Research ArticleDISEASES AND DISORDERS

Discriminative T cell recognition of cross-reactive islet-antigens is associated with HLA-DQ8 transdimer–mediated autoimmune diabetes

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Science Advances  21 Aug 2019:
Vol. 5, no. 8, eaaw9336
DOI: 10.1126/sciadv.aaw9336
  • Fig. 1 A high degree of epitope sharing and T cell promiscuity is observed between DQ8cis and DQ8trans for influenza and islet antigens.

    (A) A high degree of epitope overlap (highlighted in red) was observed between DQ8cis- and DQ8trans-restricted influenza-specific T cells. In contrast, only one DQ2cis-restricted epitope (HA102–118/HA108–124) was accommodated by DQ8cis/trans. Numbers indicate epitopes shared between DQ8cis, DQ2cis, and DQ8trans or exclusively presented by DQ8cis, DQ2cis, or DQ8trans. Each overlapping epitope (HA102–118/HA108–124, HA274–290/HA280–296, HA398–414/HA404–420, NP288–304/NP294–310, or NP480–496/NP486–498) was viewed as one epitope. (B and C) Cross-recognition of islet-specific clones was confirmed by T cell reactivity in the presence of 500 nM peptides. Black and white bars represent T cell reactivity stimulated by DQ8cis- and DQ8trans-transfected HEK293 cells, respectively. Error bars represent SDs among three experiments. SI, stimulation index.

  • Fig. 2 Direct ex vivo cross-recognition analysis of disease-relevant islet-specific T cells.

    (A) Double staining by DQ8cis and DQ8trans tetramers was observed for ex vivo polyclonal GAD65250–266-specific cells in DQ8+DQ2 participants. DQ8trans/HA102–188 tetramer was used as a control. The double staining shown here was performed in DQ8 (DQA1*0301-DQB1*0302) homozygous individuals. (B) Ex vivo sorted DQ8trans/IA-2957–972–specific clones cross-recognized DQ8cis and DQ8trans. (C and D) Comparison of total (left) and memory (right) CD4+ T cell frequencies for GAD65250–266-specific (C) and for IA-2957–972–specific (D) T cells among DQ8+DQ2 patients (closed circles), DQ8+DQ2+ patients (open circles), and DQ8+ healthy controls (gray circles). The y axis in (C) and (D) indicates T cell frequencies. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (P > 0.05). Tmr, tetramer.

  • Fig. 3 pDQ8trans most often elicit stronger disease-associated responses from cross-reactive islet-specific T cell clones.

    (A) All but one GAD65250–266-specific clones were activated more vigorously by DQ8trans in the presence of 50 and 500 nM peptides. (B) DQ8trans induced stronger responses from IA-2957–972–specific clones in the presence of 50 and 500 nM peptides. (C) Secretion of IFN-γ was significantly higher from cross-reactive T cell clones when stimulated with pDQ8trans. Peptide concentrations used in (C) were as follows: 5 nM (GAD65250–266), 50 nM (IA-2957–972), 2.5 μM (InsB11–24), which were the minimal concentrations that cytokine secretion could be detected for specific clones. Error bars in (A) and (B) represent SDs among three experiments. Comparison of T cell responses to DQ8cis and DQ8trans was evaluated by paired t tests. Statistical analysis in (A) and (B) was not performed as SIs for some clones were ≤3.

  • Fig. 4 Altered T cell avidity for DQ8trans/GAD65250–266 is associated with the absence of Arg52α residue in DQA1*0501.

    (A) 2D affinity of five antigen-reactive clones to DQ8cis (closed shapes) or DQ8trans (open shapes). 2D affinity correlates with T cell potency. MP, matrix protein. (B) Stronger responses of GAD65250–266-specific clones to pDQ8trans were correlated with higher TCR-pMHC avidity. Arg52α and, to a lesser extent, α44–50 residues for clone 07-C1 were critical to the enhanced T cell avidity toward DQ8trans/GAD65250–266. Experiments were run in duplicates with the error bars denoting the SD. Statistical significance was evaluated by extra sum-of-squares F test and listed in table S4. WT, wild-type; CI, confidence interval. (C) Amino acid sequence alignment of the core peptide-binding cleft region between the α chain of DQ8cis (DQA1*0301) and the α chain of DQ8trans (DQA1*0501). The parallel lines in DQA1*0501 indicate identical amino acid residues between two α chains. The numbers above the amino acid sequence are the sequence number of the residue in the α chain, as presented in (44), ensuring structural equivalence of the respective residues and their counterparts in HLA-DRA. The regions in which mutations were made in DQA1*0501 are highlighted in red. **P < 0.01, ****P < 0.0001.

  • Fig. 5 In silico analysis of epitope presentation in the context of different DQ molecules.

    (A) Superimposed side view of GAD65251–263, InsB12–23, and IA-2959–971 shown in stick form in the grooves of DQ8cis (nitrogen, blue; oxygen, red; sulfur, yellow; all other atoms, magenta) and DQ8trans (same atom color convention, for N, O, and S; all other atoms, green). (B) A nearly matched peptide orientation was seen in the superimposed side view of GAD65251–263, InsB12–23, and IA-2959–971 in the grooves of DQ8cis (magenta) and DQ8trans ∆α44–50, αW43_αF51insDQ8cis α44–50, αF51_αR53insR (green). Same depiction conventions and atom color codes are used as in (A). (C) TCR view of pocket 9 in complex with InsB12–23 revealed differences in residue interaction (listed in table S5). The pocket 9 view of the DQ8cis-Ins complex is rotated −60o (top half away from the viewer; bottom half toward the viewer), and the DQ8trans-Ins complex is also rotated −90o, with respect to the x axis. DQ residues are shown in stick form with the same atom color codes for N, O, and S, and carbon is in orange. The p9Arg anchor of InsB12–23 is in ball-and-stick format with carbon atoms in magenta (DQ8cis) or green (DQ8trans). All DQ residues shown in stick form are at a distance <5 Å (any atom to any atom) from the p9Arg anchor residues, with the exception of Tyr32β in DQ8trans that is shown just for comparison. Only the major interactions (highlighted in red in table S5) are shown here. The two major putative hydrogen bonds for the DQ8cis molecule and six major putative hydrogen bonds for the DQ8trans molecule are plotted. The hydrogen bond between Arg76αNη2 and Ser72αOγ of DQ8trans is not plotted because of an overlap with a covalent bond. The line indicating the cation-π interaction between p9ArgCζ and Tyr32βCγ of DQ8cis is only visible close to the latter atom and obscured around the former atom because of the depiction of covalent bonds of p9Arg. All relevant information is provided in table S5.

  • Table 1 List of unique and shared epitopes derived from common Influenza A and islet antigens for DQ8cis and DQ8trans.

    Ins, insulin; ICA69, islet cell antigen 69.

    Influenza A/New Caledonia/20/1999
    Hemagglutinin
    Influenza A/New York/318/2003
    Nucleoprotein
    PeptideSequencePeptideSequence
    Shared and cross-reactive
    between
    DQ8cis and DQ8trans
    Group I*HA102–118PENGTCYPGYFADYEELNP61–77LTIERMVLSAFDERRNK
    HA108–124YPGYFADYEELREQLSSNP288–304GYDFEKEGYSLVGVDPF
    HA114–130DYEELREQLSSVSSFERNP294–310EGYSLVGVDPFKLLQTS
    HA120–136EQLSSVSSFERFEIFPKNP306–322LLQTSQVYSLIRPNENP
    HA238–254DQEGRINYYWTLLEPGDNP468–484DERATNPIVPSFDMSNE
    HA274–290SRGFGSGIITSNAPMDENP480–496DMSNEGSYFFGDNAEEY
    HA280–296GIITSNAPMDECDAKCQNP486–498SYFFGDNAEEYDN
    HA398–414VIEKMNTQFTAVGKEFN
    HA404–420TQFTAVGKEFNKLERRM
    HA434–450IWTYNAELLVLLENERT
    Group IIHA440–456ELLVLLENERTLDFHDSNP240–256DQVRESRNPGNAEIEDL
    Group IIIHA186–202EKEVLVLWGVHHPPNIGNP175–191RSGAAGAAVKGVGTMVL
    Unique epitopes for DQ8cisHA452–468DFHDSNVKNLYEKVKSQ
    InconclusiveHA340–356IQSRGLFGAIAGFIEGG
    HA422–438NLNKKVDDGFLDIWTYN
    Islet antigens
    PeptideSequence
    Shared and cross-reactive
    between
    DQ8cis and DQ8trans
    GAD65121–140§YVVKSFDRSTKVIDFHYPNE
    GAD65250–266§AMMIARFKMFPEVKEKG
    InsB11–24§LVEALYLVCGERGF
    IA-2168–182GASSSLSPLQAELLP
    IA-2273–287FQDSGLLYLAQELPA
    IA-2761–777RSDYINASPIIEHDPRM
    IA-2957–972DQFEFALTAVAEEVNA
    ICA69297–311QEPSQLISLEEENQR
    IGRP306–320LYHFLQIPTHEEHLF
    Phogrin323–337GLSGLELDGMAELMA
    ZnT815–29AKMYAFTLESVELQQ

    *In vitro responses detected by both DQ8cis and DQ8trans tetramers.

    †In vitro responses detected only by DQ8cis tetramers.

    ‡In vitro responses detected only by DQ8trans tetramers.

    §Epitopes reported in our previous studies.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw9336/DC1

      Fig. S1. Representative tetramer-guided epitope mapping for H1HA and surface expression of DQ molecules on HEK293 cells.

      Fig. S2. Cross-reactivity of islet-specific T cell clones was investigated by staining of DQ8cis and DQ8trans tetramers and a representative ex vivo staining for DQ8trans/GAD65250–266 and DQ8trans/IA-2957–972 from one T1D and one healthy participant.

      Fig. S3. DQ8cis and DQ8trans elicit T cell responses by the same antigenic motif.

      Fig. S4. Affinity and presentation of shared epitopes.

      Fig. S5. TCR usages for GAD65250–266-specific T cell clones and investigation of cross-reactivity of islet-specific T cell clones by different DQ restriction elements.

      Table S1. List of common Influenza A epitopes presented by DQ2cis.

      Table S2. Summary of ex vivo results for GAD65250–266 and IA-2957–972.

      Table S3. List of the T cell clones, their proliferative capacity, and cytokine secretion.

      Table S4. Comparison of functional avidity EC50 between wild-type DQ8trans and other DQ8cis/trans variants in complex with GAD65250–266.

      Table S5. Predicted interactions between p9Arg/Arg76α with residues at pocket 9 of DQ8cis or DQ8trans.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Representative tetramer-guided epitope mapping for H1HA and surface expression of DQ molecules on HEK293 cells.
      • Fig. S2. Cross-reactivity of islet-specific T cell clones was investigated by staining of DQ8cis and DQ8trans tetramers and a representative ex vivo staining for DQ8trans/GAD65250–266 and DQ8trans/IA-2957–972 from one T1D and one healthy participant.
      • Fig. S3. DQ8cis and DQ8trans elicit T cell responses by the same antigenic motif.
      • Fig. S4. Affinity and presentation of shared epitopes.
      • Fig. S5. TCR usages for GAD65250–266-specific T cell clones and investigation of cross-reactivity of islet-specific T cell clones by different DQ restriction elements.
      • Table S1. List of common Influenza A epitopes presented by DQ2cis.
      • Table S2. Summary of ex vivo results for GAD65250–266 and IA-2957–972.
      • Table S3. List of the T cell clones, their proliferative capacity, and cytokine secretion.
      • Table S4. Comparison of functional avidity EC50 between wild-type DQ8trans and other DQ8cis/trans variants in complex with GAD65250–266.
      • Table S5. Predicted interactions between p9Arg/Arg76α with residues at pocket 9 of DQ8cis or DQ8trans.

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