Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides

Abstract

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) motif^2-4 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.

Extended Data Fig. 1 |. Isolation of TRBV9+ TCRs from PBMC.

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.

Extended Data Fig. 2 |. AS3.1 TCR alpha-beta pair identification.

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.

Extended Data Fig. 3 |. Development of HLA-B*27:05 yeast library.

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*27:05^3mut). 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*27:05^3mut 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 4^th round peptide-HLA-B*27:05^3mut 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 4^th 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.

Extended Data Fig. 4 |. Selection of AS TCRs on HLA-B*27:05 libraries.

a. HLA-B*27:05^3mut 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 4^th round HLA-B*27:05 9-AA libraries. HLA-B*27:05^3mut 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 4^th round HLA-B*27:05^3mut 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 4^th 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 4^th round of selection. Darker color represents greater abundance of a given amino acid at specific position.

Extended Data Fig. 5 |. Synthetic peptide activation by AS TCR expressing T cell line.

(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).

Extended Data Fig. 6 |. AS TCR tetramer staining on SCT transfectants.

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).

Extended Data Fig. 8 |. SPR sensorgram of AS TCRs and peptide-HLA-B*27:05.

(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.

Extended Data Fig. 9 |. Omit maps of AS TCR-peptide-HLA-B*27:05 complex contoured at 1 σ.

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.

Extended Data Fig. 10 |. Detailed interactions between AS TCR and peptide-HLA-B*27:05.

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.

Fig. 1 |. Identification of AS- and AAU-associated TRBV9–TRBJ2.3 TCRs.

a, Schematic of TCR isolation from PBMCs and synovial fluid (SF) cells from patients with AS, and from PBMCs and ocular fluid cells from patients with AAU. Created with BioRender.com. FACS, fluorescence-activated cell sorting; OF, ocular fluid. b, The frequency of CD8⁺ T cells containing the YSTDTQ TRBJ2.3 motif from sorted BV9⁺CD8⁺ T cells in PBMCs and synovial fluid from patients with AS. The level of detection (LoD) of blood samples is indicated by a dashed line and is the median proportion of a singleton clonotype from all blood samples. c, Unbiased single-cell TCR sequencing of ocular fluid cells and PBMCs from patients with AAU. The proportions of expanded ocular TCR clonotypes (defined as greater than 10 barcodes in the eye) are compared between the ocular fluid and corresponding PBMCs from patients with AAU. The ‘_a’ and ‘_b’- suffixes designate the first and second samplings, respectively. The level of detection of blood samples is indicated by a dashed line and is the median proportion of a singleton clonotype from all blood samples. d, Partial amino acid sequence alignment of TCRs derived from PBMCs and synovial fluid from patients with AS and TCRs derived from PBMCs and ocular fluid from patients with AAU used in this study, in RasMol colouring. The invariant 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.

Fig. 2 |. Screening TRBV9-TRBJ2.3 TCRs on HLA-B*27:05 yeast display libraries.

a, AS3.1, AS4.1, AS4.2, AS4.3 and AS4.4 TCRs selected on HLA-B*27:05 libraries. Enrichments from the naive library to the fourth round (RD4) were measured through flow cytometry and are plotted as the percentage of cells that are epitope positive. b, WebLogos representing the unique fourth-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. c, Heatmap plots showing the amino acid composition per position of the peptides enriched after the fourth round of selection. A darker colour represents a greater abundance of a given amino acid at a specific position. d, Top algorithm-predicted peptides from the human proteome based on AS3.1, AS4.1, AS4.2, AS4.3 and AS4.4 deep-sequencing data.

Fig. 3 |. Validation of TCR binding and activation by predicted human and microbial antigens.

a–m, TCRs from patients with AS or AAU were transduced into CD8⁺ SKW-3 cells and sorted for stable TCR (IP26) and CD3 (UCHT1) co-expression. Wild-type HLA-B*27:05 was transduced into K562 cells, and the cells were sorted for stable HLA molecule expression. APC lines were pulsed with 1:3 serial-diluted peptides starting from 100 μM for 2 h, co-incubated with the T cell lines for 18 h and analysed for CD69 expression by flow cytometry. AS3.1 (a), AS4.1 (b), AS4.2 (c), AS4.3 (d), AS4.4 (e), AS8.2 (f), AS8.3 (g), AS8.4 (h), AS8.5 (i), AS9.1 (j), AS9.2 (k), AU2.1 (l) and AU1.2 (m) were tested for CD69 activation. Dose–response curves for each human and common microbial stimulatory peptide are shown with means of biological triplicates with standard error of the mean. The microbial-derived peptides are in bold to differentiate them from human-derived peptides. Representative data are shown as mean ± s.d. (n = 3 biological triplicates). n, The matrix of activated peptides corresponding to each TCR included in the peptide activation assay. Check marks indicate activation of TCR by the corresponding peptide, as defined by CD69 upregulation when dosed with 100 μM (E_max) of individual peptide compared to dimethylsulfoxide (DMSO) control (n = 3 biological triplicates).

Fig. 4 |. Biophysical and structural basis of AS TCR–peptide–HLA-B*27:05 complexes.

a–d, Overview of AS3.1–PRPF3–B*27 (a), AS4.2–PRPF3/YEIH–B*27 (b), AS4.3–PRPF3/YEIH/RNASEH2B–B*27 (c) and AS8.4–YEIH–B*27 (d) complexes. The HLA-B*27:05 heavy chain, β₂m, peptide, TCRα chain and TCRβ chain are depicted in cartoon mode. HLA-B*27:05 is coloured green; β₂m is coloured cyan; peptide is coloured magenta; TCRα chain is coloured red; and TCRβ chain is coloured yellow. e–h, Top view of CDRs from AS3.1–PRPF3–HLA-B*27:05 (e), AS4.2–PRPF3/YEIH–HLA-B*27:05 (f), AS4.3–PRPF3/YEIH/RNASEH2B–HLA-B*27:05 (g) and AS8.4–YEIH–HLA-B*27:05 (h) complex crystal structures. Peptide–HLA-B*27:05 is coloured light grey and depicted in surface mode. CDR3α is coloured red and depicted in cartoon mode. CDR3β is coloured yellow and depicted in cartoon mode. i, Detailed peptide recognition through AS TCR CDR1α and CDR3β. The TCRα chain is coloured red, the TCRβ chain is coloured yellow, the peptide is coloured magenta, and HLA-B*27:05 is coloured green. j, Detailed amino acid interactions between AS TCR and peptides. The interactions between CDR1α, CDR3β and peptides are depicted as black lines. The interaction cutoff is 4 Å.