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Photocrosslinkable pMHC monomers stain T cells specifically and cause ligand-bound TCRs to be 'preferentially' transported to the cSMAC

Abstract

The binding of T cell antigen receptors (TCRs) to specific complexes of peptide and major histocompatibility complex (pMHC) is typically of very low affinity, which necessitates the use of multimeric pMHC complexes to label T lymphocytes stably. We report here the development of pMHC complexes able to be crosslinked by ultraviolet irradiation; even as monomers, these efficiently and specifically stained cognate T cells. We also used this reagent to probe T cell activation and found that a covalently bound pMHC was more stimulatory than an agonist pMHC on lipid bilayers. This finding suggested that serial engagement of TCRs is dispensable for activation when a substantial fraction of TCRs are stably engaged. Finally, pMHC-bound TCRs were 'preferentially' transported into the central supramolecular activation cluster after activation, which suggested that ligand engagement enabled linkage of the TCR and its associated CD3 signaling molecules to the cytoskeleton.

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Figure 1: Staining of T cells with a photocrosslinkable pMHC monomer.
Figure 2: T cells do not flux calcium when covalently bound to pMHC monomers in medium and instead flux calcium only after aggregation.
Figure 3: T cells flux calcium and form immunological synapses in response to covalently bound pMHC ligands on lipid bilayers.
Figure 4: A covalently bound pMHC ligand is more stimulatory than a standard agonist pMHC (without crosslinking) on planar lipid bilayers.
Figure 5: TCRs engaged by pMHC complexes rapidly migrate to the center of the immunological synapse after activation, but unengaged TCRs do not.

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Acknowledgements

We thank S. Valitutti and Y. Wong for critical reading of the manuscript; A. Ting (Massachusetts Institute of Technology) for constructs for the expression of monovalent streptavidin; A. Shaw and M. Kelly (Washington University St. Louis) for AND mouse spleen cells; and Y.-H. Chien, J. Campbell, F. Wang, J. Zhou, P. Nelida, N. Sigal and A. Girvin for discussions and/or experimental assistance. Supported by the Cancer Research Institute (J.X.), the Howard Hughes Medical Institute (M.M.D.) and the US National Institutes of Health (R01 AI022511 to M.M.D.).

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J.X. and M.M.D. conceived of the project; J.X. developed the photocrosslinkable pMHC reagent and the acid-mediated peptide-exchange method; J.B.H. optimized the lipid bilayer system and the total internal reflection fluorescence microscope system and contributed ideas; J.X. and J.B.H. worked together on live-cell total internal reflection fluorescence imaging; E.W.N., J.H., P.J.R.E. and Q.-J.L. contributed reagents and technical support; and J.X. and M.M.D. wrote the manuscript.

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Correspondence to Mark M Davis.

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Xie, J., Huppa, J., Newell, E. et al. Photocrosslinkable pMHC monomers stain T cells specifically and cause ligand-bound TCRs to be 'preferentially' transported to the cSMAC. Nat Immunol 13, 674–680 (2012). https://doi.org/10.1038/ni.2344

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