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Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response

Abstract

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8+ T cell populations specific for variants of the nonstructural protein epitope NS3133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3133-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2+ TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2+ TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

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Figure 1: TRBV bias in NS3133-DENV-specific CD8+ T cell populations.
Figure 2: Characterization of NS3133-DENV-specific CD8+ T cell clones and the binary structures of HLA-A*11:01–GTS1 and HLA-A*11:01–GTS3/4.
Figure 3: Structure of the D30 TCR bound to HLA-A*11:01–GTS1 or HLA-A*11:01–GTS3/4.
Figure 4: Interactions between the D30 TCR and HLA-A*11:01–GTS1.
Figure 5: Asn58β use in NS3133-DENV-specific CD8+ T cell populations.
Figure 6: Energetic-hotspot analysis of interactions between HLA-A*11:01—GTS1 and TRBV11-2+ TCRs.

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Acknowledgements

We thank N. Tangthawornchaikul for management of the clinical database; K. Campbell, H. Halim, A. Nguyen and staff at the Monash Macromolecular Crystallization Facility for technical support; and staff at the Australian Synchrotron for assistance with data collection. Supported by the Australian Research Council (S.G. and J.R.), the National Health and Medical Research Council, the National Institute for Health Research, the Thailand National Centre for Genetic Engineering and Biotechnology, the Thailand Tropical Disease Research Program T2 and the Wellcome Trust (D.A.P. and G.R.S.).

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Authors and Affiliations

Authors

Contributions

A.C., K.L., S.G. and J.E.M. performed experiments, analyzed data, provided intellectual input and contributed to manuscript preparation; K.L.M., C.F., H.v.d.H., E.G., W.D., A.W., T.D., P.C., W.L., S.V., P.M. and T.D. performed experiments and/or provided intellectual input; and J.R., J.M., D.A.P. and G.R.S. directed the study and wrote the manuscript.

Corresponding authors

Correspondence to Jamie Rossjohn, Juthathip Mongkolsapaya, David A Price or Gavin R Screaton.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CDR3β nucleotide alignments for public NS3133-DENV-specific TCRs.

Nucleotide alignments for the indicated public CDR3β amino acid sequences. Donor origin and HLA-A*11:01p tetramer specificity are indicated in the left column (see also Supplementary Table 2).

Supplementary Figure 2 Interactions between the D30 TCR and HLA-A*11:01–GTS3/4.

The GTS3/4 peptide (purple) is mainly contacted by the CDR3β loop (yellow) and the CDR2β loop (orange). Red dashed lines represent hydrogen bonds, and blue dashed lines represent van der Waals contacts.

Supplementary Figure 3 Structural comparison of free and bound NS3133 DENV peptides in complex with HLA-A*11:01.

(a) Superposition of HLA-A*11:01-GTS1 free (pink stick) and bound to the D30 TCR (blue stick). HLA-A*11:01 is represented in white cartoon. The D30 TCR is represented in pink cartoon with the CDR loops colored as follows: CDR1α, teal; CDR2α, green; CDR3α, purple; CDR1β, red; CDR2β, orange; CDR3β, yellow. (b) Top view of panel (a) showing the peptide alone. (c) Superposition of HLA-A*11:01-GTS3/4 free (green stick) and bound to the D30 TCR (orange stick). HLA-A*11:01 is represented in white cartoon. The D30 TCR is represented in pink cartoon with the CDR loops colored as in panel (a). (d) Top view of panel (c) showing the peptide alone.

Supplementary Figure 4 Equilibrium binding of soluble HLA-GTS1 complexes to wild-type and mutant D30 TCRs.

(a) Representative surface plasmon resonance data are shown for the indicated wild-type (WT) and mutant D30 TCRs. Two independent experiments were carried out in duplicate. Error bars indicate mean ± SEM. (b) Representative surface plasmon resonance sensorgrams are shown for the indicated wild-type (WT) and mutant D30 TCRs. Colors indicate different concentrations (1.56–400 μM) of the fluid phase analyte (HLA-A*11:01-GTS1).

Supplementary Figure 5 Equilibrium binding of soluble wild-type and mutant HLA-GTS1 complexes to the D2H, D13 and D30 TCRs.

Representative surface plasmon resonance data are shown for the indicated wild-type (WT) and mutant HLA-A*11:01-GTS1 complexes. Top: D30 TCR; middle; D13 TCR; bottom: D2H TCR. Two independent experiments were carried out in duplicate. Error bars indicate mean ± SEM.

Supplementary Figure 6 Recognition of HLA-A*11:01 and GTS1 mutants by NS3133-DENV-specific CD8+ T cell clones.

(a) Avidity of the NS3133 DENV-specific CD8+ T cell clones 44-173 13 (D13) and 44-173 30 (D30) for the indicated wild-type and mutant HLA-A*11:01-GTS1 tetramers. The TRBV7-6+ NS3133 DENV-specific CD8+ T cell clone E5 was included as a control. Data represent three independent experiments. Error bars indicate SD. gMFI, geometric mean fluorescence intensity. (b) Functional sensitivity of the NS3133 DENV-specific CD8+ T cell clones 44-173 13 (D13) and 44-173 30 (D30) for the indicated wild-type and mutant GTS1 peptides in IFN-γ ELISpot assays. The TRBV7-6+ NS3133 DENV-specific CD8+ T cell clone E5 was included as a control. Data represent three independent experiments. Error bars indicate SD. (c) Effector function profiles for the NS3133 DENV-specific CD8+ T cell clones 44-173 13 (D13) and 44-173 30 (D30) stimulated with the indicated wild-type and mutant GTS1 peptides. The TRBV7-6+ clone E5 was included as a control. Five readouts were measured by flow cytometry (CD107a, MIP-1β, TNF-α, IFN-γ, and IL-2). Data represent two independent experiments. Pie chart segments depict the fraction of cells expressing the number of functions indicated in the key.

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Culshaw, A., Ladell, K., Gras, S. et al. Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response. Nat Immunol 18, 1228–1237 (2017). https://doi.org/10.1038/ni.3850

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