BA cohort
We recruited a cohort of children from the Department of Pediatric Surgery at Guangzhou Women and Children’s Medical Center between January 2020 and January 2021. BA (n = 82) (median age: 2 months, ranging from 1 month to 7 months; male/female=0.43) was diagnosed according to intraoperative cholangiography when the intrahepatic biliary tree and/or extrahepatic bile duct could not be observed. All BA patients involved were full-term infants. For controls, children with normal liver function but requiring surgery for choledochal cyst (CC) (n = 16) (median age: 2 years 6 months, ranging from 3 month to 7 years old; male/female =0.45) were included.
Flow cytometry
Liver biopsies obtained from patients during laparoscopy or Kasai’s operation were processed according to published procedures by Wang. et al [9]. Peripheral blood mononuclear cells or hepatic lymphocytes were incubated for 45 min at 4℃ with saturating concentrations of fluorescently labeled antibodies and analyzed on a FACSAria SORP flow cytometer (BD Science). Lymphocyte gating was performed based on the forward and side scatter pattern, followed by dead cell exclusion using propidium iodide (PI) in all surface staining samples.
RRV-induced mouse model of BA and anti-PD-1 treatment
All experimental wild-type BALB/c mice were SPF rated and placed in a room with a 12-hour dark/light cycle. Mice were injected intraperitoneally (i.p.) at 12-18 hours of birth with 30μl (1.5 × 10^6 PFU/ml) rhesus rotavirus (RRV) or 0.9% saline (Control mice). RRV-infected mice that died within the first two days were excluded from the study. The clinical symptoms of BA development include yellowing of the skin and sclera, acholic stool, and growth retardation. RRV-infected mice were randomized into BA-RRV group or anti-PD-1 group. A total of four doses of anti-PD-1 antibody (50mg/kg) were given by i.p. injection every 3 days, starting on day 1 of life. Mice were sacrificed at 12 days after RRV, saline, RRV+anti-PD-1 treatment.
Liver function indices
Blood was collected by heart puncture and centrifuged at 13,000 r.p.m 4 ˚C for 5 min. Serum was collected for liver function measurement. ALT, AST, DB, TB were measured with liver function Assay (an automated clinical chemistry analyzer (Beckman Coulter AU5811, USA)).
Histopathology
The fresh liver tissues were fixed with 4% paraformaldehyde and embedded in paraffin, then sectioned (2 mm in thickness) and stained with hematoxylin and eosin (H&E) for morphological evaluation and Masson’s trichrome staining for fibrosis.
Intracellular cytokine staining
Lymphocytes isolated from liver biopsies of patients or mice models were cultured on V-bottom 96-well plates in RPMI 1640 (Gibco) medium (R10) containing 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin/streptomycin (Gibco) and 100 μM nonessential amino acids (Gibco) at 37℃ in 5% CO2. 100 ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St Louis, MO), 2 μM/ml Ionomycin (Sigma-Aldrich) and 2 μM/ml monensin (Sigma-Aldrich) were used to stimulate the lymphocytes for 4-6 hours [9]. Intracellular staining was performed using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Invitrogen) following the manufacturer’s protocol. Briefly, stimulated cells were surface stained prior to fixation on ice for 20-30 minutes. Then cells were stained for intracellular cytokines at 4℃for 1 hour and resuspended in FACS buffer (PBS+2%FBS) before analysis by flow cytometry.
Statistical analysis
Prism 7.0 (GraphPad Software) was used for statistical analysis. Unpaired t test was used for two group analysis and one-way ANOVA for three or more group analysis. Non-parametric data were analyzed by Mann-Whitney U test or Kruskal-Wallis test. For all analyses, 2-tailed p values were calculated. P values for multiple comparisons were adjusted by the Benjamini- Hochberg method (Benjamini and Hochberg, 1995). All data points are shown with central lines indicating medians unless stated otherwise.