Fig 2: Identification and phenotypic characterization of human liver-type ILC1s(A) CITE-seq analysis (protein) of hepatic Lin-CD45+ lymphocytes showing relative expression of CD49a, CD94, and NKp80 in the LT-ILC1 cluster illustrated in UMAP (only batch 2).(B) Relative expression levels of the indicated markers among each of the indicated populations as measured by CITE-seq analysis (only batch 2).(C) PhenoGraph cluster analysis of flow cytometry data from hepatic Lin-CD45+ lymphocytes displayed in UMAP. Clustering and UMAP is based on following markers: CD94, CD45, CD56, CD117, CD52, CD200R1, CRTH2, T-BET, NKp80, CD161, CD49a, EOMES, CXCR6, and CD127.(D) Heatmap showing the relative expression of the indicated markers from nine PhenoGraph clusters identified in (C) as measured by flow cytometry and calculated to a Z score for each marker.(E) Representative flow cytometry plots showing the gating strategy from singlet Lin-CD45+ lymphocytes (from healthy livers) to identify liver-type (LT)-ILC1 (pink), cNK (turquoise), CD49a+trNK cells (violet), CD49a-trNK cells (red), classical (c) ILC1 (gray), ILC2 (blue), and ILC3 (green) populations.(F) PhenoGraph cluster analysis of flow cytometry data from hepatic Lin-CD45+ lymphocytes with assignment of the indicated populations.(G) Representative histograms showing the relative expression of the indicated markers in each identified population to distinguish ILCs from NK cell populations.(H) Representative histograms showing the relative expression of the indicated markers in each identified population, each of which is elevated on LT-ILC1.See Figure S2.

Fig 3: 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.