Journal: iScience

Article Title: Single mutation tunes IRF4 function and mediates B cell character to ameliorate murine lupus

doi: 10.1016/j.isci.2025.113884

Figure Lengend Snippet: IRF4 K123R mutation in B cells ameliorated the SLE murine model Isolated splenic B cells (4 × 10 7 ) from WT or IRF4 K123R mice were co-transferred with isolated splenic T cells (2 × 10 7 ) from WT mice intravenously into RAG1 −/− mice recipients. ALD-DNA-stimulated BMDCs were injected into the reconstituted mice through tail veil. T WT B WT means WT T cells + WT B cells, and T WT B IRF4K123R means WT T cells + IRF4 K123R B cells. (A) Schematic of the RAG1 −/− mice reconstituted. (B) Serum anti-dsDNA IgG at different time points between the T WT B WT and T WT B IRF4K123R groups were measured by ELISA assay. (C) Proteinuria level at different time points was measured by colorimetry using urine protein test strip. (D) Kidneys were collected at week 20 and subjected for H&E, Masson, and periodic acid-Schiff (PAS) staining to correspondingly analyze pathological lesions, interstitial fibrosis, and glomerular damage. (E) Immunofluorescence showed the deposition of IgG and complement C3 in renal sections of the two groups. n = 5 in each group. The results are presented as the mean ± SEM from three separate experiments. ns, no significance; ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001 using nonparametric Mann-Whitney tests. Scale bars, ×400: 50 μm.

Article Snippet: IRF4 Rabbit mAb , CST , Cat#15106 RRID: AB_2798709.

Techniques: Mutagenesis, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Stripping Membranes, Staining, Immunofluorescence, MANN-WHITNEY

Journal: iScience

Article Title: Single mutation tunes IRF4 function and mediates B cell character to ameliorate murine lupus

doi: 10.1016/j.isci.2025.113884

Figure Lengend Snippet: IRF4 K123R mutation in B cells weaken the immune response in the SLE murine model Flow cytometry analysis of the immune cells from the peripheral blood of T WT B WT and T WT B IRF4K123R reconstituted lupus mice. (A) B cell and T cell subsets. (B) Immune functional markers of B cells. (C) Proportion of CD138 + plasmacytes in B cells. (D) Proportion of inhibitory markers, Tregs, and T helper cells in CD4 + T cells. The results are presented as the mean ± SEM from three separate experiments. ns, no significance; ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001 using nonparametric Mann-Whitney tests.

Article Snippet: IRF4 Rabbit mAb , CST , Cat#15106 RRID: AB_2798709.

Techniques: Mutagenesis, Flow Cytometry, Functional Assay, MANN-WHITNEY

Journal: iScience

Article Title: Single mutation tunes IRF4 function and mediates B cell character to ameliorate murine lupus

doi: 10.1016/j.isci.2025.113884

Figure Lengend Snippet: IRF4 K123R mutation reduced the immune activity of B cells (A) WT or IRF4 K123R B cells were labeled with CFSE and stimulated with LPS, and B cell proliferation was analyzed by flow cytometry. (B) Plasmacyte differentiation after LPS stimulation. (C) ELISA analysis of the anti-dsDNA IgG level of WT and IRF4 K123R B cells with or without effector T cells. (D) ELISA analysis of the cytokines (IFN-γ, IL-10, and TGF-β) from B cell supernatant. (E–F) Semi-mature activated B cells were cocultured with CFSE-labeled CD4 + T cells, in addition with CD3 agonist antibodies for 3 days. Flow cytometry was used to analyze the proliferation of CD4 + T cells (E), and cytokines of co-culture supernatant were analyzed by ELISA (F). (G) WT or IRF4 K123R B cells were stimulated with different LPS concentrations for 24 or 48 h, and B7-H1 expression was analyzed. (H) Semi-mature activated B cells were cocultured with naive CD4 + T cells under Treg polarizing condition with or without B7-H1 blocking antibody for 3 days, and Treg differentiation was analyzed by flow cytometry. (I) Semi-mature activated B cells were cocultured with CFSE-labeled CD4 + T cells, in addition to CD3 agonist antibodies with or without B7-H1 blocking antibody for 3 days. Flow cytometry analysis was used to analyze CD4 + T cell proliferation. The results are presented as the mean ± SEM from three separate experiments. ns, no significance; ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001 using nonparametric Mann-Whitney tests.

Article Snippet: IRF4 Rabbit mAb , CST , Cat#15106 RRID: AB_2798709.

Techniques: Mutagenesis, Activity Assay, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Blocking Assay, MANN-WHITNEY

Journal: iScience

Article Title: Single mutation tunes IRF4 function and mediates B cell character to ameliorate murine lupus

doi: 10.1016/j.isci.2025.113884

Figure Lengend Snippet: IRF4 K123R mutation moderated SLE depending on B7-H1 signaling (A–C) WT and IRF4 K123R B cells were stimulated with LPS for 24 h. (A) qPCR analysis of STAT3 and B7-H1. (B) Western blotting analysis of total/phosphorylated STAT3 and IRF4. (C) qPCR analysis of STAT3 and B7-H1 in B cells pretreated with or without STAT3 inhibitor. (D) B7-H1 expression in B cells pretreated with or without STAT3 inhibitor was analyzed by flow cytometry. (E–G) STAT3 inhibitor pretreated or untreated WT or IRF4 K123R B cells (4 × 10 7 ) and WT T cells (2 × 10 7 ) were intravenously injected into RAG1 −/− mice. ALD-DNA-activated BMDC was injected into the reconstituted mice through tail veil. (E). Serum anti-dsDNA IgG was measured by ELISA at week 20. (F). Proteinuria level was analyzed at week 20. (G). The deposition of IgG and C3 in renal sections was analyzed by immunofluorescence at week 20. (H) IRF4, STAT3, or phosphorylated STAT3 were pulled down from B cells by co-immunoprecipitation experiments, and western blotting was used to analyze indicated protein conjunctions. (I) Schematic of the working hypothesis. n = 5 in each group of reconstituted murine SLE model in (E–G). The results are presented as the mean ± SEM from three separate experiments. ns, no significance; ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001 using nonparametric Mann-Whitney tests. Scale bars, ×400: 50 μm.

Article Snippet: IRF4 Rabbit mAb , CST , Cat#15106 RRID: AB_2798709.

Techniques: Mutagenesis, Western Blot, Expressing, Flow Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Immunoprecipitation, MANN-WHITNEY