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How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes

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Science Immunology  05 Apr 2019:
Vol. 4, Issue 34, eaav7517
DOI: 10.1126/sciimmunol.aav7517
  • Fig. 1 Mouse and human TCR engagement of IAg7 and DQ8 with an R3-bound insulin peptide.

    (A) Schematic representations of optimal versions of the insulin B:10-23 peptide for the I.29 (left, 8E9E6ss with IAg7), 8F10 (middle, 8G9E with IAg7), and T1D3 (right, 8E9E11ss with DQ8). The core positions (p1 to p9) of the peptide in the MHCII-binding groove are numbered. Engineered disulfides are shown. Mutations from the natural sequences are in red. (B) Ribbons representing the three TCRs docked on their optimal MHCII-peptide ligands are shown for I.29 (left), 8F10 (middle), and T1D3 (right), viewed from the C-terminal end of the peptide. Colors are blue (TCR Vα), red (TCR Vβ), cyan (MHC α1), magenta (MHC β1), and yellow (peptide). (C) The footprints of the three TCRs on the solvent accessible surfaces of their MHCII-peptide ligands are shown from above: I.29, IAg7-8E9E6ss (left); 8F10, IAg7-8G9E (middle); and T1D3, DQ8-8E9E11ss (right). The surfaces are colored cyan (MHCIIα), magenta (MHCIIβ), and yellow (peptide). The surface of atoms involved in TCR contact (≤4.5 Å from a TCR atom) are colored in darker shades of the same colors. Also shown are tube representations of the tips of the TCR CDR loops: αCDR1 (green), αCDR2 (cyan), αCDR3 (blue), βCDR1 (yellow), βCDR2 (orange), and βCDR3 (red). For each complex, the arrow represents the angle of engagement of the TCR CDRs, placing αCDR2, βCDR3, and βCDR1 on one side and αCDR1, αCDR3, and βCDR1 on the other.

  • Fig. 2 TCR interactions with the peptide p8 amino acid determine type A recognition versus type B recognition.

    (A) The salt bridge between β51R of the I.29 TCR βCDR2 loop and the p8E of the 8E9E6ss peptide is shown. (B) Binding of a soluble fluorescent tetramer version of the IAg7-8E9E6ss complex to three versions of the 5KC TCR-negative T cell is shown: the untransduced 5KC control (red), 5KC transduced with the unmutated I.29 TCR (blue), and 5KC transduced with the I.29 TCR (green) bearing an R > A mutation at the CDR2 β51 amino acid. The transductants were prescreened for similar surface TCR expression. Additional data from this experiment are shown in fig. S2. (C) Extensive interaction of the 8F10 βCDR3 loop with the backbone of the 8G9E peptide in the region of p7G and p8G, approaching to within 3.1 Å at the p8G backbone nitrogen. (D) Disruption of the 8F10 T cell response to the 8G9E peptide by mutation of the p8G to L or V. Representative of three separate experiments with similar results. More data are shown in Fig. 5, C and D. (E) Extensive interaction is shown between β30R of the T1D3 TCR βCDR1 loop and the 8E9E11ss peptide from p8E to p10G, which includes a salt bridge to p8E, as well as an H-bond to Y60 of the DQ8 β-chain helix. (F) Inhibitory effect of mutating the β30R to A within T1D3 TCR βCDR1 loop on the response of the T cell to K562 cells transduced with the DQ8-8E9E11ss complex. Also shown are the changes in the responses of T1D4 and T1D10 of to the same peptide after mutating CDR2 β50R or CDR1 β30R to A, respectively. Results are the average responses and SEM seen in three separate experiments. Results in the individual experiments are also shown (open circles). Also shown are P values (Student’s two-tailed t test rounded to a single significant place) comparing the responses of the mutated and nonmutated T cells.

  • Fig. 3 Other features of the 8F10 and T1D3 TCRs.

    (A) A ribbon representation of a portion of the IAg7 α1 helix (cyan) with the rotamer changes (arrows) in α57Q and α61Q side chains from their positions before (carbons, gray) and after (carbons, cyan) engagement by the 8F10 TCR. The subsequent interaction of these amino acids with the TCR Vβ8.2 CDR2 48Y (carbon, orange) and CDR1 28N and 29N (carbon, yellow) side chains are also shown with potential H-bonds in green. (B) Left: SPR data [resonance units (RU)] for binding of various concentrations of the soluble T1D3 TCR to IAg7-8E9E11ss immobilized via a biotin tag in a BIAcore streptavidin flow cell. A flow cell containing immobilized IAg7-8G9E was used to correct the data for the fluid phase SPR signal. Standard BIAcore BIAEval software was used to fit the data to a first-order kinetic model and to calculate the kinetic association (kd, 1/M•s) and dissociation (ka, 1/s) rates. The overall equilibrium dissociation constant (KD, μM) was calculated as the kd/ka. Right: Scatchard plot of the equilibrium SPR signal obtained with each concentration of the soluble T1D3 TCR tested. The KD was calculated as the negative slope of a straight line fit to the data. Rmax (predicted RU at infinite TCR concentration) is labeled. Results were shown from a single experiment. Similar results were obtained in a second experiment. t1/2, half-life.

  • Fig. 4 Conserved features of the three TCR complexes.

    (A) Similar interactions of the TCR Vα CDR2 loops (cyan) of the I.29 (left), 8F10 (middle), and T1D3 (right) with conserved amino acids (β69E to β77T, magenta) of the IAg7 or DQ8 β-chain α helix. The view is from within the peptide-binding groove looking out to the β-chain α helix. (B) Importance of the p3Y (B:16, yellow) for interaction with the Vα CDR3s of the I.29 (left), 8F10 (middle), and T1D (right) TCRs. H-bonds are green. Also shown is how the introduced disulfide from p6 in the 8E9E6ss peptide to IAg7 α62 changes the position p3Y, realigning its hydroxyl (curved arrow) to make H-bonds both to the I.29 TCR Vα 100N and to α61Q of the IAg7 α chain. (C) The I.29 (left), 8F10 (middle), and T1D3 (right) TCRs all interact with the side chain of p-1E (B:13) of the peptide. Potential H-bonds or salt bridges are shown as green lines.

  • Fig. 5 How transpeptidation-mediated internal deletions in proinsulin could form superagonists similar to the mutant insulin peptides.

    (A) Schematic representation of how transpeptidation could cause internal deletions in proinsulin (see text for description). (B) Some potential transpeptidation protease-mediated deletions in mouse or human proinsulin that could generate superagonist fused epitopes with properties of 8E9E and 8G9E mutant peptides. (C) Peptides were synthesized, joining the highlighted donor C-peptide sequences in (B) to the appropriate truncated B-chain acceptors shown. The natural B:19C at p6 was mutated to A to prevent peptide dimerization in vitro. The figure shows the IL-2 produced in representative titrations of the mouse or human peptides with two mouse type A T cells (I.29 and 12-4.1), two mouse type B mouse T cells (8F10 and 12-4.4), and two human T cells (T1D3 and T1D4) as described in Materials and Methods. (D) All of the mouse and human T cells in Table 1 (except 7E6) were tested with the synthetic WT and fused peptides as shown in (C). Potencies (i.e., fold increase in activity of the fused peptide over the WT peptide) were calculated as described in Materials and Methods. The mouse data are from three experiments containing single titrations. The average increases in potency are shown with the SEM of the result from each experiment (open circles). The P values (Student’s two-tailed t test rounded to a single significant place) are shown for peptides with average potencies >1. The human data are from two experiments, each with triplicate titrations, which were averaged before calculating the potencies. The average overall increases in potency are shown with the result in each of the two experiments (open circles).

  • Table 1 Properties of the TCRs of the mouse and human B:9-23–specific CD4T cells.
    V α domain*V β domain
    Mouse type A
    Mouse type B
    Human “type A”

    *The Vα, Jα, Vβ, and Jβ are named using the classic nomenclature with the newer The International Immunogenetics Information System (IMGT) nomenclature for Vα and Vβ in parentheses.

    †CDR1 and CDR2 sequences are the six amino acids at the tips of these loops. Amino acids in boldface are predicted to interact similarly with peptide or MHC.

    ‡CDR3 sequences are amino acids between the conserved C at the end of the V element and the conserved F in the FGXG motif of the J element.

    • Table 2 I.29, 8F10, and T1D3 TCR contacts with their ligand.

      AA, amino acid. BSA, buried surface area, Å2 (% of total BSA).

      V domainCDR
      I.29 TCR to
      8F10 TCR to
      T1D3 TCR to
      No. of atom-to-atom
      contacts to*
      No. of atom-to-atom
      contacts to*
      No. of atom-to-atom
      contacts to*
      126D130 N126T10
      30S429 N127
      BSA104.4 (12.2)BSA14.7 (1.6)BSA165.8 (15.4)
      52N1352 N1052N17
      56 K7
      BSA135.1 (15.8)BSA152.5 (16.6)BSA173.3 (16.1)
      BSA330.8 (38.6)BSA358.1 (38.9)BSA299.6 (27.9)
      BSA175.2 (20.4)BSA228.1 (24.8)BSA116.9 (10.9)
      BSA29.6 (3.4)BSA68.1 (7.4)BSA189.6 (17.6)
      BSA82.3 (9.6)BSA99.0 (10.8)BSA130.5 (12.1)
      Total contacts1177223885981145119109
      Total BSA857.3 (100)920.4 (100)1075.7 (100)

      *Atom-to-atom distances, ≤4.5 Å.

      †Amino acids in boldface interact with p8 of the peptide.

      Supplementary Materials


        Fig. S1. Mutations to prevent I.29 and 8F10 TCR dimerization.

        Fig. S2. Extension of the mutational analysis of the I.29 TCR shown in Fig. 2B in the main manuscript.

        Table S1. Crystallography statistics.

        Table S2. Supplementary spreadsheet listing TCR- to MHC-peptide contacts.

        Table S3. Supplementary spreadsheet with raw data used to construct graphs in Figs. 2 and 5.

      • Supplementary Materials

        The PDF file includes:

        • Fig. S1. Mutations to prevent I.29 and 8F10 TCR dimerization.
        • Fig. S2. Extension of the mutational analysis of the I.29 TCR shown in Fig. 2B in the main manuscript.
        • Table S1. Crystallography statistics.

        Download PDF

        Other Supplementary Material for this manuscript includes the following:

        • Table S2 (Microsoft Excel format). Supplementary spreadsheet listing TCR- to MHC-peptide contacts.
        • Table S3 (Microsoft Excel format). Supplementary spreadsheet with raw data used to construct graphs in Figs. 2 and 5.

        Files in this Data Supplement:

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