Anti cd8a

Cells were collected from BALF and resuspended in a buffer containing 154 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA for red blood cell lysis for 5 minutes. After washing with PBS containing 2% FBS, cells were stained with anti-CD45.2 (Biolegend; 109808), anti–Siglec F (Invitrogen; 12-1702-82), anti–Gr-1 (BioLegend; 108412), anti-CD4 (Biolegend; 100516), anti-CD8a (Biolegend; 100708), anti-Mac-1 (CD11b) (Biolegend; 101208), anti-CD11c (Biolegend; 117307), anti-NK-1.1 (Ly-55) (Biolegend; 108707), anti-B220 (Biolegend; 103211), or anti-IgG2a (Biolegend; 407109) antibodies for 30 minutes on ice. To confirm the Th2 cell differentiation, cells were collected from BALF and fixed with 1% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100 and 0.2% bovine serum albumin for 3 minutes. The cells were then incubated with PE-conjugated anti-GATA3 (Invitrogen; 12-9966-42) and APC-conjugated anti-CD4 (Biolegend; 100516) antibodies for 1 hour. Flow cytometry analysis was performed using a Guava easyCyto flow cytometer (Merck Millipore).

For quantification of peripheral blood cells, 50 µl of whole blood was collected. Red blood cells were lysated and Fc- blockage was performed (Fc-Block, Pharmingen). After washing cells were stained with anti-B220 (BD Pharmingen), anti-CD8a (BioLegend), anti-CD11b (BD-Pharmingen), anti-CD4, anti-CD115, anti-Nk1.1, and anti-Ly6c (all ThermoFisher Scientific). To collect absolute numbers, CountBright Absolute Counting Beads (ThermoFisher Scientific) were added. Samples were measured on a FACSCanto II (BD Bioscience, Franklin Lakes, USA) and analyzed with FlowJo (Tree Star, Ashland, USA).

Different panels of antibody cocktails were generated from anti‐CD45 (BioLegend, 103116, 30‐F11), anti‐CD3 (BioLegend, 100218, 17A2), anti‐CD4 (BioLegend, 100421, GK1x.5), anti‐CD8a (BioLegend, 100711, 53‐6.7), anti‐NKp46 (BioLegend, 137606, 29A1.4), anti‐CD11c (BioLegend, 117307, N418), anti‐CD25 (Biolegend, 101908, 3C7), anti‐FOXP3 (Biolegend, 126404, MF‐14), anti‐granzyme B (BioLegend, 372208, QA16A02), anti‐IFNγ (BioLegend, 505849, XMG1.2), anti‐IFNγ (BioLegend, 505806, XMG1.2, anti‐CD62L (BioLegend, 104432, MEL‐14) and anti‐CD44 (BD Biosciences, 560568, IM7) antibodies, and the AmCyan Live/Dead Cell Staining Kit (Thermo Fisher Scientific). All antibodies were diluted at optimized dilutions before being used.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 10¹⁰ vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.

LPS (O111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following fluorescence-conjugated antibodies were provided from BioLegend (San Diego, CA, USA) and were used for flow cytometry analysis: anti-F4/80 (BM8), anti-CD11b (M1/70), anit-CD206 (C068C2), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-MHC class II (M5/114.15.2), anti-CD3 (17A2), anti-CD4 (RM4-5), and anti-CD8a (53-6.7).

Subsets of liver mononuclear cells were measured by flow cytometry. Before staining with a previously defined optimal dilution of monoclonal antibodies (Abs), the cells were pre-incubated with anti-CD16/32 (clone 93) to block non-specific FcRγ binding. The following Abs were used in this study: anti-CD69, anti-CD44, anti-CD3, anti-CD4, anti-CD8a, anti-CD19, (Biolegend, San Diego, CA, USA), anti-NK1.1 (eBioscience, San Diego, CA, USA) and PE-conjugated mouse CD1d tetramers loaded with PBS57 (PBS57 loaded CD1d tetramer, originally produced by the NIH tetramer facility, and supplied through Dr. David Serreze). Stained cells were assessed on a FACSCalibur (BD Biosciences, San Diego, CA, USA) using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Optimal concentrations of the mAbs were used throughout and all assays included positive and negative controls.