The PPI_15-24-specific HLA A*0201⁺-restricted CD8⁺ T cell clone, 1E6, kills unmanipulated human islets from multiple donors without any requirement for cytokine treatment or addition of exogenous cognate peptide. Percent specific lysis of human islet cells co-cultured with 1E6 (black bars) or a CD8⁺ T cell clone specific for the CMV pp65_{495 –503} peptide (white bars). Bars represent mean values ± SEM of accumulated data from 3 triplicate studies carried out using 3 different HLA A*0201⁺ organ donors. Clear killing by 1E6 is observed in the absence of cytokine treatment; this is enhanced when islets are pre-treated with cytokines to induce HLA class I expression (Supplementary Fig. 1). Parallel control experiments conducted in the presence of exogenous cognate peptide are also shown for both CD8⁺ T cell clones.

(a) The positions of the TCR CDR loops (CDR1α, red; CDR2α, green; CDR3α, blue; CDR1β, yellow; CDR2β, cyan; CDR3β, orange) in the co-complex structure between the 1E6 TCR and the preproinsulin-derived peptide ALWGPDPAAA (ALW) (yellow sticks) bound to HLA A*0201 (grey surface). The crossing angle of the TCR (58.4°) is shown. (b) View down the centre of the MHC binding groove (ALW shown in yellow sticks) illustrating the perpendicular binding conformation of the TCR CDR3α (blue) and CDR3β (orange) loops. (c) Surface representation of the binding footprint of the 1E6 TCR over A2-ALW (A2 is shown as grey surface and the ALW is shown as yellow surface). A2-ALW residues that were contacted by the 1E6 TCR are shown in red. (d) Surface representation of the binding footprint of the AS01 TCR over A2-GLC¹¹ (A2 is shown as grey surface and the GLC is shown as yellow surface). A2-GLC residues that were contacted by the AS01 TCR, calculated using a 3.2Å cut-off for H-bonds and 4Å cut-off for vdW contacts, are shown in red. The comparison of the surface contacts between the 1E6 TCR and AS01 TCR demonstrates the more focussed minimal contact zone implemented by the 1E6 TCR when binding to A2-ALW.

(a) Surface representation of the TCR residues (CDR3α, blue; CDR1β, yellow; CDR3β, orange) that contact the peptide. (b) Main contacts between the TCR and the peptide. TCR CDR3α and the TCR CDR1β and CDR3β loops made a number of vdW contacts (black dotted lines) and H-bonds (red dotted lines) with the central GPDPA motif of the ALWGPDPAAA (ALW) peptide (yellow sticks). (c) Contacts between the ALW residue Pro5 (yellow sticks) and the TCR CDR3α loop (blue sticks). The prominent exposed central position of Pro5 resulted in a number of important contacts between the TCR and the ALW peptide. (d) Contacts between the TCR CDR1β and CDR3β loops and the C-terminus of the peptide in which Asp6 was the major contact residue. Additionally, the dominant role of the TCR β-chain residue Trp97 (orange sticks) during peptide binding is illustrated. Trp97 is the only CDR3β loop residue that contacted the peptide, making a number of vdW contacts (black dotted lines) and H-bonds (red dotted lines) with Asp6, Pro7 and Ala8 in the ALW peptide.

Mutational scan of the 1E6 CD8⁺ T cell clone. Single mutations at each of the 10 positions in the ALWGPDPAAA peptide to each of the 19 other proteogenic amino acids were assessed for their ability to induce TNF secretion (data shown is average of 3 experiments). The index peptide was normalized to a response of 100 (black text with yellow background). Mutations that decreased the response to below 25 compared to index sequence are shown in pink. Nonpolar hydrophobic mutations are shown in orange, polar hydrophobic mutations are shown in green, polar uncharged mutations are shown in blue, acidic mutations are shown in red and basic mutations are shown in white.

(a) Surface representation of the TCR CDR loop (CDR3α, blue; CDR2β, cyan; CDR3β, orange) residues that contact the MHC surface. (B) Contacts between the TCR and the MHCα1 domain. The TCR CDR3α and the TCR CDR2β and CDR3β loops made a number of vdW (black dotted lines) and H-bond (red dotted lines) contacts with the MHCα1 central domain, including residue Arg65, which is part of the MHC restriction triad ref. (c) Contacts between the TCR β-chain CDR3β loop and the MHCα2 domain. These important stabilizing interactions included a vdW contact with the gatekeeper residue Gln155.