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: bioRxiv

Article Title: Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

doi: 10.1101/2025.03.10.642367

Figure Lengend Snippet: (A) Schematic of domains of the IRF7 protein (top): DNA binding domain (DBD), trans-activation domain (TAD), IRF-association domain (IAD), and nuclear export signal (NES). (bottom) Schematics of the alternatively spliced variants indicating the translation start sites in exon 1 and retained intron (green boxes), and stop codon in intron 1 (red octogon). Isoform-specific amino acids preceding the DBD are indicated in blue for exIRF7 and red for cIRF7. (B) Plot of IRF7 intron 1 retention across human cell types from public GTEx data. Blue bars indicate percent of total IRF7 expression that retains intron 1. Red area indicates percent of cIRF7 isoform. (C) RT- PCR analysis of IRF7 intron 1 alternative splicing in various human cell types under different stimulation conditions. (D) Quantification of the RT-PCR data in panel C. Conditions that preferentially express exIRF7 are shown in blue, conditions that preferentially express cIRF7 are in red. Black bars indicate near equal expression of both isoforms.

Article Snippet: Antibodies used to detect protein expression levels are as follows: IRF7 (Abcam, ab238137), pIRF7 (Cell Signaling, 5184S), FLAG (Cell Signaling, 14793S), IRF3 (Cell Signaling, 11904S), NFkB-p65 (Cell Signaling, 8242T), NFkB-p50 (Cell Signaling, 12540S), ATF2 (Cell Signaling, 35031S), c-Jun (Cell Signaling, 9165T), HMGA1 (Cell Signaling, 12094S), and GAPDH (Cell Signaling, 2118S), anti-rabbit HRP-linked secondary antibody (Cell signaling, 7074S).

Techniques: Binding Assay, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing

Journal: bioRxiv

Article Title: Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

doi: 10.1101/2025.03.10.642367

Figure Lengend Snippet: ( A ) Design of CRISPR-Cas9 genome editing to create cell lines expressing either cIRF7 or exIRF7. ( B ) Validation of CRISPR tools at both RNA and protein levels: (top) RT-PCR confirms the expression of IRF7 isoforms at the RNA level compared to WT cells. (bottom) Western blot analysis reveals the protein expression levels of exIRF7 and cIRF7 relative to WT and IRF7 KO clones. ( C ) RT-qPCR analysis of selected differentially expressed genes (DEGs) from RNA-seq (n=3, mean ± SD) under conditions of IFNα and pIC, with and without ruxolitinib treatment. Differences that are significant are indicated (p<0.05, unpaired student t-test). ( D-F ) Scatter plots based on RNA-seq data showing differentially expressed genes (DEGs) in exIRF7 and cIRF7 compared to IRF7 KO cells after induction with pIC (10 µg/ml for 6 hours, D, F ) or IFNα (500 U/ml for 6 hours, E ). Panel F is the same as panel D but with different gene groupings indicated. Significant DEGs were identified with a base read minimum of 50, a minimum log2-fold change of 1 and a p-value threshold of 0.05.

Article Snippet: Antibodies used to detect protein expression levels are as follows: IRF7 (Abcam, ab238137), pIRF7 (Cell Signaling, 5184S), FLAG (Cell Signaling, 14793S), IRF3 (Cell Signaling, 11904S), NFkB-p65 (Cell Signaling, 8242T), NFkB-p50 (Cell Signaling, 12540S), ATF2 (Cell Signaling, 35031S), c-Jun (Cell Signaling, 9165T), HMGA1 (Cell Signaling, 12094S), and GAPDH (Cell Signaling, 2118S), anti-rabbit HRP-linked secondary antibody (Cell signaling, 7074S).

Techniques: CRISPR, Expressing, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Quantitative RT-PCR, RNA Sequencing

Journal: bioRxiv

Article Title: Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

doi: 10.1101/2025.03.10.642367

Figure Lengend Snippet: ( A ) Luciferase assay with cells co-expressing IRF7 isoforms, a NanoLuc internal control, and firefly luciferase under the control of an IFNβ promoter segment, after exposure to an innate immune trigger, sendai virus infection (500HAU/ml for 6hrs). Luciferase values are normalized to the NanoLuc control and shown relative to untreated cIRF7-expression. Significance shown was determined by unpaired t-test, each point is an independent biological replicate. ( B ) Sequence of IFNb promoter used in reporter assays and EMSAs (panels D-E). ( C ) Western blot analysis of Flag-affinity purified recombinant IRF7 proteins used in gel-shift assays. Proteins were purified from Jurkat cells stably expressing each isoform after treatment with pIC (10 µg/ml for 6 hours). The blot illustrates their relative expression levels, phosphorylation status, and absence of known IRF7 co-factors. ( D ) Representative native gel-shift assay (EMSA) using indicated amounts of IRF7 proteins from panel C incubated with a radiolabeled DNA oligo corresponding to core IFNβ promoter as shown in panel B (green sequence). Migration of free DNA probe and protein:DNA complexes is indicated. The blue arrows highlight the unique DNA:protein complex observed with exIRF7. Replicate gels with independent protein preps shown in Supplemental Fig S6. ( E ) EMSA as in panel D but with IRF7 protein held constant and antibodies to IRF7, known co-factors, and IgG control added.

Article Snippet: Antibodies used to detect protein expression levels are as follows: IRF7 (Abcam, ab238137), pIRF7 (Cell Signaling, 5184S), FLAG (Cell Signaling, 14793S), IRF3 (Cell Signaling, 11904S), NFkB-p65 (Cell Signaling, 8242T), NFkB-p50 (Cell Signaling, 12540S), ATF2 (Cell Signaling, 35031S), c-Jun (Cell Signaling, 9165T), HMGA1 (Cell Signaling, 12094S), and GAPDH (Cell Signaling, 2118S), anti-rabbit HRP-linked secondary antibody (Cell signaling, 7074S).

Techniques: Luciferase, Expressing, Control, Virus, Infection, Sequencing, Western Blot, Affinity Purification, Gel Shift, Purification, Stable Transfection, Phospho-proteomics, Incubation, Migration

Journal: bioRxiv

Article Title: Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

doi: 10.1101/2025.03.10.642367

Figure Lengend Snippet: (A) Mass photometry traces of cIRF7 (top) and exIRF7 (middle). Mass of major populations are indicated as determined by fitting guassian curves as described in Methods. Bottom plot is the overlap of the individual traces . (B) Percent dimer of exIRF7 (blue) and cIRF7 (red) as calculated across a range of protein concentrations. Data points that differ significantly (p<0.001, unpaired student t-test) between the isoforms are indicated by ****. Kd calculated by curve fitting the data is shown. (C) Representative native gel-shift assay (EMSA) using 200 ng of purified IRF7 protein, and/or 20 or 40 ng of purified recombinant IRF3, incubated with a radiolabeled DNA oligo corresponding to core IFNβ promoter as shown in . Migration of free DNA probe and protein:DNA complexes is indicated. The blue arrow highlights the unique exIRF7 complex, the black arrow indicates the IRF3 and IRF3/IRF7 complexes. A replicate gel with independent protein preps shown in Supplemental Fig S6.

Article Snippet: Antibodies used to detect protein expression levels are as follows: IRF7 (Abcam, ab238137), pIRF7 (Cell Signaling, 5184S), FLAG (Cell Signaling, 14793S), IRF3 (Cell Signaling, 11904S), NFkB-p65 (Cell Signaling, 8242T), NFkB-p50 (Cell Signaling, 12540S), ATF2 (Cell Signaling, 35031S), c-Jun (Cell Signaling, 9165T), HMGA1 (Cell Signaling, 12094S), and GAPDH (Cell Signaling, 2118S), anti-rabbit HRP-linked secondary antibody (Cell signaling, 7074S).

Techniques: Gel Shift, Purification, Recombinant, Incubation, Migration

Journal: bioRxiv

Article Title: Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

doi: 10.1101/2025.03.10.642367

Figure Lengend Snippet: ( A ) The CancerSplicingQTL database quantification of IRF7 intron 1 retention in different genotypes of SNP rs12290989. Each dot represents a single individual with the given genotype. Box plot represents average and distribution of intron retention. (B) Representative images from A549 cells with the indicated genotype infected for 24 hours with GFP-labeled PIV3 at MOI of 0.25. (C) Quantification of automated microscopy images as in panel B for PIV3 across multiple MOI of infection.

Article Snippet: Antibodies used to detect protein expression levels are as follows: IRF7 (Abcam, ab238137), pIRF7 (Cell Signaling, 5184S), FLAG (Cell Signaling, 14793S), IRF3 (Cell Signaling, 11904S), NFkB-p65 (Cell Signaling, 8242T), NFkB-p50 (Cell Signaling, 12540S), ATF2 (Cell Signaling, 35031S), c-Jun (Cell Signaling, 9165T), HMGA1 (Cell Signaling, 12094S), and GAPDH (Cell Signaling, 2118S), anti-rabbit HRP-linked secondary antibody (Cell signaling, 7074S).

Techniques: Infection, Labeling, Microscopy