Clone mopc 21

Cryopreserved PBMCs from healthy women were thawed and seeded at a density of 2 × 10⁵ cells per well in an anti-CD3 (10 µg/ml, clone OKT3, BioLegend) coated 96-well plate. Cells were supplemented with soluble anti-CD28 (2.5 µg/ml, clone CD28.2, BioLegend) and anti-CD99 (10 µg/ml, clone hec2, BioLegend) or respective isotype control (10 µg/ml, clone MOPC-21, BioLegend). Samples were incubated for 72 h at 37 °C and 5% CO₂, stained with antibodies listed in Table S4 and analyzed by flow cytometry after 2, 24, 48 and 72 h.

Antibodies to the surface epitopes CD14 (clone HCD14, Pacific Blue-conjugated; and clone M5E2, Brilliant Violet 510-conjugated), CD16 (clone 3G8, PE-conjugated), TLR2 (clone TL2.1, PE-conjugated) and TLR4 (clone HTA125, Brilliant Violet 421-conjugated) as well as isotype controls (clone MOPC-173, clone MOPC-21, clone RTK2071) were purchased from BioLegend. For intracellular cytokine staining, antibodies to TNF-α (clone MAb11, PerCP/Cy5-5-conjugated), IL-1β (clone H1b-98, Alexa Fluor 647-conjugated), IL-8 (clone E8N1, Alexa Fluor 488-conjugated) and IL-10 (clone JES3-9D7, PE/Cy7-conjugated) were also obtained from BioLegend. Fixable viability stain (eFluor® 780, APC-H7-conjugated) was purchased from eBioScience (San Diego, CA).

Resting NK cells (2 × 10⁵) were immunostained for 20 min on ice with 5 µg/mL of purified mouse anti-human NKp30 antibody (Clone P30-15; BioLegend) or its concentration-matched isotype control (Clone MOPC-21; BioLegend). After a single wash in PBS, samples were resuspended in PBS/1% bovine serum albumin (BSA) containing 20% (vol/vol) goat serum (Sigma-Aldrich) and incubated for 20 min on ice, after which cells were washed in PBS and pellets were resuspended in PBS/1%BSA containing 10 µg/mL of FITC goat anti-mouse IgG (Clone Poly4053; BioLegend). After a 20-min incubation on ice, cells were washed in PBS and analyzed on a CyAN_ADP cytometer, where 10 000 NK cells were assessed.

Antibodies anti-Human CD81 mouse mAb (clone 1.3.3.22, Santa Cruz Biotechnology, Santa Cruz CA); Anti-human CD4 mouse mAb (clone SK3) and an isotype control (clone MOPC-21, Biolegend, San Diego CA); Anti-human TLR3 rabbit mAb (clone D10F10, Cell Signaling Technology, Danvers MA); Anti-human TLR7 rabbit pAb (#2633S, Cell signaling Technology); Anti-human TLR8 rabbit mAb (clone D3Z6J, Cell Signaling Technology); Anti-human TLR9 rabbit mAb (clone D2C9, Cell Signaling Technology); Anti-human MyD88 rabbit mAb (clone D80F5, Cell Signaling Technology); Anti-human TRIF/TICAM-1 (clone MAB6216, R&D Systems, Minneapolis MN) mouse mAb; Anti-human NALP3 rabbit pAb (Imgenex, San Diego CA); Anti-human RIG-I (D14G6) rabbit mAb (clone D14G6, Cell Signaling Technology); Anti-human AIM2 rabbit pAb (Abcam, Cambridge, MA) and Anti-human Actin (A2066, Sigma-Aldrich) were purchased from respective vendors. Secondary Antibodies anti-mouse IgG-HRP goat polyclonal (HAF007, R&D Systems), anti-rabbit IgG HRP-linked (7074, Cell Signaling Technology) were also purchased.

Murine cells were generally infected or stimulated at a density of 1.5 × 10⁵ host immune cells/well in a 96-well flat-bottom plate. Only for ROS measurements, cell density was reduced to 5 × 10⁴ cells/well. BMDCs and neutrophils were seeded in antibiotic-free RPMI 1640 supplemented with 10% FCS, whereas BMDMs were seeded in antibiotic-free DMEM supplemented with 10% FCS. Unless indicated otherwise, cells were infected overnight with the indicated bacterial strains at various multiplicities of infection (MOIs), transfected with 2 μg/ml bRNA complexed with Lipofectamine 2000 at a ratio of 1 μl Lipofectamine 2000 per 1 μg RNA or stimulated with Pam₃CSK₄ (1 μg/ml), R848 (1 μg/ml) and LPS (100 ng/ml). For TLR2 blocking experiments, BMDMs were pre-treated with antibodies against mouse TLR2 (Clone T2.5, 5 μg/ml, Biolegend, San Diego, CA) or the appropriate control IgG (Clone MOPC-21, 5 μg/ml, Biolegend, San Diego, CA) 45–60 min prior to infection. 90 min after infection, extracellular bacteria were killed by adding penicillin/streptomycin.

Rhesus wt^allo and HIP^allo were cultured on diluted laminin-coated (Thermo Fisher Scientific) T75 flasks in PluriSTEM Media (MilliporeSigma). Medium was changed every 24 h, and Accutase was used for cell passaging at a ratio of 1:8 to 1:10 every 3 d. RevitaCell supplement was added for the first 24 h. FLuc transduction was performed as outlined above for human cells. Cells were harvested and labeled with APC-conjugated anti-HLA-A,B,C antibody (clone G46_2.6, 555555, 1:20 dilution, BD Biosciences; clone G46_2.6 has shown cross-reactivity with rhesus macaque MHC class I) or APC-conjugated IgG1 isotype-matched control antibody (clone MOPC-21, 554681, 1:5 dilution, BD Biosciences). Cells were labeled with Alexa Fluor 647-conjugated anti-HLA-DR,DP,DQ antibody (clone Tu39, 563591, 1:20 dilution, BD Biosciences; clone Tu39 has shown cross-reactivity with rhesus macaque MHC class II) or Alexa Fluor 647-conjugated IgG2a isotype-matched control antibody (clone G155-178, 565357, 1:60 dilution, BD Biosciences). Also, cells were labeled with FITC-conjugated anti-CD47 antibody (clone CC2C6, 323106, 1:20 dilution, BioLegend) or FITC-conjugated mouse IgG1k isotype-matched control antibody (clone MOPC-21, 400110, 1:20 dilution, BioLegend). Representative histograms are shown. Gating strategies are provided in Supplementary Fig. 18.