Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region–complementary-determining region 3β (BV9–CDR3β) motif2,3,4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide–MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9–CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.
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The protein sequences used for target prediction were obtained from Uniprot (http://www.uniprot.org/). The human proteome ID is UP000005640. The Chlamydia proteome ID is UP000049987. The Salmonella proteome ID is UP000000541. The Shigella proteome ID is UP000001006. The Klebsiella proteome ID is UP000019183. The Yersinia proteome ID is UP000000815. The processed and raw deep-sequencing data have been deposited in the GEO database under the accession code GSE215018. The structures have been deposited in the RCSB protein data bank with the accession codes 7N2N, 7N2O, 7N2P, 7N2Q, 7N2R, 7N2S and 8CX4. There is no restriction on data availability. Source data are provided with this paper.
Custom Perl scripts for the deep-sequencing data processing are available from https://github.com/jlmendozabio/NGSpeptideprepandpred.
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The study received support from the National Institutes of Health (NIH; 5R01AI103867 (K.C.G.) and U19AI057229 (K.C.G.)), the Howard Hughes Medical Institute (K.C.G.), the UK Medical Research Council (MR/M019837/1; G.M.G., A.J.M. and P.B.), Oxford University McMichael Trust Fund (G.M.G and L.I.G), the Rosetrees Trust (M455; G.M.G.), Oxford University John Fell Fund and Medical Sciences Division Internal Funds (G.M.G. and L.I.G.), the UK National Institute for Health Research Oxford Biomedical Research Centre (BRC) (P.B.), the Ministry of Science and Higher Education of the Russian Federation (075-15-2019-1789; E.A.K., I.V.Z. and D.M.C.), the Rheumatology Research Foundation (M.A.P.), the Arthritis National Research Foundation (M.A.P.), the Rheumatic Diseases Research Resource-based Center at Washington University (NIH P30-AR073752; M.A.P. and W.M.Y.), the Bursky Center for Human Immunology and Immunotherapy Programs (W.M.Y.) and the Barnes-Jewish Hospital Foundation (W.M.Y.). The views expressed are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of sequencing data and J. Webber (Cell Sorting Facility, Kennedy Institute of Rheumatology, University of Oxford) for conducting cell sorting. We also thank N. Ternette, W. Paes and R. Inman for data analysis advice and scientific discussions. The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The ALS-ENABLE beamlines are supported in part by the NIH, National institute of General Medical Sciences (grant P30 GM124169). The schematic cartoon figures in Fig. 1 and Extended Data Fig. 1 were created with BioRender.com.
M.A.P. has received research support from Eli Lilly and Company paid to the university and served as a consultant for AbbVie, Priovant Therapeutics and JK MarketResearch. The remaining authors declare no competing interests.
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Extended data figures and tables
a. Schematic view of TRBV9+ TCRs isolation strategy from patients’ PBMC. Created with BioRender.com. b. Weblogo of CDR3β variants from HLA-B*27:05+ AS patient PBMC samples. c. Sorting strategy for scRNAseq library generation for Round 1 samples (HLA-B*27:05+ AS patient PBMC with overnight rest, post thaw). Sample AS1541 is shown. Final panel shows BV9-PerCP-Cy5.5 isotype control staining against CD8-PE. d. Representative sorting data for scRNAseq library generation. Sample AS1541 from Round 1 (HLA-B*27:05+ AS patient PBMC with overnight rest, post thaw) and sample AS1455B2 from Round 2 (HLA-B*27:05+ AS patient PBMC, 28 days expansion with αCD3 and IL-2 followed by magnetic depletion of CD4+, CD19+ and CD14+ cells) are shown. CD8+ T cell frequencies (as a % of total live cells) and BV9+ frequencies (as a % of CD8+ T cells) for each sample are as follows: Round 1 samples (CD8+ %/BV9+ %): AS1802 14.9/3.97; AS1311 16.4/10.7; AS1541 10.1/4.37. Round 2 samples: AS1455 82.9/5.60; AS1567 31.4/1.16; AS1661 66.7/7.21; AS1803 49.4/12.2. e. Enrichment of TRBV9 during first round of scRNAseq library generation by sorting. Pie charts show percentage of BV9+ cells in each sample pre- and post-mixing of sorted fractions. f. Partial amino acid sequence alignment of five AS patient PBMC-derived TCRs in this study, in RasMol colouring. The CDR1 and CDR2 sequences are shown for TRAV21 and TRBV9 in the top row. CDR3 amino acid sequences are shown in the bottom rows along with corresponding variable and joining gene usage. Structurally important bulky residues are marked with asterisks.
a. Correlation of the top100 TCR α and β nucleotide sequence proportion in two independent samples of CD3+CD8+TRBV9+ synovial fluid T cells of patient P. Confirmation of the frequency-based paired TCR α and β with single-cell VDJ-sequencing of an independent sample of CD3+CD8+TRBV9+ synovial fluid T cells from the patient. b. Enrichment of the BV9–Y/FSTDTQ–BJ2-3 motif in the joint compared to the blood in AS. Bulk TCR sequencing was performed on CD8+ T cell cDNA from the blood and synovial fluid. The graph displays the proportion of TCR sequence reads containing the BV9–Y/FSTDTQ–BJ2-3 motif in paired blood and synovial fluid of three Ankylosing Spondylitis patients. Level of detection (LoD) of blood samples is indicated by a dashed line and is the median proportion of a single sequence read from all blood samples
a. GRb TCR tetramer staining of wildtype SRY-β2M-HLA-B*27:05 single chain trimer expressed on yeast surface. Single chain trimer expression was monitored by anti-C-Myc-488. Streptavidin-647 (SA-647) was used to tetramerize and label the GRb TCR. b. Schematic of the SRY-β2m-HLA-B*27:05 single chain trimer on yeast surface. Error prone PCR introduced random mutations in SRY peptide, β2m and HLA-B*27:05 heavy chain. Sequencing of the enriched SRY-β2M-HLA-B*27:05 error prone library identified mutations located on HLA-B*27:05 heavy chain, shown as cartoon diagram and colored green. Bona fide mutations are shown as stick diagram and colored red. c. GRb TCR tetramer staining was rescued by M5L, H114Y and A153D mutation of the SRY-β2m-HLA-B*27:05 single chain trimer (HLA-B*27:053mut). Single chain trimer expression was monitored by anti-C-Myc-488. Streptavidin-647 (SA-647) was used to tetramerize and label the GRb TCR. d. Location of M5L, H114Y, and A153D mutations shown on the HLA-B*27 ribbon structure. e. Library design shows the anchor residue preference for the HLA-B*27:053mut library at the P2 and PΩ. For other positions, the NNK codon allowed all 20 amino acids and stop codons. Nucleotide abbreviations for codon usage are listed according to the IUPAC nucleotide code. Library diversities were estimated based on SDCAA plate colony numbers under serial dilution. f. GRb TCR tetramer staining of 4th round peptide-HLA-B*27:053mut yeast-display library, indicating successful enrichment. g. Deep sequencing counts on the most enriched peptides after 4 rounds of selection with the GRb TCR. h. WebLogo plots of the top 100 peptides after 4th round of selection with the GRb TCR, the two predominant clusters within the selection, and the sequence of the cognate peptide recognized by the GRb TCR in the abundance at the given position among the unique peptides.
a. HLA-B*27:053mut 10mer library enrichment per round of selection by the five AS TCRs as measured by flow cytometry. b. AS4.1, AS4.2, AS4.3 and AS4.4 TCR tetramer staining on the 4th round HLA-B*27:05 9-AA libraries. HLA-B*27:053mut expression was monitored by anti-C-Myc-488. Streptavidin-647 (SA-647) was used to tetramerize and fluorescently label the AS TCRs. c. AS3.1, AS4.1, AS4.2, AS4.3 and AS4.4 TCR tetramer staining on the 4th round HLA-B*27:053mut 10-AA libraries. HLA-B*27:05 expression was monitored by anti-HA-488. Streptavidin-647 (SA-647) was used to tetramerize and fluorescently label the AS TCRs. d. WebLogos representing the unique 4th round selected peptides for each AS TCR based on deep sequencing reads. The size of each amino acid letter represents its abundance at the given position among the unique peptides. e. Heatmap plots showing the amino acid composition per position of the peptides enriched after the 4th round of selection. Darker color represents greater abundance of a given amino acid at specific position.
(a-j) CD8+ SKW-3 cells were transduced with AS TCRs via lentivirus and sorted for stable TCR (IP26) and CD3 (UTCH1) co-expression. K562 cells were transduced with wild type HLA-B*27:05 via lentivirus and sorted for stable HLA molecule expression. The antigen-presenting cell line was pulsed for 2 h with 100 µM peptides and co-incubated with the T cell lines for 18 h then analyzed for CD69 expression by flow cytometry. (a-e) AS3.1, AS4.1, AS4.2, AS4.3 and AS4.4 TCR were tested for CD69 activation by yeast-selected mimotopes from 9-AA libraries. (f-j) AS3.1, AS4.1, AS4.2, AS4.3 and AS4.4 TCR were tested for CD69 activation by yeast-selected mimotopes from 10-AA libraries. Red dots show activation, as defined by CD69% at least 2-fold greater than negative controls (DMSO and null peptide, n = 1).
a. Representative flow cytometry showing AS8.4 cells alone, AS8.4 T cells + K562 (HLA-B*27:05+), AS8.4 T cells + K562 (HLA-B*27:05+ GPER1+), AS8.4 T cells + K562 (HLA-B*27:05+ YEIH+) and AS8.4 T cells + K562 (HLA-B*27:05+ 1uM YEIH peptide). b. Plots of percentage of CD69% positive AS8.4 T cells alone, AS8.4 T cells + K562 (HLA-B*27:05+), AS8.4 T cells + K562 (HLA-B*27:05+ GPER1+), AS8.4 T cells + K562 (HLA-B*27:05+ YEIH+) and AS8.4 T cells + K562 (HLA-B*27:05+ 1uM YEIH peptide). Data are shown as mean±s.d., n = 3 biological replicates. NS, not significant, P = 0.5453; ****P<0.0001 (one-way ANOVA). c. Representative flow cytometry histograms showing AS TCR tetramer staining for SCT transfected b2m KO 293T cells. Red histograms: negative controls (irrelevant p-HLA-B*27:05 construct); Blue histograms: potential arthritogenic peptide identified in this study.
Extended Data Fig. 7 Differential activation of AS TCRs by potential arthritogenic peptides on HLA-B*27:05 and HLA-B*27:09 backgrounds.
a. AS8.4 T cells incubated with GPER1-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. b. AS8.2 T cells incubated with GPER1-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. c. Structure models showing how the GPER1 P9 Arg interacts with HLA-B*27:05 Asp at 116 and HLA-B*27:09 His at 116. The peptide Arg 9 is colored magenta, Asp 116 is colored green, and His 116 is colored cyan. d. AU2 T cells incubated with RNASEH2B-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. e. AS4.3 T cells incubated with RNASEH2B-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. f. Structure models showing how the RNASEH2B P9 Arg interacts with HLA-B*27:05 Asp at 116 and HLA-B*27:09 His at 116. The peptide Arg 9 is colored magenta, Asp 116 is colored green, and His 116 is colored cyan. g. AU2 T cells incubated with PRPF3-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. h. AS4.3 T cells incubated with PRPF3-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. i. Structure models showing how the PRPF3 P9 Lys interacts with HLA-B*27:05 Asp at 116 and HLA-B*27:09 His at 116. The peptide Lys 9 is colored magenta, Asp 116 is colored green, and His 116 is colored cyan. j. AS8.4 T cells incubated with YEIH-pulsed K562 cells, express HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. k. AS8.2 T cells incubate with YEIH-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. l. Structure models showing how the YEIH P9 Phe interacts with HLA-B*27:05 Asp at 116 and HLA-B*27:09 His at 116. The peptide Phe 9 is colored magenta, Asp 116 is colored green, and His 116 is colored cyan. m. AU2 T cells incubate with MPP4-pulsed K562 cells, expressing HLA-B*27:05 or HLA-B*27:09, respectively, were tested for CD69 up-regulation. Data are shown as mean±s.d., n = 3 biological replicates. n. Structure models showing how the MPP4 P9 His interacts with HLA-B*27:05 Asp at 116 and HLA-B*27:09 His at 116. o. Thermal stability of refolded peptide/HLA-B*27 complex as determined by differential scanning fluorimetry. The melting temperature, Tm, of each complex is plotted as a bar graph with mean±s.d. (n = 3 technical triplicates).
(a-p) Binding analysis of AS TCRs AS3.1, AS4.1, AS4.2, AS4.3 and AS8.4 to GPER1/PRPF3/RNASEH2B/YEIH-HLA-B*27:05 molecules. Increasing concentrations of each TCR was injected over immobilized GPER1/PRPF3/RNASEH2B/YEIH-HLA-B*27:05 complexes. The response unit vs concentration plots were fitted to a steady state affinity model. The SPR measurements were performed once for each TCR-pMHC interaction.
a. Simulated annealing composite omit map of the entire AS4.3 TCR-RNASEH2B-HLA-B*27:05 complex contoured at 1 σ. The TCRα chain is colored red; the TCRβ chain is colored yellow; the peptide is colored magenta; the β2M is colored cyan; the HLA-B*27:05 heavy chain is colored green. (b-h) Simulated annealing composite omit maps of the AS4.3 TCR-RNASEH2B-HLA-B*27:05, AS3.1 TCR-PRPF3-HLA-B*27:05, AS4.2 TCR-PRPF3-HLA-B*27:05, AS4.2 TCR-YEIH-HLA-B*27:05, AS4.3 TCR-PRPF3-HLA-B*27:05, AS4.3 TCR-YEIH-HLA-B*27:05 and AS8.4 TCR-YEIH-HLA-B*27:05 complex interfaces contoured at 1 σ. The TCR CDR1α is colored red; the TCR CDR3β is colored yellow; the peptide is colored magenta; the HLA-B*27:05 heavy chain is colored green.
a. Side view of superimposed AS TCR-peptide-HLA-B*27:05 complexes. b. Top view of superimposed interface between AS TCR CDR loops and peptide-HLA-B*27:05. c. Superimposed complex structures reveal CDR3β interaction with peptides and HLA-B*27:05. d. Superimposed complex structures reveal CDR2β interaction with HLA-B*27:05. (e-k) Peptide recognition by CDR1α and CDR3β in AS TCR-peptide-HLA-B*27:05 complexes. The upper panels are ‘Sticks’ indicating TCR residues and peptide residues making contacts. CDR1α is colored red or orange and depicted in cartoon. CDR3β is colored yellow or wheat and depicted in cartoon. Peptide is colored magenta and HLA-B*27:05 is colored green and depicted in cartoon. The lower panels show CDR1α, CDR3β and peptide sequences and atomic interactions. The black lines indicate van der Waals contacts; red dashed lines indicate hydrogen bonds.
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Yang, X., Garner, L.I., Zvyagin, I.V. et al. Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides. Nature 612, 771–777 (2022). https://doi.org/10.1038/s41586-022-05501-7
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Nature Reviews Immunology (2022)