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: Defective ILC3 function in IRF4-deficient mice drives C. rodentium and C. albicans infection in a cell-intrinsic manner (A–F) NCG mice were adoptively transferred with 80,000 intestinal ILC3s (Lin − CD127 + CD27 − KLRG1 − ) sorted from the small intestine of Irf4 f/f and Irf4 f/f Rorc cre mice or PBS as control after being treated with ABX for 1 week. ILC3s were stimulated with IL-23 and IL-1β for 30 min before injected into NCG mice through the tail vein. NCG mice were orally inoculated with C. rodentium 24 h after adoptive transfer. (A, B) Measurements (A) and statistical analysis (B) of the colon lengths from NCG recipients ( n = 4 control, n = 5 WT transferred, n = 7 KO transferred). (C) CFUs in the feces of NCG recipients 9 days after infection ( n = 4 control, n = 5 WT transferred, n = 7 KO transferred). (D) Changes in body weight were recorded at the indicated time points ( n = 4 control, n = 5 WT transferred, n = 7 KO transferred). (E) H&E staining of colon tissue sections. (F) Pathological score of colon histology. (G–L) Irf4 f/f or Irf4 f/f Rorc cre ILC3s were adoptively transferred into NCG mice with C. albicans infection. (G and H) Measurements (G) and statistical analysis (H) of the colon lengths from NCG recipients ( n = 2 control, n = 4 transferred). (I) Fecal fungal burden ( n = 3 control, n = 4 transferred). (J) Variations of body weight were shown each day ( n = 4). (K) Pathological score of colon histology. (L) Histological analysis of colonic tissues by H&E staining. The data are representative of at least two independent experiments (A–L). Bar graphs are presented as mean ± SEM. A two-tailed Student’s t test was performed for comparisons. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also .

Article Snippet: PE CD127 Monoclonal Antibody (A7R34) , eBioscience , Cat# 12-1271-82; RRID: AB_465844.

Techniques: Infection, Control, Injection, Adoptive Transfer Assay, Staining, Two Tailed Test

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: IRF4 deficiency compromises MHC class Ⅱ expression and further restrains ILC-mediated apoptosis of effector CD4 + T cells both in vitro and in vivo (A) Violin plots visualizing the expression of MHC-class-Ⅱ-related signature genes. (B) FACS analysis and MFI of MHC class Ⅱ expression in ILC3s isolated from Irf4 f/f and Irf4 f/f Rorc cre mice ( n = 6). ILC3 subsets were gated as Lin − RORγt + and then CCR6 + NKp46 − , CCR6 − NKp46 + , or CCR6 − NKp46 − . The lineage cocktail included TCRγδ, CD3ε, CD19, CD5, CD11c, Gr-1, and Ter119. (C and D) Activated OT-Ⅱ CD4 + T cells were cultured ex vivo with purified ILC3s from the siLP of Irf4 f/f and Irf4 f/f Rorc cre mice in the presence or absence of Ova peptide or the anti-MHC class Ⅱ neutralizing antibody. (C) Quantification of OT-Ⅱ T cells recovery (%). (D) Quantification of Annexin-V + OT-Ⅱ T cells. (E and F) Naive CD4-positive T cells (gating as CD4 + CD25 − CD62L hi CD44 lo cells) were sorted from OT-Ⅱ mice and pre-activated overnight. After pre-activation, CD4 positive T cells were transplanted into recipient Irf4 f/f and Irf4 f/f Rorc cre mice along with OVA peptide administration every 2 days following transfer. Nine days later, mice were sacrificed for further analysis of OT-Ⅱ CD4 + T cells (gating as CD8 − CD4 + TCRβ + Vβ5 + ) transferred in the spleen, mLN, siLPL, and cLPL of recipient mice. (G) One hundred thousand intestinal ILC3s (Lin − CD127 + CD27 − KLRG1 − ) were sorted from Irf4 f/f or Irf4 f/f Rorc cre mice and transferred with 500,000 activated OT-ⅡCD4 + T cells into NCG mice, flowing by OVA peptide i.p. every 2 days. Nine days later, survived OT-ⅡCD4 + T cells were quantified. ( n = 2 APC, n = 5 transferred group). 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 (B–G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also .

Article Snippet: PE CD127 Monoclonal Antibody (A7R34) , eBioscience , Cat# 12-1271-82; RRID: AB_465844.

Techniques: Expressing, In Vitro, In Vivo, Isolation, Cell Culture, Ex Vivo, Purification, Activation Assay, Two Tailed Test

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 CD127 Monoclonal Antibody (A7R34) , eBioscience , Cat# 12-1271-82; RRID: AB_465844.

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

Journal: iScience

Article Title: PARP1-NFATc1-PD1 pathway of maturation and stability of CD8 + T cells is beneficial against chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2025.112926

Figure Lengend Snippet: Splenic CD8 + T cell activation profile in mice infected with T. cruzi (±PARP1) Splenocytes of non-infected and chronically-infected WT and Parp1 −/− mice were labeled with fluorescent-conjugated antibodies and analyzed by flow cytometry. (A–E) CD3 + CD8 + T cells were examined for surface expression of CD25, CD27, CD44, CD62L, and CD127 to differentiate effector (Teff) subsets (early: Teff-E, intermediate: Teff-I, late: Teff-L) and memory/effector memory (Tmem) subsets (central memory: Tcm, early: Tem-E, late: Tem-L). The tSNE plots, generated with non-linear reduction method to visualize the color-coded CD8+T cell subsets, are shown in A. Splenic percentages of CD8+Teff (total, Teff-E, Teff-I, Teff-L; B and C) and CD8 + Tmem (total, Tcm, Tem-E, Tem-L; D and E) subsets are shown. (F–L) Each CD8 + T subset was analyzed for intracellular markers by flow cytometry. The tSNE plots visualizing CD8 + Teff and Tmem subsets producing IFNγ, perforin (PRF1) and granzyme B (GZM) are shown in F. Percentage frequencies of CD8 + Teff-E (G), Teff-I (H), Teff-L (I), Tcm (J), Tem-E (K), and Tem-L (L) subsets producing IFNγ, PRF1, and GZM are shown. In bar graphs, individual data point and mean ± SEM values ( n ≥ 20-mice/group for A–E, and n = 5-mice/group for F–L) are plotted. Significance (∗ p value ≤0.05) two groups was analyzed by Student’s unpaired t test or Mann-Whitney U test and identified by a horizontal bar. Gating strategy detailed data , and schematics of development of T cell response in Parp1 −/− -vs-WT mice infected with T. cruzi are presented in supplementary file.

Article Snippet: PE-Cyanine7 Rat anti-mouse CD127 , ThermoFisher/eBioscience , RRID: AB_2573394 ; 25-1273-82, SB/199, 569/780.

Techniques: Activation Assay, Infection, Labeling, Flow Cytometry, Expressing, Generated, MANN-WHITNEY

Journal: iScience

Article Title: PARP1-NFATc1-PD1 pathway of maturation and stability of CD8 + T cells is beneficial against chronic Trypanosoma cruzi infection

doi: 10.1016/j.isci.2025.112926

Figure Lengend Snippet: Metabolic status of CD8 + T lymphocytes in mice infected with T. cruzi (±PARP1) (A–L) Splenic cells of WT and Parp1 −/− mice (± Tc for 150-days) were labeled with fluorescent-conjugated antibodies for CD3, CD4, CD8a, CD27, CD44, CD62L, and CD127 antigens along with fluorescence probes for lipids, thiols, and glucose. Cells were acquired by flow cytometry and analyzed by FlowJO software. Histograms and median fluorescence intensities (MFI) of lipids-, thiols-, and glucose-metabolizing CD8 + Teff total (A–C), Teff-E (D), Teff-I (E), Teff-L (F), Tmem total (G–I), Tcm (J), Tem-E (K), and Tem-L (L) subsets are shown. In bar graphs, individual data point and mean ± SEM values are plotted from n = 5-mice/group (duplicate recordings/sample). Significance (∗ p value ≤ 0.05) comparing two groups was analyzed by Student’s unpaired t test or Mann-Whitney U test and identified by a horizontal bar. Detailed data are available in .

Article Snippet: PE-Cyanine7 Rat anti-mouse CD127 , ThermoFisher/eBioscience , RRID: AB_2573394 ; 25-1273-82, SB/199, 569/780.

Techniques: Infection, Labeling, Fluorescence, Flow Cytometry, Software, MANN-WHITNEY