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Systems Imaging of the Immune Synapse

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The Immune Synapse

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1584))

Abstract

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Rachel Ambler and Xiangtao Ruan are shared first authors.

Robert F. Murphy and Christoph Wülfing are shared senior authors.

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References

  1. Monks CR, Kupfer H, Tamir I, Barlow A, Kupfer A (1997) Selective modulation of protein kinase C-theta during T-cell activation. Nature 385(6611):83–86

    Article  CAS  PubMed  Google Scholar 

  2. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machinery controlling T cell activation. Science 285:221–226

    Article  CAS  PubMed  Google Scholar 

  3. Schmick M, Bastiaens PI (2014) The interdependence of membrane shape and cellular signal processing. Cell 156(6):1132–1138. doi:10.1016/j.cell.2014.02.007

    Article  CAS  PubMed  Google Scholar 

  4. Angermann BR, Klauschen F, Garcia AD, Prustel T, Zhang F, Germain RN, Meier-Schellersheim M (2012) Computational modeling of cellular signaling processes embedded into dynamic spatial contexts. Nat Methods 9(3):283–289. doi:10.1038/nmeth.1861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Purvis JE, Lahav G (2013) Encoding and decoding cellular information through signaling dynamics. Cell 152(5):945–956. doi:10.1016/j.cell.2013.02.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mast FD, Ratushny AV, Aitchison JD (2014) Systems cell biology. J Cell Biol 206(6):695–706. doi:10.1083/jcb.201405027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. DeFord-Watts LM, Dougall DS, Belkaya S, Johnson BA, Eitson JL, Roybal KT, Barylko B, Albanesi JP, Wulfing C, van Oers NS (2011) The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse. J Immunol 186(12):6839–6847. doi:10.4049/jimmunol.1002721

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ksionda O, Saveliev A, Kochl R, Rapley J, Faroudi M, Smith-Garvin JE, Wulfing C, Rittinger K, Carter T, Tybulewicz VL (2012) Mechanism and function of Vav1 localisation in TCR signalling. J Cell Sci 125(Pt 22):5302–5314. doi:10.1242/jcs.105148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liang Y, Cucchetti M, Roncagalli R, Yokosuka T, Malzac A, Bertosio E, Imbert J, Nijman IJ, Suchanek M, Saito T, Wulfing C, Malissen B, Malissen M (2013) The lymphoid lineage-specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells. Nat Immunol 14(8):858–866. doi:10.1038/ni.2634

    Article  CAS  PubMed  Google Scholar 

  10. Paster W, Brockmeyer C, Fu G, Simister PC, de Wet B, Martinez-Riano A, Hoerter JA, Feller SM, Wulfing C, Gascoigne NR, Acuto O (2013) GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte development. J Immunol 190(7):3749–3756. doi:10.4049/jimmunol.1203389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Singleton KL, Gosh M, Dandekar RD, Au-Yeung BB, Ksionda O, Tybulewicz VL, Altman A, Fowell DJ, Wülfing C (2011) Itk controls the spatiotemporal organization of T cell activation. Sci Signal 4(193):ra66. doi:10.1126/scisignal.2001821

  12. Roybal KT, Sinai P, Verkade P, Murphy RF, Wülfing C (2013) The actin-driven spatiotemporal organization of T-cell signaling at the system scale. Immunol Rev 256(1):133–147. doi:10.1111/imr.12103

    Article  CAS  PubMed  Google Scholar 

  13. Roybal KT, Mace EM, Mantell JM, Verkade P, Orange JS, Wulfing C (2015) Early signaling in primary T cells activated by antigen presenting cells is associated with a deep and transient lamellal actin network. PLoS One 10(8):e0133299. doi:10.1371/journal.pone.0133299

    Article  PubMed  PubMed Central  Google Scholar 

  14. Purtic B, Pitcher LA, van Oers NS, Wülfing C (2005) T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells. Proc Natl Acad Sci U S A 102(8):2904–2909

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sinai P, Dozmorov IM, Song R, Schwartzberg PL, Wakeland EK, Wulfing C (2014) T/B-cell interactions are more transient in response to weak stimuli in SLE-prone mice. Eur J Immunol 44(12):3522–3531. doi:10.1002/eji.201444602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sinai P, Nguyen C, Schatzle JD, Wülfing C (2010) Transience in polarization of cytolytic effectors is required for efficient killing and controlled by Cdc42. Proc Natl Acad Sci U S A 107(26):11912–11917. doi:10.1073/pnas.0913422107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Singleton KL, Parvaze N, Dama KR, Chen KS, Jennings P, Purtic B, Sjaastad MD, Gilpin C, Davis MM, Wülfing C (2006) A large T cell invagination with CD2 enrichment resets receptor engagement in the immunological synapse. J Immunol 177(7):4402–4413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Singleton KL, Roybal KT, Sun Y, Fu G, Gascoigne NR, van Oers NS, Wülfing C (2009) Spatiotemporal patterning during T cell activation is highly diverse. Sci Signal 2(65):ra15

    Google Scholar 

  19. Roybal KT, Buck TE, Ruan X, Cho BH, Clark DJ, Ambler R, Tunbridge HM, Zhang J, Verkade P, Wulfing C, Murphy RF (2016) Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics. Sci Signal 9(424):rs3. doi:10.1126/scisignal.aad4149

  20. Negulescu PA, Krasieva TB, Khan A, Kerschbaum HH, Cahalan MD (1996) Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4(5):421–430

    Article  CAS  PubMed  Google Scholar 

  21. Mustelin T, Tasken K (2003) Positive and negative regulation of T-cell activation through kinases and phosphatases. Biochem J 371(Pt 1):15–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The original research upon which these protocols are based was supported in part by the National Institutes of Health grant P41 GM103712, by National Science Foundation grants MCB1121793 and MCB1121919, and by the European Research Council grant PCIG11-GA-2012-321554.

Author information

Authors and Affiliations

  1. School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TD, UK

    Rachel Ambler & Christoph Wülfing

  2. Computational Biology Department, School of Computer Science, Carnegie Mellon University, 7723 Gates-Hillman Center, Pittsburgh, PA, 15213, USA

    Xiangtao Ruan & Robert F. Murphy

  3. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

    Robert F. Murphy

  4. Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

    Robert F. Murphy

  5. Department of Machine Learning, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

    Robert F. Murphy

Authors
  1. Rachel Ambler
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  2. Xiangtao Ruan
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  3. Robert F. Murphy
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  4. Christoph Wülfing
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Corresponding authors

Correspondence to Robert F. Murphy or Christoph Wülfing .

Editor information

Editors and Affiliations

  1. Department of Life sciences, University of Siena, Siena, Siena, Italy

    Cosima T. Baldari

  2. University of Oxford, Kennedy Institute of Rheumatology, Headington, Oxford, United Kingdom

    Michael L. Dustin

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Ambler, R., Ruan, X., Murphy, R.F., Wülfing, C. (2017). Systems Imaging of the Immune Synapse. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_25

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  • DOI: https://doi.org/10.1007/978-1-4939-6881-7_25

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6879-4

  • Online ISBN: 978-1-4939-6881-7

  • eBook Packages: Springer Protocols

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