Fig 2: Hepatic IFN-?3 responsive macrophages are present in vivo. IFNL1, IFNL2/3, and IFNLR1 mRNA expression was measured in healthy, NAFLD/NASH, HBV, and HCV infected liver tissue (n = 9/group) (A). To examine IFNLR1 expression in vivo, biopsy sections obtained from healthy, autoimmune hepatitis, and HCV infected liver tissue were labeled with monocyte/M? markers CD11b and CD68 as well as IFNLR1 antibodies, and examined by confocal microscopy (B). To assess the responsiveness of liver M?s to IFN-?3, immune cells were isolated from fresh liver tissue, sorted by FACS and cultured in the presence of IFN-?3. Live CD45+ immune cells were sorted based on the expression of CD3 and CD56 into NK and T cell subsets, and CD14 and CD68 into M? subsets (C). IFNLR1 expression, as determined based on IFNLR1 MFI was compared among liver immune cell subsets, and was significantly higher in CD14+, CD68+ liver M?s, as compared to NK (CD56+), NKT (CD3+, CD56+), and T cells (CD3+, CD56-) (D) (n = 6). Sorted T cells and M?s were cultured with 100 ng/ml IFN-?3 for 10 h and ISG mRNA expression was compared to mock treated cells (E) (n = 8). Scale bars represent 100 µm. Data are representative of one cell sorting experiment. Wilcoxon matched pairs signed rank test, *p < 0.05, **p < 0.01, and ***p < 0.001 (mean ± SE).

Fig 3: Defining the molecular signature of FL-derived HSCs with the highest engraftment potential.a Engraftment data demonstrating superior engraftment of the GPI-80+ fraction 16 weeks post transplantation. Five NSG mice were transplanted per condition with 10,000 cells each (4000 cells for the GPI-80+ condition) and engraftment was assessed by determining the ratio (%) of peripheral blood cells expressing human CD45 versus total CD45 expressing cells. The mean is shown with error bars representing the standard error of the mean. One-way analysis of variance (ANOVA) was performed with Tukey test to account for multiple comparisons and obtain adjusted p-values: GPI-80+ vs. CD34+bulk p = 0.002, GPI-80+ vs. CD34-GYPA- p = 0.001 (**p < 0.01). Source data are provided as a Source Data file. b Violin plots of differentially expressed genes between HSCs/MPPs in the GPI-80+ and CD34+ bulk fractions (left) and bar plots illustrating the percentage of cells expressing these genes per fraction (right). c UMAP representation showing the clusters identified in the CD34+ HSC/MPP population. d UMAP representation of the HSCs/MPPs corresponding to the CD34+ bulk sample, retaining the cluster definitions established in panel c. e UMAP representation of the HSCs/MPPs corresponding to the GPI-80+ sample, retaining the cluster definitions established in panel c. Additional labeling with one of the top most enriched genes is shown for each cluster (see Table S4). f Cluster dynamics upon GPI-80-enrichment showing the proportion of each cluster before (CD34+bulk) and after (GPI-80+) functional enrichment in a stacked bar plot. Percentages of cells in each cluster are shown on the left. g UMAP overlays illustrating mRNA expression patterns of genes enriched in different GPI-80+ clusters. The scale bar shows scaled expression from 0-Max. h Regulon specificity score (RSS) plot showing the regulons with the highest specificity for a given cluster within the GPI-80+ fraction. The scale bar represents z-scores and dot sizes reflect the RSS for each regulon. i Overlay of the expression patterns of cluster-specific regulons onto the GPI-80+ UMAP.

Fig 4: An improved PSuv/PM2 AND-NOT logic circuit (v2) rewires p53-deficient tumor cells to produce IFN?.a–c In a, the p53-tet H1299 cells (±10 ng/ml DOX) were introduced with the circuit shown on the left (“v2”). The PSuv-driven CRISPRa is programmed to activate transcription of endogenous IFNG, whereas PM2-ACRmax forms an inhibitory module (compared to a non-expression construct, PM2-Con). The non-targeting sgNC was used as a negative control for circuit actuation. The cell lysates were harvested 48 h after transfection and were subjected to IB. The data shown are representative of two independent experiments. b The conditioned media from the cells were also collected and were added to freshly prepared PBMC for 24 h. The cells were subjected to flow cytometry of Class II HLA levels (CD45+CD11b+-gated). The relative MFI values are marked on the histograms. The dotted lines denote the control levels. In c, p53-tet cells were transfected with the circuit at low confluency (~20%). For target activation, a construct containing a tandem of U6-dependent IFNG-targeting sgRNAs (sgIFN3 + 1) was used. Ninety-six hours after transfection, the cells were subjected to MTT assay. A quantitative summary from three independent experiments is presented (mean ± SEM, two-sided t-test, P values provided). d Co-cultures containing LLC cells (p53-deficient, labeled with mCherry) and their p53+ knock-in derivatives in equal proportion were established. The mixed cells were transfected with the PSuv-CRISPRa/PM2-ACRmax circuit (v2) targeting EGFP (sgTa: sgTGTa) for 24 h. Cells were subjected to flow cytometry analyses for mCherry and EGFP fluorescence (pseudo-color). The EGFP+ subpopulation in either the mCherry+ and mCherry- gates are further resolved in an EGFP fluorescence histogram, respectively in red and blue. The insets in the histograms show the averaged EGFP levels (EGFP+%×MFI) in these different gates from two independent experiments. e The parental and p53+ LLC cells were respectively transfected with the PSuv/PM2 circuit (v2) for conditional mouse Ifng activation by CRISPRa. The mRNA levels for IFN? and its target gene Irf1 are shown. Cdkn1a levels report p53 status. These qPCR results are presented as mean ± SEM (n = 3 biological replicates). Source data are provided in the Source Data file.

Fig 5: The crosstalk between fetus and mothers leads to high levels of RANKL/RANK expression at the maternal–fetal interface. (a) RANKL expression in villi and decidua of normal pregnancy (n=12) by immunohistochemistry. Original magnification: × 200. (b) RANKL secretion by primary trophoblasts (1 × 105 cells per well) and DSCs (1 × 105 cells per well) (n=6) from normal pregnant women by ELISA after culture for 24–96 h (left). RANKL production by trophoblasts alone, DSCs alone and the co-culture of trophoblasts and DSCs (n=6) for 48 h (right). (One-way ANOVA). (c) We isolated primary PBMCs from peripheral blood (n=6) and DLC from deciduas of normal pregnant women (n=6), and then analyzed RANK expression on pMo and dMf from normal pregnant women by labeling with anti-CD14, RANK and CD45 antibodies. (Student’s t-test). (d) Further analysis of the phenotype of RANK+ and RANK- pMo and dMf from normal pregnant women (n=24) by FCM. (One-way ANOVA). (e and f) Trophoblasts, DSCs and/or dMf (n=6) were co-cultured at a 1 : 1 : 1 ratio for 48 h, and then RANKL expression on CK7+ trophoblasts and CK7- DSCs, and RANK on CD14+ dMf were evaluated by FCM, respectively. (One-way ANOVA). Tro: human trophoblasts. pMo: human peripheral blood monocytes; dMf: human dMf. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001. NS: no statistical difference