Journal: Science immunology

Article Title: A multimorphic mutation in IRF4 causes human autosomal dominant combined immunodeficiency

doi: 10.1126/sciimmunol.ade7953

Figure Lengend Snippet: (A) 293T cells were transfected with an empty vector (EV) or a vector-expressing IRF4WT or IRF4T95R. Nuclei were stained with DAPI (blue), cytoplasm with phalloidin (red), and IRF4 with an anti-IRF4 antibody (green). Left: representative images. Right: a summary of randomly chosen cells. (B) Ratio of nuclear to cytoplasmic IRF4 in Raji cells transduced with retrovirus expressing IRF4WT or IRF4T95R. Left: representative immunoblot. Right: mean ± SD of three independent experiments. PARP, poly(adenosine diphosphate–ribose) polymerase. (C) IRF4T95R showed increased affinity for an ISRE, two AICEs, and an EICE site. (D) Fractions of all bound SiR-HaloTag- IRF4WT and IRF4T95R molecules (left) and molecules long bound for >2 s (right) as determined by single-molecule fluorescence microscopy with interlaced time-lapse illumination. (E) HEK293 cells were transfected with control plasmid (−), IRF4WT, or IRF4T95R. Nuclear extracts were analyzed by EMSA using 3xGAAA ISRE. Supershifts (ss) of WT and T95R extracts using HA-tag antibody or IgG control are shown at the far right. Note that IRF4T95R binds more strongly to ISRE compared with IRF4WT. Dashed lines indicate cuts of the scan for presentation. (F) Top: IRF4WT (left) and IRF4T95R (right) motifs found in the HT-SELEX data. Bottom: 8-nucleotide oligomer containing GAAA (left) or GATA (right) enriched in IRF4WT (x axis) or IRF4T95R (y axis). (G) 293T cells were transfected with a TK-cypridina luciferase vector (an internal control) and either a canonical (ISRE)1–driven luciferase vector (top) or a noncanonical (ISRE)1–driven luciferase vector (bottom), together with a pFLAG-CMV empty vector (400 ng) or increasing amounts of plasmids encoding IRF4WT or IRF4T95R. The luciferase activity was compared with that induced by the empty vector, which was set to 1. Mean ± SD of two to four independent experiments is shown. (H) ChIP-seq analysis of immortalized B cells from P3 compared with a HC. Top left: overlay of IRF4 ChiP-seq peaks in EBV-B cells of P3 and HC. From left to right: ISRE, AICE, and EICE motifs found in IRF4T95R, IRF4WT, AICE, or EICE ChIP-seq data (indicated at the left of the motifs). The importance of each motif toward the IRF4T95R-specific (purple), IRF4WT-specific (green), or common (gray) component of the ChIP-seq data is shown to the right of each motif. Noncanonical motifs are surrounded by a purple line. (I) Normalized IRF4WT-specific (green, top), common (gray, middle), and IRF4T95R-specific (purple, bottom) ChIP-seq peak counts (y axis) for different groups of differentially expressed genes. (J) HEK293 cells were transfected with AP-1 (JUNB and BATF) with or without IRF4WT or IRF4T95R, as indicated. Nuclear extracts were analyzed for binding to various CXCL13 sites, as indicated. Note that IRF4T95R shows strongly increased (CXCL13-A) or exclusive (CXCL13-C) binding compared with IRF4WT. (K) HEK293 cells were transfected with CXCL13 reporter construct encompassing CXCL13 sites A and B together with AP-1 (JUNB and BATF) and IRF4 variants, as indicated. Luciferase activity is shown as fold activation compared with control transfected cells (far left), which is set as 1. Mean ± SD of three independent experiments is shown. (L) FC of CXCL13 levels in serum or plasma from P3 to P7 compared with HC. Statistical significance was determined by one-tailed Welch’s t test (A, B, D, and G) and by Tukey’s post hoc test (K). *P < 0.05, **P < 0.01, and ****p < 0.0001.

Article Snippet: 4′,6-diamidino-2-phenylindole (DAPI; Chemometech, #910-3018) and phalloidin (Cell Signaling, #8940) were added for 40 min. After several washes, fluorescence was detected with a confocal Leica SP8 microscope.

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Transduction, Western Blot, Fluorescence, Microscopy, Control, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Construct, Activation Assay, Clinical Proteomics, One-tailed Test