Skip to main content
SpringerLink
Log in

Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers

  • Protocol
  • First Online:
The Immune Synapse

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

Abstract

Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples. Successful experiments might then require further days to fully analyze. We present methods for automation of many steps in SLB formation, imaging in 96-well glass bottom plates, and analysis that enables >100-fold increase in throughput for fixed samples and wide-field fluorescence. This increased throughput will allow better coverage of relevant parameters and more comprehensive analysis of aspects of the immunological synapse that are well reconstituted by SLB.

*Salvatore Valvo and Viveka Mayya are shared first authors. Daniel Ebner and Michael L Dustin are shared last authors.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Zinkernagel RM, Doherty PC (1974) Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251(5475):547–548

    Article  CAS  PubMed  Google Scholar 

  2. Poo WJ, Conrad L, Janeway CA Jr (1988) Receptor-directed focusing of lymphokine release by helper T cells. Nature 332(6162):378–380

    Article  CAS  PubMed  Google Scholar 

  3. Norcross MA (1984) A synaptic basis for T-lymphocyte activation. Ann Immunol (Paris) 135D(2):113–134

    CAS  Google Scholar 

  4. Paul WE, Seder RA (1994) Lymphocyte responses and cytokines. Cell 76:241–251

    Article  CAS  PubMed  Google Scholar 

  5. Dustin ML, Olszowy MW, Holdorf AD, Li J, Bromley S, Desai N, Widder P, Rosenberger F, van der Merwe PA, Allen PM, Shaw AS (1998) A novel adapter protein orchestrates receptor patterning and cytoskeletal polarity in T cell contacts. Cell 94:667–677

    Article  CAS  PubMed  Google Scholar 

  6. Dustin ML, Colman DR (2002) Neural and immunological synaptic relations. Science 298(5594):785–789

    Article  CAS  PubMed  Google Scholar 

  7. Geiger B, Rosen D, Berke G (1982) Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells. J Cell Biol 95(1):137–143

    Article  CAS  PubMed  Google Scholar 

  8. Carpen O, Virtanen I, Saksela E (1982) Ultrastructure of human natural killer cells: nature of the cytolytic contacts in relation to cellular secretion. J Immunol 128(6):2691–2697

    CAS  PubMed  Google Scholar 

  9. Schmidt RE, Caulfield JP, Michon J, Hein A, Kamada MM, MacDermott RP, Stevens RL, Ritz J (1988) T11/CD2 activation of cloned human natural killer cells results in increased conjugate formation and exocytosis of cytolytic granules. J Immunol 140(3):991–1002

    CAS  PubMed  Google Scholar 

  10. Kupfer A, Singer SJ (1989) Cell biology of cytotoxic and helper T cell functions: immunofluorescence microscopic studies of single cells and cell couples. Annu Rev Immunol 7:309–337

    Article  CAS  PubMed  Google Scholar 

  11. Stinchcombe JC, Bossi G, Booth S, Griffiths GM (2001) The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15(5):751–761

    Article  CAS  PubMed  Google Scholar 

  12. Batista FD, Iber D, Neuberger MS (2001) B cells acquire antigen from target cells after synapse formation. Nature 411(6836):489–494

    Article  CAS  PubMed  Google Scholar 

  13. Carroll-Portillo A, Spendier K, Pfeiffer J, Griffiths G, Li H, Lidke KA, Oliver JM, Lidke DS, Thomas JL, Wilson BS, Timlin JA (2010) Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces. J Immunol 184(3):1328–1338

    Article  CAS  PubMed  Google Scholar 

  14. Goodridge HS, Reyes CN, Becker CA, Katsumoto TR, Ma J, Wolf AJ, Bose N, Chan AS, Magee AS, Danielson ME, Weiss A, Vasilakos JP, Underhill DM (2011) Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature 472(7344):471–475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395(6697):82–86

    Article  CAS  PubMed  Google Scholar 

  16. 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 

  17. 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(5425):221–227

    Article  CAS  PubMed  Google Scholar 

  18. Yokosuka T, Kobayashi W, Sakata-Sogawa K, Takamatsu M, Hashimoto-Tane A, Dustin ML, Tokunaga M, Saito T (2008) Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation. Immunity 29(4):589–601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tseng SY, Waite JC, Liu M, Vardhana S, Dustin ML (2008) T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase Ctheta. J Immunol 181(7):4852–4863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Campi G, Varma R, Dustin ML (2005) Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J Exp Med 202(8):1031–1036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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

    Article  CAS  PubMed  Google Scholar 

  22. Varma R, Campi G, Yokosuka T, Saito T, Dustin ML (2006) T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25(1):117–127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bunnell SC, Hong DI, Kardon JR, Yamazaki T, McGlade CJ, Barr VA, Samelson LE (2002) T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J Cell Biol 158(7):1263–1275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Al-Alwan MM, Rowden G, Lee TD, West KA (2001) The dendritic cell cytoskeleton is critical for the formation of the immunological synapse. J Immunol 166(3):1452–1456

    Article  CAS  PubMed  Google Scholar 

  25. Al-Alwan MM, Rowden G, Lee TD, West KA (2001) Fascin is involved in the antigen presentation activity of mature dendritic cells. J Immunol 166(1):338–345

    Article  CAS  PubMed  Google Scholar 

  26. Comrie WA, Li S, Boyle S, Burkhardt JK (2015) The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility. J Cell Biol 208(4):457–473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Natkanski E, Lee WY, Mistry B, Casal A, Molloy JE, Tolar P (2013) B cells use mechanical energy to discriminate antigen affinities. Science 340(6140):1587–1590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kumari S, Depoil D, Martinelli R, Judokusumo E, Carmona G, Gertler FB, Kam LC, Carman CV, Burkhardt JK, Irvine DJ, Dustin ML (2015) Actin foci facilitate activation of the phospholipase C-gamma in primary T lymphocytes via the WASP pathway. eLife 4. 10.7554/eLife.04953

  29. Kumari S, Vardhana S, Cammer M, Curado S, Santos L, Sheetz MP, Dustin ML (2012) T Lymphocyte myosin IIA is required for maturation of the immunological synapse. Front Immunol 3:230. doi:10.3389/fimmu.2012.00230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Nair PM, Ngu H, Torres E, Marsters S, Lawrence DA, Stephan JP, Komuves L, Ashkenazi A (2015) Membrane display and functional analysis of juxtacrine ligand-receptor signaling. Biotechniques 59(4):231–238 240

    Article  CAS  PubMed  Google Scholar 

  31. Mayya V, Neiswanger W, Medina R, Wiggins CH, Dustin ML (2015) Integrative analysis of T cell motility from multi-channel microscopy data using TIAM. J Immunol Methods 416:84–93. doi:10.1016/j.jim.2014.11.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Goktug AN, Chai SC, Chen T (2013) Data analysis approaches in high throughput screening. In: El-Shemy HA (ed) Drug discovery. InTech, 2013. Rijeka, Croatia

    Google Scholar 

  33. Hu J, Gondarenko AA, Dang AP, Bashour KT, O’Connor RS, Lee S, Liapis A, Ghassemi S, Milone MC, Sheetz MP, Dustin ML, Kam LC, Hone JC (2016) High-throughput mechanobiology screening platform using micro- and nanotopography. Nano Lett. doi:10.1021/acs.nanolett.5b04364

    Google Scholar 

  34. Torres AJ, Contento RL, Gordo S, Wucherpfennig KW, Love JC (2013) Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells. Lab Chip 13(1):90–99. doi:10.1039/c2lc40869d

    Article  CAS  PubMed  Google Scholar 

  35. Johnston SC, Dustin ML, Hibbs ML, Springer TA (1990) On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules. J Immunol 145(4):1181–1187

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank M. Santos and S. Davis for sharing methods for protein production using the lentiviral system. Wellcome Trust grant 100262/Z/12/Z and the Kennedy Trust for Rheumatology Research supported this work.

Author information

Author notes

    Authors and Affiliations

    1. Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK

      Salvatore Valvo, Viveka Mayya, Jehan Afrose, Hila Novak-Kotzer & Michael L. Dustin

    2. Target Discovery Institute, Nuffield Department of Medicine, The University of Oxford, Oxford, OX3 7FZ, UK

      Elena Seraia & Daniel Ebner

    Authors
    1. Salvatore Valvo
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Viveka Mayya
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Elena Seraia
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Jehan Afrose
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Hila Novak-Kotzer
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Daniel Ebner
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Michael L. Dustin
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Daniel Ebner or Michael L. Dustin .

    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

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2017 Springer Science+Business Media LLC

    About this protocol

    Cite this protocol

    Valvo, S. et al. (2017). Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers. 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_26

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-6881-7_26

    • Published:

    • 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

    Publish with us

    Policies and ethics

    Search

    Navigation