Sa bv421

BW58.MAIT and M12.C3.MR1 cells (10⁵ each) were co-cultured in 96-well U-bottom plates in the presence of 5-OP-RU, Ac-6-FP, or media controls. For MR1 blocking anti-MR1 mAb 8F2.F9 (30 (link)) or isotype control, MHC I reactive mAb W6/32, purified in-house, were added at 40 μg/ml for 1 h prior to addition of compounds. After overnight incubation, cells were stained with live/dead FVD-e780 (Invitrogen, 1:1000) in PBS, blocked with 2.4G2 hybridoma supernatant for 15 min at room temperature, then stained with biotinylated anti-MR1 (8F2.F9, 5.8 μg/ml), CD137-PE (17B5, Biolegend, 1:200), and TCRβ-APC (H57-597, Biolegend, 1:200), followed by SA-BV421 (Biolegend, 1:1000) in PBS containing 2% fetal calf serum and 2 mM EDTA, before acquisition on a BD Fortessa flow cytometer using Diva Software (https://www.bdbiosciences.com/en-au/products/software/instrument-software). Analysis was performed using FlowJo software (https://www.flowjo.com/) (v10) and activation of BW58.MAIT cells measured by an increase in CD137 and decrease in TCRβ surface expression.

The recombinant SARS-CoV-2 prefusion stabilized S protein and RBD were biotinylated using an EZ-Link Micro Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Probes were generated by coupling biotinylated proteins to fluorophore-conjugated streptavidin (SA) molecules for detection by flow cytometry (SA-BV421, SA-APC, and SA-PE, BioLegend). Isolated PBMCs were stained by a panel of antibodies listed in Table S3 as well as S protein and RBD probes (S-APC, S-PE, and RBD-BV421, 100 ng each). The samples were analyzed on a BD Aria III Fusion cell sorter (weeks 6 and 30) or a BD LSRFortessa flow cytometer (all other time points). At weeks 6 and 30, memory B cells (CD3⁻ CD11c⁻ CD14⁻ CD16⁻ CD123⁻ HLA-DR⁺ CD20⁺ IgM⁻ IgG⁺) double-positive for S protein binding were single cell sorted into 96-well plates and frozen immediately on dry ice for subsequent B cell receptor (BCR) amplification. Data were analyzed using FlowJo v.10.7.1 (FlowJo).

PBMCs were stained for surface markers CD4 and CADM1 following LIVE/DEAD cell staining (Invitrogen, L34976). Then the viral protein Tax was stained intracellularly with Foxp3 staining kit (eBioscience, 00-5523-00) (
Figure 2a). CADM1 staining was included in order to obtain an equivalent number of HTLV-1-infected cells in the Tax
^– population
^{20 (link)}. HTLV-1-infected T cell clones were stained with LIVE/DEAD and anti-Tax antibody (
Figure 2b). The antibodies used were: mouse anti-CD4-PE (clone RPA-T4; BioLegend, 300507; concentration used, 0.8 μg/ml); chicken anti-CADM1-biotin (clone 3E1; MBL, CM004-6; 20 μg/ml) in combination with SA-BV421 (BioLegend, 405226; 1 μg/ml); mouse anti-Tax-Cy5 or anti-Tax-AF647 (clone LT-4; 0.4 μg/ml)
^{21 (link)}. Cell sorting was carried out with a BD FACSAria III.