Brilliant violet 785

Antibodies and clones against the following proteins were used: CD3 (UCHT1, PE-Cy5, Beckman Coulter), CD14 (MϕP9, Horizon V500, BD Biosciences), CD16 (3G8, Brilliant Violet 711 or Brilliant Violet 785, Biolegend), CD19 (HIB19, Horizon V500, BD Biosciences), CD38 (HIT2, Brilliant Violet 711, BD Biosciences), CD45 (HI30, Alexa Fluor 700, Biolegend), CD49a (TS2A, AlexaFluor 647, Biolegend), CD56 (N901, ECD, Beckman Coulter, or HCD56, Brilliant Violet 711, Biolegend), NKG2A (Z1991.10, APC-A780, Beckman Coulter), CD69 (TP1.55.3, ECD, Beckman Coulter), CXCR3 (G025H7, PE-Cy7, Biolegend). After washing twice, cells were stained with streptavidin Qdot 605 or Qdot 585 (both Invitrogen) and Live/Dead Aqua (Molecular probes, Life Technologies). After surface staining, PBMC were fixed and permeabilized using FoxP3/Transcription Factor staining kit (eBioscience). For intracellular staining, the following antibodies were used: granzyme B (GB-11, PE-CF594, BD Biosciences), IFN-γ-Brilliant Violet 570 (4S.B3, Brilliant Violet 570, Biolegend), Ki67 (B56, A700, BD Biosciences), perforin (dG9, PE, Biolegend), and TNF (MAb11, Brilliant Violet 421, Biolegend). IAV infection was monitored using anti-influenza A nucleoprotein-1 (431, Abcam). Samples were analyzed on a BD LSRFortessa equipped with 5 lasers (BD Biosciences), and data were analyzed using FlowJo versions 9.5.2 and 10.5.3 (Tree Star Inc).

Flow cytometric analysis was performed as previously described [19 (link)]. Briefly, harvested BMDM were incubated in 1 μg/mL of anti-mouse Fc receptor antibody in 100 mL PBS containing 0.5% BSA plus 0.02% NaN₃ (FACS buffer) for 15 min on ice. Subsequently, single-cell suspensions were stained for 15 min at 4°C with blue-fluorescent reactive dye, L23105 (Life Technologies) to discriminate dead cells. After washing, 1-3 × 10⁶ cells were surface-stained in FACS buffer for 15 min at 4°C with antibodies recognizing CD11b (Alexa Fluor 700, BioLegend), F4/80 (Brilliant Violet 785, BioLegend), CD86 (Brilliant Violet 421, BioLegend), PD-L1 (PE-Cy7, BioLegend), and PD-L2 (PE, BioLegend). Surface-stained cells were washed three times with FACS buffer and treated with Fix/Perm reagent according to the protocol of the cytofix/cytoperm kit (BD Biosciences, San Jose, CA, USA). The cells were intracellularly stained in FACS buffer containing anti-Nos2 (PE, eBiosciences) and anti-h/m arginase 1 (APC, R&D systems) for 30 min at 4°C and further collected on an LSR II cytofluorometer (BD, Franklin Lakes, NJ). Stained cells were gated according to size (SSC-A) and forward scatter (FSC-A) to eliminate debris. Doublets were excluded from the analysis by using forward scatter height (FSC-H) and FSC-A. Data analysis was performed using FlowJo Software (FlowJo, LLC).

Sixteen weeks after transplantation, peripheral blood was collected from the tail vein of recipient mice. Collection was repeated at 4‐week intervals. Then, the erythrocytes were lysed using erythrocyte lysis buffer (Shanghai Yeasen Biotech Co. Ltd), and the remaining cells were stained with antibodies against the following markers: anti‐CD3 – PerCP/Cyanine5.5 (Biolegend, 100,218), anti‐CD45R/B220 – APC (Biolegend, 103,212), anti‐CD11b – Brilliant Violet 421 (Biolegend, 101,236), anti‐Ly‐6G/Ly‐6C (Gr‐1) – Brilliant Violet 421 (Biolegend, 108,434), anti‐CD45.2 – Brilliant Violet 605 (Biolegend, 109,841), anti‐CD45.1 – Brilliant Violet 785 (Biolegend, 110,743).

Cells were washed with FACS buffer (PBS buffer supplemented with 1% FCS and 1 mM EDTA) by centrifugation and stained with the respective antibodies. Reactions were incubated for 30 min at 4 °C. Following two washes with FACS buffer, samples were resuspended in 200 μl of FACS buffer and data was acquired using a Fortessa analyser (BD Bioscience). All data were processed with FlowJo (v9.9, TreeStar). Antibodies used in this study: anti-human CD4, APC (BioLegend; Clone: OKT4, Catalog No: 317416, Dilution: 1:100), anti-human CD45RA, Brilliant Violet 785 (BioLegend; Clone: HI100, Catalog No: 304140, Dilution: 1:100), anti-human CD45RO, PE-Cyanine7 (BioLegend; Clone: UCHL1, Catalog No: 304229, Dilution: 1:100), anti-human CD197 (CCR7), (BD Bioscience; Clone: 150503, Catalog No: 561271, Dilution: 1:100), anti-human IFN gamma, PE-Cyanine7 (eBioscience; Clone: 4s.B3, Catalog No: 25-7319-82, Dilution: 1:50), anti-human IL-9, PE (BD bioscience; Clone: MH9A3, Catalog No: 560814, Dilution: 1:50).

Splenocytes were isolated and prepared for flow cytometry as described previously (35 (link)). Splenocytes were stimulated overnight with clinical M. avium isolates from patients 9 and 13 at an MOI of 1. Concanavalin A (2.5 μg/ml; Sigma) stimulation was used as a positive control, and unstimulated cells served as a negative control. A protein transport inhibitor (eBioscience) was added during the last 4 h of stimulation. Surface antigens were stained with monoclonal antibodies against CD3 (fluorescein isothiocyanate [FITC; BioLegend]), CD4 (Brilliant Violet 605 [BioLegend]), and CD8 (Brilliant Violet 785 [BioLegend]). After fixation (2% paraformaldehyde; Sigma) and permeabilization (0.5% saponin; Sigma), intracellular cytokine staining was performed for IFN-γ (phycoerythrin [BioLegend]) and TNF-α (allophycocyanin [BioLegend]). Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo (FlowJo, LLC) and GraphPad Prism (GraphPad Software, Inc.) software.