Spv l3

Ten μg/mL of DONQ52, control Ab (anti-KLH antibody, clone IC17-SG181), DQN0139, SPV-L3 (anti-pan HLA-DQ antibody, Beckman Coulter), Tu39 (anti-HLA-DR, DP, DQ antibody, BioLegend), or MOPC-173 (mouse IgG2a isotype antibody, BioLegend) was incubated with 1 × 10⁵ cells/100 μL/well Ba/F3 cells expressing HLA II in FACS buffer for 30 min at room temperature, followed by antibody detection with ×50 diluted Goat F(ab′)2 anti-Human IgG, Mouse ads-PE (Southern Biotech), or Goat F(ab′)2 anti-Mouse IgG2a, Human ads-PE (Southern Biotech) by flow cytometry (BD LSRFortessa X-20, Becton, Dickinson and Company). Gating strategy to determine the MFI is shown in Supplementary Fig. 11. MFI was calculated using BD FACSDiva Ver.8.0.1 software (Becton, Dickinson and Company). Staining antibodies used in flow cytometry experiments with experimental dilutions are listed in Supplementary Table 8.

After resuspending PBMC to 0.9 - 1.8 million per ml in RPMI/10% Human AB serum, two 2 ml fractions were transferred to fresh tubes and incubated with anti-HLA-DQ antibody at 10 μg/ml (clone SPVL3; Beckman Coulter) or anti-HLA-DR antibody at 10 μg/ml (clone L243; BioLegend) for one hour at 37°C. Test conditions were assessed in duplicate wells.

PBMCs were cultured in complete RPMI in flat-bottom 96 well plates, at 37°C in a 5% CO₂ atmosphere. 500,000 PBMCs were stimulated for 18 h with lysate of infected (iRBCs) or uninfected RBCs (uRBCs) in a ratio of 3 RBCs per PBMC, in the presence of anti-human leukocyte antigen HLA-DQ (SPVL-3; Beckmann Coulter, USA), anti-HLA-DP (B7/21; abcam, USA) and anti-HLA-DR (L243; Biolegend, USA), or in the presence of respective isotype controls. Following stimulation, cells were centrifuged and supernatants were recovered for cytokine analysis.