Clone 26

PBMCs (10⁶) were cultured with fixed E. coli using the indicated amounts of BpC in the presence or absence of NHS, hiNHS or different amounts of purified IgG (Intratect) in R10 medium. Alternatively, bacteria pre‐opsonized in the presence of hiNHS or purified IgG (as described above) were used at the indicated BpC numbers. After 20–22 h of culture, MAIT cell activation was analyzed by flow cytometry. Alternatively, THP‐1 cells or BCLs were preincubated with paraformaldehyde‐fixed E. coli or IgG‐ E. coli in the indicated amounts of BpC for 5 or 24 h at 37°C. After the incubation, enriched CD8 T cells were added at a ratio of 1:2 and co‐cultured for further 20 h at 37°C. Blocking antibodies against IL‐12p40 (5 μg mL⁻¹; Clone C.8.6; Biolegend, San Diego, CA, USA) and IL‐18 (5 μg mL⁻¹; Clone KU18.81; Biolegend), MR1 (5 μg mL⁻¹; Clone 26.5; Biolegend), TNF (5 μg mL⁻¹; Miltenyi Biotec), isotype IgG1 (5 μg mL⁻¹; Clone 11711; R&D Systems, Minneapolis, MN, USA) or isotype IgG2a (5 μg mL⁻¹; Clone 20102; R&D Systems) were added at the beginning of the co‐cultures if indicated. The final 4 h of the co‐cultures were in the presence of Brefeldin A (eBioscience, Waltham, MA, USA). Activation and cytokine production of MAIT cells were analyzed by flow cytometry.

Primary macrophages cells were isolated from PBMC by adhesion to tissue culture plastic plates as described previously (21 (link)). Macrophages were >90% pure as determined by staining with CD14 and CD68. Isolated primary macrophages were harvested using a rubber policeman and were then infected with H. pylori at MOI of 20. For blocking experiments, H. pylori-infected macrophages were pre-treated with anti-MR-1 mAb (10 μg/ml, clone 26.5, Biolegend) or their matched isotype control mouse IgG2a (10 μg/ml, MOPC-173, Biolegend) before coculture with autologous effector cells.

MAIT cell function was determined in vitro using paraformaldehyde-fixed E. coli stimulation (one shot top10, Life Technology, multiplicity of exposure 10) in the presence of 1.25 μg/ml anti-CD28 mAb (clone L293, BD Biosciences)[24 (link)] or ZIKV at a MOI of 5 (without anti CD28 mAb). E. coli was fixed for 5 minutes in 1% paraformaldehyde. PBMCs were further cultured for 24 hours at 37°C/5% CO₂ in RPMI medium supplemented with 10% fetal bovin serum. Monensin (Golgi Stop, BD Biosciences) was added during the last 6 hours of the stimulation. In some experiments blocking antibodies against MR-1 (5μg/ml, clone 26.5, Biolegend), IL-12p70 (10μg/ml, clone 24910, R&D systems), and IL-18 (10μg/ml, clone 125-2H, MBL International, Woburn, MA, USA) were added.