Ly6g 1a8

Cellular phenotypic analysis was carried out by direct immunofluorescence with the following antibodies: CD11b M1/70 (eBioscience), Ly6g 1A8 (eBioscience) F4/80 BM8 (Biolegend), MHCII 2G9 (Biosciences), NK1.1 S17016D (Biolegend), CD3 145-2C11 (eBioscience), CD31 390 (Biolegend), αSMA (α smooth muscle actin) 1A4 (eBioscience), CD62-E REA369 (Miltenyi Biotec), CD62P RMP-1 (Biolegend), CD54 (Biolegend), CD106 (Biolegend), CD192 (CCR2) REA538 (Miltenyi Biotec)., PSGL-1 34 RA10 (Thermofisher), CD44 IM7.8.1 (Miltenyi Biotec), CD11a 121.7 (Thermofisher), CD49d 9C10 MFR4.B (Biolegend). All incubations were done in the presence of 50 μg/ml mouse IgG or human IgG. The same isotype control antibody was always included as a negative control, and dead cells were excluded by Annexin V staining (Sigma, USA). Flow cytometry was performed with a 10 color Gallios device (Beckman Coulter, USA), cells were counted using Flow-Count fluorospheres (Beckman Coulter, USA), following the manufacturer’s instructions, and data were analyzed using Flowjo software (Tree Star, Inc, USA).

A single cell suspension prepared from colonic tissues as described above was fixed using PBS containing 1% paraformaldehyde and stained on ice for 25 min with antibodies in PBS containing 2% heat-inactivated fetal calf serum. The antibodies used were fluorescein isothiocyanate (FITC)-labeled anti-murine Gr-1 (RB6-8C5, eBioscience), and allophycocyanin-labeled anti-murine CD11b (M1/70, BD Pharmingen). Data collection and analysis were performed on a fluorescence-activated cell sorting (FACS) Calibur flow cytometer using CellQuest software (Becton Dickinson). For cell sorting, a single cell suspension from the colon was stained with antibodies and sorted on a FACS AriaII (Becton Dickinson). The antibodies used for sorting were those described above plus FITC-labeled anti-murine T-cell receptor (TCR)-β (H57-597, eBioscience), Ly-6G (1A8, eBioscience), CD19 (1D3. eBioscience) or CD3 (145-2C11, eBioscience), PE-labeled anti-murine CD49b (DX5, Biolegend), CD11c (N418, BD Pharmingen), or F4/80 (BM8, Biolegend).

Blood was collected from tail veins in the presence of 5 mM EDTA, incubated with BD Fc Block for 10 min, stained with mAbs specific to CD45 (clone 30-F11, BD Bioscience), CD11b (M1/70, BD Bioscience), Ly6C (AL-21, BD Bioscience), Ly6G (1A8, eBioscience), NK1.1 (PK136, eBioscience), CD3 (17A2, eBioscience), CD19 (1D3, BD Bioscience), TER119 (TER119, eBioscience), CD4 (RM4-5, BD Bioscience), CD8 (53-6.7, BD Bioscience), Gr1 (RB6-8C5, BD Bioscience) for 20 min at room temperature. The blood was then fixed and lysed using BD FACS Lysing solution (BD Bioscience). After fixation, cells were washed and resuspended in 2% FBS/PBS and then immediately analyzed by FACSSymphonyA3 and FlowJo software. AccuCheck counting beads were used for quantification of absolute cell numbers. Gating strategy for counting beads is shown in Supplementary Figure S1.

Whole lung was finely minced with scissors and enzymatically digested using Liberase (Sigma). Single-cell suspensions were prepared from homogenized lung, BAL, and spleen by straining through a 40 μM mesh filter. The red blood cells were lysed with lysis buffer (ACK lysis buffer) and the white blood cells stained with respective fluorescent-labeled anti-mouse antibodies for flow cytometric analysis: CD45 (30-F11), TCRγδ (GL3; BioLegend), CD3 (145–2C11), CD8 (53–6.7), CD44 (IM7), CD69 (H1.2F3), CD19 (1D3), NK1.1 (PK136) (BD Bioscience), CD80 (16-10A1), CD4 (GK1.5), CD11b (M1–70), CD11c (N418), F4/80 (BM8), and Ly6G (1A8; Invitrogen). Within each antibody cocktail mixture, cells were incubated with CD16/32 (2.4G2) to block Fc-mediated adherence of the antibodies. Cell viability was determined by the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). Total mtROS were assessed using the fluorescent dye-based MitoSOX™ Red with the absorption/emission: 510/580 nm. Markers for apoptosis and necrosis involved staining with Annexin-V (BioLegend) and PI (Invitrogen). A minimum of 100,000 events were acquired for each sample on the FACSAria II (BD Biosciences) and data analysis carried out using FlowJo software. Manual clustering of multidimensional flow cytometry was guided by isotype controls and/or untreated samples.

Whole lung was finely minced with scissors and enzymatically digested using liberase (Sigma-Alrich, United States). Single cell suspensions were prepared from homogenised tissue straining through a 40 μM mesh filter. The red blood cells were lysed with lysis buffer (ACK lysis buffer) and the WBC stained with respective fluorescent-labelled anti-mouse antibodies for flow cytometric analysis: leukocytes (CD45; 30-F11), T cells CD3 (145-2C11), CD8 (53–6.7), activation marker (CD69; H1.2F3) and neutrophils (Ly6G; 1A8) (Invitrogen, United States). Tetramer staining of virus-specific CD8⁺ T cells was performed using the peptides D^bNP₃₆₆ (ASNENMETM) that was synthesised at Biomolecular Resource Facility, Australian National University, Australia. Within each antibody cocktail mixture, cells were incubated with CD16/32 (2.4G2) to block Fc-mediated adherence of the antibodies. Cell viability was determined by LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). Total mitochondrial ROS was assessed using the fluorescent dye-based MitoSOX™ Red with the absorption/emission: 510/580 nm. A minimum of 100,000 events were acquired for each sample on the FACSAria II (BD Biosciences, United States) and data analysis carried out using FlowJo (United States) software. Manual clustering of multidimensional flow cytometry was guided by isotype controls and/or untreated samples.