Fig 2: 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.

Fig 3: 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 4: Differential repolarization of M1 macrophages by aged and infected PMNs and direct F. tularensis infection. Surface markers of M1 and M2 polarized MDMs and M1 macrophages that ingested F. tularensis, aged PMNs or iPMNs were analyzed at 12 and 48 h by flow cytometry. (A–D) Geometric mean fluorescent intensity (GMI) of M1 surface markers CD38, CD86, CD64, and CD80, respectively as the mean ± SD (n=4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons post-test. (E, F) GMI of M2 surface markers CD200R, CD206, MERTK and CD163, respectively, as the mean ± SD (n=4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons post-test.

Fig 5: Validation of macrophage polarization. MDMs were left untreated (M0) or were stimulated for up to 48 h with IFNγ + LPS or IL-4 to induce M1 or M2 polarization, respectively, prior to surface marker quantitation by flow cytometry. Graphs show geometric mean intensity (GMI) as the average ± SD from 4 independent experiments. (A) CD38. (B) CD86. (C) CD64. (D) CD80. (E) CD200R. (F) CD206. (G) MERTK. (H) CD163. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by ANOVA with Tukey’s multiple comparisons post-test.