Journal: iScience

Article Title: The transcription factor IRF4 regulates the homeostasis and function of intestinal ILC3s

doi: 10.1016/j.isci.2025.112800

Figure Lengend Snippet: Cleavage under targets and tagmentation sequencing of IRF4 binding in intestinal ILC3 subsets (A) Heatmap showing the genome-wide distribution of IRF4-binding signals at peak centers in ILC3 subsets sorted from Irf4 f/f and Irf4 f/f Rorc cre mice by CUT&Tag. (B) Occupancy of IRF4 at all gene promoter regions (±5 kb of TSS). (C) Donut chart showing the percentages of IRF4 binding at exon regions, intron regions, or intergenic regions. (D) Venn plot displaying the overlap of the IRF4-regulated genes from pairwise comparisons of NKp46 + ILC3s, NKp46 − CCR6 − ILC3s, and CCR6 + ILC3s. (E) Gene set enrichment analysis (GSEA) of NKp46 + ILC3 signature gene sets enriched in shared IRF4-modified genes. (F and G) IGV visualizes the indicated gene locus containing ATAC-seq and IRF4-binding peaks in ILC3 subsets. IRF4 CUT&Tag sequencing data are from two independent replicates. (H–K) Rescue experiments. Retroviruses were generated by transfection of pMX-IRES-GFP plasmids containing the indicated genes into Plat-E cells using PolyJet. CLPs (Lin − CD127 + c-Kit int Sca-1 int Flt3 + ) were sorted from the bone marrow from Irf4 f/f and Irf4f/f Rorc cre mice and transfected with retroviral supernatants. Retrovirus-transfected CLPs were then collected and adoptively transferred into sublethally irradiated CD45.1 + wild-type recipient mice through intravenous tail vein injection. Transduced cells were transferred with CD45.1 + wild-type bone marrow cells to help the engraftment of the CLPs. After 2 weeks, recipient mice were sacrificed, and organs were collected for analysis. (H) Flow cytometry of CD45.2 + GFP + ILC3 subsets isolated from the siLP in the indicated recipient mice (upper). MHC class II expression of CD45.2 + GFP + ILC3 subsets isolated from the siLP in the indicated recipient mice (lower). (I) Cytokine production in siLP CD45.2 + GFP + ILC3s from the indicated recipient mice. (J) The percentages of the indicated subsets were compared. (K) The percentages of MHC class II + ILC3s, IL-17A + ILC3s, and IL-22 + ILC3s were compared. Bar graphs are presented as mean ± SEM. A two-tailed Student’s t test was performed for comparisons. The data are representative of at least three independent experiments. See also .

Article Snippet: PE-Cyanine5 CD135 (Flt3) Monoclonal Antibody (A2F10) , eBioscience , Cat# 15-1351-82; RRID: AB_494219.

Techniques: Sequencing, Binding Assay, Genome Wide, Modification, Generated, Transfection, Retroviral, Irradiation, Injection, Flow Cytometry, Isolation, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Suppression of melanoma by mice lacking MHC-II: Mechanisms and implications for cancer immunotherapy

doi: 10.1084/jem.20240797

Figure Lengend Snippet: H2-Aa –deficient cDC2 are necessary for melanoma inhibition in H2-Aa cit/cit mice. Tumor growth curves of B16F10 melanoma after s.c. inoculation on day 0 into the flank of mice. No PD1 antibody was administered. (A and B) Lethally irradiated WT or H2-Aa cit/cit (cit) recipients of WT (CD45.1), or H2-Aa cit/cit ( cit , CD45.2), or a 1:1 mixture of H2-Aa cit/cit and WT bone marrow were inoculated with B16F10 cells 12 wk after bone marrow transfer ( n = 7 or 8 recipients per group). (C–F) Mice in which H2-Aa was deleted in (C) DC ( H2-Aa flox/flox ;Cd11c-cre), (D) B cells ( H2-Aa flox/flox ;Cd19-cre), (E) macrophages ( H2-Aa flox/flox ;LysM-cre), or (F) cDC1 ( H2-Aa flox/flox ;Xcr1-cre). (C–F, n = 5–10 mice per group). H2-Aa cit/cit ;Xcr1-cre mice in F were checked by flow cytometric analysis to confirm the absence of undesired H2-Aa KO in B cells (pre-experiment) and cDC2 cells (post-experiment) to exclude any H2-Aa germline deletion due to Xcr1-cre leakage ( ; ; ). Data are representative of two independent experiments (A–F). WT C57BL/6J mice from JAX (C–F) were used as controls. Error bars indicate SEM (A–F). P values were determined by two-way ANOVA with post-hoc Tukey test (A–F). *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: To stain the progenitors of DC, bone marrow was isolated and stained with antibodies against lineage markers (CD3, CD19, NK1.1, and Ter-119), CD11c, Flt3 (clone A2F10; eBioscience), Sirpα, Ly6C, and Siglec-H (clone 551; BioLegend) at a 1:200 dilution for 1 h at 4°C.

Techniques: Inhibition, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Suppression of melanoma by mice lacking MHC-II: Mechanisms and implications for cancer immunotherapy

doi: 10.1084/jem.20240797

Figure Lengend Snippet: H2-Aa cit/cit cDC2 have increased cross-priming activity. (A–D) Antigen uptake assays of BMDC ( n = 4 or 5 per group). Frequency of FITC-positive BMDC (A and B) or CellTrace Violet (CTV)-positive BMDC (C and D) after incubation with FITC-labeled OVA (A and B) or after co-culture with CTV-labeled B16F10 cells (C and D). BMDC were induced from bone marrow cells by GM-CSF (A and C) or Flt3L (B and D). (E and F) In vitro cross-priming by cDC2 (E) and cDC1 (F). Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture (3 days) with DC purified from draining lymph nodes on day 6 after inoculation of mice with B16F10-OVA tumors. cDC2 cells were sorted as Lin− CD45 + Ly6C− CD11c+ Xcr1− CD11b+ and cDC1 cells were sorted as Lin− CD45 + Ly6C− CD11c+ Xcr1+ CD11b−. Inset, ratio of peak area for G0, G1, G2, or G3/total peak area (G0+G1+G2+G3). (G) In vivo cross-priming by DC. WT and H2-Aa cit/cit mice were injected intravenously with equal amounts of CTV-labeled OT-I CD8 T cells, and 1 day later, recipients received OVA by i.p. injection. 4 days after OVA injection, splenocytes were collected and analyzed by flow cytometry. CTV mean fluorescence intensity (MFI, left), and the total number of OT-I CD8 T cells in the spleen (right). Three mice per group are shown (E–G). Data points represent individual mice (A–G). Data are representative of two independent experiments (A–G). WT littermates were used as controls (A–G). Error bars indicate SD (A–G). P values were determined by Student’s t test (A–G); no differences between genotypes were found in A–D and F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To stain the progenitors of DC, bone marrow was isolated and stained with antibodies against lineage markers (CD3, CD19, NK1.1, and Ter-119), CD11c, Flt3 (clone A2F10; eBioscience), Sirpα, Ly6C, and Siglec-H (clone 551; BioLegend) at a 1:200 dilution for 1 h at 4°C.

Techniques: Activity Assay, Incubation, Labeling, Co-Culture Assay, In Vitro, Purification, In Vivo, Injection, Flow Cytometry, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Suppression of melanoma by mice lacking MHC-II: Mechanisms and implications for cancer immunotherapy

doi: 10.1084/jem.20240797

Figure Lengend Snippet: Elevated cDC2 cross priming activity, cell counts, and MHC-I expression in H2-Aa cit/cit mice. (A) In vitro cross-priming by macrophages. Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture (3 days) with macrophages purified from draining lymph nodes on day 6 after inoculation of mice with B16F10-OVA tumors. Macrophages were sorted as Lin− CD45 + CD11b+ F4/80+. Inset: Ratio of peak area for G0, G1, G2, or G3/total peak area (G0+G1+G2+G3). (B and C) In vitro cross-priming by cDC2. CTV-labeled naïve OT-I CD8 T cells were co-cultured (3 days) with OVA (100 μg/ml) and cDC2 purified from Flt3L (100 ng/ml) induced WT or H2-Aa cit/cit BMDCs. (B) Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture. cDC2 were sorted as Lin− CD45 + Ly6C− CD11c+ CD11b hi Xcr1 lo . Right: Ratio of peak area for G0, G1, G2, G3, or G4/total peak area (G0+G1+G2+G3+G4). (C) ELISA analysis of IFNγ secreted by OT-I CD8 T cells after co-culture. (D) The total number of cDC1 in spleens of WT and H2-Aa cit/cit mice. (E) The frequencies of splenic cDC1 in mice of the indicated genotypes. (F) Representative flow cytometry plots of pre-cDC in the bone marrow. Bone marrow cells were gated on Lin− CD11c+ CD172a− Flt3+. Unc pre-cDC, uncommitted pre-cDC. (G) Intracellular Irf8 mean fluorescence intensity (MFI) in splenic cDC1. (H) Representative flow cytometry plots of the indicated cDC populations in mesenteric lymph nodes (MLN) of WT and H2-Aa cit/cit mice. Cells were gated on Lin− CD45 + Ly6C− CD11c+. (I) The total number of cDC2 in MLN of WT and H2-Aa cit/cit mice. (J) MHC-I and CD80 MFI on cDC2. (K) Frequency of H2-K b -SIINFEKL tetramer positive tumor infiltrated CD8 T cells in WT or H2-Aa cit/cit mice on day 9 after s.c inoculation with B16F10-OVA cells. Data points represent individual mice (A–E, G, and I–K). Data are representative of two independent experiments (A–K). WT littermates (A–D and F–K) and WT C57BL/6J (E) from JAX were used as controls. Error bars indicate SD (A–E, G, and I–K). P values were determined by Student’s t test (A–E, G, and I–K). n = 3 per group (A–C, F, and G), n = 4 per group (D, E, and H−K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To stain the progenitors of DC, bone marrow was isolated and stained with antibodies against lineage markers (CD3, CD19, NK1.1, and Ter-119), CD11c, Flt3 (clone A2F10; eBioscience), Sirpα, Ly6C, and Siglec-H (clone 551; BioLegend) at a 1:200 dilution for 1 h at 4°C.

Techniques: Activity Assay, Expressing, In Vitro, Labeling, Co-Culture Assay, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Suppression of melanoma by mice lacking MHC-II: Mechanisms and implications for cancer immunotherapy

doi: 10.1084/jem.20240797

Figure Lengend Snippet: H2-Aa deficiency increased cDC2 and altered their transcriptional program to promote cross-presentation. (A) Representative flow cytometry plots of the indicated cDC populations in spleens of WT and H2-Aa cit/cit mice. Cells were gated on Lin− CD45 + Ly6C− CD11c+. (B) The total number of cDC2 in spleens of WT and H2-Aa cit/cit mice. (C) MHC-I and CD80 mean fluorescence intensity (MFI) on cDC2. (D and E) Frequency (D) and MHC-I MFI (E) of cDC2 among tumor-infiltrating lymphocytes isolated from B16F10 melanomas collected on day 11 after B16F10 inoculation into the flank of mice. (F) Immunoblot analysis of Nlrc5 in lysates of panDC enriched from spleens of WT and H2-Aa cit/cit (cit/cit) mice. α-tubulin was used as a loading control. (G) Uniform Manifold Approximation and Projection (UMAP) clustering of scRNA-seq data from splenic DC sorted from 3 naïve H2-Aa cit/cit mice (right) and 3 naïve WT littermates (left), showing 10 color-coded clusters at a resolution of 0.2. (H) Proportion of each cell cluster identified in G. (I) KEGG pathway enrichment analysis of genes significantly increased in H2-Aa cit/cit relative to WT Ccr2+ cDC2 (adjusted P ≤ 0.05, n = 410). One-sided hypergeometric test was used to determine the statistical significance of enrichment. (J) Volcano plot showing differentially expressed genes in WT versus H2-Aa cit/cit (cit) Ccr2+ cDC2 (adjusted P ≤ 0.05, n = 854). Shaded areas contain genes with Log 2 Fold change (FC) > 0.4 and Log 2 FC less than −0.4. Data points represent individual mice with four mice per group (B–E). Data are representative of one experiment (G–J) or two independent experiments (A–F). WT littermates were used as controls (A–H). Error bars indicate SD (B–E). P values were determined by Student’s t test (B–E). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: .

Article Snippet: To stain the progenitors of DC, bone marrow was isolated and stained with antibodies against lineage markers (CD3, CD19, NK1.1, and Ter-119), CD11c, Flt3 (clone A2F10; eBioscience), Sirpα, Ly6C, and Siglec-H (clone 551; BioLegend) at a 1:200 dilution for 1 h at 4°C.

Techniques: Flow Cytometry, Fluorescence, Isolation, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Suppression of melanoma by mice lacking MHC-II: Mechanisms and implications for cancer immunotherapy

doi: 10.1084/jem.20240797

Figure Lengend Snippet: Analysis of the MHC-II monoclonal antibody HB12-18. (A and B) Anti-MHC-II (M5/114, 25 μg/g body weight) or control IgG were injected i.p. into WT mice on days 0, 4, 7, 10, 13, and 16 after B16F10 inoculation on day 0 into the flank of mice ( n = 5 per group). (A) Tumor growth curve. (B) Frequencies of myeloid DC (CD11b+CD11c+) in the peripheral blood on day 15. (C) Splenocytes from C57BL/6J, H2-Aa cit/cit , or BALB/cJ mice were incubated with different concentrations of HB12-18 ( n = 3 per group). MHC-II expression (HB12-18 reactivity) on CD19 + B cells was determined by flow cytometry. MFI, mean fluorescence intensity. (D) WT (CD45.1) mice were injected i.v. with CellTrace Violet (CTV)-labeled OT-I CD8 T cells (CD45.2) and CellTrace Far Red (CTF)-labeled OT-II CD4 T cells (CD45.2), and 1 day later, recipients were mock injected (left panel), or injected i.p. with OVA+IgG (middle panel) or OVA+HB12-18 (200 μg/mouse, right panel) ( n = 3 per group). Representative flow cytometry plots of CTV-positive OT-I CD8 T cells (CD45.2) and CTF-positive OT-II CD4 T cells (CD45.2) in the spleens of WT recipients (CD45.1) 4 days after immunization. Data points represent individual mice (B). Data are representative of two independent experiments (A–D). Error bars indicate SD (B and C) or SEM (A). P values were determined by Student’s t test (B) or two-way ANOVA with post-hoc Tukey test (A); no difference between treatments was found in A. ****P < 0.0001.

Article Snippet: To stain the progenitors of DC, bone marrow was isolated and stained with antibodies against lineage markers (CD3, CD19, NK1.1, and Ter-119), CD11c, Flt3 (clone A2F10; eBioscience), Sirpα, Ly6C, and Siglec-H (clone 551; BioLegend) at a 1:200 dilution for 1 h at 4°C.

Techniques: Control, Injection, Incubation, Expressing, Flow Cytometry, Fluorescence, Labeling

Journal: Aging Cell

Article Title: Rejuvenation of the reconstitution potential and reversal of myeloid bias of aged HSCs upon pH treatment

doi: 10.1111/acel.14324

Figure Lengend Snippet: Reagents and tools table.

Article Snippet: Anti‐Flk2 , eBioscience , clone A2F10.

Techniques: Recombinant, Sequencing, Suspension, DNA Library Preparation, Modification, Software