Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: Design and characterization of mouse T cell-targeted TGF- β agonists. a. Schematic of the plasmid construct encoding the MSA–TGM1-targeting-arm fusion protein. b. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. c. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. d. Representative flow cytometry plots showing mouse CD4 and CD8α expression in mouse CD4- or CD8α-transduced HEK293 cells. e. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. f. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. g. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. h. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. i. Dose-dependent curves showing Foxp3 cell percentages in in vitro -cultured CD4 T cells, and Granzyme B–producing cell percentages in in vitro -cultured CD8 T cells treated with the indicated proteins on day 3. Data are presented as mean ± s.e.m.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Plasmid Preparation, Construct, SDS Page, Molecular Weight, Recombinant, Flow Cytometry, Expressing, In Vitro, Cell Culture

Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: a. Frequencies of immune cell subsets. b. SCENIC prediction of IRF4 and Blimp1 transcription factor activity. c. Immunoglobulin gene expression. d. UMAP plots showing cell cycle gene signature scores. e. Quantification of cell cycle gene signature scores. f. Scaled signature scores for TGF-β–inducible genes, senescence genes, and apoptosis-related genes, aggregated by sample.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Activity Assay, Gene Expression

Journal: STAR Protocols

Article Title: Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq

doi: 10.1016/j.xpro.2025.104184

Figure Lengend Snippet: Generation of the IRF4 Bio mouse strain and in vitro differentiation of primary T cells into Th17 cells (A) Schematic overview of the bacterial artificial chromosome (BAC) transgene production for the generation of the IRF4 Bio mouse strain. (B) Cross-breeding of animals: IRF4 Avi mice, containing the BAC transgene, are crossed with ROSA26 BirA mice, ubiquitously expressing BirA ligase under the ROSA26 promoter, resulting in offsprings that express in vivo biotinylated IRF4 (IRF4 Bio ). (C) Western blot analyses of full cellular lysates and nuclear extracts demonstrate successful in vivo biotinylation of IRF4 (left, anti-IRF4 antibody; right, horseradish peroxidase-conjugated streptavidin [SA-HPO]). (D) Workflow for in vitro differentiation of primary Th17 cells.

Article Snippet: For example, in the case of the TF IRF4, good results were achieved using the μMACS FactorFinder Kit by Miltenyi, which has been discontinued.

Techniques: In Vitro, Expressing, In Vivo, Western Blot

Journal: STAR Protocols

Article Title: Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq

doi: 10.1016/j.xpro.2025.104184

Figure Lengend Snippet: Integrated analysis of IRF4 interactors and double-motif occurrence (A) Double-motif occurrence of IRF4 interactors in Th17 ChIP-seq peaks. Heatmap shows normalized co-occurrence frequencies (0–1) for IRF4 together with transcription factors detected by AP-MS, considering only motifs occurring within <5 bp spacing in the same peak. Asterisk (∗): GTF2IRD1-isoform2. (B) Volcano plot of IRF4 interactors identified by AP-MS comparing biotinylated IRF4 (“Bio”) and control (“Ctrl”) conditions in Th17 cells. Labeled interactors indicate TFs with double-motif occurrences in Th17. (C) Sequence logos of the individual TF motifs used for double-motif annotation. (D) STRING-based IRF4 interaction network displaying interactors classified as TFs according to the criteria by Lambert et al. (STRING DB default settings). See also .

Article Snippet: For example, in the case of the TF IRF4, good results were achieved using the μMACS FactorFinder Kit by Miltenyi, which has been discontinued.

Techniques: ChIP-sequencing, Protein-Protein interactions, Control, Labeling, Sequencing

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: Spleen B cells from naïve WT or IRF4 K123R mice and spleen T cells from naïve WT mice were either purified by negative selection using microbeads or depleted (B-depleted) using anti-B220 or anti-CD3 microbeads using AutoMACS separation (Milteny Biotech).

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: Spleen B cells from naïve WT or IRF4 K123R mice and spleen T cells from naïve WT mice were either purified by negative selection using microbeads or depleted (B-depleted) using anti-B220 or anti-CD3 microbeads using AutoMACS separation (Milteny Biotech).

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: Spleen B cells from naïve WT or IRF4 K123R mice and spleen T cells from naïve WT mice were either purified by negative selection using microbeads or depleted (B-depleted) using anti-B220 or anti-CD3 microbeads using AutoMACS separation (Milteny Biotech).

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: Spleen B cells from naïve WT or IRF4 K123R mice and spleen T cells from naïve WT mice were either purified by negative selection using microbeads or depleted (B-depleted) using anti-B220 or anti-CD3 microbeads using AutoMACS separation (Milteny Biotech).

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

Journal: Biomolecules

Article Title: The miR-125a-5p/IRF4 Axis Mediates Sodium Arsenite-Induced M2 Macrophage Polarization

doi: 10.3390/biom15111630

Figure Lengend Snippet: Prediction results of miR-125a-5p target genes. ( a ) Venn diagram shows that three miRNA binding sites were used to predict miRDB, Targetscan, and miRTarBase finding miR-125a-5p target proteins. ( b , c ) DAVID database GO enrichment analysis and visual display. ( d ) Targetscan database predicting binding sites of miR-125a-5p and IRF4.

Article Snippet: Antibodies for VEGF, iNOS , Arg1, CD206 and IRF4 were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay

Journal: Biomolecules

Article Title: The miR-125a-5p/IRF4 Axis Mediates Sodium Arsenite-Induced M2 Macrophage Polarization

doi: 10.3390/biom15111630

Figure Lengend Snippet: MiR-125a-5p overexpression reverses NaAsO 2 -induced IRF4 upregulation in macrophages. ( a ) Western Blot analysis of IRF4 protein expression in THP-1-derived macrophages induced by different concentrations of NaAsO 2 . ( b ) RT-qPCR confirmed successful miR-125a-5p overexpression with mimic transfection. ( c ) Western Blot was used to detect the expression of IRF4 protein in each treatment group after transfection with miR-125a-5p NC or mimic. The original images can be found in . * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control/control -miR-NC group; ## p < 0.01, ### p < 0.001 vs. NaAsO 2 /IL-4-miR-NC group. Data = mean ± SD (n = 3); ns: not significant.

Article Snippet: Antibodies for VEGF, iNOS , Arg1, CD206 and IRF4 were purchased from Proteintech (Wuhan, China).

Techniques: Over Expression, Western Blot, Expressing, Derivative Assay, Quantitative RT-PCR, Transfection, Control