Fig 2: Targeted HSPCs Repopulate the Immune Cell Lineages(A) Experimental scheme of mCherry insertion into the Lmnb1 gene in HSPCs that were transplanted into irradiated Rag2-/-c?-/- mice. Hematopoietic cell lineages were analyzed by flow cytometry 8 weeks (n = 4) or 16 weeks (n = 3) after reconstitution.(B) FACS analysis showing the percentages of donor cells (GFP+) in the bone marrow (BM) and spleen of the recipient mice 8 weeks after reconstitution.(C) Frequency of mCherry+ cells among GFP- (host) or GFP+ (donor) cells in the bone marrow and spleen of the recipients determined by flow cytometry (left). The graph depicts percentages of mCherry+ cells in the bone marrow (black) and spleen (orange) of the recipient animals 8 weeks (n = 4) and 16 weeks (n = 3) after reconstitution.(D) LSK gating (left) and frequency of the mCherry+ cells among the LSK cells 8 and 16 weeks after reconstitution (right). The graph (right) shows the data from 4 recipient animals (8 weeks) or 3 recipient animals (16 weeks).(E) Absolute numbers of mCherry+ T, B, NK, and Gr-1+ cells in the bone marrow and spleen of the recipient animals 8 weeks (black) or 16 weeks (orange) after reconstitution.(F) FACS analysis showing frequencies of mCherry+ cells among the HSC/MPP1, MPP2, and MPP3/4 LSK subpopulations in the bone marrow of the recipient mice 8 and 16 weeks after reconstitution.(G) Graphs depict percentages of LSK subsets (top) and percentages of mCherry+ cells within each subset (bottom).

Fig 3: Viable aged neutrophils are cleared through activation of program cell removal. a Analysis of neutrophil and macrophage cell populations after thioglycollate injection to WT and MRP8-Bcl2 mice. Peritoneal cells were collected and analyzed at 0, 2, 4, 8, 12, 24, and 72 h after thioglycollate injection. Similar lifespan was observed for WT and Bcl2 macrophages and neutrophils. Macs, macrophage;s WT, wild-type; PMN, neutrophil/polymorphonuclear cell. Results are representatives of three independent experiments. n = 3 mice for each time point. Error bars represent standard deviation. Cells were identified by surface markers such as CD11b+F4/80-GR1++ (Neutrophils), CD11b+F4/80+ (macrophages). Percentage reactive cells, the % of the total viable cells found in the peritoneum after induction of peritonitis. b WT but not Bcl2 neutrophils undergo cell death. WT and Bcl2 neutrophils were collected and cultured in vitro for 72 h. Cell viability was examined by AnnexinV and DAPI staining. Cells that were AnnexinV-DAPI- were considered as viable cells. n = 3. **P < 0.01 (t-test) for viability between WT and Bcl2 neutrophils. Error bars represent standard deviation. c A schematic showing peritonitis in WT and MRP8-Bcl2 mice. WT neutrophils undergo both programmed cell death (PCD) and programmed cell removal (PrCR) after maturation while Bcl2 neutrophils are resistant to PCD but maintain PrCR programs. d RNAseq anaylsis of 4 groups of neutrophils, including (1) WT neutrophils from bone marrow (BM), (2) WT neutrophils recruited to peritoneum (PT), (3) MRP8-Bcl2 neutrophils from bone marrow, and (4) MRP8-Bcl2 neutrophils recruited to peritoneum. PT, peritoneal; BM, bone marrow. e Distribution among different cellular pathways (y-axis) of: (blue) 333 genes that are associated with increased susceptibility to PrCR and (red) genes in the human genome. RNAseq analysis was performed on 4 groups of neutrophils, as described in d. RNAseq analysis was used to identify genes upregulated in the maturation process (BM to peritoneum) in both WT and MRP8-Bcl2 mice, which are likely associated with susceptibility to PrCR

Fig 4: Mll-AF4+VEC-Cre+ aging mice have a higher proportion of CD3+ T-lymphoid cells in the spleen. (A) Experimental layout. Proportions of (B) CD11b+Gr1+, (C) B220+CD19+IgM+, (D) B220+CD19+IgM–, and (E) CD3+ cells in the peripheral blood of the control and Mll-AF4+VEC-Cre+ aged cohort at two time points. Proportions of (F) CD11b+Gr1+, (G) B220+CD19+IgM+, (H) B220+CD19+IgM–, and (I) CD3+ in the bone marrow and spleen of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months old). Peripheral blood counts of (J) white blood cells, (K) red blood cells, and (L) platelets of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months). Statistical differences were assessed using a nonparametric Mann–Whitney U test with a bilateral p value (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 5: MHC class II+ ILC3s limit airway Th17 cell responses to microbes upon HDM exposure(A) Representative plots and quantitative graphs of the frequency and number of Th17 cells in lung parenchyma and MedLN from H2-Ab1fl/fl control mice and MHC class II?ILC3 mice after exposure to HDM or PBS. Cells were gated on live CD45+ CD19- CD3/CD5+ CD4+ (data pooled from 4 independent assays, n = 6).(B) Quantitative graph of the number of neutrophils in lung parenchyma from mice in (A). Neutrophils were identified as live CD45+ CD19- TCRß- CD11b+ Ly6G+ (data pooled from 4 independent assays, n = 7).(C) ELISA analysis for IL-5 and IL-17A levels from supernatants of microbiota-extract-restimulated MedLN cells from HDM-exposed MHC class II?ILC3 mice (data pooled from 2 independent assays).(D) Quantitative graphs of the number of Th17 cells in lung parenchyma and MedLNs from H2-Ab1fl/fl control mice and MHC class II?ILC3 mice after exposure to sterile papain (data pooled from 2 independent assays, n = 4).(E) Bar graphs of major bacterial composition in HDM from four different vendors. Data are represented as mean ± SEM. Statistics are calculated by two-way ANOVA. See also Figure S4.