True nuclear buffer kit

For flow cytometric staining of IL‐17 and IL‐22, LT and LN single‐cell suspensions were adjusted to 1 × 10⁷ cells mL⁻¹ and incubated for 4 h with PMA (0.1 μg mL⁻¹; Sigma‐Aldrich) and ionomycin (0.75 μg mL⁻¹; Sigma‐Aldrich) in the presence of Golgi‐Plug (BD). After blocking (human TruStain FcX; BioLegend) and viable stain (Pacific Orange; Thermo Fisher), True Nuclear Buffer kit (BioLegend) was used for washing and cell permeabilisation throughout flow cytometry staining (for antibodies, staining panels and analysis strategy, see Supplementary figure 2). Data were acquired using a FACS Canto (BD) and analysed using FlowJo software (version 10; Treestar).

Following processing, single cell suspensions underwent surface and TF/intracellular cytokine staining (ICS). Prior to staining, cells were incubated at 37°C in 5% CO₂ in RPMI supplemented with 1%HEPES, 1% L-glutamine, 10% human AB serum, and 0.1% brefeldin A (Golgiplug; BD Biosciences, San Jose, CA) for 3 h. Cells were stained with a viability dye (Zombie NIR) followed by surface stains (Table S3) using standard protocols. TF and ICS was performed following permeabilization using True-Nuclear buffer kit according to the recommended protocol (True-Nuclear Transcription Factor Buffer Set; BioLegend, San Diego, CA). Samples were acquired on a Cytek Aurora (Cytek, Bethesda, MD) and analyzed using FlowJo Software (BD Biosciences) (Figure S2) and positive staining was verified against unstained controls. Only samples with >50 flow events in the parent population were reported. For analysis of CD3⁻CD20⁻ or CD20⁺ cells, animal 6319 was excluded due to poor staining with the anti-CD20 antibody. In total, 88 granuloma samples were taken for flow cytometric analysis (early = 24, mid = 31, and late = 33) representing 94% (average) of the original lung granulomas isolated from animals at the time of necropsy (Table S2).