Fig 2: Tumor ILC3s, especially NKp44+ ILC3s are negatively correlated with pathological stage. Distribution of ILCs and ILC subtypes by FACS. MNCs from tumor, proximal, and distal regions of 58 patients with colon cancer were prepared. (A) The gating used to define ILC subtypes: MNCs were stained for Lin cocktail (CD3, CD14, CD19, and CD20), CD94, CD34, CD1a, TCRa/ß, TCR?/d, CD45, CD127, CRTH2, and CD117. Total ILCs were identified as Lin- CD94- CD34- CD1a- TCRa/ß- TCR?/d- CD45+ CD127+, ILC1s were identified as Lin- CD94- CD34- CD1a- TCRa/ß- TCR?/d- CD45+ CD127+ CRTH2- CD117-, ILC2s were identified as Lin- CD94- CD34- CD1a- TCRa/ß- TCR?/d- CD45+ CD127+ CRTH2+, and ILC3s were identified as Lin- CD94- CD34- CD1a- TCRa/ß- TCR?/d- CD45+ CD127+ CRTH2- CD117+. ILC3s were further divided into NKp44+/- ILC3s. (B) ILC levels among the CD45+ cells in the indicated tissues. (C) Percentage of ILC1s, ILC2s, and ILC3s among CD45+ cells and total ILCs in the indicated tissues. (D) Percentage of NKp44+/- ILC3s among CD45+ cells in the indicated tissues. (E, F) Correlation between the percentage of ILC3s or NKp44+/- ILC3s in tumor (T) versus distal (D) tissue and the pathological stage of cancer. The distal tissue was considered normal tissue and was used for normalization to the background, here T/D. (G) Percentage of ILC3s among CD45+ cells in colon glandular cancer and mucous carcinoma tissue. In (B–D, G), each symbol represents the indicated tissue from one patient (circle, tumor; square, proximal region; triangle, distal region). In (A, E, F), each dot represents one patient. A paired t-test and Spearman test were used for statistical comparison. *P < 0.05; **P < 0.01; ***P < 0.001.

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.

Fig 4: 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 5: LT-ILC1 cells accumulate in fibrotic area of the human liverRepresentative images of three cirrhotic (A) and two control livers (C) generated with PhenoCycler (formerly Codex) with a selection of markers such as CD56, CD94, aSMA, CD36, and CD32, as well as DAPI for nucleus staining (A, C). An overview (left) and magnification (right) are shown side by side with scale bar and indicated diameters. Fibrotic regions were recognized and delineated with a white dashed line using aSMA-staining. The red arrows indicate LT-ILC1 cells automatically detected by the HALO image analysis software by CD45+CD3-CD56+CD94+EOMES-KLRG1+ definition (see staining in Figure S5). Images in (B) and (D) were artificially generated as cell contours using HALO image analysis and correspond to the respective section from (A) and (C). Only the identified LT-ILC1 cells were highlighted for enhanced visibility (surface is green, nucleus is blue and indicated with red arrows) with visualization of the non-fibrotic (NF) and fibrotic (F) regions.