Fig 2: Ag85B is not essential for the induction of iBALT organogenesis by HPIV2. (A–D) Mice were intra-nasally administered HPIV2, two times at a 1-week interval. Seventeen days after the final immunization, lung samples were examined by means of immunohistochemistry (A, B) and flow cytometry (C, D). (A, B) Lung tissues were prepared and stained with the indicated antibodies and reagent. Data are representative of two independent experiments. Scale bars: 300 µm (A) and 100 µm (B). Dotted circles in (A) indicate individual iBALT structures. Arrows in (B) indicate CD11b+CD11c+ cells. (C) Representative flow cytometric data of the lung inoculated with either HPIV2 or PBS as a control for the evaluation of cytokine production. Zombie-TCRß+CD4+ populations are shown. (D) Numbers of Th17 (gated on Zombie-TCRß+CD4+IL-17A+) and Th1 (gated on Zombie-TCRß+CD4+IFN?+) cells in the lung were determined on the basis of total cell numbers and flow cytometric data. Data are combined from two independent experiments, and each point represents data from an individual mouse. Horizontal lines indicate medians. Statistical significance was evaluated by using the Mann–Whitney test; **P < 0.01; N.S., not significant.

Fig 3: A population of macrophages reside around the bile duct in the healthy murine liver(A) UMAP of murine myeloid cells (71,261 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Expression of Gpnmb and Cd207.(E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy. Capsule macs identified by white arrows. Scale bars, 50 µm.(F) Molecular Cartography of indicated genes at liver capsule.(G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy. Scale bars, 100 µm.(H) Molecular Cartography of indicated genes at portal triad. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to markers. Images are representative of 2–4 mice.(I and J) Top GO terms for KCs (I) and bile-duct LAMs (J).(K) Representative image showing expression of VSIG4 (red) CD19 (yellow) and CD3E (magenta) by MICS analysis (left) and % of B or T cells found with/without a KC per field of view (right). Data are pooled from multiple fields of view in 2 mice. ***p < 0.001 Student’s t test.(L) Mice (29-week-old) were treated with 3.5 mg/kg LPS or PBS and 2 h later, livers were harvested without the capsule. KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10, and Il18 was examined by qPCR, compared with b-actin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni post-test. See also Figure S3 and Table S2.