Fig 2: Liver sinusoidal endothelial cells supported the differentiation of liver-type ILC1s(A) Sorted peripheral blood ILCPs (Figure S5A; Lin-CD45+CD127+CD94-NKp80-CD45RA+CD117+CD294-KLRG1-NKP44-) from four healthy donors were cultured for 2 weeks on OP9-DL4 feeder cells or LSECs with IL-7 +/- TGF-ß1 as shown to the left. The dot plots to the right show the expression patterns of CD56 and CD94 among Lin- lymphocytes derived in the indicated culture conditions.(B) Frequencies of CD200R1+CD49a+EOMES- cells among total Lin-CD45+CD94+CD56+ cells derived in vitro following culture of ILCPs (n = 8) as in (A).(C) Representative flow cytometry analyses of EOMES, T-BET, perforin, CXCR6, NKp80, CD200R1, and CD49a expression by Lin-CD45+CD94+CD56+ cells derived from ILCPs in the indicated culture conditions. Fluorescence minus one (FMO) control with the respective lacking specific antibody is shown as black dotted lines in each histogram.(D) Intracellular flow cytometry analysis of IFN-?, IL-2, GM-CSF, TNF-a, and IL-22 production by in vitro derived Lin-CD45+CD94+CD56+ cells generated from ILCPs as in (A) and following stimulation for 5 h with PMA and ionomycin. Unstimulated cells were set as controls (dotted black line).(E) Representative flow cytometry analyses showing expression of the indicated markers by total Lin-CD45+ cells obtained after 2 weeks culture of primary sorted liver-type hepatic cNK, CD49a+trNK, or CD49a-trNK cells on OP9-DL4 feeder cells or LSECs with IL-7 +/- TGF-ß1 as shown to the left. Data are representative of at least three independent experiments.(F) Representative flow cytometry analyses showing expression of the indicated markers by total Lin-CD45+ cells obtained after 2-week culture of primary sorted liver-type ILC1s (LT-ILC1s) or hepatic NK cells (sorted as Lin-CD56+CD94+NKp80+) with OP9-DL4 cells and IL-7. Data are representative of at least three independent experiments.(G) Frequencies of CD200R1+CD49a+EOMES-cells among total Lin-CD45+CD94+ lymphocytes obtained from cultures initiated with either total hepatic NK cells or liver-type ILC1s (F).*p < 0.05, **p < 0.01, ***p < 0.001, error bars represent SEM. See Figure S6.

Fig 3: Liver-type ILC1s are increased in cirrhotic liver tissues(A) Comparison of LT-ILC1 (Lin-CD45+CD94+NKp80-CD200R1+CD49a+) frequencies calculated as percentages among total Lin- lymphocytes between control livers (perfusate n = 7), control livers (non-perfusate; n = 8), and cirrhotic livers (n = 29) (patient characteristics shown in Table S2).(B) Correlation matrix estimated for LT-ILC1s (percent of Lin-), age, AST, ALT, gamma-GT, total bilirubin, MELD score (Model of End Stage Liver Disease), IFNG (mRNA), IL13 (mRNA), IL1B (mRNA), IL2 (mRNA), IL7 (mRNA), and TGFB1 (mRNA). LT-ILC1 frequencies were obtained from 25 cirrhotic livers. Spearman correlation coefficients (R) are given as color code. Statistically significant correlations are indicated with *p < 0.05, **p < 0.01, ***p < 0.001.(C) Representative expression of the indicated markers by human LT-ILC1 (pink), cNK cells (turquoise), CD49a+trNK cells (violet), CD49a-trNK cells (red), cILC1s (gray), ILC2s (blue), and ILC3s (green), comparing populations derived from healthy and cirrhotic livers. Data are representative of at least three independent experiments.(D) Comparison of IFN-? production (healthy, n = 7; cirrhotic, n = 9), IL-2 production (healthy, n = 4; cirrhotic, n = 4), CD107a degranulation (healthy, n = 3; cirrhotic, n = 5), TNF-a production (healthy, n = 7; cirrhotic, n = 5), and GM-CSF production (healthy, n = 4; cirrhotic, n = 3) between healthy and cirrhotic livers related to LT-ILC1 (pink), cNK cells (turquoise), CD49a+trNK cells (violet), CD49a-trNK cells (red), and ILC3s (green), from healthy and cirrhotic livers following 5 h PMA and ionomycin stimulation and measured by intracellular flow cytometry.(E) Volcano plot showing DEG differences based on scRNA-seq data between LT-ILC1s comparing “control versus cirrhotic liver tissues” from batch 2.*p < 0.05, ***p < 0.001, error bars represent SEM. See Figure S4.