Fig 2: CAF-derived TGF-ß1 up-regulates Ln-?2 expression in tumor cells.(A) Left: On the basis of an analysis of the GEO database, the depletion of CAFs decreased the expression of Ln-?2 in a mouse pancreatic ductal adenocarcinoma model (GSE52812, n = 3). Right: Coculture with primary CAFs significantly up-regulated the expression of Ln-?2 in basal breast cancer cells (GSE41678, n = 4). The statistical analysis was performed using the unpaired two-tailed t test with Welch’s correction. Data are presented as means ± SD. *P < 0.05 and ***P < 0.001. (B) The expression of Ln-?2 (green) in A549 and KYSE510 cells was determined using double IF staining after coculture with or without CAFs (a-SMA+, red) in vitro. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 50 µm. (C) Western blotting showed that treatment with CAF-conditioned media (CAF M) significantly increased Ln-?2 levels compared with treatment with paired NF-conditioned media (NF M; n = 2). (D) Levels of the TGF-ß1 protein in CAF M and NF M were analyzed by Western blotting after concentration using an Amicon Ultra centrifugal filter (3 kDa, #UFC200324, Millipore) (n = 2). Total proteins were stained with Coomassie Brilliant Blue R-250. (E) Western blot showing the levels of the Ln-?2 protein in A549 cells, KYSE510 cells, and three primary CAF lines. (F) IF staining showed that the increase in Ln-?2 expression observed in A549 and KYSE510 cells after coculture with CAFs was blocked by galunisertib (10 µM, #S2230, Selleck). Scale bar, 50 µm. (G) Western blotting confirmed that galunisertib and the TGF-ß1–neutralizing antibody (anti–TGF-ß1; 1 µg/ml, #521707, BioLegend) inhibited the increase in the Ln-?2 levels induced by treatment with CAF-conditioned media. In (C), (E), and (G), ß-tubulin was analyzed as a loading control.

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.