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.
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 & Movie).
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).
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).
Prof. Dr. Vigo Heissmeyer; Ludwig Maximilian University, Institute for Immunology, Munich
1. M. Pesic et al., J. Clin. Invest. 123, 1192 (2013).