Systems Imaging of the Immune Synapse
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.
Key wordsLive cell imaging Computational image analysis Spatiotemporal distributions Immunological synapse T cell activation
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.
- 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 CrossRefPubMedPubMedCentralGoogle 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 CrossRefPubMedGoogle 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 CrossRefPubMedPubMedCentralGoogle 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
- 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 CrossRefPubMedPubMedCentralGoogle 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):ra15Google 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