Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

Nature Nanotechnology volume 13, pages 610–617 (2018)Cite this article

Subjects

Abstract

Elucidating the rules for receptor triggering in cell–cell and cell–matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: TCR triggering by surface-bound ligands.
Fig. 2: Small clusters of 37 ligands are optimal for TCR triggering.
Fig. 3: Ligand spacing has distinct effect on TCR triggering on 2D and 3D arrays.
Fig. 4: T cells sense ligand position locally.
Fig. 5: Ligand position affects T cell spreading.
Fig. 6: Axial positioning of ligand controls CD45 exclusion.

References

  1. Iwashima, M., Irving, B. A., van Oers, N. S., Chan, A. C. & Weiss, A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263, 1136–1139 (1994).

    Article  Google Scholar 

  2. Liaunardy-Jopeace, A., Murton, B. L., Mahesh, M., Chin, J. W. & James, J. R. Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells. Nat. Struct. Mol. Biol. 24, 1155–1163 (2017).

    Article  Google Scholar 

  3. Sherman, E. et al. Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35, 705–720 (2011).

    Article  Google Scholar 

  4. Pageon, S. V. et al. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination. Proc. Natl Acad. Sci. USA 113, E5454–E5463 (2016).

    Article  Google Scholar 

  5. Hui, E. & Vale, R. D. In vitro membrane reconstitution of the T-cell receptor proximal signaling network. Nat. Struct. Mol. Biol. 21, 133–142 (2014).

    Article  Google Scholar 

  6. Chang, V. T. et al. Initiation of T cell signaling by CD45 segregation at ‘close contacts’. Nat. Immunol. 17, 574–582 (2016).

    Article  Google Scholar 

  7. Davis, S. J. & van der Merwe, P. A. The kinetic-segregation model: TCR triggering and beyond. Nat. Immunol. 7, 803–809 (2006).

    Article  Google Scholar 

  8. Cochran, J. R., Cameron, T. O., Stone, J. D., Lubetsky, J. B. & Stern, L. J. Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation. J. Biol. Chem. 276, 28068–28074 (2001).

    Article  Google Scholar 

  9. Deeg, J. et al. T cell activation is determined by the number of presented antigens. Nano Lett. 13, 5619–5626 (2013).

    Article  Google Scholar 

  10. Matic, J., Deeg, J., Scheffold, A., Goldstein, I. & Spatz, J P. Fine tuning and efficient T cell activation with stimulatory aCD3 nanoarrays. Nano Lett. 13, 5090–5097 (2013).

    Article  Google Scholar 

  11. Delcassian, D. et al. Nanoscale ligand spacing influences receptor triggering in T cells and NK cells. Nano Lett. 13, 5608–5614 (2013).

    Article  Google Scholar 

  12. Choudhuri, K., Wiseman, D., Brown, M. H., Gould, K. & van der Merwe, P. A. T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436, 578–582 (2005).

    Article  Google Scholar 

  13. Carbone, C. B. et al. In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase. Proc. Natl Acad. Sci. USA 114, E9338–E9345 (2017).

    Article  Google Scholar 

  14. Chen, B. M. et al. The affinity of elongated membrane-tethered ligands determines potency of T cell receptor triggering. Front. Immunol. 8, 793 (2017).

    Article  Google Scholar 

  15. Irles, C. et al. CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling. Nat. Immunol. 4, 189–197 (2003).

    Article  Google Scholar 

  16. James, J. R. & Vale, R. D. Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012).

    Article  Google Scholar 

  17. Schmid, E. M. et al. Size-dependent protein segregation at membrane interfaces. Nat. Phys. 12, 704–711 (2016).

    Article  Google Scholar 

  18. Wu, Y., Vendome, J., Shapiro, L., Ben-Shaul, A. & Honig, B. Transforming binding affinities from three dimensions to two with application to cadherin clustering. Nature 475, 510–513 (2011).

    Article  Google Scholar 

  19. Schvartzman, M. & Wind, S. J. Robust pattern transfer of nanoimprinted features for sub-5-nm fabrication. Nano Lett. 9, 3629–3634 (2009).

    Article  Google Scholar 

  20. Cai, H. et al. Molecular occupancy of nanodot arrays. ACS Nano 10, 4173–4183 (2016).

    Article  Google Scholar 

  21. Mossman, K. D., Campi, G., Groves, J. T. & Dustin, M. L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  Google Scholar 

  22. Roman, G., Martin, M. & Joachim, P. S. Block copolymer micelle nanolithography. Nanotechnology 14, 1153 (2003).

    Article  Google Scholar 

  23. Schoen, I., Hu, W., Klotzsch, E. & Vogel, V. Probing cellular traction forces by micropillar arrays: contribution of substrate warping to pillar deflection. Nano Lett. 10, 1823–1830 (2010).

    Article  Google Scholar 

  24. Bettinger, C. J., Langer, R. & Borenstein, J. T. Engineering substrate topography at the micro- and nanoscale to control cell function. Angew. Chem. Int. Ed. 48, 5406–5415 (2009).

    Article  Google Scholar 

  25. Vardhana, S., Choudhuri, K., Varma, R. & Dustin, M. L. Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32, 531–540 (2010).

    Article  Google Scholar 

  26. Cai, E. et al. Visualizing dynamic microvillar search and stabilization during ligand detection by T cells. Science 356, eaal3118 (2017).

    Article  Google Scholar 

  27. Shi, X. et al. Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 493, 111–115 (2013).

    Article  Google Scholar 

  28. Gagnon, E., Schubert, D. A., Gordo, S., Chu, H. H. & Wucherpfennig, K. W. Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain. J. Exp. Med. 209, 2423–2439 (2012).

    Article  Google Scholar 

  29. Minguet, S., Swamy, M., Alarcón, B., Luescher, I. F. & Schamel, W. W. A. Full activation of the T Cell receptor requires both clustering and conformational changes at CD3. Immunity 26, 43–54 (2007).

    Article  Google Scholar 

  30. Tolar, P., Sohn, H. W. & Pierce, S. K. The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer. Nat. Immunol. 6, 1168–1176 (2005).

    Article  Google Scholar 

  31. Shah, N. H. et al. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor. eLife 5, e20105 (2016).

    Article  Google Scholar 

  32. Su, X., Ditlev, J. A., Rosen, M. K. & Vale, R. D. Reconstitution of TCR signaling using supported lipid bilayers. Methods Mol. Biol. 1584, 65–76 (2017).

    Article  Google Scholar 

  33. Stone, M. B., Shelby, S. A., Nunez, M. F., Wisser, K. & Veatch, S. L. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes. eLife 6, e19891 (2017).

    Article  Google Scholar 

  34. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  Google Scholar 

  35. Lohmuller, T. et al. Supported membranes embedded with fixed arrays of gold nanoparticles. Nano Lett. 11, 4912–4918 (2011).

    Article  Google Scholar 

  36. Manz, B. N., Jackson, B. L., Petit, R. S., Dustin, M. L. & Groves, J. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters. Proc. Natl Acad. Sci. USA 108, 9089–9094 (2011).

    Article  Google Scholar 

  37. Taylor, M. J., Husain, K., Gartner, Z. J., Mayor, S. & Vale, R. D. A DNA-based T cell receptor reveals a role for receptor clustering in ligand discrimination. Cell 169, 108–119 (2017).

    Article  Google Scholar 

  38. Jensen, M. C. & Riddell, S. R. Designing chimeric antigen receptors to effectively and safely target tumors. Curr. Opin. Immunol. 33, 9–15 (2015).

    Article  Google Scholar 

  39. Guest, R. D. et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J. Immunother. 28, 203–211 (2005).

    Article  Google Scholar 

  40. Li, J. et al. Membrane-proximal epitope facilitates efficient t cell synapse formation by anti-FcRH5/CD3 and is a requirement for myeloma cell killing. Cancer Cell 31, 383–395 (2017).

    Article  Google Scholar 

  41. Gradisar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362–366 (2013).

    Article  Google Scholar 

  42. Goodman, R. P. et al. Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication. Science 310, 1661–1665 (2005).

    Article  Google Scholar 

  43. Dustin, M. L., Starr, T., Varma, R. & Thomas, V. K. Supported planar bilayers for study of the immunological synapse. Curr. Protoc. Immunol. 18, 11–35 (2007).

    Google Scholar 

  44. Cai, H. et al. Spatial control of biological ligands on surfaces applied to T cell activation. Methods Mol. Biol. 1584, 307–331 (2017).

    Article  Google Scholar 

  45. Vasiliver-Shamis, G., Cho, M. W., Hioe, C. E. & Dustin, M. L. Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse. J. Virol. 83, 11341–11355 (2009).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank S. Curado for coordination and S. Davis for insightful comments. This work was supported primarily by the National Science Foundation under award no. CMMI-1300590, by the National Institutes of Health Common Fund Nanomedicine programme, grants PN2 EY016586, R37 AI043542 and P01 A1080192; and Wellcome Trust and Kennedy Trust for Rheumatology Research PRF 100262Z/12/Z. The Columbia Nano Initiative provided cleanroom and processing facilities. We thank M. Cammer of NYULMC OCR for microscopy and analysis support.

Author information

Author notes

  1. Haogang Cai

    Present address: Center for Nanoscale Materials, Argonne National Laboratory, Argonne, IL, USA

Authors and Affiliations

  1. Department of Mechanical Engineering, Columbia University, New York, NY, USA

    Haogang Cai

  2. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    James Muller & Michael L. Dustin

  3. The Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

    David Depoil, Viveka Mayya & Michael L. Dustin

  4. Mechanobiology Institute of Singapore, National University of Singapore, Singapore, Singapore

    Michael P. Sheetz

  5. Department of Biological Sciences, Columbia University, New York, NY, USA

    Michael P. Sheetz

  6. Department of Applied Physics and Applied Mathematics, Columbia University, New York, NY, USA

    Shalom J. Wind

Authors
  1. Haogang Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James Muller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Depoil
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Viveka Mayya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael P. Sheetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael L. Dustin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shalom J. Wind
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.C. carried out nanofabrication. H.C. and D.D. contributed equally to development of the bilayer backfill methodology. H.C. and J.M. contributed equally to the data acquisition and analysis. V.M. provided purified ICAM1. S.J.W., M.L.D. and M.P.S. designed the experiments. H.C., J.M., M.L.D. and S.J.W. interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Michael L. Dustin or Shalom J. Wind.

Ethics declarations

Competing Interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–13, Supplementary Tables 1–2, Supplementary References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cai, H., Muller, J., Depoil, D. et al. Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering. Nature Nanotech 13, 610–617 (2018). https://doi.org/10.1038/s41565-018-0113-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-018-0113-3

This article is cited by

Search

Advanced search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research