Mouse fc block

Tumor tissues were collected from each mouse on Day 8 or 9 after treatment, dissociated into single cells by using a Tumor Dissociation Kit and a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). In cell mixtures, leukocytes positive for CD45 were isolated with mouse TIL (CD45) microbeads (Miltenyi Biotec) by using an OctoMACS Separator (Miltenyi Biotec). After washing and filtration, cells were blocked with Mouse BD Fc Block (BD Biosciences, San Jose, CA, USA), and stained with the antibodies listed in S1 Table and with DAPI (4′,6-diamidino-2-phenylindole, Dojindo, Kumamoto, Japan). The gating strategy is shown in S1 Fig. To detect IFN-γ-producing activated CD8⁺ T cells, we incubated CD45⁺ cells with RPMI1640 containing with 10% FBS, PMA/Ionomycin (Sigma, St. Louis, Missouri, USA), and BD GolgiPlug (BD Biosciences) for 4 h at 37 °C, and then stained them with antibodies against CD45, CD3 (Thermo Fisher Scientific, Waltham, MA, USA), CD4, and CD8 (BD Biosciences). The cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) and then stained with antibody against IFN-γ or GzmB (BD Biosciences). The cells were sorted with a BD FACS AriaII SORP or LSRFortessa X-20 (BD Biosciences) and the data were analyzed by using FlowJo v10 (BD Biosciences) or Cytobank (Cytobank, Inc., Santa Clara, CA, USA).

The cells were dissociated with Accutase for 15 minutes. The detached cells were diluted in 1% bovine serum albumin/PBS and centrifuged at 900 rpm for 4 minutes. The cell pellets were resuspended in PBS and blocked with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block) (1 : 50; BD Biosciences, San Jose, CA; 5543142) for 10 minutes on ice. The cells were permeabilized and fixed with Cytofix/Cytoperm fixation and permeabilization solution (BD Biosciences; 51-2090KZ) for 20 minutes, incubated with the primary antibodies for 30 minutes, and, if necessary, incubated with the secondary antibodies for 30 minutes on ice. These cells were analyzed on a FACSAria II flow cytometer (BD Biosciences). To quantify the TTF1-positive cells, the cells were stained with a mouse anti-TTF1 antibody conjugated to APC (1 : 50; Abcore, Ramona, CA; AC12-0362-03). An anti-mouse IgG1 isotype control antibody (APC) (1 : 50; Abcore; 1A-632) was used as an isotype control. For pro-SPC, the cells were stained with a rabbit anti-pro-SPC antibody (2 : 50; Abcam; ab170699), goat anti-rabbit IgG (APC) (1 : 50; Abcam; ab130805), and an anti-rabbit IgG isotype control antibody (APC) (1 : 50, Abcam; ab130805).

The mouse cell depletion kit (Miltenyi), EpCAM positive cell selection kit (Miltenyi), and EasySep EpCAM positive cell selection kit (STEMCELL technologies) were used according to protocols. For FACS, 10 million cells were blocked in 1 mL of FACS buffer by the addition of 50 μL of Human TruStain FcX^™ (BioLegend) and 25 μL of Mouse BD Fc Block^™ (BD Pharmingen) for 20 minutes at room temperature, then stained for 1 hour at 4°C with 5 μL of PE mouse anti-mouse H-2K^d (1:200 dilution) (BD Pharmingen; clone SF1-1.1) and 5 μL of APC mouse anti-human CD326 (1:200 dilution) (Miltenyi; clone HEA-125). Single color staining dilutions were previously determined and single color controls were included in all experiments. Cells were washed 3 times with 5 mL of FACS buffer and then re-suspended in 1 mL of FACS counterstain buffer containing 200 ng/mL DAPI (Lonza) and 25 μg/mL DNAse I and DNAse I buffer additives (New England Biolabs). Cells were sorted on BD FACSAria II instrument in the MSKCC Flow Cytometry Core Facility.

For flow cytometry analysis, cells were washed and incubated with Fc-receptor blockage using CD16/CD32 (Mouse BD Fc Block) (clone 2.4G2 (RUO), BD Pharmingen) and 5% rat serum (Invitrogen, Carlsbad, CA, USA) for 10 min prior to labeling. Subsequently, the cells were incubated with labelling antibodies for 20 min, washed and analyzed on the Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). The following fluorescent-labeled antibodies were used for cell labeling: Ly6G (clone: 1A-8), ICAM-1 (CD54) (clone: 3E2), CD62L (L-Selectin) (clone: MEL-14), CXCR1 (clone: U45-632), all from BD Biosciences and CD11b (clone: M1/70) and CXCR2 (clone: SA044-G4), from BioLegend (San Diego, CA, USA). Neutrophil subsets were characterized using CD62L (L-Selectin) and ICAM-1 (CD54). Neutrophils were considered immature when expressing a CD62L + /ICAM-1(CD54)-phenotype and mature when expressing CD62L + /ICAM-1(CD54) + or CD62L-/ICAM-1(CD54) + ^{21 (link),22 (link)}.

Brains of adult mice were minced and disaggregated in Type IV collagenase (400 U/ml, Worthington), Dispase (1.2 U/ml, Worthington), and DNase I (32 U/ml, Worthington) in PBS with Ca²⁺ and Mg²⁺ (GIBCO) at 37°C for 60 min, with gentle trituration every 10 min. After that, the disaggregated cells were washed with cold PBS and filtered through a 40‐μm mesh. After centrifugation, cell pellets were resuspended in 10 ml 20% BSA and centrifuged at 1000 g at 4°C for 25 min to remove the myelin. The pellets were then resuspended in PBS/3% FBS/0.1% NaN3/2 mM EDTA and blocked rat anti‐mouse CD16/32 (Mouse BD Fc Block, BD Pharmingen) for 5 min on ice and labeled with antibodies according to our previous study¹⁰ for 1 h at 4°C. The stained cells were sorted into EGM‐2/MV medium (Clontech) with an Aria II sorter (BD) or analyzed with an LSRII analyzer (BD) at Fluorescence‐activated cell sorting (FACS) Facility, and FACS data were analyzed using FlowJo software (TreeStar).

Liver NPCs were washed twice with FACS buffer (PBS, pH 7.4, with 1% BSA and 0.05% Sodium Azide) and incubated with nonspecific IgG to assess background fluorescence. Cells were then incubated with anti‐FcγR II/III antibody (Mouse BD Fc Block; BD Pharmingen) for 15 min on ice, followed by staining with a mixture of fluorescence labeled antibodies including PerCP anti‐CD45 (BD bioscience 557235), Alexa Flour 700 anti‐Ly6G (Biolegend 127622), APC‐cy7 anti‐CD11B (Biolegend 101226), and PE‐cy7 anti‐F4/80 (eBioscience 25–4801‐82) for 30 min on ice. Subsequently, cells were washed and permeabilized for 30 min on ice. For Intracellular markers, PE‐Dazzle594 anti‐TNFα (Biolegend 506345), PE anti‐IL1B antigen (Thermo Fisher Scientific), and APC IL6 antigen (BD Bioscience) were stained for 30 min on ice. Cells were washed before measurement. Flow cytometry was performed using MoFlo Astrios EQ (Beckman Coulter Life Sciences) and data was analyzed using FlowJo software (Tree Star Inc.).