Abstract

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

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Acknowledgements

The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).

Author information

Author notes

    • Veronica T Chang

    Present address: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

    • Veronica T Chang
    • , Ricardo A Fernandes
    • , Kristina A Ganzinger
    •  & Steven F Lee

    These authors contributed equally to this work.

Affiliations

  1. Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK.

    • Veronica T Chang
    • , Ricardo A Fernandes
    • , Yuan Lui
    • , Elizabeth Huang
    •  & Simon J Davis
  2. Department of Chemistry, University of Cambridge, Cambridge, UK.

    • Kristina A Ganzinger
    • , Steven F Lee
    • , James McColl
    • , Peter Jönsson
    • , Matthieu Palayret
    •  & David Klenerman
  3. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

    • Christian Siebold
    • , Karl Harlos
    • , Charlotte H Coles
    • , E Yvonne Jones
    • , Robert J C Gilbert
    •  & A Radu Aricescu

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Contributions

V.T.C., R.A.F., K.A.G., S.F.L., E.Y.J., R.J.C.G., D.K., A.R.A. and S.J.D. designed experiments; V.T.C., R.A.F., K.A.G., S.F.L., J.M., P.J., M.P., K.H., C.S., C.H.C., Y.L., E.H., R.J.C.G. and A.R.A. performed experiments; R.A.F., K.A.G., S.F.L., C.S., E.Y.J., R.J.C.G., V.T.C., A.R.A., D.K. and S.J.D. drafted and/or edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David Klenerman or A Radu Aricescu or Simon J Davis.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1 and 2

Videos

  1. 1.

    Calcium signaling in mLck-expressing J.CaM1.6 cells.

    Representative movie (raw data) showing the influx of calcium (change in Fluo-4 intensity) in cells expressing mLck upon interaction with an ‘activating’ surface (OKT3-coated glass) at 20°C. Movie is part of the data presented in Figure 5g. The movie is recorded as 300 ms frames every 1 second and played at 33 frames per second. Scale bar, 10 μm.

  2. 2.

    Calcium signaling in CD45 RABCLck-expressing J.CaM1.6 cells.

    Representative movie (raw data) showing the influx of calcium (change in Fluo-4 intensity) in cells expressing RABCLck upon interaction with an ‘activating’ surface (OKT3-coated glass) at 20°C. Movie is part of the data presented in Figure 5g. The movie is recorded as 300 ms frames every 1 second and played at 33 frames per second. Scale bar, 10 μm.

  3. 3.

    Segregation of CD45 and mLck on SLBs.

    Representative movie (raw data) showing that mLck and CD45 segregate immediately upon contact of Jurkat T cells expressing CD48 with a rCD2-containing SLB. CD45 is labeled with Alexa Fluor 488-tagged Fab (green) and mLck is labeled with TMR via a HaloTag® (red). The movie is played back 10-fold faster than real-time (10 frames per second). Scale bar, 2 μm.

  4. 4.

    Contact dependence of CD45 exclusion on SLBs.

    Representative movie (raw data) showing that CD45 exclusion on rCD2-containing SLBs is contact dependent. Normal Jurkat T cells (i.e. cells not expressing CD48) do not form a stable cell-SLB contact: rCD2 in the SLB (left panel) does not accumulate and CD45 expressed by the cells (right panel) is evenly distributed (i.e. there is no exclusion). CD45 is labeled with Alexa Fluor 488-tagged Fab (green) and rCD2 is tagged with Alexa Fluor 647 (red). At 0.18 a white light transmission image of the same area is shown indicating the presence of multiple Jurkat T cells above the SLB. The movie is played back 10-fold faster than real-time (10 frames per second). Scale bar, 2 μm.

  5. 5.

    Zap70 recruitment to contacts depleted of CD45 in Jurkat T cells.

    Representative movie showing simultaneous CD45 exclusion and Zap70 cluster formation in a CD48-expressing Jurkat T cell interacting with a rCD2-containing SLB. The movie combines raw data for the CD45 channel (i.e. CD45 labeled with Alexa Fluor 488-tagged Fab (green), left) with a movie of the Zap70 channel (i.e. Zap70 labeled with HaloTag® TMR, red) prior to (raw data, middle) and post application of a bandpass filter (right; Zap70 clusters identified by the analysis software are marked with a circle and running number). The movie is played back 5-fold faster than real-time (10 frames per second). Scale bar, 2 μm.

  6. 6.

    Zap70 recruitment in contacts depleted of CD45 in J.RT3-T3.5 T cells.

    Movie collage analogous to Supplementary Video 5 for a CD48-expressing J.RT3-T3.5 T cell. The movie is played back 5-fold faster than real-time (10 frames per second). Scale bar, 2 μm.

  7. 7.

    TCR triggering without ligands (an animation).

    The animation shows the resting T cell surface and the changes in organization of signaling proteins that occur when the cell interacts with an SLB containing the cell adhesion molecule CD2, leading to ligand-independent TCR triggering. The early stages of the animation strive to portray the rapid movements of the proteins and the crowded nature of the T cell surface. The interaction of CD2 (in the SLB) with a non-signaling form of CD48 (on the T cell) results in close-contact formation and the exclusion of CD45. Because contact with the SLB is over a large area, the exclusion of CD45 results in strong TCR phosphorylation by non-excluded Lck kinase, leading to ZAP70 recruitment and calcium signaling in the absence of TCR ligands. The animation finishes with an image of an SLB-contacting Jurkat T cell from the present study showing the distribution of CD45 (green) and Lck (red; see Supplementary Fig. 6f). The molecules shown are: the TCR (terracotta; based on a model using TCRαβ PDB accession code 1AO7, CD3ɛδ PDB 1XIW, CD3ɛγ PDB 1JBJ and CD3ξξ PDB 2HAC), CD45 (light green; based on a model using the CD45 ECD structure described herein and the CD45 phosphatase domain PDB 1YGR); Lck in an active conformation (red; based on Src PDB 1Y57 and the zinc-clasp motif in PDB 1Q68); CD48 (dark green) and CD2 (yellow) modeled on human CD2 (using PDB 1HNF); CD48/CD2 complexes based on a model of the CD2/CD58 complex (using PDB 1QA9); and ZAP-70 (purple; PDB 4K2R). All transmembrane regions and CD3 cytoplasmic regions were modeled as α-helixes in the Build Structure function in UCSF Chimera (http://www.cgl.ucsf.edu/chimera/), using the native protein sequences. A probe radius of 8 Å was used to generate all model surfaces. The animation was built using Blender software (http://www.blender.org).

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https://doi.org/10.1038/ni.3392

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