Ethics Statement

For the use of human specimens, all protocols involving human participants recruited at Kumamoto University, Kyushu Medical Centre, Kyushu University, Tokyo Metropolitan Cancer and Infectious Diseases Centre, Komagome Hospital were reviewed and approved by the Institutional Review Boards of Kumamoto University (approval numbers 2066 and 461). All human participants provided written informed consent.

Collection of human PBMCs

Human PBMCs were obtained from thirty-one early pandemic convalescents (median age: 48, Range: 19–77, 74% male) (Table 1); twenty-four HLA-C*12:02⁺ convalescents (median age: 42.5, Range: 23-86, 58.3% male), thirty-eight HLA-C*12:02^– convalescents (median age: 43.5, Range: 25–88, 63.2% male), five HLA-C*12:02⁺ seronegative (median age: 24, Range: 23–40, 80% male) and four HLA-C*12:02^– seronegative donors (median age: 24, range: 20–24, 25% male) (Table 2). PBMCs were purified by a density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Life Sciences, Cat# 17-1440-03) and stored in liquid nitrogen until further use.

Cell culture

A549 cells stably expressing human ACE2 and HLA-A*24:02-IRES-GFP or C*1202 or C*1402-IRES-NGFR were generated by electroporated transduction and were maintained in Ham’s-F12 (Wako, Cat# 080-08565) containing 10% foetal bovine serum (FBS) (Sigma-Aldrich, Cat#172012-500 ML). C1R cells expressing HLA-A*24:02⁵⁶, -B*52:01⁵⁷, or -C*12:02¹² were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS.

Virus

Four clinically isolated SARS-CoV-2 lineages were used: SARS-CoV-2 Wuhan strain [SARS-CoV-2/Hu/DP/Kng/19-020 (DDBJ Accession ID: LC528232)] was provided by the Kanagawa Prefectural Institute of Public Health. B.1.1.529 (Omicron/BA.1) lineage [hCoV hCoV-19/Japan/TKYX00012/2021 (GISAID Accession ID: EPI_ISL_8559478)] and B.1.1.529 (Omicron/BA.5) lineage [hCoV-19/Japan/TKYS14631/2022 (GISAID Accession ID: EPI_ISL_12812500)] was provided by the Tokyo Metropolitan Institute of Public Health, Tokyo, Japan. B.1.1.529 (Omicron/BA.2) lineage [hCoV hCoV-19/Japan/TY40-385-P1/2022 (GISAID Accession ID: EPI_ISL_9595859)] was provided by the National Institute of Infectious Diseases, Tokyo, Japan.

Analysis of SARS-CoV-2 genomic variation

To investigate genomic mutations in SARS-CoV-2, we downloaded 17,163,274 genome sequences from the GISAID database as of March 9, 2025⁵⁸. Among these, 5,553,216 sequences containing no ambiguous or degenerate bases were obtained. After further filtering for sequences that 1) were derived from human hosts, 2) exceeded 29,000 nucleotides in length, and 3) exhibited less than 2% nucleotide divergence across the genome, a total of 5,378,593 genome sequences were retained for mutation analysis (accessible at https://doi.org/10.55876/gis8.250519sk). For each sequence, SGV-caller was used to identify genomic mutations at nucleotide and amino acid levels⁵⁹.

ELISpot assay

Ex vivo IFN-γ ELISpot assay was performed using the following antibodies and reagents: anti-human IFN-γ mAb 1-D1K, purified (Mabtech, Code:3420-3-1000, 1/500 dilution), anti-human IFN-γ mAb 7-B6-1, biotinylated (Mabtech, Code: 3420-6-250, 1/2000 dilution), Streptavidin-ALP (Mabtech, Code:3310-8-1000), 25×AP Colour Development Buffer (BIORAD), AP Conjugated Substrate Kit (BIORAD, Cat# 1706432) according to the manufacturer’s protocol. Pools of 15-mer peptides, overlapping by 11 amino acids and spanning the SARS-CoV-2 nucleocapsid protein (SCRUM Inc.). Briefly, after washing a MultiScreen 96-well plate with sterilised PBS and blocked with RPMI 1640medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS, 2 × 10⁵ defrosted PBMCs per well were pulsed with each SARS-CoV-2 prototype nucleocapsid overlapping peptide (2 μg/ml) and cultured for 20 hours. The responses were defined as the number of spot-forming cells (SFC)/10⁶ calculated by ImmunoSpot (Cellular Technology Limited). When the number of spots for the negative control (no peptide) was 0, thresholds for positive responses were defined as more than 2 spots, and when spots were more than one spot for the negative control, the responses were defined as more than twice.

Tetramer staining

PE-conjugated KF9 peptide-loaded HLA-C*12:02 tetramers were generated by NIH (National Institutes of Health). SARS-CoV-2-derived peptides-loaded MHC class I tetramers were generated by QuickSwitchTM Quant HLA-A*24:02 Tetramer Kit-PE (MBL International Corporation, Cat# TB-7302-K1) according to the manufacturer’s protocol. PBMCs were stained with tetramers for 30 min at room temperature. After tetramer staining, surface staining with the following antibodies: CD3 BV421 (UCHT1, 1/50 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution), CCR7 BV510 (G043H7, 1/25 dilution), CD45RA APC (HI100, 1/100 dilution; Biolegend) was performed. Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and levels of protein expression were analysed by flow cytometry using a FACS Canto II (BD Biosciences) and Cytek Northern Lights (Cytek Japan) followed by analysis using FlowJo v10 software (BD Biosciences).

Activation-Induced Marker Assay

An activation-induced marker assay was performed as previously described^19,20,46. Briefly, human PBMCs were pulsed with 100 nM of the peptides (KF9: KAYNVTQAF, residues 266-274 of the SARS-CoV-2 nucleocapsid protein, and QI9: QYIKWPWYI, residues 1208-1216 of the SARS-CoV-2 spike protein; Genscript) maintained in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS and 30 U/ml recombinant human IL-2 (Peprotec, Cat# 200-02) for 14 days. SARS-CoV-2 Wuhan strain (5 × 10⁵ PFU/ml) was inactivated with 0.5% Beta-propiolactone (Cat#H0168, Tokyo Chemical Industry Co., Ltd) at 56 °C 45 min as previously described²¹. Human PBMCs were stimulated with the peptide pools of SARS-CoV-2 spike (Miltenyi Biotec, Cat#130-126-701), nucleocapsid (Miltenyi Biotec, Cat#130-126-698,) and membrane protein (Miltenyi Biotec, Cat#130-126-703) or 3 μl of the inactivated virus particles and cultured for 14 days. The in vitro expanded CD4⁺ or CD8⁺ T cells (i.e., T-cell lines) were restimulated with or without the peptide. After incubation at 37 °C for 24 h, the cells were washed and surface stained with following antibodies: CD3 FITC (UCHT1, 1/100 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/ Cy5.5 (HIB19, 1/100 dilution), CD25 PEcy7 (M-A251, 1/50 dilution) and CD137 APC (4B4-1, 1/50 dilution; Biolegend). Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min on ice, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and levels of protein expression were analysed by flow cytometry using a FACS Canto II (BD Biosciences) and Cytek Northern Lights (Cytek Japan) followed by analysis using FlowJo v10 software (BD Biosciences).

Intracellular cytokine staining

Intracellular cytokine staining was performed as previously described^19,20,46. Briefly, A549-ACE2-C1202 cells (3 × 10⁵ cells) were transfected with 2 μg of plasmids expressing prototype nucleocapsid using PEI Max (Polysciences, Cat# 24765-1) following the manufacturer’s protocol. At 2 days post-transfection, the transfectants were harvested and mixed with the T-cell lines generated from HLA-C*12:02⁺ convalescents (see above) and cocultured with RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS, 5 μg/ml brefeldin A (Sigma-Aldrich, Cat# B7651) in a 96-well U plate at 37 °C for 5 h. The cells were washed and stained with the following antibodies: CD3 FITC (UCHT1, 1/100 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/ Cy5.5 (HIB19, 1/100 dilution), and CD107a-BV421 (H4A3, 1/100 dilution; Biolegend). Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation at 4 °C for 20 min, the cells were fixed and permeabilised with a Cytofix/ Cytoperm Fixation/Permeabilisation solution kit (BD Biosciences, Cat# 554714) and were stained with IFN-γ PE (4S.B3, 1/100 dilution; BD). After incubation at room temperature for 30 min, the cells were washed, and levels of protein expression were analysed by flow cytometry using a FACS Canto II (BD Biosciences) and Cytek Northern Lights (Cytek Japan), followed by analysis using FlowJo v10 software (BD Biosciences).

TCR cDNA amplification from single T cells and construction of TCR expression vector

The cryopreserved PBMCs were stained with KF9/C12 tetramers PE (10 μg/ml), CD8 APCcy7 (RPA-T8, 1/100 dilution; Biolegend), and 7-amino-actinomycin D (7-AAD), and then tetramer⁺CD8⁺7-AAD^– cells were sorted into 96-well plates (NIPPON Genetics, Cat# 4ti-0770/C) by using an FACS Aria II (BD Biosciences). TCRα and TCRβ cDNA pairs were amplified from single T cells by a one-step multiplex RT-PCR method described in our previous study⁶⁰. The DNA sequences of the PCR products were then analysed by direct sequencing, and the TCR repertoire by IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/vquest). The amplified TCRα and TCRβ cDNA fragments were connected to the missing constant region and linked to the blasticidin S resistance (BlaR) gene by the Gibson assembly method with P2A ribosomal skipping sequences. The resultant TCRβ-P2A-TCRα-P2A-BlaR DNA was cloned into the PiggyBac vector (SBI, Cat# PB530A-2) by the Gibson assembly method.

TCR sensitivity assay

The plasmid PB TCR-P2A-BlaR was electroporated into Jurkat∆-Luc with Transposase vector (SBI, Cat# PB200PA-1) using Neon® Transfection System (Thermo Fisher Scientific) under the condition 1200 v, 5 ms, 5 pulses. After 48 h, Jurkat∆-Luc cells stably expressing TCRs were selected with RPMI medium containing 10 μg/ml of blasticidin-S for 10-14 days. These cells were cocultured with A549-ACE2-C1202 cells expressing each spike protein with an E:T ratio of 2:1 and incubated with RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS at 37 °C for 6 h. The mixture was measured for luciferase production using a luminescent substrate (Promega, Cat#E2510) by a CentroXS3 plate reader (Berthold Technologies).

The peptide-dependent stabilization assay

TAP-deficient MCF7 cells (MCF7ΔTAP1/2) were generated by using the CRISPR‒Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology, Cat#sc-42981-SH and sc-42983-SH) at the University of Toyama. MCF7ΔTAP1/2 were enriched by staining the cells with an HLA-A, B, C mAb (1/50 dilution, W6/32, BioLegend, Cat#311410) and sorting HLA class I-negative cells using a FACSAria. MCF7ΔTAP1/2 expressing HLA-C*12:02-IRES-NGFR MCF7ΔTAP1/2-C1202) were generated using Neon® Transfection System (Thermo Fisher Scientific) under the condition 1250 v, 20 ms, 2 pulses. A total of 1 × 10⁵ cells of MCF7ΔTAP1/2-C1202 cells were pulsed with peptides and then incubated at 26 °C overnight as previously described⁶¹. At the end of the incubation, unbound peptides were removed, and cells were stained with Fixable Live/Dead Violet Dye (1/50 dilution, Invitrogen, Cat#L34964), PE-labelled HLA class I-specific mAb TP25.99 (1/50 dilution, Thermo Fisher Scientific, Cat#MA5-44116), APC-labelled NGFR mAb (1/50 dilution, BioLegend, Cat#345108) and analysed by Cytoflex (Beckman Coulter, Inc.), followed by analysis using CytExpert2.6 and FlowJo v10 software (BD Biosciences). The mean fluorescence intensity (MFI) was calculated, and the mean values of triplicates are presented. Relative HLA expression was calculated as the ratio of the MFI of peptide-pulsed MCF7ΔTAP1/2-C1202 cells to that of control (non-peptide-pulsed) cells kept at 26 °C.

T-cell proliferation assay

T cell proliferation assay was performed using Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557) following the manufacturer’s protocol. Briefly, defrosted PBMCs were washed with sterilised PBS, and 1 × 10⁶ cells/ml PBMCs were labelled with 1 μM CTV (Celltrace violet). Labelled PBMCs were pulsed with either prototype nucleocapsid whole peptide or KF9 peptide and cultured in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS for 1 week. The in vitro expanded PBMCs were stained with C12/KF9 tetramers for 30 min at room temperature. After tetramer staining, surface staining with the following antibodies: CD3 BV421 (UCHT1, 1/50 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution), CCR7 BV510 (G043H7, 1/25 dilution), CD45RA APC (HI100, 1/25 dilution; Biolegend) was performed. Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and levels of protein expression were analysed by flow cytometry using a Cytek Northern Lights (Cytek Japan). The data obtained by flow cytometry were analysed with FlowJo v10 software (BD Biosciences).

Retrovirus Production

Phoenix-A cells were used to produce retrovirus for transducing TCR genes into human peripheral blood mononuclear cells (PBMCs). Phoenix-A (3.5 × 10⁵) were cultured in a 6-well collagen-coated plate (Iwaki, Cat# 4810-010) with 2 ml of DMEM culture medium one day before transfection. TCR expression vectors were transfected into the cells using PEI Max transfection reagent (Polysciences, Cat# 24765-1) according to the manufacturer’s instructions. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. At 48 hours post-transfection, the culture supernatant was harvested and filtered through a 0.45-μm membrane filter (Merck Millipore, Burlington, MA, USA). The collected retroviral supernatant was concentrated using the Retro-X Concentrator (Takara, Cat# 631455) according to the manufacturer’s instructions.

Retroviral Transduction of TCRs into Primary CD8⁺ T Cells

For retroviral TCR transduction, 1 × 10⁶ CD8⁺ T cells, isolated from PBMCs by using MACS with CD8 microbeads (Miltenyi Biotec, Cat# 130-045-201), were stimulated with CD3/CD28 Dynabeads™ (Thermo Fisher Scientific) in the presence of recombinant human IL-2 (PeproTech, Cat# 200-02) for two days. The PBMCs were harvested and resuspended at 5 × 10⁵ cells/ml in RPMI 1640 containing 10% FBS in the presence of human IL-2 (30 U/ml). Meanwhile, the wells of 24-well plates were coated with 0.3 ml of RetroNectin (50 μg/ml, Takara, Kyoto, Japan) and incubated at 4 °C overnight. TCR-encoding retroviruses were adhered to the wells by centrifugation for 2 h at 2,000×g and 32 °C. The stimulated CD8⁺ T cells were then added to the virus-coated wells and centrifuged at 500×g for 10 minutes at room temperature, followed by overnight incubation at 37 °C in a 5% CO₂ atmosphere. The next day, the cells were transferred to freshly prepared retrovirus-coated plates and incubated at 37 °C in a 5% CO₂ atmosphere for another 24 hours to perform a second round of infection. Following transduction, CD8⁺ T cells were expanded in the presence of IL-2 (30 U/ml) for an additional 2–3 days. RatCD2⁺ CD8⁺ T cells were sorted by MACS using an anti-ratCD2-FITC antibody (OX-34, BioLegend, Cat# 201303, 1:100 dilution) and anti-FITC microbeads (Miltenyi Biotec, Cat# 130-048-701). The resulting TCR-transduced CD8⁺ T cells were used for functional assays on days 12–14. After tetramer staining above, surface staining with the following antibodies: ratCD2 FITC (OX-34, 1/100 dilution), CD3 BV510 (UCHT1, 1/50 dilution; Biolegend), CD8 APCcy7 (RPA-T8, 1/100 dilution) was performed. Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and levels of protein expression were analysed by flow cytometry using a Cytek Northern Lights (Cytek Japan) followed by analysis using FlowJo v10 software (BD Biosciences).

Plasmid Construction

The plasmid expressing the SARS-CoV-2 nucleocapsid proteins of the parental (D614G-bearing B.1 lineage) was prepared using forward primer (5-AAA GGT ACC GCC GCC ACC ATG AGC GAT AA-3) and reverse primer (5-TTT GCG GCC GCT TAC TTT TCA AAC TGC GG-3). The resulting PCR fragment was digested with KpnI (New England Biolabs, Cat# R0142S) and NotI (New England Biolabs, Cat# 1089S) and inserted into the corresponding site of the pCAGGS vector. Nucleotide sequences were determined by Genetic Analyser 3500xL (Applied Biosystems), and the sequence data were analysed by GENETYX v12 (GENETYX Corporation).

Live virus suppression assay

A live virus suppression assay by T cells was performed as previously described²⁰. A549 cells expressing ACE2/C1202 (1 × 10⁴ cells) were infected with each SARS-CoV-2 lineage at an MOI of 0.1 for 120 min at 37 °C. Cells were washed and cocultured with T cells at an E:T ratio of 2:1 and 1:1. Control wells containing virus-infected targets without T cells were also included. After 72 h incubation, the culture supernatant was collected and subjected to real-time RT-PCR. 5 μl of culture supernatant was lysed in an equal amount of buffer composed of 2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl (pH 7.4), 40% glycerol, and 0.4 U/μl recombinant RNase inhibitor (Promega, Cat# N2615) and then incubated at room temperature for 10 min. 90 μl of RNase-free water (Nacalai Tesque, Cat# 06442-95) was added, and 3 µl of diluted sample was used as the template. Real-time RT-PCR analyses for viral RNA copy number were carried out with One Step PrimeScript™ III RT-qPCR Mix (Takara, Cat# RR600B), and reactions were performed by using LightCycler® 96 System (Roche Diagnostics GmbH, Mannheim, Germany). For the primer, Primer/Probe N2 (2019-nCoV) (Takara, Cat# XD0008) were used as follows: NIID_2019-nCOV_N_ forward, 5-AAATTTTGGGGACCAGGAAC-3; NIID_2019-nCOV_N_ reverse, 5-TGGCAGCTGTGTAGGTCAAC-3; and NIID_2019-nCoV_N_ probe, 5-FAM-ATGTCGCGCATTGGCATGGA-BHQ3. The viral RNA copy number was standardised with a Positive Control RNA Mix (2019-nCoV) (Takara, Cat#XA0142). The relative viral copy was calculated as the viral RNA copy number obtained by virus-infected targets without T cells, normalised to 1.

pHLA protein production

The experiments were performed following our published methodology⁶². DNA plasmids in a pET30 vector encoding the HLA-C*12:02 heavy chain and β2-microglobulin were individually transformed into BL21 Escherichia coli (E. coli) competent cells (RIL strain). Each protein was expressed as inclusion bodies and purified from the transformed E. coli cells. Soluble peptide-HLA complexes were produced by refolding inclusion bodies in the following amounts: 90 mg of α-chain, 20 mg of β2-microglobulin, and 10 mg of peptide (Genscript, Piscataway, NJ, USA). The refolded mixture was dialysed into 10 mM Tris-HCl pH 8.0, and pHLA was purified using anion exchange chromatography (Cytiva, Marlborough, Massachusetts, USA).

TCR protein production

Similarly, with HLA protein production, the experiments for TCR protein production were performed following our published methodology⁶. DNA plasmids in pET30 or pET28 vectors encoding GV37β and GV37α were individually transformed into BL21 Escherichia coli (E. coli) competent cells (RIL strain). Each protein was expressed as inclusion bodies and purified from the transformed E. coli cells. Soluble TCR complexes were produced by refolding inclusion bodies in the following amounts: 50 mg of α-chain and 50 mg of β-chain. The refolded mixture was dialysed into 10 mM Tris-HCl pH 8.0, and TCR was purified using anion exchange chromatography (Cytiva, Marlborough, Massachusetts, USA).

Crystallisation and structure determination

Crystals of both complexes were grown via the sitting-drop, vapour diffusion method at 20 °C with a protein: reservoir drop ratio of 1:1, at a concentration of 3 mg/mL in 10 mM Tris-HCl pH 8.0, 150 mM NaCl. Crystals of HLA-C*12:02 in complex with the KF9 peptide (N_266-274) were grown in 2% polyethylene glycol (PEG) 400 (Sigma-Aldrich, Cat# 91893) and 20% PEG3350 (Sigma-Aldrich, Cat# 202444). Crystals of GV37 TCR-HLA-C*12:02-KF9 complex were grown in 0.2 M Magnesium formate dihydrate (Hampton Research, Cat# HR2-245) and 20% PEG3350. The crystals were cryoprotected in 30% PEG3350 added to the mother liquor, and flash-frozen in liquid nitrogen. The data were collected on the MX2 beamline at the Australian Synchrotron⁶³, processed using XDS⁶⁴, and scaled using the CCP4 suite⁶⁵. Both HLA-C*12:02-KF9 structures with and without the GV37 TCR were determined by molecular replacement using the PHASER programme⁶⁶ from the CCP4 suite with a model of HLA-C*14:02 without the peptide (derived from PDB ID: 7WJ2⁶⁷), and the GV37 TCR model was built using AlphaFold2⁶⁸. COOT⁶⁹ was used to build in the peptide based on the electron density, and the structures were refined using PHENIX⁷⁰. The final models have been validated and deposited using the wwPDB OneDep system, and the final refinement statistics PDB codes are summarised in Table 3. All molecular graphics representations were created using PyMOL (version 1.20; copyright, Schrodinger, LLC).

Differential Scanning Fluorimetry

Thermal stability was measured using the ViiA 7 Real-Time PCR machine (Thermofisher, Scoresby, Australia), where pHLA samples were heated from 25 to 95 °C at a rate of 0.5 °C/min with excitation and emission channels set to yellow (excitation 549.5 ± 10 nm and detection at 586.5 ± 10 nm). The experiment was performed at two concentrations of pHLA (5 μM and 10 μM) in duplicate with 10X SYPRO Orange dye (Thermofisher, Scoresby, Australia, Cat# 6650). HLA-A*02:01-M1_58-66 was used as a positive control⁷¹, Fluorescence intensity data were normalised and plotted using GraphPad Prism 10 (version 10.0.3). The Tm value for each pHLA is determined to be the temperature at which 50% of maximum fluorescence intensity is reached.

Surface plasmon resonance (SPR)

SPR experiments were conducted at 25 °C on the BIAcore T200 instrument in 10 mM Tris-HCl pH 8.0 (Thermo Fisher Scientific, Cat# BP152-5), 150 mM NaCl (Chem Supply, Cat# SA046), 0.005% surfactant P20 (Cytiva, Marlborough, Massachusetts, USA, Cat# BR100054), and 0.5% BSA (Sigma-Aldrich, St Louis, MO, USA, Cat# A7006). CM5 chips were used to immobilise GV37 TCR (coupled at ~2000 response units) in 10 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) (Sigma-Aldrich, St Louis, MO, USA, Cat# H4034) and 150 mM NaCl pH 7.0. The first flow cell was loaded with HLA-A*02:01-M1 (negative control). The experiments were conducted with ten serial dilutions of the HLA-C*12:02-KF9 complex starting at 200 μM. All the experiments were conducted in duplicate (n = 2 independent experiments). BIAevaluation (v.3.1) and GraphPad Prism 10 were used for data analysis reported in Fig. 3f and 3g.

Statistics and reproducibility

Data and statistical analysis were performed using Prism 10 (GraphPad Software). For two-way comparison, the matched-paired Wilcoxon signed-rank test (Fig. 1b, 1d, Fig. 2f, Fig. 5b, 5c, Fig. 6c, d and e) or unpaired Mann–Whitney t-test (Fig. 2e,  2f, Fig. 5e and Fig. 6b) was used.

In Fig. 2b, c, d, g, h, Fig. 3c, d, Fig. 5a–e, Supplementary Fig. 1k, Supplementary Fig. 4g, and Supplementary Fig. 5c assays were performed in triplicate. Data are representative of two or three independent experiments. In Fig. 3c, representative blots of three independent experiments were shown.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.