Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Patients with biallelic IRF7 variants. (A) Pedigrees of the six kindreds containing seven patients with life-threatening viral infections (P1–P7) bearing rare biallelic IRF7 variants. Solid black symbols indicate patients with critical viral infections. The IRF7 genotype is indicated under each symbol. (B) The plot depicts the population frequency of IRF7 missense and pLoF variants (gnomAD v2.1.1) against CADD score (v1.6, GRCh37). Symbols indicate a total of 463 variants, 4 identified exclusively in patients and 459 present in the gnomAD database. The patient-derived variants reported in this study are highlighted, with pLoF and missense variants colored black and red, respectively. The population-derived homozygous IRF7 missense variants are highlighted in blue. (C) Schematic representation of IRF7. The lower part represents the genomic organization of the IRF7 locus, with black rectangles indicating the exons of the gene according to different transcripts. Below, a track indicates vertebrate nucleotide conservation across the IRF7 locus. The upper part shows the primary protein domain structure of IRF7. The N-terminal portion contains an α-helical DNA binding domain, followed by domains implicated in transactivation, autoinhibition, and regulation, as indicated. The positions of the patient-derived biallelic and population-derived homozygous IRF7 variants are indicated. A blue dotted line indicates the linkage of the IRF7 K179E and Q412R variants. Hmz, homozygous; Htz, heterozygous.

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: Derivative Assay, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Expression and activity of novel IRF7 variants. (A) HEK293T cells were transiently transfected with WT or mutant forms of IRF7 . IRF7 levels were assessed by Western blotting with antibodies against the N-terminus of IRF7 or an N-terminal FLAG tag. GAPDH was used as a loading control. Representative immunoblots from at least three independent experiments are shown. EV, empty vector. (B) HEK293T cells were transiently transfected with WT or mutant forms of IRF7 , together with an IFN-β luciferase reporter and a constitutively expressed reporter. Cells were either left untreated or infected with Sendai virus for 24 h before the assessment of normalized luciferase activity. The significance of differences between variants and the WT (mean ± SEM of n ≥ 3 independent experiments) was determined by two-way ANOVA (*, P < 0.05). Source data are available for this figure: .

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: Expressing, Activity Assay, Transfection, Mutagenesis, Western Blot, FLAG-tag, Control, Plasmid Preparation, Luciferase, Infection, Virus

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Patients with the newly discovered IRF7 variants do not produce IRF7 protein or IFN-α upon pDC stimulation. (A) Protein levels for IRF7 in PBMCs with and without IFN-β stimulation for 24 h; comparison of patients and healthy controls (HC). Actin staining was used as a loading control. Representative immunoblots (IB) from single independent experiments per patient are shown. (B and C) Frequency of IFN-α– and TNF-producing pDCs (live Lin – CD11c – HLA-DR + CD303 + CD123 + ) after 6 h of stimulation with imiquimod for fresh (B) and thawed (C) PBMCs. (D and E) Frequency of IFN-α– and TNF-producing pDCs (live Lin – CD11c – HLA-DR + CD303 + CD123 + ) after 6 h of stimulation with CpG ODN for fresh (D) and thawed (E) PBMCs. For P5 and P6, cells were assessed at two independent time points 6 mo apart. (A–E) All data are presented as the frequency of responding cells minus the frequency of the corresponding unstimulated controls. Box plots are bound by the 25th and 75th percentiles. The median is marked, and the whiskers indicate the minimum and maximum. Individual values are plotted. Unpaired t tests were performed to compare healthy controls (HC; n = 8–9) with patients. *, P < 0.05; ****, P < 0.0001. Source data are available for this figure: .

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: Comparison, Staining, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: IFN and inflammatory responses in IRF7-deficient pDCs challenged with SARS-CoV-2 and IAV. RNA-seq analysis of isolated pDCs from P2 and a healthy control (HC) either unstimulated or cultured with SARS-CoV-2 or IAV. (A–C) Genes in the annotated interest groups with expression >2.5-fold higher or lower in P2 vs. HC after viral culture were plotted as a heatmap of expression z-score (A) and expression fold-change in P2 vs. HC (B and C). Data are representative of a single experiment and reanalyzed from .

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: RNA Sequencing, Isolation, Control, Cell Culture, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Patients with newly discovered deleterious IRF7 variants produce normal levels of TNF after the TLR3-mediated stimulation of BDCA3 + DCs. (A and B) Frequency of TNF-producing BDCA3 + DCs (live Lin – CD123 – HLA-DR + CD141 + ) after 6 h of stimulation with poly(I:C), for fresh (A) and thawed (B) PBMCs. All data are presented as the frequency of responding cells minus that of the respective unstimulated controls. Box plots are bound by the 25th and 75th percentiles. The median is marked, and the whiskers indicate the maximum and minimum. Individual values are plotted. Unpaired t tests were performed to compare healthy controls (HC; n = 8–9) and patients. FSC, forward scatter.

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Genetic, demographic, and clinical features of IRF7-deficient patients

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: Virus

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: IRF7-deficient patients have enhanced CD4 + T cell responses to influenza and coronaviruses. (A and B) Frequency of IFN-γ–producing memory CD4 + (live Lin – CD3 + CD4 +CD8– CCR7 – CD95 + ; A) or CD8 + (live Lin – CD3 +CD8+ CD4 – CCR7 – CD95 + ; B) T cells after stimulation with peptides or PMA + ionomycin for 6 h. Unstimulated controls are shown on the left of the graph. Box plots are bound by the 25th and 75th percentiles. The median is marked, and the whiskers indicate the maximum and minimum. Individual values are plotted. Unpaired t tests were performed to compare healthy controls (HC; n = 9–14) and patients (P5 and P6). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

doi: 10.1084/jem.20220202

Figure Lengend Snippet: Large numbers of influenza-specific CD4 + T cell blasts in IRF7-deficient patients. (A and B) Whole-blood CD4 + (CD3 + CD4 + ) and CD8 + (CD3 + CD4 – ) T cell blast responses to influenza split-virus vaccine (A) or SARS-CoV-2–inactivated virus (B) after 6 d of stimulation; comparison of healthy controls (HC, n = 91 [A] or n = 46 [B]) with patients. For P6, cells were assessed at two independent time points 1 yr apart. Box plots are bound by the 25th and 75th percentiles with the median marked. The whiskers indicate the 5th and 95th percentiles, and outlier values are plotted. Unpaired t tests with Welch’s correction were performed to compare the HC and patient groups. ****, P < 0.0001. (C–E) VirScan assay showing the presence of antibodies against viruses in the serum samples from patients and controls, with the analysis focusing on IAV (D) and coronaviruses (E). Data are representative of a single experiment. Adj Sp_score, adjusted species score; spp_RF, significant species response frequency; AbbPep, abbreviation of peptide (SpeciesName_ProteinName_UniprotID_start_end).

Article Snippet: IRF7 levels were assessed by Western blotting using anti-IRF7 antibody (clone D2A1J; Cell Signaling) and anti-FLAG antibody (clone M2; Sigma-Aldrich).

Techniques: Virus, Comparison

Journal:

Article Title: Downregulation of IRF-3 Levels by Ribozyme Modulates the Profile of IFNA Subtypes Expressed in Infected Human Cells

doi: 10.1128/JVI.75.6.3021-3027.2001

Figure Lengend Snippet: DNA binding of in vivo-phosphorylated IRF-3. Whole-cell extracts were prepared from 2fTGH and 2fTGH/IRF-7 (clone 7/9) cells infected with Sendai virus 4 h after infection and incubated (200 μg) with magnetic beads containing either IFNA1 or IFNA2 VRE for 2 h at 4°C as described previously (Au et al., submitted). After extensive washing, proteins retained on the beads were separated on SDS–10% polyacrylamide gels and transferred to membranes, and IRF-2, IRF-3, and IRF-7 were identified by immunoblotting with specific antibodies. The relative levels of IRF-2, IRF-3, and IRF-7 in total cellular lysates were determined by Western blotting.

Article Snippet: IRF-3 antibodies were previously described ( 8 ), and IRF-2 antibodies were from Santa Cruz. (C) The relative levels of ectopic IRF-7 proteins were not affected in 2fTGH cells cotransfected with IRF-7 expression plasmid and pU1/IRF-3 ribozyme.

Techniques: Binding Assay, In Vivo, Infection, Incubation, Magnetic Beads, Western Blot

Journal:

Article Title: Downregulation of IRF-3 Levels by Ribozyme Modulates the Profile of IFNA Subtypes Expressed in Infected Human Cells

doi: 10.1128/JVI.75.6.3021-3027.2001

Figure Lengend Snippet: Cleavage of IRF-3 transcripts in vitro and downregulation of IRF-3 protein levels in vivo. (A) The in vitro-transcribed 32P-labeled 470-nucleotide (nt) IRF-3 transcript containing the GUC catalytic site was incubated alone (lane 2) or with an equal amount (lane 3) or molar excess of pU1/IRF-3 ribozyme (lane 4). The incubation conditions were described previously (3). Under these conditions, no degradation of template transcripts or ribozyme RNA (lanes 2 and 1, respectively) was observed. When IRF-3 template was incubated with pU1/IRF-3 ribozyme, two RNA fragments were produced, and their sizes (300 and 170 nt) were consistent with the predicted site for cleavage. (B) The relative levels of IRF-3 proteins were reduced in 2fTGH cells cotransfected with IRF-3 expression plasmid and pU1/IRF-3 ribozyme. Fifteen micrograms of whole-cell lysate of 2fTGH cells cotransfected with IRF-3 (3 μg) and pU1/IRF-3 (3 and 6 μg; lanes 2 and 3, respectively) were analyzed on an SDS-7.5% PAGE gel, and the relative levels of IRF-3 were identified by immunobloting with IRF-3 antibodies. The levels of IRF-2 were determined as a control of ribozyme specificity. IRF-3 antibodies were previously described (8), and IRF-2 antibodies were from Santa Cruz. (C) The relative levels of ectopic IRF-7 proteins were not affected in 2fTGH cells cotransfected with IRF-7 expression plasmid and pU1/IRF-3 ribozyme. Twenty micrograms of whole-cell lysate of 2fTGH cells cotransfected with IRF-7 (3 μg) and pU1/IRF-3 (3 and 6 μg; lanes 2 and 3, respectively) were analyzed on an SDS–10% PAGE gel, and the relative levels of IRF-7 and endogenous IRF-3 were determined by immunoblotting. IRF-7-specific antibody was from Santa Cruz. Relative levels of endogenous IRF-3 in these cell lysates are shown for comparison

Article Snippet: IRF-3 antibodies were previously described ( 8 ), and IRF-2 antibodies were from Santa Cruz. (C) The relative levels of ectopic IRF-7 proteins were not affected in 2fTGH cells cotransfected with IRF-7 expression plasmid and pU1/IRF-3 ribozyme.

Techniques: In Vitro, In Vivo, Labeling, Incubation, Produced, Expressing, Plasmid Preparation, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT

doi: 10.1186/s40478-025-02173-y

Figure Lengend Snippet: Decitabine reduces DNA methylation levels and synergizes with RG2833 to modulate gene expression. A Boxplots depicting genome-wide MML in DMSO (pink) and DAC (blue) for 4 ATRT cell lines. From left-to-right: CHLA02, CHLA05, CHLA06, and CHLA266. Center line represents the median, box is the interquartile range (IQR), and whiskers are 1.5 × IQR. B Heatmaps showing the top 150 most differentially expressed genes in 4 ATRT cell lines (from left to right: CHLA02, CHLA05, CHLA06, and CHLA266). Treatment order is as follows from left to right: DMSO, 5 µM RG2833, 100 nM DAC, combination). All heat maps are divided into 5 clusters. Red, upregulated; blue, downregulated. C Top 4 Gene Ontology enrichments from GOrilla in CHLA06 for all genes upregulated in combination treatment relative to DMSO. All enrichments were significant (gene ontology, y-axis; -log10(p-value), x-axis) (top graph). P -values, FDR q-values and enrichment scores are listed in the bottom table for the top 4 enrichments. N, total number of genes in set; B, total number of genes associated with a gene ontology term; n, number of genes in the top of the upregulated gene set list; b—number of intersecting genes; enrichment = (b/n)/(B/N). D Immune response (top) and defense response (bottom) enrichments from Gorilla for all 4 ATRT cell lines, from top to bottom: CHLA02, CHLA05, CHLA06, and CHLA266 (cell line, y-axis; -log10(p-value), x-axis). E “MEISSNER_BRAIN_HCP_WITH_H3K4ME3_AND_H3K27ME3” enrichments from GSEA for all 4 ATRT cell lines: CHLA02, CHLA05, CHLA06, and CHLA266 (cell line, y-axis; -log10(p-value), x-axis). F Volcano plots of differentially expressed genes for all 4 cell lines (CHLA02, top left; CHLA05 top right; CHLA06, bottom left; CHLA266, bottom right) in DMSO vs. combination treatment comparisons. Universally upregulated genes include VCX/VCX2/VCX3A/VCX3B, and CDKN2A. PDK1 is universally downregulated. Genes are represented as red points if the FDR < 0.05. G Western blots of IRF7 (top) and STING (bottom) protein expression in 4 cell lines. From left to right: CHLA02, CHLA05, CHLA06, and CHLA266. GAPDH is used as the loading control. Treatment conditions from left to right are: DMSO, 5µM RG2833, 100 nM DAC, combination

Article Snippet: Protein levels were assessed using the following antibodies: IFITM3 (1:1000; Cell Signaling Technology; #59212S), IRF-7 (1:200; Cell Signaling Technology #13014), GAPDH control (1:10,000; Santa Cruz; #SC-47724), STING (1:200; Cell Signaling Technology, #13647S), alpha-tubulin (1:1,000; #2144S) and p16 INK4A (1:100, Cell Signaling Technology #80772S).

Techniques: DNA Methylation Assay, Gene Expression, Genome Wide, Western Blot, Expressing, Control

Journal: Acta Neuropathologica Communications

Article Title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT

doi: 10.1186/s40478-025-02173-y

Figure Lengend Snippet: DAC and RG2833 upregulate the expression of CDKN2A and lead to a reduction in cell viability. A GBiB tracks of dMML of ATRT-883, ATRT-884, and ATRT-900 for ATRT vs. Normal comparisons over the CDKN2A locus, replicated from Fig. e for comparison (top). GBiB tracks of dMML for CHLA02, CHLA05, CHLA06, and CHLA266 vs. DMSO treatment over the CDKN2A locus (bottom). B Box plots demonstrating mRNA count (n = 3) for CHLA02, CHLA05, CHLA06, and CHLA266. Treatment conditions are DMSO (red), 5 µM RG2833 (green), 100 nM DAC (blue), and combination (purple). Center line, median; box; IQR, whiskers: 1.5 × IQR. C Western blots of CDKN2A protein expression in CHLA02, CHLA05, and CHLA06. GAPDH is used as the loading control. Treatment conditions from left to right are DMSO, 5 µM RG2833, 100 nM DAC, combination. D Bliss synergy results from treatment of CHLA02, CHLA05, and CHLA06 with escalating concentrations of DAC (0 nM, 25 nM, 50 nM, 75 nM, 100 nM) and/or RG2833 (0 nM, 500 nM, 1 µM, 1.5 µM, and 2 µM). Shown are dose–response density graphs, 3D response plots, and a dose–response matrix based on inhibition (x-axis = RG2833, y-axis = decitabine)

Article Snippet: Protein levels were assessed using the following antibodies: IFITM3 (1:1000; Cell Signaling Technology; #59212S), IRF-7 (1:200; Cell Signaling Technology #13014), GAPDH control (1:10,000; Santa Cruz; #SC-47724), STING (1:200; Cell Signaling Technology, #13647S), alpha-tubulin (1:1,000; #2144S) and p16 INK4A (1:100, Cell Signaling Technology #80772S).

Techniques: Expressing, Comparison, Western Blot, Control, Inhibition