Knocking at the brain's door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures

Abstract

Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease.

Fig. 1

a, b Comparison between conventional one-photon excitation versus two-photon excitation. a During conventional excitation, a photon activates a fluorescence molecule (left), whereas during two-photon excitation, two photons can sum up their energy to activate a fluorescence molecule (right). After nonradiative relaxation (dotted line), a fluorescence molecule emits fluorescence, which is common in both conventional and two-photon excitation. b Conventional laser can excite a fluorescence molecule on its way (indicated as black area in left). However, fluorescent molecules can be activated only at the focus point since two-photon effect needs energy accumulation (indicated as black circle in right). c CNS intravital imaging setup. The animal is anesthetized and respiration is controlled during the entire imaging session. Additionally, body temperature is kept at 37°C and ECG is monitored. Two-photon laser is routed to xy-scanner via EOM to control laser power. Thereafter, laser is focused by objective lens and reaches the sample. Emission is collected by the objective lens and detected by NDDs which are located immediately behind it

Fig. 2

Intravital recording of T_MBP-GFP cells (green) in vessels of the CNS (a) and the ear (b). Vessels were marked by i.v. injection of Texas Red dextran conjugates (red). Thirty-minute movies, consisting of 60 time points, were time-projected. Crawling T cells appear as continuous lines, whereas rolling cells appear as round-shaped dots. Scale bar 30 μm

Fig. 3

Schema of the invasion steps of T_MBP-GFP cells into the CNS parenchyma. T_MBP-GFP cells arrive at CNS leptomeningeal vessels and roll on endothelial cells (1). This is followed by intraluminal crawling directed both upstream and downstream of blood flow (2). After the extravasation (3), T_MBP-GFP cells continue to crawl to the abluminal surface of vessels (4a). A part of T cells dive to the Robin Virchow space (RV; 4b). T cells meet local antigen-presenting cells at the perivascular space of leptomeningeal vessels (5a) or parenchyma (5b) and are activated. Activated T cells are allowed to penetrate within the CNS parenchyma (6). Finally, T cells can be further activated by antigen-presenting cells in the CNS parenchyma (7)