Primary hepatocyte culture

Primary hepatocytes were isolated from naïve H-2^bxd mice as described (40). Mice were anesthetized with ketamine, and using septic technique, angiocatherization into the portal vein was performed. Steady-state perfusion of liver was performed first with 1× PBS (5mL/min for 5 minutes), then Liver Perfusion Medium (5mL/min for 3 minutes, Gibco, ThermoFisher Scientific, Carlsbad, CA), and finally Liver Digest Medium (3mL/minute for 5-8 minutes, Gibco). Digested liver was excised and a single cell suspension made, suspended in wash solution composed of DMEM (Gibco), 10% FBS, and 1× antibiotic/mycotic solution (containing penicillin, streptomycin and amphotericin B, Sigma-Aldrich, St. Louis, MO). Non-hepatocytes and debris were then removed with a 50% Percoll (GE Healthcare, Little Chalfont, United Kingdom) gradient. Remaining cells were counted and resuspended in DMEM with 10% FBS and 1× antibiotic/mycotic. 2 × 10⁴ cells were plated on collagen-coated 96-well plates in 100 μL volume and incubated overnight in 37°C, 5% CO₂ incubator. Primary hepatocyte cultures were infected with 2 × 10⁴ PbANKA-GFP or CTV-labeled sporozoites per 96 well (MOI ~1), and plate was spun 10 minute, 2400 RPM, no brake centrifugation. Cultures were further incubated for 20 or 40 hours to allow infection and liver-stage parasite development. Infection was confirmed by analyzing GFP⁺ or CTV⁺ hepatocytes by inverted fluorescent microscopy (when cultures infected with GFP+ sporozoites) or flow cytometry (when cultures were infected with 5mM CTV-labeled sporozoites). Wells were washed twice with 1× PBS and then exposed to enriched splenic-derived CD8+ T cells from DC-LM vaccinated BALB/cJ x BALB.b F1 mice in DMEM supplemented with 10% FBS and 1× antibiotic/mycotic medium. CD8+ T cell enrichment from DC-LM vaccinated mouse spleens was performed using EasySep CD8+ T cell isolation kit (StemCell Technologies, Vancouver, Canada). 8 × 10⁴ antigen-specific CD8+ T cells with 1× Brefeldin A (Biolegend) were added to 96-wells based on calculations of 90% CD8+ T cell enrichment, and pre-harvest peripheral blood CD8⁺ tetramer⁺ staining of donor cell population. CD8+ T cells were also added to non-infected hepatocyte culture wells with or without 5mM exogenous peptide to serve as positive and negative controls, respectively. CD8+ T cells added onto hepatocyte cultures were incubated for an additional 6 hours at 37°C, 5% CO_2. IFN-γ⁺ CD8+ T cells were detected following intracellular cytokine staining and flow cytometry to determine the proportion of IFN-γ-producers in well containing sporozoite-infected hepatocytes (for 20 or 40 hours) relative to wells stimulated with exogenous peptide.