Fcr block

After polarization of THP1 cells into M2-like macrophages, cells were digested using Accutase (BioLegend, 423201). After centrifugation, cells were resuspended using 100 μl PBS, and incubated for 5-10 min at 4 °C using FcRblock (1 μg/test, 101319, BioLegend). Subsequently, staining was performed using APC anti-human CD11b (0.5 μg/test, 101212, BioLegend), PE/Dazzle™ 594 anti-human CD86 (5 μl/test, 305433, BioLegend), and PE anti-human CD206 (5 μl/test, 321105, BioLegend) and incubated at 4 °C for 60 min. Wash the cells 2 ~ 3 times, add 500 μL cell staining buffer to resuspend the cells, and detected with FACSAria™ III (BD Biosciences CA, USA).

The MACS CD206⁺ enriched population of lung resident macrophages were incubated with FcR Block (422302; BioLegend) for 5 min and stained at a dilution of 1:50 with one of two panels of directly conjugated antibodies for 30 min at 4°C: anti-human CD45 (563792; BD), CD204 (371906; BioLegend), CD206 (321132; BioLegend), CD14 (562698; BD Biosciences), CD16 (302028; BioLegend), ACE2 (FAB933P; R&D), HLA-DR (307618; BioLegend), CD11b (393114; BioLegend), CD11c (301644; BioLegend); anti-human CD45 (324016; BioLegend), CD204 (371904; BioLegend), and CD206 (321103; BioLegend). Stained cells were then washed with FACS buffer (2% FBS in PBS) three times, and then incubated with cell viability marker propidium iodide (PI, 1 μg/ml, 421301; BioLegend). Flow cytometry was performed on a FACS Aria II (BD Biosciences).
Living (PI⁻) single, immune (CD45⁺), and lung resident macrophages (CD206⁺) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206⁺CD204^hi and CD206⁺CD204^lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (GE Healthcare Biosciences, UK) gradient centrifugation, cryopreserved in fetal bovine serum (FBS) containing 10% dimethyl sulfoxide and stored in liquid nitrogen until use. Total CD19+ B cells were isolated from cryopreserved PBMCs using CD19+ positive magnetic bead isolation (Miltenyi Biotec, Auburn, CA). Purified B cells were stained with viability dye eFluor™ 506 (Invitrogen, Waltham, MA) in phosphate buffer saline (PBS) for 10 mins at room temperature (RT), washed, followed by incubation with FcR Block (BioLegend, San Diego, CA) for 15 mins at 4°C. The cells were then incubated with fluorochrome-labelled monoclonal antibodies against CD19 PE (clone HIB19), CD20 Pacific Blue (clone 2H7), CD24 PerCP-Cy5.5 (clone ML5), CD38 AF700 (clone HB-7), CD27 FITC (clone H-T27I), TIM-1 APC (clone ID12), and TIGIT PeCy7 (Clone A15153G) all obtained from BioLegend (San Diego, CA) for 30 mins at 4°C. Cells were washed and sorted on FACS Aria III (Becton Dickenson Biosciences, San Diego, CA) 70 uM filter, 50 psi. Population purity was >95%. The absolute cell count of each B cell subtype was calculated based on the volume of blood obtained from each donor.

Cells were surface stained, fixed and permeabilized as below. Cells were stained with LIVE/DEAD (Invitrogen), blocked with mouse serum and FcR-Block (clone 2.4G2, Biolegend), stained for cell surface markers (See Table 1 for list of antibodies used), and then fixed and permeabilized for intra-cellular staining with 1 × fixation-permeabilization buffer (eBioscience). Ki67 was detected using antibody clone B56 from BD Biosciences or antibody clone REA183 from Miltenyi Biotech, both of which allow the detection of Ki67^hi cells. In Fig. 7: Lineage= CD19/TCRβ/Ly6G/Ly6C/F4/80/CD11c/FcɛR1α/NK1.1/Ter119/CD5/CD11b/CD49b. Samples were acquired using a BD Fortessa and analysed with FlowJo software (Tree Star). Fluorescence activated cell sorting was performed using a BD FACSAria.

Blood, collected from buccal sinus and treated with heparin (10 U/mL), was incubated with 1× RBC lysis buffer (Biolegend, San Diego, CA, USA) and then was resuspended in 100 μL of FACS buffer (PBS with 2% FBS), with Fixable Live/Dead Stain (Life Technologies, Carlsbad, CA, USA) and an appropriate combination of fluorescent antibodies specific to CD45, F4/80, CD3, CD45R (Thermo Fischer, Waltham, MA, USA), and FcR block (Biolegend, San Diego, CA, USA). After that, cells were incubated with BTN-Kat and ITN-Kat on ice without permeabilization, or stained by intracellular cytokine staining protocol using BD Fixation and Permeabilization Solution Kit with BD GolgiPlug™ (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using CytExpert 2.0 (Beckman Coulter, Brea, CA, USA).

Cells and tissue sections were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X-100, and incubated with FcR block (Biolegend—Cat: 422302) and endogenous peroxidase block (Vector laboratories—Cat: SP-1000-600). The cells were stained with the following FITC- or biotin-labeled primary antibodies: anti-CD19 (HIB19), anti-IgM (MHM-88), anti-Ki67 (SolA15), anti-Cit-H3 (ab5103), anti-TNF (MAb11). HRP-conjugated anti-FITC (AB_2314402) and anti-biotin (AB_2339006) antibodies were used with tyramide signal amplification reagent (Thermo Fisher – Cat: B40953, B40957) to detect secondary antibodies.