Divergent T-cell receptor recognition modes of a HLA-I restricted extended tumour-associated peptide

Abstract

Human leukocyte antigen (HLA)-I molecules generally bind short peptides (8-10 amino acids), although extended HLA-I restricted peptides (>10 amino acids) can be presented to T cells. However, the function of such extended HLA-I epitopes in tumour immunity, and how they would be recognised by T-cell receptors (TCR) remains unclear. Here we show that the structures of two distinct TCRs (TRAV4⁺TRAJ21⁺-TRBV28⁺TRBJ2-3⁺ and TRAV4 ⁺ TRAJ8⁺-TRBV9⁺TRBJ2-1⁺), originating from a polyclonal T-cell repertoire, bind to HLA-B*07:02, presenting a 13-amino-acid-long tumour-associated peptide, NY-ESO-1_60-72. Comparison of the structures reveals that the two TCRs differentially binds NY-ESO-1_60-72-HLA-B*07:02 complex, and induces differing extent of conformational change of the NY-ESO-1_60-72 epitope. Accordingly, polyclonal TCR usage towards an extended HLA-I restricted tumour epitope translates to differing TCR recognition modes, whereby extensive flexibility at the TCR-pHLA-I interface engenders recognition.

Fig. 1

Functional reactivity of CD8⁺ T cells binding to HLA-B*07:02 presenting NY-ESO-1_60–72. a Flow cytometry analysis of melanoma patient’s PBMCs showing CD8⁺ T-cell expansion pre- (Day 0) and post- (Days 71 and 197) NY-ESO-1-ISCOMATRIX^TM vaccination following in vitro stimulation with NY-ESO-1(55–72) peptide as assayed by intracellular cytokine staining (ICS) measuring interferon-γ (IFN-γ) production. b Flow cytometry analysis showing a distinct NY-ESO-1_60–72-HLA-B*07:02 reactive CD8⁺ T-cell population following vaccination and in vitro stimulation with NY-ESO-1(55–72) peptide. Four functionally distinct NY-ESO-1_60–72-HLA-B*07:02-specific CD8⁺ T-cell clones were identified from the melanoma patient’s PBMCs. c NY-ESO-1_60–72-HLA-B*07:02 tetramer-positive CD8⁺ T-cell clones were generated from single-cell cloning. d Four NY-ESO-1_60–72-HLA-B*07:02-specific TCRs were retrovirally reconstituted in SKW3 cells and recognised NY-ESO-1_60–72-HLA-B*07:02 presented on peptide-pulsed HLA-B*07:02⁺ antigen-presenting cells (APCs). The overlaid flow cytometry histograms of CD69 up-regulation in TCR-transduced SKW3 cells following co-incubation with NY-ESO-1_60–72 peptide-pulsed (blue histogram) or non-pulsed APCs (red histogram). The SKW3 subsets were gated on live lymphocytes, CD3^hi GFP^hi markers and were subsequently displayed as % of max (Y-axis) versus CD69 (X-axis). LC13 TCR-transduced SKW3 cells served as a TCR positive but nonreactive control as we have shown it recognises the EBNA-3A_339–347 peptide-pulsed HLA-B*08:01⁺ APCs and up-regulated CD69 surface marker. A second independent retroviral transduction experiment was performed for the KFJ4 (TRAV13), KFJ4 (TRAV26), KFJ37 and LC13 TCRs, with consistent results, see Supplementary Fig. 4. e Four different NY-ESO-1_60–72-HLA-B*07:02-specific T-cell clones were individually co-incubated with HLA-B*07:02⁺ and NY-ESO-1-expressing SK-Mel-14 cells in 1:2 ratio in the presence of 10 μg mL⁻¹ of Brefeldin A for 5 h to detect naturally presented NY-ESO-1_60–72-HLA-B*07:02. CD8⁺ T-cell clone recognition of naturally presented NY-ESO-1_60–72-HLA-B*07:02 on NY-ESO-1-expressing melanoma cell line, SK-Mel-14. CD8⁺ T-cell responses were assayed by ICS measuring IFN-γ production. f Functional sensitivity of the CD8⁺ T-cell clones to exogenous NY-ESO-1_60–72 peptide at different concentrations. CD8⁺ T-cell clones that are sensitive to lower levels of antigen are considered to possess high avidity

Fig. 2

NY-ESO-1_60–72 peptide specificities and steady-state affinities for CD8⁺ T-cell clones. a Normalised data for IFN-γ production by different CD8⁺ T-cell clones when stimulated with 10⁻⁷ M concentration of native NY-ESO-1_60–72 peptide (APRGPHGGAASGL) or single alanine-substituted peptides. CD8⁺ T cell responses stimulated by native peptide for each clones were accorded as 100%, and other CD8⁺ T cell responses stimulated by single alanine-substituted peptides were calculated as a relative percentage to the native peptide responses. CD8⁺ T cell responses stimulated by different single alanine-substituted peptides concentrations are shown in Supplementary Fig. 2. Surface plasmon resonance sensorgrams (b) and equilibrium binding curves (c) showed that the in vitro refolded and purified recombinant TCRs (immobilised at ~300–350 response units) interacted with NY-ESO-1_60–72-HLA-B*07:02 (screened at serial dilutions from 200 to 0 µM). SPR sensorgrams are representative of a single experiment. Equilibrium binding curves were derived from two replicate experiments with K_D, k_on, k_off and equilibrium curve means calculated from the duplicate experiments (error and error bars denote SEM). d The observed electron density (2mFo-DFc), shown as blue mesh, for the NY-ESO-1_60–72 peptide contoured to 1σ illustrates the structural plasticity and flexibility of the central peptide residues (P6-His-P8-Gly). e NY-ESO-1_60–72 adopts a bulged conformation upon presentation by HLA-B*07:02. Comparison of the NY-ESO-1_60–72-HLA-B*07:02 bulged peptide presentation with the RL9-HLA-B*07:02 peptide presented in a canonical fashion (PDB: 5EO1). The heavy chain is shown in grey with the NY-ESO-1_60–72 and RL9 peptides shown in black and red, respectively

Fig. 3

Overview of the ternary complexes. a KFJ37 TCR-NY-ESO-1_60–72-HLA-B*07:02, b KFJ5 TCR-NY-ESO-1_60–72-HLA-B*07:02 and c SB27 TCR-LPEP-HLA-B*35:08 (PDB: 2AK4) with the TCR α- and β-chains coloured lighter and darker shades of blue, green and yellow, respectively. The HLA-B heavy chain and β₂-microglobulin molecules are shown as light and dark grey, respectively. Middle and lower panels show the TCR–HLA interfaces (d–f) and footprints (g–i). The complementarity determining region (CDR) loops are coloured as follows; CDR1α light blue, CDR2α green, CDR3α teal, CDR1β orange, CDR2β red, CDR3β yellow and framework (FW) contacts are coloured pink over the HLA-B*07:02 binding groove with the NY-ESO-1_60–72 peptide shown as black sticks. The centre of mass of the respective Vα and Vβ domains are shown as grey and black spheres, respectively. In the lower panel the molecular surface of the HLA-B molecules is shown with atomic contacts coloured according to the respective CDR-loop mediated interactions

Fig. 4

Molecular interactions at the TCR-NY-ESO-1_60–72–HLA-B*07:02 interface. a KFJ37 α-chain and b β-chain interactions with HLA-B*07:02 and NY-ESO-1_60–72 shown as grey cartoon and black sticks, respectively. c Interaction between the KFJ37 TCR and the NY-ESO-1_60–72 peptide. Interaction between the KFJ5 α-chain (d) and β-chain (e) with HLA-B*07:02. f Interaction between the KFJ5 TCR and the NY-ESO-1_60–72 peptide. The CDR loops are coloured according to Fig. 3. Hydrogen, van der Waals and salt-bridge contacts are shown by black, yellow and red dashed lines, respectively

Fig. 5

Conformational plasticity of the NY-ESO-1_60–72 peptide dictates TCR recognition modes. a Side view of the NY-ESO-1_60–72 peptide as presented for KFJ37 TCR-mediated recognition and b top view. c Side view of the NY-ESO-1_60–72 peptide as presented for KFJ5 TCR-mediated recognition and d top view. HLA-B*07:02 shown as grey cartoon with the NY-ESO-1_60–72 peptide shown as sticks coloured, blue and green for the KFJ37 TCR and KFJ5 TCR recognised epitopes. e Overlay of the two NY-ESO-1_60–72 peptide conformers with the carbon-a deviations shown for the entire peptide (f). The conserved interaction codon of the TRAV4*01 variable domain with the KFJ37 TCR and KFJ5 TCR coloured blue and green, respectively. Hydrogen and van der Waals bonds are shown by black and yellow lines with the conserved non-germline encoded Asp/Glu108α salt bridge also shown, in red