18 Flow cytometry at day 11 confirmed depletion efficiencies of >95%. Following cardiac perfusion with phosphate-buffered saline, livers were aseptically removed and mechanically disrupted between sterile frosted microscope slides. Cell suspensions were passed twice through 70 μm filters before cell isolation. Liver CD11b+ cells were isolated using anti-CD11b magnetic beads and positive selection columns (Miltenyi) per the manufacturer’s protocol. Gr1+ cells were isolated using phycoerythrin-tagged

anti-Gr1 (RB6-8C5; eBioscience) and positive immunomagnetic separation using a phycoerythrin selection kit (StemCell Technologies, Inc.). CD11b+Ly6GhiLy6Clo cells were isolated via positive selection employing biotinylated anti-Ly6G and anti-biotin find more magnetic microbeads (Miltenyi). CD11b+Ly6G−Ly6Chi cells were isolated by negative selection of Ly6G− cells followed by positive selection with biotinylated anti-Ly6C and anti-biotin magnetic microbeads (Miltenyi). Flow cytometry verified that all cell isolations yielded >90% pure populations. Bead-isolated CD4+ T cells were

cultured for 3 days with plate-bound anti-CD3ε (10.0 μg/mL), soluble anti-CD28 (1.0 μg/mL; BD Biosciences) recombinant interleukin-12 (IL-12) (10 ng/mL; Peprotech) and anti-IL-4 (10 μg/mL; NCI). Th1 effector development was confirmed by intracellular IFN-γ staining. Cells were incubated with Fc Block (anti-CD16/CD32; eBioscience)

for 20 minutes at 4°C then washed twice. Cells were stained with antibodies to CD4, CD11b, Gr1, F4/80, programmed Quizartinib ic50 death ligand 1 (PD-L1; eBioscience, San Diego), Ly6G (Clone 1A8), Ly6C (Clone 1G7.G10), major histocompatibility complex class II (BD Biosciences), or CD14 (Biolegend) and acquired on either BD FACSCalibur (eBiosciences) or Accuri C6. Data analysis was performed with FlowJo, version 8.8.6 (Tree Star) software. Cells obtained from suppression assay cultures at 48 hours were surface stained as described above, fixed and permeabilized (CytoFix/CytoPerm; BD Biosciences), stained with rabbit anti-mouse iNOS (BD Biosciences), followed Tyrosine-protein kinase BLK by blocking with 10% normal goat serum, and secondary staining with goat anti-rabbit (Jackson ImmunoResearch). Surface-marker-appropriate isotype and intracellular staining with secondary antibody alone served as negative control. RAW 264.7 cells cultured for 24 hours with lipopolysaccharide and IFN-γ served as positive control. Cells were acquired by flow cytometry. Prior to inclusion in cocultures, bead-isolated CD4+ or CD8+ T cells from wild-type mouse spleens were stained with 5.0 μM 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Invitrogen) for 10 minutes and quenched by washing twice in Roswell Park Memorial Institute medium 1640/10% fetal bovine serum. Isolated Tgfb1+/− or Tgfb1−/− CD11b+ cells were added at 3.0 × 105 (“300K”) or 1.