Emeritus Group Neuroimmunology

Genetic markers of immune cell activation

Once activated, T cells pass the blood-brain barrier (BBB) to induce clinical disease. Recent studies indicated that T cell migration across the BBB requires a complex set of dialogues between the activated T cells and the surrounding stromal cells, first, in the peripheral immune tissues and later in the CNS.

To follow T cell activation at real-time in the living animal, we have developed two activation probes indicating distinct phases of lymphocyte activation. A calcium sensor indicates early/immediate changes of intracellular calcium levels, and a fluorescent nuclear factor of activated T cells (NFAT) analog represents relatively late stages of T cell activation, namely translocation of the transcription factor from cytoplasm to nucleus.

<p><strong>Fig. 1.</strong> <strong>The Twitch calcium-sensing protein.</strong> (A) Schematic representation of the calcium indicator Twitch containing the donor fluorophore CFP, the acceptor cpCitrine, and the calcium-sensitive domain of Troponin C (TnC) before and after the binding of calcium. (B) Spectrometric analysis of purified Twitch protein before (black) and after (red) the addition of 1 mM calcium. Excitation 430 nm. FI, fluorescence intensity; AU, arbitrary units.</p> Zoom Image

Fig. 1. The Twitch calcium-sensing protein. (A) Schematic representation of the calcium indicator Twitch containing the donor fluorophore CFP, the acceptor cpCitrine, and the calcium-sensitive domain of Troponin C (TnC) before and after the binding of calcium. (B) Spectrometric analysis of purified Twitch protein before (black) and after (red) the addition of 1 mM calcium. Excitation 430 nm. FI, fluorescence intensity; AU, arbitrary units.

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a) TWITCH, an intracellular calcium sensor

Intracellular calcium was detected by a Fluorescent Resonance Energy Transfer (FRET)-based protein sensor. The censor contains two fluorescent proteins, cyan fluorescent protein (CFP) and cpCitrine (YFP), which are connected via a troponin C domain. In low calcium environment, the protein emits blue light. The increased calcium ion binds troponin C domain and change protein confirmation, resulting in the emission of yellow light. The radiometric analysis of these colors indicates intracellular calcium (1) (Fig. 1).

Collaborators:  
Dr. Oliver Griesbeck; Max Planck Institute of Neurobiology, Research Group – Cellular Dynamics, Martinsried

1.M. Mues et al., Nature Med. 16, 778 (2013).

b) NFAT translocation as transcriptional T cell activation marker

As a complementary T cell activation marker, a truncated NFAT-GFP fusion protein (ΔNFAT-GFP) was used. In resting T cells, ΔNFAT-GFP locates in the cytosol. However, upon stimulation via T cell receptor and subsequent intracellular calcium signaling ΔNFAT-GFP translocates rapidly into the nucleus, like its wildtype analog (Fig. 2).

<strong>Fig. 2.</strong> <strong>The T cell activation sensor &Delta;NFAT-GFP.</strong> (A) Schematic of na&iuml;ve and truncated version of NFAT. Numbers indicate amino acid positions. (B) Three possible patterns of &Delta;NFAT-GFP (green) and SNARF-1 (cell body staining, red) in double-labeled T cells. Cytosolic (resting), nuclear (activated), and nuclear/cytosolic (intermediate). Scale bar: 5 &micro;m. Zoom Image
Fig. 2. The T cell activation sensor ΔNFAT-GFP. (A) Schematic of naïve and truncated version of NFAT. Numbers indicate amino acid positions. (B) Three possible patterns of ΔNFAT-GFP (green) and SNARF-1 (cell body staining, red) in double-labeled T cells. Cytosolic (resting), nuclear (activated), and nuclear/cytosolic (intermediate). Scale bar: 5 µm. [less]

The use of ΔNFAT-GFP labeled MBP specific T cells (TMBP-NFAT-GFP cells) now allows determining at real-time the sites where encephalitogenic T cells are activated on their way from the blood circulation into the CNS (1).

Collaborators:  
Prof. Dr. Vigo Heissmeyer; Ludwig Maximilian University, Institute for Immunology, Munich

1. M. Pesic et al., J. Clin. Invest. 123, 1192 (2013).


 
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