Fig 2: Rab27-dependent transfer of miR-146a to myeloid and T cells in vivo.(A) Schematic of BM chimera and representative flow plots of CD45.2+CD3+ sorted cells from the mLN. (B) miR-146a expression within sorted mLN and cLP CD11b+ cells, as well as mLN and cLP CD4+ T cells (n = 6). One-way ANOVA with Tukey’s multiple-comparison test comparing the column means. (C) Schematic of mTmG gut explant experiment with ImageStream technology. (D) Representative images of green puncta on mTomato+CD3+CD4+ cells from CD11c-Cre mTmG mLNs. Representative images of green puncta on mTomato+ CD11b+ cells from CD11c-Cre mTmG mLNs. (E) Representative flow cytometry plots of P3/4 monocytes from F. (F) Percentage of mGFP+mTomato+ myeloid populations within the mLNs and cLP. (G) Representative flow cytometry plots of CD4+ T cells from H. (H) Percentage of mGFP+mTomato+ lymphocyte populations within the mLNs and cLP. Unpaired 2-tailed t test for each cell type in F and H. *P < 0.05, **P < 0.005, ***P < 0.0005. See also Supplemental Figure 4.

Fig 3: Strongyloides venezuelensis-experienced mice develop an increased pulmonary inflammation and a resistance against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection. S. venezuelensis (Sv)-infected or uninfected mice were inoculated with 500 N. brasiliensis (Nb) L3 at day 28. B6; C57BL/6, WT; wild type, sac; sacrificed. (B) The number of lung stage N. brasiliensis larva. Two days after N. brasiliensis infection, migrating larva were isolated from the lungs and counted (n = 11). cont, control (uninfected at day 0). Pooled data from two independent experiments are shown (Mean ± SD). (C) The numbers of worms in the intestine were counted at indicated days (n = 5). (D) The numbers of eggs per gram feces (EPG) from each group at day 7 post-N. brasiliensis infection (n = 7). (E–G) The numbers of eosinophils and ILC2s (E), the amounts of IL-5 and IL-13 in the BALF (F), and the Il5, Il13, and Il33 mRNA expression levels in the lungs as analyzed by Q-PCR (G) from day 35 are shown (n = 4–5). uninf.; uninfected at day 28. Statistical analyses were conducted using two-way ANOVAs with Bonferroni post-hoc tests (B,E,G), Wilcoxon matched-pairs signed rank tests, and two-tailed (C) or unpaired t-tests (F). Data are representative of two independent experiments. (H) C57BL/6 WT mice (n = 5–6) were infected with 5000 L3 S. venezuelensis at day 0. The numbers of ILC2s, eosinophils, CD3+/B220+ cells, and monocytes among the BALF cells at the indicated days were analyzed by flow cytometry (FACScalibur). Cell populations were defined as follows: ILC2s, FSCloSSCloLin(CD3, CD4, CD8, CD19, NK1.1, IgE, Gr-1, siglecF)-Sca-1+ST2+; Eosinophils, CD45+CD3-B220-CCR3+, CD3/B220: CD45+CCR3-CD3+/B220+; and Monocytes, CD45+Autofluorescencehigh. Data were compared with day 0 data by one-way ANOVA. Data are representative of two independent experiments.

Fig 4: The bone marrow within the imaging volume reaches steady-state comparable to homeostasis 28 days after LIMB implantation. a Immunofluorescence analysis of bone sections after removal of the LIMB implant over the time course of 4 weeks. ECM formation was identified by the marker Laminin (Lam). Stem-cell antigen 1 (Sca-1) is highly expressed in arterioles. The leukocyte marker CD45 indicates localization of inflammatory cells adjacent to the window cavity (wc). Lam is highly expressed around the implant during the first week and completely normalizes after 2–4 weeks. CD45+ cell accumulations are found during the first weeks in proximity to the wc. b Movat’s pentachrome stain detects connective tissues and reveals remodeling of bone primarily on the periosteal interface near the fixation plate. a, b Images are representative for 3–5 mice per time point post-surgery. c Overview immunofluorescence images of the femoral bones from an individual mouse 3 days post-surgery. Note the specific reaction to the implant-bone marrow interfaces indicated by accumulations of CD45+ cells, and Lam+ and Sca1+ arteries (yellow). bm bone marrow, cb cortical bone. d Immunofluorescence image of the region around the endoscope tubing in a LIMB-implanted femur 7 days post-surgery, including the bone cortex and periosteum under the plate. The presence of various blood vessel subsets indicated by CD31 and Emcn demonstrates intact blood supply to the periosteum and to the bone. Scale bar = 100 µm. e Blood supply is intact throughout the marrow cavity, indicated by CMTPX-labeled splenocytes, which localize in the bone marrow 4 h after transplantation in both contralateral and LIMB-implanted femurs at 42 days post-surgery (n = 3 mice). f Histological DAPI stain (gray) shows intact tissue structure, with no separation of the bone marrow and the vasculature by the screws or endoscope tubing. g Flow cytometry analysis of femurs with the LIMB implant, their contralateral femurs and femurs of control mice. Similar frequencies and cell counts of various cell populations shows no effect of the LIMB implant on bone marrow cell composition (n = 8 LIMB-implanted mice, n = 8 controls, two independent experiments). Error bars represent s.e.m. values. Statistical analysis was performed using t-test

Fig 5: Kmt2c deletion impairs IL-1ß-driven myeloid differentiation and conveys a selective advantage to cycling, IL-1ß-stimulated HSCs(A–C) HSC, MPP, and HPC numbers in wild-type and Kmt2c?/? mice after treatment with PBS or IL-1ß for 3 days. n = 8–9.(D) CD45.2+ donor leukocyte chimerism in peripheral blood from primary recipients transplanted with 20 wild-type and Kmt2c?/? HSCs, after a 21-day treatment with vehicle (PBS) or IL-1ß. n = 10–13.(E) Phenotypic HSC subpopulations after IL-1ß treatment based on EPCR and CD34 expression.(F) Percent of HSCs with Kmt2c deletions after one cycle of PBS or 5-FU, with or without concomitant IL-1ß or TNF. n = 6–20. Error bars reflect standard deviation.*p < 0.05, **p < 0.01, ***p < 0.001; comparisons were made by two-tailed Student’s t test or one-way ANOVA with Holm-Sidak post hoc test (multiple comparisons).