Fig 1: HpBARI binds to murine ST2, blocking IL-33 ligation.(A) HpBARI or HpARI (1 µg/ml each) were coated onto wells of an ELISA plate, followed by addition of mouse ST2-Fc (ST2) (top panel) or mouse IL-33 TRAP (bottom panel) or IgG controls, followed by detection using anti-human IgG-HRP. Representative of 3 repeat experiments. Two-way ANOVA comparing HpBARI-mST2 to HpARI-mST2. Error bars show SEM. (B) Mouse ST2-Fc, mouse IL-33 TRAP or IgG were bound to protein G-coated beads, and used to immunoprecipitate HpBARI. Anti-myc western blots shown. Representative of 2 repeat experiments. (C–D) Bone marrow cells of wild type (C–D) or ST2-deficient mice (C) were cultured for 5 days with IL-2, IL-7 and IL-25 (all 10 ng/ml), then incubated with biotinylated HpARI or HpBARI (0.1 µg/ml) for 20 min at 4°C, followed by detection of biotin with avidin-PE. Representative plots gated on ICOS+lineage–CD45+ ILC2. In (D), where indicated samples were treated with HES (10 µg/ml) or unlabelled HpBARI (10 µg/ml) for 20 min prior to HpBARI staining (D). Representative of at least two repeat experiments. (E) Surface plasmon resonance of HpBARI binding to chip-coated mouse ST2 (left panel) or mouse IL-33 TRAP (right panel). (F) Anti-IL-33 western blot, after immunoprecipitation of mouse IL-33 with mouse ST2-Fc or mouse IL-33 TRAP-Fc on protein G dynalbeads, in the presence of HpBARI or heat-treated HpBARI (HT). Representative of 2 repeat experiments.
Fig 2: HpBARI suppresses ST2 detection, and suppresses IL-33 responses in vitro.(A) Naive murine lung cells were cultured at 37°C overnight with HES or recombinant HpBARI, and ST2 expression measured by flow cytometry. (B–D) Naive murine bone marrow cells cultured for 5 days with IL-2, IL-7 and IL-33 (all at 10 ng/ml) followed by ELISA of cell-free supernatants for IL-5 (B), IL-6 (C) and IL-13 (D). Dotted line indicates levels with IL-2, IL-7 and IL-33 alone. All data are representative of >3 repeat experiments, with three technical replicates per measurement. Error bars show SEM.
Fig 3: Pcgf1-deficient hematopoietic stem and progenitor cells (HSPCs) undergo myeloid reprogramming.(A) Principal component analyses (PCA) based on total gene expression obtained by RNA-seq of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), pre-GM, and granulocyte-macrophage progenitors (GMPs) from control and Pcgf1?/? mice. Magnified view of the boxed part is depicted on the right. (B) Gene set enrichment analysis (GSEA) using RNA-seq data. Summary of GSEA data of representative gene sets is shown. Normalized enrichment scores (NES), nominal p-values (NOM), and false discovery rates (FDR) are indicated. The gene sets used are indicated in Supplementary file 1. (C) Quantitative RT-PCR analysis of Cebpa in LSK cells and GMPs. Hprt1 was used to normalize the amount of input RNA. Data are shown as the mean ± SEM (n = 3). ***p<0.001 by the Student’s t-test. (D) Effects of Cebpa overexpression on HSPC differentiation in vitro. LSK cells were transduced with either control (GFP) or Cebpa retrovirus, then cultured on TSt-4 stromal cells in the presence of IL-7 (upper). The proportions of myeloid (Mac1+ and /or Gr-1+), B cells (CD19+), and others (Mac1-Gr-1-CD19-) among CD45.2+GFP+ hematopoietic cells on day 17 of culture are indicated (lower left; n = 4 each). RT-qPCR analysis of Cebpa in LSK cells transduced with control or Cebpa retrovirus on day 14 of culture (n = 3). Hprt1 was used to normalize the amount of input RNA (lower right). Each symbol is derived from an individual culture. Data are shown as the mean ± SEM. **p<0.01, ***p<0.001 by the Student’s t-test. (E) Impact of Cebpa haploinsufficiency on myeloid-biased differentiation of Pcgf1?/? HSPCs. LSK cells from Rosa26CreERT, Rosa26CreERT;Pcgf1fl/fl and Rosa26CreERT;Pcgf1fl/fl;Cebpafl/+ mice were treated with 4-OHT (200 nM) for 2 d in culture to delete Pcgf1 and Cebpa. Cells were further cultured on TSt-4 stromal cells in the presence of IL-7 (upper). The proportions of myeloid (Mac1+ and /or Gr-1+), B cells (CD19+), and others (Mac1-Gr-1-CD19-) among CD45.2+GFP+ hematopoietic cells on day 17 of culture are indicated (lower; n = 4 each;* versus control). Each symbol is derived from an individual culture (lower left). RT-qPCR data of Cebpa in LSK cells on day 14 of culture (n = 3). Hprt1 was used to normalize the amount of input RNA (lower right). Data are shown as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by the Student’s t-test (lower left) or the one-way ANOVA (lower right). Figure 2—source data 1.Raw data for Figure 2.
Fig 4: reMAIT cell differentiation from MAIT-iPSCs on OP9/DLL1 monolayer(A) OP9/DLL1 monolayer on day 4 after seeding on gelatin-coated dish. This picture shows optimal confluency for initiation of reMAIT cell differentiation.(B) After the medium change on day 3, a rapid morphological change is observed, and by day 5, mesodermal cell masses with raised center are formed.(C) Mesodermal cells differentiate into lymphoblasts on new OP9/DLL1 in the presence of FLT3-L (Day 8). Lymphoblasts have a grape cluster-like morphology.(D) Lymphoblasts differentiate into reMAIT cells and reMAIT cells are expanding in the presence of FLT3-L and IL-7.(E) The proliferating reMAIT cells can be transferred onto a new OP9/DLL1 monolayer (10-cm culture dish) from 6-well plate and let them grow until they cover the entire surface of OP9/DLL1. (A-E) Each bar in the images indicates 200 μm.(F) Almost all T cells induced from MAIT cell-derived iPSCs are MR1-tetramer positive and TCRβ+ cells (but not recognized by 6-FP-loaded MR1 tetramer).
Fig 5: High-GATA3 expression induced by G3SE is essential for the development from ILCP to ILC2.a Flow cytometry analysis of BM cells. Frequencies and numbers of CLPs (Lin−CD127+CD135+LPAM-1− cells), PD-1−ST2− (Lin−CD127+CD135−LPAM-1+PD-1−ST2− cells), IL17RB−PD-1+ (Lin−CD127+CD135−LPAM-1+PD-1+ST2−IL17RB− cells), IL17RB+PD-1+ (Lin−CD127+CD135−LPAM-1+PD-1+ST2−IL17RB+ cells), and ST2+ ILC2s (Lin−CD127+CD135−LPAM-1+PD-1−ST2+ cells) were evaluated; n = 6. Lin: CD3ε, CD4, CD5, CD8a, CD11b, CD11c, CD19, NK1.1, Gr1, B220, Ter119. b, c Sorted ILCPs from the BM of WT mice and G3SEKO mice were cultured on OP9-DL1 cells in the presence of IL-7 and SCF for 5 days. Frequencies of Lin−CD25+PD-1− cells, Lin−CD25−PD-1− cells, Lin−CD25−PD-1+ cells (b), and Lin−CD25+GATA3+ cells (c) were evaluated, n = 3. d ILCPs sorted from the BM of WT mice and G3SEKO mice were cultured on OP9-DL1 cells. Cells were infected with GATA3-expressing or empty retrovirus on day 1. The infected cells (Thy1.1+ cells) were analyzed on day 6. WT, G3SEKO+empty-RV: n = 5, G3SEKO+Gata3-RV: n = 4. Each data point indicates one mouse from six (a) or three (b–d) independent experiments. Data are presented as mean ± SE. Statistical analysis was performed using unpaired, two-sided Welch’s t-test. p values are shown on the graphs. Source data are provided as a Source Data file.
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