Il 2 jes6 5h4

The following antibodies were used in this study: CD4 (RM4–5; Biolegend), CD8⍺ (53–6.7; Biolegend), Thy1.1 (OX-7; Biolegend), CD44 (1M7; Biolegend), CD69 (H1.2F3; Biolegend), CD103 (2E7; Biolegend), V⍺2 (B20.1; Biolegend), IFNγ (XMG1.2; Biolegend), TNF⍺ (MP6-XT22; Biolegend), IL-2 (JES6–5H4; Biolegend). MHC-II tetramers were obtained from the National Institutes of Health tetramer core facility. Tetramer staining was performed for 1 hour at 37° C and a tetramer loaded with a human CLIP peptide was used as a negative staining control. All other staining was performed for 15–30 minutes at 4° C. Intravascular labeling was performed as described previously (25 (link)). Briefly, 3 μg of anti-V⍺2 was injected i.v. in 200 μl of PBS, and tissues were harvested 3 minutes later. Data were acquired using a LSRII Flow Cytometer (BD) in the OHSU Flow Cytometry Core Facility. Flow cytometry data were analyzed using FlowJo software (BD) version 9.9.6 or 10.5.3.

1 x 10⁶ liver NPC cells were incubated with 100 μl of various antibodies diluted at optimal concentrations for 40 min at 4 °C. The following fluorochrome-conjugated antibodies were used: CD45.2 (104; Biolegend, 109831), CD45.1 (A20; Biolegend, 110741), CD45 (30-F11; Biolegend, 103126) F4/80 (BM8; Biolegend, 123114), CD11b (M1/70; Biolegend, 101227), CD9 (MZ3; Biolegend, 124805), GPNMB (CSTREVL; Thermo Fisher, 50-5708-82), CD90.2 (30-H12; Biolegend, 105335), CD4 (GK1.5; Biolegend, 100407), CD8 (53-6.7; Biolegend, 100734), PD-1 (29F.1A12; Biolegend, 135218), IL-2 (JES6-5H4; Biolegend, 503807). Liver macrophages were gated as CD45+F4/80^hiCD11b^int and CD45+F4/80^intCD11b^hi for KC or MDM, respectively. For intracellular staining, 1 x 10⁶ NPCs were cultured in complete RMPI 1640 medium with Brefeldin A and PMA/ionomycin for 6 hours before harvested for flow staining. After surface staining, fixed cells were permeabilized using Invitrogen transcription factor staining buffer set according to manufacturer’s protocol. Samples were analyzed using BD LSR cell analyzer at the Vision Research Core Facility at the University of Michigan Medical School or Attune NXT4 Flow Cytometer at MCDB research core facility at the University of Michigan. Data were analyzed using the CellQuest software (BD Biosciences) or Attene NXT software and Flowjo (Flowjo.com).