Ghost dye violet 510 viability dye

Biotinylated HLA-B*15-monomers loaded with NQKLIANQF (SARS-CoV-2) and NQKLIANAF (CCCoV) versions of the peptide were tetramerised using TotalSeq-C-0951-PE-Streptavidin (Biolegend 405261, 0.5 mg/mL) and TotalSeq-C-0956-APC-Streptavidin (Biolegend 405283, 0.5 mg/mL). 60 μL of HLA-monomers (500 nM) were mixed with 1 μL of PE-conjugated (B15_NQKLIANQF) or APC-conjugated (B15_NQKLIANAF) streptavidin reagents and incubated for 1 hour in the dark on ice. Jurkat 76.7 cells expressing the potentially cross-reactive TCR were stained with 1 μL Ghost Dye Violet 510 Viability Dye (Tonbo Biosciences 13-0870-T100) and 5 μL of each MHC-tetramer for 30 minutes on ice. Flow cytometry data were analyzed using FlowJo software (TreeStar). Cross-reactivity of the Jurkat 76.7 T cell line was determined by co-staining of the live cells with PE and APC-labeled MHC-tetramers.

Donor PBMCs were thawed and resuspended in 100 μL FACS buffer (PBS, 0.5% BSA, 2 mM EDTA). Cells were stained with 5 μL Fc-block (Human TruStain FcX, Biolegend) and 1.5 μL of S_167-180 HLA class II PE-conjugated tetramer for 30 minutes on ice. After the incubation a cocktail of fluorescently-labeled surface antibodies (2 μL of each: Ghost Dye Violet 510 Viability Dye, Tonbo Biosciences; anti-human CD3 PerCP Cy5.5-conjugated, Biolegend, clone OKT3; anti-human CD4 BV711-conjugated, Biolegend, clone OKT4; anti-human CD45RA BV421-conjugated, Biolegend, clone HI100; and anti-human CCR7 FITC-conjugated, Biolegend, clone G043H7) was added. Samples were incubated for an additional 20 minutes on ice. Single, Live, CD3-positive, CD4-positive, tetramer-positive cells were sorted on the Sony SY3200 into 384-well plates with premixed SuperScript VILO cDNA Synthesis mix (Invitrogen) for subsequent scTCR sequencing. scTCR library preparation and sequencing was performed as previously described (Wang et al., 2012 ). In brief, cDNA underwent two rounds of nested multiplex PCR amplification with a forward primer mix specific for V-segments and reverse primers for C-segments of TCRalpha and TCRbeta and sequenced on Illumina MiSeq platform (2x150 read length). TCR sequences with undefined alpha-chain were excluded from the analysis. Resulting TCR sequences can be found in Table S2.

Jurkat 76.7 cells expressing TCRs 4.1 and 6.3 (2.5x10⁵) were co-cultured with PBMCs from SARS-CoV-2 naive DPB1^∗:04:01-positive donor (6x10⁵) pulsed with 1 μM of peptide, 1 μg/mL each of anti-human CD28 and CD49d (BD Biosciences). An unstimulated (CD28, CD49d) and positive control (CD28, CD49d, 1X Cell Stimulation Cocktail, PMA/ionomycin; eBioscience) were included in each assay. Cells were incubated for 18 hours (37 ^°C, 5% CO₂). After the incubation cells were washed twice with FACS buffer (PBS, 2% FBS, 1 mM EDTA), resuspended in 50 μL of FACS buffer, and then blocked using 1 μL human Fc-block (BD Biosciences). Cells were then stained with 1 μL Ghost Dye Violet 510 Viability Dye (Tonbo Biosciences) and a cocktail of fluorescent antibodies: 1 μL each of anti-human CD3 (Brilliant Violet 421-conjugated, Biolegend, clone SK7), anti-human CD69 (PerCP-eFluor710-conjugated, eBioscience, clone FN50), and anti-mouse TCRβ chain (APC/Fire750-conjugated, Biolegend, clone H57-597). Cells were incubated for 20 minutes at room temperature and then washed with a FACS buffer. Cells were analyzed by flow cytometry on a custom-configured BD Fortessa using FACSDiva software (Becton Dickinson). Flow cytometry data were analyzed using FlowJo software (BD Biosciences). Responsiveness to peptide stimulation was determined by measuring frequency of NFAT-GFP, CD69 and αβTCR expression.