Brilliant violet 510

Fluorochrome-labelled antibodies for CD14 (PE), CD40 (Alexa Fluor 700), CD83 (PE/Dazzle), CD11c (APC), and CD86 (Brilliant Violet 510) were purchased from Biolegend (USA). Antibodies for HLA-DR (PE-Cy7) were purchased from Invitrogen (USA). Collected moDCs were first incubated with viability dye FVD780 (eBioscience, USA) for 15 minutes on ice and washed twice with DPBS. Fc blocker (Biolegend, USA) was added for another 15 minutes of incubation to exclude unspecified antigen-binding. Then, the mixture of targeted antibodies was added and incubated for 30 minutes. All samples were processed with the Fortessa flow cytometer (BD, USA), and data were analysed with FlowJo. Unstained controls were used to set up instruments and determine voltages, single stained samples were also prepared for compensation controls and to reveal the fluorescence spillover. In addition, fluorescence minus one (FMO) controls were included in the protocol to help define positive from negative populations.

Isolation of synovium was performed according to published methods (18 (link)–20 (link)). Briefly, immediately after euthanasia, the synovial tissues around the knee joints were collected and pooled from 3 animals per group. The synovium was minced and digested in a 1 mg/ml collagenase type I (Sigma-Aldrich) for 1 h at 37°C and rinsed through a 70-μm filter (BD Biosciences). The isolated synoviocytes were suspended in phosphate-buffered saline (PBS) containing 20 μg/ml of antibody cocktail. Brilliant Violet 510- and phycoerythrin-conjugated antibodies against mouse CD45.2 and F4/80 were obtained from Biolegend (Chaoyang, Beijing, China). For isotype control, Brilliant Violet 510- or phycoerythrin-conjugated non-specific mouse or rat IgG2a were substituted for the primary antibody, respectively. After incubating with antibody cocktails for 30 min at 4°C, the cells were washed with PBS and resuspended in PBS and macrophages were sorted using FACS and RNA was isolated from these sored cells.

Frozen PBMCs were thawed and washed with RPMI 1640 supplemented with 10% foetal calf serum (FCS), 2 mM L-Glutamine, 10 mM HEPES, and 50 U/ml Penicillin-Streptomycin (all from Thermo Fisher). Cells were stained with Viability Dye eFluor 780 (eBioscience) and antibodies against CD3 (PE-labelled, clone HIT3a, BD Biosciences), CD4 (BrilliantViolet510-labelled, clone OKT4, BioLegend), CD8 (PerCP-Cy5.5-labelled, clone HIT8a, BioLegend), CD25 (PE-Cy7-labelled, clone 2A3, BD Biosciences) and CD127 (AlexaFluor647-labelled, clone HIL-7R-M21, BD Biosciences). After cell wash, PBMCs were fixed with Fixation Buffer (BioLegend) and subsequently analysed using a BD LSRFortessa flow cytometer (BD Biosciences). Gating procedures are depicted in S3 Fig. For detection of mIL-7R in the second independent cohort of tuberculosis patients and healthy contacts we used the CD127 antibody clone A019D5 (BioLegend). Comparison of both antibody clones revealed similar T-cell binding pattern as well as percentages of mIL-7R_high and mIL-7R_low T cells.

Multicolor flow cytometry of peripheral blood cells was performed at Chungnam National University Hospital (CNUH). To exclude dead cells, single-cell suspensions were incubated for 20 min with a viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). For neutrophil analysis, cells were stained with fluorochrome-conjugated antibodies, including anti-CD66b (Brilliant Violet 421; BD Biosciences), anti-CD56 (Brilliant Violet 510; BD Biosciences), anti-CD3, anti-CD19, anti-CD20 (Brilliant Violet 510; BioLegend), anti-PD-L1 (Brilliant Violet 711; BioLegend), anti-CD45 (Brilliant Violet 786; BioLegend), anti-CD10 (VioBright FITC; BioLegend), and anti-CD16 (APC-Cy7; BioLegend). For T-cell analysis, the cells were stained with fluorochrome-conjugated antibodies, including anti-CD14, anti-CD19 (Brilliant Violet 510; BD Biosciences), anti-CD4 (Brilliant Violet 876; BD Biosciences), anti-CD45RA (APC-R700; BD Biosciences), anti-CD3 (APC-Cy7; BD Biosciences), anti-CD8 (VioBright FITC; BioLegend), and anti-CCR7 (PerCP-Cy5.5; BioLegend). Flow cytometry was performed using a BD LSR Fortessa X-20 flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). When gating subpopulations of cells, the distinction between positive and negative was based on Fluorescence Minus One (FMO) control.

Neutrophils were incubated with MVs isolated from PLY-or PBS (Control; Ctr)- treated MitoTracker Deep Red (APC)-labeled A549 (4 × 10⁶ cells) at 37 °C or 4 °C. After 1 h of incubation, neutrophils were washed, stained with CD11b (Brilliant Violet 510, Biolegend) and 7-AAD (Biolegend), and analyzed by FACS. For each sample, an average of 5000 cells was analyzed and the percentage of CD11b + /7-AAD−/APC + cells was recorded.