Mhcii bv421

Liver non-parenchymal cells were isolated as previously described (Melino et al., 2016 (link)). Briefly, tissue disaggregation was performed by finely chopping liver samples (∼1-2 g) in 10 ml digestion solution containing 1 mg/ml Collagenase IV (Worthington) and 20 μg/ml DNAse1 (Roche) and incubating at 37°C for 45 min on a rocking platform before mashing through a 70 μm filter (Falcon). The cell pellet was collected by centrifugation (400 g) and resuspended in an isotonic 30% Percoll solution to separate hepatocytes and non-parenchymal cells. Cells were stained for a panel of myeloid markers [F4/80-AF647 (1:150), Cd11b-BV510 (1:200), Ly6G-BV785 (1:200), MHCII-BV421 (1:200), Tim4-PE-Cy7 (1:300), Ly6C-PE (1:300) (Biolegend)] in buffer containing 2.4G2 supernatant to block Fc binding, washed and resuspended in buffer containing viability dye 7AAD (Life Technologies) for acquisition using a Cytoflex (Becton Dickinson). Live single cells were identified for phenotypic analysis by excluding doublets (FSC-A>FSC-H), 7AAD+ dead cells and debris. Single colour controls were used for compensation and unstained and fluorescence-minus-one controls were used to confirm gating. Data were analysed using FlowJo 10 (Tree Star). Cell counts were calculated by multiplying the frequency of the cell type of interest by the total mononuclear cell yield/g of disaggregated tissue.

Tumor samples were collected individually, and cell suspension was obtained after mechanical dissociation and incubation with collagenase D (Roche, Basiléia, Swiss) at a concentration of 0.22 U/mL per sample at 37 ºC for 40 min. Then, samples were washed twice with PBS 1X (pH 7.4), filtered in a 70 µm cell strainer (BD Biosciences), and resuspended in solutions containing anti-CD45-PerCpCy5.5 (BioLegend), anti-CD11c-PE (BD Biosciences), MHC-II-BV421 (BioLegend), F4/80-FITC (Thermo Fischer Scientific), anti-CD86-APC (BioLegend), anti-CD11b-Alexa-Fluor488 (BioLegend), anti-Gr-1-PE (BD Biosciences), CD8a-BV605 (BioLegend), and anti-IFN-γ-PE (BioLegend) mAbs for 30 min at 4ºC. For analysis of E7-specific intratumoral CD8 T cells, samples were stained with the APC-labeled H-2Db E7-specific dextramer (Immudex, Copenhage, Denmark), and subsequently stained with anti-CD8a-Pacific Blue (BioLegend). After two washes, cells were resuspended in PBS, acquired in a LSR Fortessa flow cytometer (BD Biosciences), and analyzed using the Flow Jo software (BD Biosciences).