation, and reverse nuclear-cytoplasmic (n-c) transport after ionomycin washout, in representative resting in vitro TMBP-NFAT-GFP cells. Bright field images (gray) were overlaid with pseudocolor images depicting GFP intensity distribution, from blue (low intensity) to red (high intensity). Numbers denote the relative time after addition (top) or washing (bottom) of ionomycin. Scale bars: 5 μm. (D) Cytoplasmic-nuclear ΔNFAT-GFP translocation times after addition of ionomycin, and nuclear-cytoplasmic translocation times after ionomycin washout. Each symbol represents a single cell; results are the sum of at least 3 independent experiments per condition. (E) EAE clinical course induced by transfer of TMOG-GFP or TMBP-GFP cells as well as TMOG-NFAT-GFP or TMBP-NFAT-GFP cells. Mean ± SD from at least 3 animals per group are shown. Representative data from 3 independent experiments per cell line.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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