T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex

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Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iTreg) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iTreg TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

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Figure 1: Functional and phenotypic properties of proinsulin-reactive iTreg clones.
Figure 2: Reversed iTreg TCR–pMHC class II docking topology.
Figure 3: Interatomic contacts at the iTreg TCR–pMHC class II interface.
Figure 4: T cell signaling.
Figure 5: T cell signaling in response to C19 proinsulin peptide mutants or with FS18 TCR mutants.

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We thank staff at the Australian synchrotron, the Leiden University Medical Center, the Monash Macromolecular Crystallization Facility, T. Beddoe, J. Teeler, C. van der Torren, S. Scally, M. Tran, S. Gras, K. Ladell, G. Dolton, R. Ayala-Perez and R. Berry for assistance. This work was supported by the Australian Research Council (ARC) and the National Health and Medical Research Council of Australia (NHMRC). A.P.U. is an ARC Future Fellow, D.I.G. is an NHMRC Senior Principal Research Fellow, D.A.P. is a Wellcome Trust Senior Investigator, K.J.R. is an NHMRC Career Development Fellow, A.W.P. is an NHMRC Senior Research Fellow, T.T. is an NHMRC Principal Research Fellow and J.R. is an NHMRC Australia Fellow. F.S.K., A.J. and B.O.R. are supported by the Netherlands Organization for Scientific Research (VICI 918.86.611), T.N. and S.L. are supported by the European Union 7th Framework Program (FP7/2007-2013) under Grant No. 241447 (Novel Immunotherapies for Type I Diabetes (NIAMIT)) and G.D. and A.R.v.d.S. are supported by the National Diabetes Expert Center funded by the Dutch Diabetes Research Foundation (DFN) and the Diabetes Research Netherlands (DON) Foundation.

Author information

D.X.B., F.W. and F.S.K. performed experiments, provided intellectual input and analyzed data; A.R.v.d.S. , K.L.L., J.P., N.L.D., G.D., S.L., A.J., J.P.V., Z.C., A.P.U., D.I.G., J.M., D.A.P., K.J.R., A.W.P. and T.N. either performed experiments, analyzed data, provided reagents and/or provided intellectual input; H.H.R., T.T., B.O.R. and J.R. led the investigation and, with T.N., wrote the manuscript. B.O.R. and J.R. conceived the study.

Correspondence to Hugh H Reid or Tony Tiganis or Bart O Roep or Jamie Rossjohn.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Phenotype and function of polyclonal T cell lines stimulated with autologous proinsulin C19-A3-loaded tDC (Treg) or inflammatory moDC (Teff).

(A) Expression of CD25, CD127, FoxP3, Granzyme B, IL-10 and IFNγ, associated with different subtypes of regulatory T cells, by Treg and Teff lines. (B) Treg-line but not Teff-line suppressed naive T cells proliferation (TnCFSE). Details of the suppression assay are described in the Methods section

Supplementary Figure 2 Phenotypic analyses of iTreg clones

(A) Mean fluorescence intensity expression of surface markers by iTreg clones (green) compared to effector T cell clones (red). Green circle shows a representative staining for iTreg clone FS18. Box and whiskers were generated according to Tukey. ***p<0.001, **p<0.01; 2-way ANOVA (for CD4, αβTCR, CD28 and GITR) and Mann-Whitney test (for CD62L). (B) Proliferation of clones derived from the Treg line upon stimulation with pro-insulin-loaded PBMC (black bars) or CMV-loaded PBMC (white bars). (C) IL-10 production by iTreg clones FS25 and FS17 upon stimulation with PBMC loaded with different concentrations of pro-insulin peptide.

Supplementary Figure 3 Electron density maps for the proinsulin C19-A1 peptide

An SA-omit map (a) was calculated using two round of simulated annealing in phenix.refine with a model without the peptide. The main features of the peptide are clearly visible in the SA-omit map. (b) After refinement in phenix.refine and buster the electron density of the peptide is well defined. Both maps were contoured at 1.2σ.

Supplementary Figure 4 Electron density snapshots of FS18

Electron density snapshots of FS18 TCR at a contour level of 1.2σ. The α-chain is in green and the β-chain in gray.

Supplementary Figure 5 Similar FS17 TCR and FS18 TCR docking

The FS17 HLA-DR4pro-insulin was superposed on the FS18 HLA-DR4pro-insulin by aligning the HLA-DRA domains in coot. FS18 is shown as a transparent cartoon. The TCR α chain is colored in lime green and the β chain in gray. The CDRs are colored as follows: CDR1α in red, CDR2α in pink, CDR3α in cyan, CDR1β in orange, CDR2β in purple, CDR3β in blue.

Supplementary Figure 6 CD69 upregulation in SKW3.FS18 cells

1x105 BLCL9031 APCs and either (a) 2x105 SKW3.SP3.4 or (b) 2x105 SKW3.FS18 T cells were incubated with or without the indicated amounts of a) DQ8-glia-α1 or b) C19-peptide in the presence or absence of 20μg/ml of blocking antibodies against HLA-DR4, HLA-DQ8, MHC-I and MHC-II for 16 hours at 37°C. Cells were harvested and stained with phycoerythrin-conjugated mouse anti-human CD3 (UCHT1; BD Pharmingen) and allophycocyanin-conjugated mouse anti-human CD69 (FN50; BD Pharmingen) and analyzed by flow cytometry. Representative CD3 versus CD69 plots of three independent experiments are shown.

Supplementary Figure 7 CD69 upregulation in SKW3.FS17 cells

1x105 BLCL9031 APCs and 2x105 SKW3.FS17 T cells were incubated without or with 50 μg/ml C19-(pro-insulin)-peptide in the presence or absence of 20 μg/ml of blocking antibodies against HLA-DR, HLA-DQ, MHC-I and MHC-II for 16 hours at 37°C. Cells were harvested and stained with V450-conjugated mouse anti-human CD3 (UCHT1; BD Pharmingen) and allophycocyanin-conjugated mouse anti-human CD69 (FN50; BD Pharmingen) and analyzed by flow cytometry.

Supplementary Figure 8 pMHC-restricted TCR signaling in SKW3.SP3.4 and SKW3.HA1.7 T cells

2x105 BLCL9031 APCs were incubated with or without 50 μg/ml of (a) DQ8-glia-α1 or (b) hemagglutinin (HA)-peptide for 1 hour at 37°C. BLCL9031 cells were washed twice to remove excess amounts of peptide and antibody and incubated with 1x105 (a) SKW3.SP3.4 T cells or (b) SKW3.HA1.7 T cells. Cells were briefly spun down at 4°C to allow for cell-cell conjugation. T cells were the incubated with or without peptide-pulsed BLCL9031 APCs for the indicated times at 37°C. Un-stimulated cells were left on ice (untreated) in the presence of peptide-free BLCL9031 cells. Cells were fixed and permeabilized and stained with fluorochrome-conjugated antibodies for p-(Y418) SFK, p-(Y142) CD3ζ and p-(T202/Y204) ERK1/2 and analyzed by flow cytometry. Representative histograms of two independent experiments are shown.

Supplementary Figure 9 TCR-CD3 surface expression on SKW3-FS18 cell lines

1x105 SKW3.FS18 T cells, wt and mutants, were stained with V450-conjugated mouse anti-human CD3 (UCHT1; BD Pharmingen) and analyzed by flow cytometry. MFI of CD3-V450 was calculated in FlowJo and plotted in Prism (GraphPad Software). Quantified results are the means ± SD of four replicates and representative of three independent experiments.

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Beringer, D., Kleijwegt, F., Wiede, F. et al. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex. Nat Immunol 16, 1153–1161 (2015) doi:10.1038/ni.3271

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