Fig 2: Tumor necrosis factor receptor signaling controls PNAd expression in most TEC but not in LN BEC. C57BL/6 WT or TNFR1/2-/- mice were I.P. injected with B16-OVA cells and tumors were harvested 14 days later. LN were harvested from non-tumor-bearing mice. Resected tissues were prepared for flow cytometry as described in Methods. Representative flow cytometry plot, percentages, and gMFI of PNAd on CD45neg Ter119neg PDPNneg CD31+ endothelial cells in CD45+ depleted LN and I.P. tumors suspensions. PNAd gMFIs were calculated on cells gated above FMO control. Data represents one experiment, n = 5 LN or tumors per group. Results are mean ± SD analyzed by unpaired Welch’s t-test. ns: p>0.05, *p<0.05, **p<0.01.

Fig 3: Transplanted mLNs retain normal morphology, cell distribution and lymphatics. (A) H&E stainings of transplanted vs. non-transplanted mLNs reveal a regular immune architecture with B cell follicles and some germinal centers (arrowheads) in both settings in the cortex and T cell zones (top). Lymph node capsules (arrow) display signs of capsule fibrosis in transplanted mLNs. Immunohistochemical analysis of CD19+ B cells confirm proper B cell follicles in the cortex and distribution of CD3+ T cells dominating in the paracortical areas. CD31 staining on LECs in the subcapsular sinus (asterisk) revealed an intact structure of the mLN capsule, 5-6 weeks after mLN transplantation. (B) Donor mLNs lymph vessels conjoin to recipient’s intestinal lymphatic vasculature after transplantation. Evans blue injected into the iliac Peyer’s patches drains into the transplanted mLNs, 5 weeks after mLN transplantation.

Fig 4: Most of the mesenchymal cells, and very few ATIIs, are GHR positive in normal lung but not in IPF fibrotic area.(A) Representative confocal microscopy images of GHR and COL1A1 coimmunostaining for healthy and IPF human lung tissues. (B) Quantification of GHR and COL1A1 coexpression by colocalization calculated from staining in (A). (C) GHR and HTII-280 coimmunostaining reveals that a small portion of HTII-280+ATII cells are GHR positive in normal human lungs and no GHR+HTII-280+ cells in fibrotic human lungs. (D) Quantification of GHR and HTII-280 coexpression by flow cytometry or by colocalization calculated from staining in (C). (E) Rare CD31+GHR+ endothelial cells were found in normal lungs but not IPF lungs. (F) Quantification of GHR and CD31 coexpression by colocalization calculated from staining in (E). Arrows show the coexpressing cells. Scale bars, 100 µm (A to C, lower power images) and 10 µm (A to C, higher power images). Bars represent means ± SEM, n = 7 biological replicates. ****P < 0.0001 by unpaired two-tailed Student’s t test.

Fig 5: The IL33-containing BAC in transgenic mice encodes human-specific regulatory patterns and demonstrates the importance of the 5 kb noncoding segment for proper IL33 expression.a Schematic of human BAC clone RP11-725F15 (166 kb) spanning the entire coding region of IL33 and its upstream region including the 20 kb asthma-associated interval and the 5 kb region of interest shaded in blue (black bars). To produce a human IL33 reporter strain, a cassette containing E2-Crimson with a stop sequence was inserted into exon 2, in frame with the IL33 translational start site (red dotted line). Transgenic mice were generated with either the full BAC (hIL33CrimBAC) or a BAC containing a deletion of the 5 kb interval within the LD block (hIL33Crim BAC5kdel). b, c Immunofluorescence staining of mouse peripheral lymph node sections (left panels) and trachea tissue sections (right panels) of E2-Crimson in hIL33Crim BAC mice (b) or hIL33Crim BAC5kdel (c). Representative founder BAC transgenic lines are shown. Sections were stained with anti-E2-Crimson (red) and the mouse endothelial cell marker CD31 (green). Hoechst 33342 staining for nuclei is in blue. Pie charts show the distribution of Crimson expression (Crm) in each “humanized” BAC mouse line. d qPCR analysis of E2-Crimson mRNA obtained from lymph node, heart, and lung from both BAC strains is shown. Violin plot shows average dCT values (Crimson/Ppia) obtained from animals containing either the full BAC or the 5 kb deletion (n = 10 biologically independent animals for full BAC and n = 6 biologically independent animals for 5 kb deletion). Center line: median; box limits: upper and lower quartiles; whiskers: 1.5 × interquartile range. **p = 0.0017 (lymph node); **p = 0.0066 (heart), ***0.0001; one-way ANOVA with post hoc Sidak multiple comparison test. Source data are provided as a Source Data file.