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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zinkernagel RM, Doherty PC (1974) Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251(5475):547–548
Poo WJ, Conrad L, Janeway CA Jr (1988) Receptor-directed focusing of lymphokine release by helper T cells. Nature 332(6162):378–380
Norcross MA (1984) A synaptic basis for T-lymphocyte activation. Ann Immunol (Paris) 135D(2):113–134
Paul WE, Seder RA (1994) Lymphocyte responses and cytokines. Cell 76:241–251
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
Dustin ML, Colman DR (2002) Neural and immunological synaptic relations. Science 298(5594):785–789
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
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
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
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
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
Batista FD, Iber D, Neuberger MS (2001) B cells acquire antigen from target cells after synapse formation. Nature 411(6836):489–494
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights 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