Isolation and Characterization of Intrahepatic Lymphocytes

The spleen and thymus were mechanically dissociated. The supernatant was collected and incubated with red blood cell lysis buffer (BD Biosciences) to remove red blood cells. IHLs were isolated according to our previous method^{52 (link)}. Briefly, liver tissue was pressed and digested with collagenase IV (0.05%, Roche Applied Science, Indianapolis, IN) at 37 °C for 30 min. After digestion, cell suspensions were passed through 70-μm nylon cell strainers to yield single-cell suspensions. Cells were purified by centrifugation (400 × g) at room temperature for 30 min over a 30%/70% discontinuous Percoll gradient (Sigma-Aldrich). Then cells were collected from the interphase, thoroughly washed, and re-suspended in complete RPMI-1640 medium containing 10% FBS. The relative percentages of lymphocyte subpopulations were measured by flow cytometry, and their absolute numbers were calculated according to their percentages and the total numbers.