Anti cd11b

We carried out flow cytometric (FCM) analyses using a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo 7.6.5 software (TreeStar Inc., Ashland, OR, USA). Based on a previous study,23 the monoclonal antibodies (mAbs) used for surface staining of lymphocytes to assess the phenotypic properties of NK cells were as follows: fluorescein isothiocyanate (FITC)‐conjugated anti‐CD3 (HIT3a), anti‐CD56 (B159), anti‐CD19 (HIB19); phycoerythrin (PE)‐conjugated anti‐NKp30 (p30‐15), anti‐NKp46 (9E2), anti‐CD122 (Mik‐β3), anti‐CD56 (B159), and anti‐CD11b (ICRF44); and allophycocyanin (APC)‐conjugated anti‐CD3 (HIT3a), anti‐natural‐killer group 2, member D (NKG2D; 1D11), purchased from Becton Dickinson (San Jose, CA, USA); PE‐conjugated anti‐tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL; RIK‐2; eBioscience, Santa Clara, CA, USA); and PE‐conjugated anti‐signal regulatory protein β (SIRPβ; B4B; BioLegend, San Diego, CA, USA). Mouse immunoglobulin (Ig)G1κ was used as an isotype‐matched control. Dead cells were excluded from the analysis by light scatter analysis and propidium iodide staining.

Mice were anesthetized and liver samples were collected and washed with ice-cold PBS. Harvested liver samples were either embedded in an optimum-cutting temperature (OCT) medium for cryostat section (7 µm) or fixed in 10% neutral buffered formalin 24 h for paraffin section (4 µm). Sections were incubated with 10% fetal bovine serum (FBS) for non-specific binding for 1 h at room temperature. Primary antibodies [anti-F4/80 (1:50; Bio-Rad: #MCA497, anti-CD11b (1:100; BD: # 550282), anti-CD45 (1:200; BD: # 550539), anti-Ly6G (1:150; Abcam: # ab25377), and αSMA (1:900; Abcam: # ab124964)] were diluted in 10% FBS and were incubated for 2 h at room temperature. Sections were washed with PBS and signals were amplified by secondary antibodies [ImmPRESS-HRP anti-rabbit IgG (Vector Labs # MP-7401) or ImmPRESS-AP anti-rat IgG (Vector Labs: # MP-5444)] for 40 min at room temperature. Immunogenic reaction was visualized by Alkaline Phosphatase (AP) Substrate (Vector Labs: # SK-5105), ImmPACT Vector Red Alkaline reagent (Vector Labs: # SK-5105) or DAB (Zsbio: # ZLI-9018) or with DAPI (1:2000) and mounted. All IHC images were quantified with ImageJ (NIH) in 5 random views at 40 × magnification as percentage of positive pixels in a blinded fashion.

Splenocytes were harvested from 10-week-old WT and single TCR T cell mice. Erythrocytes were lysed using ACK buffer (0.15 M Ammonium chloride, 1 M potassium bicarbonate, 0.1 M EDTA in water) for 5 minutes at room temperature. Splenocytes were then enriched for T cells by using biotin conjugated anti-CD11b (BD Biosciences), anti-CD11c (eBiosciences), anti-B220 (eBiosciences), and anti-biotin magnetic beads (Miltenyi Biotec) before being passed through magnetic columns (Miltenyi Biotec). DNA was then purified from T cell-enriched cells using the DNeasy kit and protocol (Qiagen). Purified DNA was shipped to ImmunoSEQ (Seattle, WA) for TCRβ sequencing. Between 200,000 and 450,000 total sequences were obtained from each sample resulting in between 10,000 and 28,000 unique productively rearranged sequences per mouse. All ImmunoSEQ data are subject to quality control processing in efforts to minimize intrinsic amplification and sequencing errors [16 (link)]. The ImmunoSEQ data are contained in S1 Table.

Total lung cells were harvested at day 7 p.i. after mock or RSV infection as previously described (17 (link), 19 (link)). Isolated cells were incubated with anti-FcγRII/FcγRIII mAb (24G2; BD Biosciences). For cell-surface marker staining, an aliquot of cells was stained with the following anti-mouse antibodies: anti-CD11c, anti-F4/80, anti-CD11b, and anti-Gr-1 (all from BD-Pharmingen). Samples were stained at 4°C in PBS with 1 % FBS and analyzed with a FACS Canto flow cytometer equipped with BD FACSDiva software (both from Becton Dickinson Immunocytometry Systems). Analysis was performed using WinMDI2.8 (Scripps).

A section of each tumor was fixed in 4% paraformaldehyde and then embedded in paraffin for histological hematoxylin–eosin (H&E) (Zhongshanjinqiao, Beijing, China), anti-S100A4 (Abcam, Cambridge, UK) and anti-Ki67 (BD Pharmingen, San Diego, CA) immunohistochemical staining.
For double staining, frozen tumor sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-CD11b (BD Pharmingen, San Diego, CA), anti-F4/80 (BD Pharmingen, San Diego, CA) and anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by staining with Alexa Fluor 488- or 555-conjugated secondary antibodies (Invitrogen, Grand Island, NY). Sections were evaluated under the microscope (DP71, OLYMPUS) using both bright-field and fluorescence microscopy.