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-Cyanine7 KLRG1 Monoclonal Antibody (2F1) , eBioscience , Cat# 25-5893-82; RRID: AB_1518768.

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-Cyanine7 KLRG1 Monoclonal Antibody (2F1) , eBioscience , Cat# 25-5893-82; RRID: AB_1518768.

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