Abstract
Immune cells need to communicate with each other via direct cell contact formation. The contact zone has similar functions as a neuronal synapse and is therefore named immune synapse. Supramolecular activation clusters consisting of a variety of surface receptors and cytoplasmic proteins are formed within the immune synapse, which are pivotal for T-cell activation. Thus, a malfunction of immune synapse formation has detrimental effects on the healthiness of the individual.
Classical confocal microscopy to analyze the supramolecular cluster formation and maturation of the immune synapse between primary human T-cells and antigen-presenting cells is time consuming and the number of cells that can be analyzed is limited. Therefore, we have established an InFlow microscopy approach for the analysis of immune synapses. InFlow microscopy is a hybrid method combining fluorescence microscopy and flow cytometry. Our InFlow microscopy method allows quantifying protein distribution in immune synapses of several hundred or even thousand cell couples in one sample. Importantly, comparisons of different samples with a strong statistical power are possible with InFlow microcopy.
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References
Samstag Y, John I, Wabnitz GH (2013) Cofilin: a redox sensitive mediator of actin dynamics during T-cell activation and migration. Immunol Rev 256:30–47. doi:10.1111/imr.12115
Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227
Delon J, Germain RN (2000) Information transfer at the immunological synapse. Curr Biol 10:R923–R933
Yokosuka T, Sakata-Sogawa K, Kobayashi W, Hiroshima M, Hashimoto-Tane A, Tokunaga M, Dustin ML, Saito T (2005) Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6:1253–1262. doi:10.1038/ni1272
Seminario MC, Bunnell SC (2008) Signal initiation in T-cell receptor microclusters. Immunol Rev 221:90–106. doi:10.1111/j.1600-065X.2008.00593.x
Wabnitz GH, Lohneis P, Kirchgessner H, Jahraus B, Gottwald S, Konstandin M, Klemke M, Samstag Y (2010) Sustained LFA-1 cluster formation in the immune synapse requires the combined activities of L-plastin and calmodulin. Eur J Immunol 40:2437–2449. doi:10.1002/eji.201040345
Eibert SM, Lee KH, Pipkorn R, Sester U, Wabnitz GH, Giese T, Meuer SC, Samstag Y (2004) Cofilin peptide homologs interfere with immunological synapse formation and T cell activation. Proc Natl Acad Sci U S A 101:1957–1962. doi:10.1073/pnas.0308282100
Hosseini BH, Louban I, Djandji D, Wabnitz GH, Deeg J, Bulbuc N, Samstag Y, Gunzer M, Spatz JP, Hammerling GJ (2009) Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy. Proc Natl Acad Sci U S A 106:17852–17857. doi:10.1073/pnas.0905384106
Martin-Cofreces NB, Baixauli F, Sanchez-Madrid F (2014) Immune synapse: conductor of orchestrated organelle movement. Trends Cell Biol 24:61–72. doi:10.1016/j.tcb.2013.09.005
Chang JT, Palanivel VR, Kinjyo I, Schambach F, Intlekofer AM, Banerjee A, Longworth SA, Vinup KE, Mrass P, Oliaro J, Killeen N, Orange JS, Russell SM, Weninger W, Reiner SL (2007) Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315:1687–1691
Wabnitz GH, Kocher T, Lohneis P, Stober C, Konstandin MH, Funk B, Sester U, Wilm M, Klemke M, Samstag Y (2007) Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25. Eur J Immunol 37:649–662. doi:10.1002/eji.200636320
Dustin ML, Depoil D (2011) New insights into the T cell synapse from single molecule techniques. Nat Rev Immunol 11:672–684. doi:10.1038/nri3066
Wabnitz GH, Michalke F, Stober C, Kirchgessner H, Jahraus B, van den Boomen DJ, Samstag Y (2011) L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells. Eur J Immunol 41:3157–3169. doi:10.1002/eji.201041366
Schulte B, John I, Simon B, Brockmann C, Oelmeier SA, Jahraus B, Kirchgessner H, Riplinger S, Carlomagno T, Wabnitz GH, Samstag Y (2013) A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition. J Biol Chem 288:29430–29439. doi:10.1074/jbc.M113.479766
Feldmeyer N, Wabnitz G, Leicht S, Luckner-Minden C, Schiller M, Franz T, Conradi R, Kropf P, Müller I, Ho AD, Samstag Y, Munder M (2012) Arginine deficiency leads to impaired cofilin dephosphorylation in activated human T lymphocytes. Int Immunol 24:303–313. doi:10.1093/intimm/dxs004
Hoffmann SC, Wabnitz GH, Samstag Y, Moldenhauer G, Ludwig T (2011) Functional analysis of bispecific antibody (EpCAMxCD3)-mediated T-lymphocyte and cancer cell interaction by single-cell force spectroscopy. Int J Cancer 128:2096–2104. doi:10.1002/ijc.25556
Hoffmann S, Hosseini BH, Hecker M, Louban I, Bulbuc N, Garbi N, Wabnitz GH, Samstag Y, Spatz JP, Hammerling GJ (2011) Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells. Immunol Lett 136:13–20. doi:10.1016/j.imlet.2010.11.005
Hochweller K, Wabnitz GH, Samstag Y, Suffner J, Hammerling GJ, Garbi N (2010) Dendritic cells control T cell tonic signaling required for responsiveness to foreign antigen. Proc Natl Acad Sci U S A 107:5931–5936. doi:10.1073/pnas.0911877107
Klemke M, Wabnitz GH, Funke F, Funk B, Kirchgessner H, Samstag Y (2008) Oxidation of cofilin mediates T cell hyporesponsiveness under oxidative stress conditions. Immunity 29:404–413
Acknowledgements
The authors thank Henning Kirchgessner for his continuing support during the establishment of InFlow microscopy protocols and for critical reading of this method. This work was supported by the German research foundation (SFB938).
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Wabnitz, G.H., Samstag, Y. (2016). Multiparametric Characterization of Human T-Cell Immune Synapses by InFlow Microscopy. In: Barteneva, N., Vorobjev, I. (eds) Imaging Flow Cytometry. Methods in Molecular Biology, vol 1389. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3302-0_10
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DOI: https://doi.org/10.1007/978-1-4939-3302-0_10
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