Fig 2: The density of proliferating CD8+ T-lymphocytes varies between tissues and individual patients. Representative multiplex immunofluorescence images of CD8+ (white) and Ki67+ (red) cells in (A) normal human tonsil, (B) thymus, (C) sialadenitis, (D) lichen sclerosus, (E) Microsatellite stable colorectal cancer (MSI- CRC) and (F) Microsatellite instable colorectal cancer (MSI+ CRC). The visualizations of the digital image analysis highlight the subset of Ki67+CD8+ proliferating cytotoxic T-cells (blue).

Fig 3: Effect of HOPX on proliferation and differentiation in MLE12 epithelial cells in vitro. Evaluation of si-RNA-based Hopx knockdown in MLE12 lung epithelial cells with (A) qRT-PCR of Hopx, (B) Western blotting of HOPX, (C) quantification of Western blotting data. (D) EdU assay of MLE12 lung epithelial cells co-stained with HOPX antibody in vitro. Alexa Fluor 647-EdU+ cells and Alexa Fluor 488-HOPX+ cells were evaluated by FCM (Representative image of n = 2). (E) Quantification of HOPX+ EdU+ cells and HOPX-EdU+ cells in MLE12 cells (n = 2). (F,G) Cellular scratch assay, qRT-PCR analysis of (H) Mki67 and (I) Sftpc, and (J) WST assay of MLE12 cells transfected with control siRNA and Hopx siRNA. (J) The ratio of Ki67 positive/negative cells with HOPX co-expression in IF. *p < 0.05, **p < 0.01, ***p < 0.005.

Fig 4: Treatment with hzVSF at the high dose or with TAF, alone and together with hzVSF at an intermediate dose, reduces the presence of intrahepatic immune cell subsets and lowers liver replenishment by new hepatocytes. Comparison of mean percentages of macrophages, CD3+ T-cells, and Ki67+ hepatocytes of (a) Groups 1–2 treated with placebo or hzVSF at a high dose, (b) Groups 3–6 treated with placebo or hzVSF at a low, intermediate, and high dose, and (c) Groups 7–8 treated with TAF, alone or in combination with hzVSF at an intermediate dose, relative to the pretreatment baseline (week –4). Vertical lines indicate the standard error of the mean. Horizontal dotted lines present the mean percentage of Groups 1–2, Groups 3–6, or Groups 7–8 at baseline. Macrophages in Group 3 and Group 4 were significantly increased compared to the baseline after the EOS during weeks 17–19 (+: p < 0.05). Compared to Group 3 and Group 6, macrophages were significantly increased in Group 5 at week 6 (*: p < 0.05). CD3+ T-cells in Group 3 were significantly increased compared to the baseline after the EOS during weeks 17-19 (+: p < 0.05). Ki67+ hepatocytes in Group 6 were significantly reduced compared to the baseline at week 12 (+: p < 0.05). Compared to Group 7, Ki67+ hepatocytes were significantly reduced in Group 8 at week 12 (*: p < 0.05). # The mean Ki67+ hepatocyte percentage in Group 4 was 15.5%.

Fig 5: Expression and localization of HOPX in control (donor) and IPF lungs. In silico analysis from Lung Genomics Research Consortium (LGRC) of (A) HOPX mRNA expression in organ donor (control) and IPF lungs, (B) correlation between HOPX mRNA expression and DLco (blue dots: control, red dots: IPF). (C) Correlation between HOPX mRNA and MMP7 mRNA in control (blue dots) and IPF (red dots) in silico. QRT-PCR analysis of (D) HOPX mRNA, (E) SFTPC mRNA, and (F) ACTA2 mRNA from whole lung homogenate of control and IPF lungs in our cohort. (G) Immunohistochemical staining of serial sections of control and IPF lung tissue for KRT5, proSP-C, HOPX, KRT7, and Ki67. In IPF lungs, proSP-C+/KRT7+ cells revealed cytoplasmic or in part nuclear expression of HOPX without nuclear Ki67 (Indicated by arrows in panels 1–4). ProSP-Clow/KRT7+ AECs of IPF-lungs also indicated robust HOPX-immunoreactivity with nuclear Ki67 expression (asterisks in panel 2 and 4). Some proSP-C+/KRT7+ cells without HOPX immunoreactivity were observed in the area of relatively preserved alveolar septa (white arrowheads in panel 5). KRT5+/KRT7+ bronchiolar basal cells did not express HOPX, and indicated nuclear expression of Ki67 (Indicated by black arrowheads in panel 2). (H) Immunohistochemical staining of serial sections of control donor lung tissues for KRT5, proSP-C, HOPX, KRT7, and Ki67. In donor lungs, HOPX expression was present in proSP-C+/KRT7+ ATII (arrows) and proSP-Clow/KRT7+ AECs (asterisks) (panels 2 and 3 in Fig. 4O), and faint in normal bronchiolar epithelium. ProSP-Clow/KRT7+ AECs expressing HOPX also revealed nuclear Ki67 expression (indicated by asterisks). In silico single cell RNA sequence analysis of (I) HOPX, (J) SFTPC, (K) KRT7, (L) CTNNB1, and (M) CTND1 in EpCAM+/HTII280+ cells. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001.