Abstract
A keystone of antiviral immunity is CD8+ T cell recognition of viral peptides bound to MHC-I proteins. The recognition modes of individual T cell receptors (TCRs) have been studied in some detail, but the role of TCR variation in providing a robust response to viral antigens is unclear. The influenza M1 epitope is an immunodominant target of CD8+ T cells that help to control influenza in HLA-A2+ individuals. Here we show that CD8+ T cells use many distinct TCRs to recognize HLA-A2–M1, which enables the use of different structural solutions to the problem of specifically recognizing a relatively featureless peptide antigen. The vast majority of responding TCRs target a small cleft between HLA-A2 and the bound M1 peptide. These broad repertoires lead to plasticity in antigen recognition and protection against T cell clonal loss and viral escape.
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Acknowledgements
This work was supported by the NIH (grants AI038996 (to L.J.S.), AI49320 (to L.K.S.), and AI109858 (to L.J.S. and L.K.S.)) and the Nebraska Research Initiative (grant to D.G.). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility. We thank J. Birtley and Z. Maben for assistance with crystallization, freezing, and shipping of crystals; P. Trehn for advice on model analysis; W. Uckert (Max Delbruck Center, Berlin, Germany) for TCRα/β− Jurkat J76 cells transfected with human CD8α; and P. Thomas for technical advice on single-cell PCR.
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I.Y.S. conceived the project, designed the experimental approach, performed single-cell sequencing experiments, characterized TCR transfectants, determined crystal structures, and wrote the manuscript. A.G. performed NGS analyses, performed single-cell sequencing experiments, and edited the manuscript. R.M. performed NGS analyses and edited the manuscript. D.G. performed NGS analyses and edited the manuscript. L.K.S. conceived the project, designed the experimental approach, supervised TCR sequencing analyses, and wrote the manuscript. L.J.S. conceived the project, designed the experimental approach, supervised cellular and molecular studies, and wrote the manuscript.
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Supplementary Figure 1 Diversity of CD8+ T cell repertoire, dominant usage of TRAV38, and CDR3α and CDR3β sequence motifs in the HLA-A2–M1-specific response: unique clonotypes.
(a) HLA-A1/M1-specific TRBV repertoires assayed for donors 185, 215, 250, 264 directly ex vivo or after culture are similar (Pearson’s correlation coefficient). CD8 T cells isolated from PBMC were stained with M1 tetramer and Vb-specific mAb or were expanded and analyzed by NGS as described in methods. (b-h) As Figure 1 except frequencies are expressed in terms of numbers of unique sequences. (i) Frequencies of xGxY or xRS/Ax motif-harboring TCRβ in the total M1-specific TRBV19 repertoires. Frequencies of xRSx motif are shown in white in each bar and accumulated frequencies of xRSx and xGxY motifs are shown above each bar. (j) Shannon-Weaver and Simpson Diversity index of the TCRα and TCRβ of the six donors.
Supplementary Figure 2 Diverse TRAV pairing with xRSx TRBV19, and restricted TRAV38-TRAJ52 pairing with xGxY TRBV19.
(a) Schematic diagram. HLA-A2/M1-dextramer-specific CD8 T cells from PBMC were sorted directly ex vivo into single wells for TCRα and TCRβ sequencing. (b) TRAV usage of amplified productive TCRα (n=34) and TCRβ (n=82) sequences. TR gene usage and CDR3 sequences of 13 selected TCRα/TCRβ pairs, categorized into four groups, are shown in Supplementary Table I.
Supplementary Figure 3 Inconspicuous HLA-A2-restricted M1 peptide.
(a) Structures of representative HLA-A2/peptide complexes shown in surface representation with peptides colored and N-termini at left. (b) Exposed surface areas of individual residues of 131 peptides (see Methods for PDB IDs) bound in unligated HLA-A2/peptide complex structures. (c) Total exposed peptide surface area for 131 HLA-A2/peptide complexes. Average of all peptides is shown as horizontal line. Peptides from panels a and b are shown as colored dots. (d) Total buried peptide surface area for HLA-A2/peptide complexes as in (c).
Supplementary Figure 4 Surface expression, MHC tetramer binding, and T cell activation by paired TCRα/β chains.
(a) TCR surface expression of transiently- transfected TCR-J76-CD8+ cells. (b) Concentration-dependent binding of HLA-A2/M1 tetramer to stably-transfected T cell lines. (c) TCR surface expression of stably-transfected TCR-J76-CD8+ cells with selected paired TCRα/β. (d) Paired TCRα/β can initiate T cell signaling after M1-peptide stimulation. J76-CD8 stably expressing paired TCRα/β were stimulated with antigen presenting cells loaded with M1 (black bars), irrelevant HLA-A2-restricted peptides (hatched bar: BMLF1, tyrosinase) or no peptide (open bar). CD69 expression after peptide stimulation was compared to corresponding unstimulated controls. Representative FACS plots for one TCR (LS12) are shown at right. Error bars represent standard deviations from three independent experiments.
Supplementary Figure 5 Electron density and peptide conformation.
(a) Electron density for LS01-M1-HLA-A*02 and LS10-M1-HLA-A*02 complexes in the region of TCR-peptide-MHC interaction. Composite omit maps were built with final models of each TCR/pMHC complexes and reflection data. Top views from TCR around M1-peptides of final models are shown in the composite omit maps. Yellow: M1-peptide (stick), red: HLA-A*02 (line), orange: TCRa (line), green: TCRb (line). Sigma levels of contour of maps were set as 1.5. (b) Overlaid M1 peptides from LS10-HLA-A2/M1 complex (deep blue) and HLA-C*08/M1 complex, from PDB 4NT6 (green).
Supplementary Figure 6 Functional characterization of residues in TCR-pMHC contact region, and TCR-pMHC contact maps for LS01, LS10, and JM22 TCRs.
(a-c) Effects of alanine mutation of CDR3 residues on HLA-A2/M1- LS10 TCR binding are illustrated in bar graphs with positions of mutated residues marked as red dots above sequence logos. (d) TRAV38-specific residues (Asp31α of CDR1α, Glu52α and Tyr54α of CDR2α) from the LS10 TCR interact with a rarely-contacted MHC residue (Arg157MHC). (e) Mutation of LS10 interface residues Phe98β, Gln100β and Arg101β in CDR3β to alanines abolished M1-tetramer binding. (f) Comparison of TRAJ52-containing CDR3α from different TCR structures. Alanine and tyrosine are underlined in the sequences and depicted as sticks in line representation. (g) Gln155MHC is stabilized by Tyr31α from CDR1α of LS10 TCR. (h) Mutation of LS10 Tyr31α and Asn95α abolished M1-tetramer binding (i) Effect of mutation of CDR1β and CDR2β residues (Asp32β, Gln52β, Ile53β) on HLA-A2/M1 tetramer binding for three TCR. For JM22 all three residues are essential (Ishizuka, J. et al., Immunity 28, 171–182, 2008). (j) Contact maps for three TCR-HLA-A2/M1 complexes. Contact residues from TCR are listed vertically and from pMHC horizontally, with residue numbers shown. Grid boxes are filled with corresponding CDR color if residues from pMHC and TCR contact. Error bars in (a-c,e,h,i) represent standard deviation of duplicate measurements from each of two independent samples.
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Song, I., Gil, A., Mishra, R. et al. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8+ T cell epitope. Nat Struct Mol Biol 24, 395–406 (2017). https://doi.org/10.1038/nsmb.3383
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DOI: https://doi.org/10.1038/nsmb.3383
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