Abstract
To study T cell activation in vivo in real time, we introduced a newly developed fluorescence resonance energy transfer–based, genetically encoded calcium indicator into autoantigen-specific and non–autoantigen-specific CD4+ T cells. Using two-photon microscopy, we explored the responses of retrovirally transduced calcium indicator–expressing T cells to antigen in the lymph nodes and the central nervous system. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around antigen-presenting cells and an instant rise of cytosolic calcium. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium concentrations while still meandering through the space. This approach enabled us to follow the migration and activation patterns of T cells in vivo during the course of the disease.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 571, projects B6 and C6), the German Competence Network on Multiple Sclerosis (KKNMS), the Novartis Foundation for Therapeutic Research and the Max Planck Society. O.G. and T.T. were supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 870) and the EuroV1sion EU FP7 grant. We thank I. Arnold-Ammer, L. Penner, and B. Kunkel for technical assistance.
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Contributions
M.M. established the calcium indicator expression in T cells and performed all experiments except two-photon imaging. I.B. performed the mouse surgery and in vivo two-photon imaging. M.M. and I.B. designed experiments and analyzed data. T.T. and O.G. generated the higher-affinity calcium indicator Twitch-1. H.W., N.K. and G.K. supervised the study. M.M., G.K. and H.W. wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–12 and Supplementary Methods (PDF 6279 kb)
Supplementary Video 1
Visualization of calcium influx in EL4 cells by TN-XXL (related to Supplementary Figure 2). EL4 lymphoma cells stably expressing TN-XXL were imaged in vitro before and after the addition of 4 μM ionomycin. (MOV 1234 kb)
Supplementary Video 2
CD3 binding triggers a strong calcium flux in T cells (related to Figure 2). TN-XXLCD–expressing 2D2 T cells interacting with anti-CD3/CD28 beads were imaged in vitro. (MOV 6089 kb)
Supplementary Video 3
APC encounter triggers strong calcium flux in T cells (related to Figure 2). TN-XXLCD–expressing 2D2 T cells interacting with recombinant MOG-pulsed IgHMOG B cells were imaged in vitro. (MOV 2279 kb)
Supplementary Video 4
T cells respond strongly to antigen presented by DCs (related to Figure 3). Twitch-1CD–expressing OT-II T cells were adoptively transfered into WT recipient hosts before the subcutaneous injection of SNARF-1–labeled DCs. After one day, in vivo two-photon microscopy of the popliteal lymph node was performed before and after the i.v. injection of 100 μg of OVA peptide. (MOV 9970 kb)
Supplementary Video 5
All T cells displaying strong calcium signals are engaged with DCs (related to Figure 3). The experimental settings are the same as in Supplementary Video 4 after the injection of antigen but with highlighting the positions of DCs. (MOV 9922 kb)
Supplementary Video 6
Encephalitogenic T cells also respond strongly to antigen presented by DCs (related to Supplementary Figure 10). The experimental settings are the same as in Supplementary Video 4 but with 2D2 T cells and injection of 100 μg of NF-M peptide. (MOV 9869 kb)
Supplementary Video 7
CNS-resident APCs efficiently present antigen to infiltrating T cells (related to Figure 4). Twitch-1CD–expressing OT-II T cells were adoptively transferred into MOG peptide–immunized WT mice, followed by two-photon microscopy of the spinal cord, before and after the i.v. injection of 100 μg of OVA peptide and Texas red–dextran. (MOV 9987 kb)
Supplementary Video 8
Extravasated encephalitogenic T cells showing calcium oscillations in the CNS (related to Figure 5).Twitch-1CD–expressing 2D2 T cells were adoptively transfered into Rag2−/− recipients, and two-photon microscopy of the spinal cord was performed at the onset of EAE (score 0.5). (MOV 9838 kb)
Supplementary Video 9
Differential calcium signaling in encephalitogenic T cells during peak EAE (related to Figure 5). The experimental settings are the same as in Supplementary Video 8, but the imaging was performed at the peak of EAE (score >2). (MOV 9922 kb)
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Mues, M., Bartholomäus, I., Thestrup, T. et al. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nat Med 19, 778–783 (2013). https://doi.org/10.1038/nm.3180
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DOI: https://doi.org/10.1038/nm.3180
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