Fig 2: Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit+) cells from WT and Becn1vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34-LSK) from WT and Becn1vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit+ cells were treated with ABT263 (10 µM) plus S63845 (10 µM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit+ cells with or without apoptosis induction. WT c-Kit+ cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 µM) plus S63845 (10 µM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 105 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry+ cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 105 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.

Fig 3: AID Reporter System Identifies LCMV-Specific B Cells in a Polyclonal Response(A–C) We infected AIDrep mice with rCl13 or VSV on day 0 or left them uninfected. TAM was administered on days 0, 5, and 10, as schematically shown in (A). LCMV-NP binding by splenic EYFP+ GL7+ B cells was analyzed on day 50 (B and C). Panel (B) shows a representative FACS plot with numbers indicating the percentage of gated cells, as quantified in (C). For gating strategy, see Figure S2.(D–F) We infected AIDrep mice with rCl13 or Vacc on day 0 and treated them with tamoxifen on days 0, 5, and 10, as schematically shown in (D). On day 30, we purified B cells from spleen by magnetic cell separation and transferred them into syngeneic C57BL/6 recipients, which had been infected with rCl13 6 days before (day 24) or with Vacc 3 days earlier (day 27). Six days after transfer (day 36), we measured the expansion and plasma cell differentiation of proliferated (CTVlo) adoptively transferred EYFP-reporting B cells in the spleen (E and F). Two distinct EYFP+ ASC populations in (E) correspond to different stages of maturation. EYFP-reporting cells were enumerated in (F). Representative FACS plots are gated on EYFP+ B220+ CD138- GL7+ B cells (B) and on recipient (CD45.2+) lymphocytes in black with EYFP+ CTVlo donor cells overlaid in green (E), respectively (see Figure S2). Numbers in FACS plots represent percentages of gated cells among EYFP+ B220+ CD138- GL7+ B cells.Bars represent means ± SEM, n = 3–4 (C) and n = 4 (F). N = 2. One-way ANOVA with Bonferroni’s post-test for multiple comparisons (C). Two-way ANOVA with Bonferroni’s post-test for multiple comparisons (F). **p < 0.01 comparing B cells; ##p < 0.01 comparing ASCs, respectively. See also Figure S2.

Fig 4: Effects of the Npc1 loss-of-function mutation in hematopoietic cells on blood, splenic, and bone marrow myeloid cells in WTD-fed Ldlr-/- mice. Ldlr-/- mice were transplanted with Npc1wt or Npc1mut BM and fed a chow diet for 9 weeks, followed by WTD for 9 weeks. A: Total monocytes, monocyte subsets, and neutrophils in blood from mice fed WTD for 4 weeks, assessed by flow cytometry. BM (B and C) and spleen (D and E) were collected after 9 weeks of WTD feeding, and monocytes and neutrophils were assessed by flow cytometry. B and D: Representative flow cytometry plots of monocytes and neutrophils as percentage of total CD45+ leukocytes and Ly6Clo and Ly6Chi monocyte subsets as percentage of CD115+ monocytes in BM (B) and spleen (D). Neu denotes neutrophils. Total monocytes, monocyte subsets, and neutrophils in BM (C) and spleen (E). n = 15–16 mice per genotype. Data are shown as mean ± SEM. ***P < 0.001 by t-test.

Fig 5: DL1-NOTCH2 Optimizes the Differentiation of cDC1s from the BMTotal murine BM cells were cultured in the presence of FLT3L alone (FL), FLT3L with control OP9 cells (FL+OP9) or FLT3L with OP9 cells expressing Notch ligand DL1 (FL+Notch). OP9 cells were added on day 3, and BM cells were analyzed on day 7 of differentiation.(A) Representative staining plots of differentiated BM cells. The top row shows total live cells with B220+ CD11clo pDCs and B220- CD11chi cDCs highlighted; the other rows show gated cDCs with CD11bhi cDC2s and/or CD11b- cDC1s highlighted.(B) The expression of CD8a on gated cDCs in BM cultures differentiated as in (A).(C) The subset composition of differentiated BM cells. Shown are fractions of pDCs (of total live cells) and cDC subsets (of gated B220- CD11c+ MHC class II+ cDCs) and the absolute number of these subsets per 105 initial BM cells. cDC1s were defined either as CD24+ or CD8a+. Data points represent values from BM cultures of individual mice pooled from 2 experiments; bars represent mean.(D) Representative expression of the indicated surface markers on gated CD24+ cDC1s from FL or FL+Notch cultures. The expression of cDC1 markers on cDC2s is included as a control.(E) Representative expression of CD11c and MHC class II on DC subsets from FL or FL+Notch cultures. Subsets were gated, omitting the marker that is shown in each case.(F) Representative expression of Esam on cDC2s from FL or FL+Notch cultures. The expression on cDC1s is included as a control; the dotted line represents negative staining.(G) The effect of NOTCH2 blockade on the expression of CD8a on cDC1s and of Esam on cDC2s. BM cells were differentiated in FL or FL+Notch cultures in the presence of control immunoglobulin G (IgG) or anti-N2; the fluorescence intensity of marker expression in the indicated subsets is shown. Data represent mean ± SD of 5 parallel cultures for anti-N2 and IgG and 9 cultures pooled from 2 experiments for controls.(H) The effect of transcription factor deletion on DC differentiation. BM from control wild-type (WT) mice or mice with DC-specific deletion of Irf8 (Irf8 conditional knockout, cKO) or with germline deletion of Irf8 or Batf3 were cultured in FL or FL-Notch cultures. Shown is the fraction of the indicated DC subsets among total live cells; data points represent values from individual mice pooled from three (for Irf8) or two (for Batf3) experiments; bars represent mean.Statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05.