Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies binding to IFNα 2 , IFNβ 1b and IFNω in patients with H7N9 infection and healthy controls. (A) Age and sex distribution of the three study groups. For each age group, the number of individuals positive for autoantibodies neutralising at least one tested IFN-I (IFNα 2 , IFNβ 1b , IFNω) at low concentrations is indicated in red. M, male; F, female; nAb+, positive for IFN-I-neutralising autoantibodies. (B) Detection of IgG autoantibodies binding to IFNα 2 , IFNβ 1b or IFNω in serum samples by multiplex bead-based assay. Samples with a Z-score >7 were considered positive for IFN-I-binding autoantibodies. Measurements were performed without technical replicates because of limited sample availability. (C) Prevalence of IFN-I-binding autoantibodies by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies binding to at least one of the tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies binding to both IFNα 2 and IFNω.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Binding Assay, Infection, Multiplex Assay, Bead-based Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies neutralising IFN-I in patients with H7N9 infection and healthy controls. (A) Luciferase-based reporter assay to assess the capacity of autoantibody positive sera to neutralise IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Each sample was tested in biological duplicates and the mean values are shown. Samples were classified as neutralising if the mean of the relative luciferase activities was below 25% (dotted line) of the mean of the negative pool (four autoantibody-negative control sera). All sera positive for IFN-I-binding autoantibodies were tested; numbers are indicated above the graphs. Lines connect measurements of neutralising activity from the same serum sample at low and high IFN concentrations. (B) Prevalence of autoantibodies neutralising low IFN concentrations (IFNα 2 : 0.5 ng/ml, IFNβ 1b : 0.25 ng/ml; IFNω: 0.2 ng/ml) by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies neutralising at least one tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies neutralising both IFNα 2 and IFNω. (C) Area-proportional Venn diagrams illustrating the absolute numbers of samples with autoantibodies neutralising high and low concentrations of IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Venn diagrams were created with BioVenn ( https://www.biovenn.nl/index.php ).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Luciferase, Reporter Assay, Negative Control, Binding Assay, Activity Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Association between the presence of IFN-I-neutralising autoantibodies and H7N9 infection. (A) The association between age, sex and IFN-I-neutralising autoantibodies in patients with H7N9 infection or in the two control groups combined (poultry workers + close contacts) was assessed using Firth's penalised logistic regression. Predicted probabilities for the presence of autoantibodies with 95% confidence intervals (CIs, shaded areas around the curve) are shown across participant age for men and women. To visualise the modelled probabilities in relation to the underlying data, we overlaid sex-specific age density distributions beneath the predicted probability curves. (B) Odds ratios (OR) with 95% CIs for the presence of autoantibodies neutralising low IFN concentrations in patients compared to healthy controls, adjusted for age and sex, determined by Firth’s penalised logistic regression models. See also for the results of the logistic regression analyses and for unadjusted estimates. IFNα 2 ± IFNω ± IFNβ 1b , positive for autoantibodies neutralising at least one of the tested IFN-I; IFNα 2 ± IFNω, positive for autoantibodies neutralising IFNα 2 and/or IFNω; ∗∗∗∗, p < 0.0001 (Firth’s penalised logistic regression).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Control

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Neutralising sera block the antiviral effect of IFNα 2 in cell culture infected with IAV. Antiviral activity of IFNα 2 (5 ng/ml) against IAV (PR8-GFP, MOI 1) alone or in the presence of serially diluted IFN-I-neutralising sera (n = 19), autoantibody-negative sera (n = 4), or a monoclonal anti-IFNα 2 antibody in A549 cells. Infection rates (GFP + /DAPI + cells) at 7 h post-infection were normalised to untreated, infected cells. The dotted line indicates the reduction of infected cells after IFN treatment alone. If possible, the mean of two independent experiments is shown. Sufficient material was available for 12 out of 19 samples.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Blocking Assay, Cell Culture, Infection, Activity Assay

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A interacts with IRF3 and is associated with colorectal cancer (A and B) Whole-cell lysates (WCL) from RKO and HCT116 cells were collected for IP using anti-IRF3 or anti-KMT5A antibodies, followed by IB analysis. (C) Cytoplasmic and nuclear proteins from RKO were collected for IP using anti-KMT5A antibodies, followed by IB analysis. (D) Boxplots were used to compare KMT5A mRNA expression in COAD and READ, with Student’s two-tailed t test, p < 0.0001. (E) Disease-specific survival analysis of TCGA-COAD and READ based on KMT5A expression levels. (F) Protein expression of KMT5A was analyzed by IB in colorectal tumor and paired adjacent normal tissues. (G) mRNA was extracted from colorectal tumors and paired adjacent normal tissues, and KMT5A mRNA levels were analyzed by qPCR. (H) The levels of IFN-β colorectal tumor and paired adjacent normal tissues were measured using ELISA. For (D)–(H) statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Data are presented as mean ± SD. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. All immunoblotting experiments were performed independently three times with similar results.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A affects immune infiltration in colorectal tumors (A) To establish tumor models, MC38 cells with either control or mKMT5A knockdown via shRNA were injected subcutaneously into C57BL/6 mice. (B and C) Tumor mass and volume derived from the experiments in (A) were quantified. (D and E) Flow cytometry was conducted to analyze CD8 + T cell infiltration in the tumors from (A). (F) The levels of IFN-β in the tumors from (A) were measured using ELISA. (G and H) Activated T cells co-cultured with pre-treated RKO cells. Representative images (G) and statistical analysis (H) are shown. (I) The levels of IFN-β in the cell culture supernatant from (G) were measured using ELISA. For (B), (E), (F), (H), and (I) statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. For (C), two-way ANOVA with Tukey’s post-hoc test was used. Data are presented as mean ± SD. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Control, Knockdown, shRNA, Injection, Derivative Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A is associated with the type I interferon pathway Control and KMT5A knockdown RKO cells were subjected to RNA sequencing (RNA-seq) analysis. (A) Volcano plot depicting differentially expressed genes, with upregulated genes in the knockdown group compared to the control highlighted in red. (B) Heatmap illustrating the expression patterns of the differentially expressed genes. (C) KEGG pathway enrichment analysis of significantly altered pathways in KMT5A-knockdown cells based on transcriptomic data. (D) Gene Ontology (GO) analysis of significantly differentially expressed genes in KMT5A-knockdown cells, categorizing them into biological processes (BP), cellular components (CC), and molecular functions (MF). (E) Gene set enrichment analysis (GSEA) of gene sets related to the upregulation of interferon BETA-associated pathways.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Control, Knockdown, RNA Sequencing, Expressing

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A inhibits phosphorylation activation of IRF3 (A) RKO cells were treated with poly(I:C) and protein samples were collected at 0, 8, 16, and 24 h for Western blot (WB) analysis. (B) Quantification of p-IRF3, RIG-1, and MDA5 protein levels relative to control in the samples collected in (A). (C) IFN-β secretion levels at different time points from (A) were measured using ELISA. (D, E) RKO and HCT116 cells transfected with either an empty vector or a KMT5A overexpression plasmid were treated with poly(I:C), followed by WB analysis of whole-cell lysates. (F) RKO cells transduced with control shRNA (shNC) or KMT5A-specific shRNAs (#1 and #2) were treated with poly(I:C) and analyzed by WB using whole-cell lysates. (G) RKO cells from (F) were co-transfected with the IFN-β-Luc reporter plasmid and pRL-TK plasmid, and after 24 h, luciferase activity was measured using a dual-luciferase assay kit. (H) The relative mRNA levels of KMT5A and INF-β in RKO cells from F were quantified using qPCR. (I) IFN-β levels in the RKO cells from (F) were measured using ELISA. (J) RKO cells, either wild-type or stably expressing HA-KMT5A, were treated with poly(I:C) and whole-cell lysates were collected at 0, 8, 16, and 24 h for WB analysis. (K) Quantification of p-IRF3 protein levels relative to control in samples collected in (J). For (G)–(I), statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. For (K), two-way ANOVA with Tukey’s post-hoc test was used. Data are presented as mean ± SD. Statistical significance is indicated as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All western blot analyses were performed independently three times, yielding consistent results.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Over Expression, Transduction, shRNA, Luciferase, Activity Assay, Stable Transfection, Expressing

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A induces mono-methylation of lysine 193 on IRF3 (A) HEK293T cells were transfected with FLAG-IRF3 and/or HA-KMT5A plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads. Subsequent analysis was conducted via IB. (B) Whole-cell lysates were extracted from RKO cells with either control (shNC) or KMT5A shRNA (#1 and #2) silencing. IP was performed using anti-IRF3 antibodies, followed by WB analysis. (C) RKO cells were transfected with HA-KMT5A WT or HA-KMT5A D338A, IP was performed using anti-IRF3 antibodies, followed by WB analysis. (D) RKO cells were treated with either DMSO or UNC0379. IP was performed using anti-IRF3 antibodies, followed by IB analysis. (E) RKO cells were treated with either DMSO or varying concentrations of UNC0379. IP was conducted using anti-IRF3 antibodies, followed by IB analysis. (F) The levels of IFN-β in RKO cells from experiment (E) were quantified using ELISA. Data were analyzed using one-way ANOVA with Tukey’s post-hoc test, presented as mean ± SD. Statistical significance was defined as ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. (G) In vitro methylation assays were conducted by incubating purified His-IRF3 with KMT5A in the presence of S-adenosyl-L-methionine, followed by IB analysis. (H) Secondary mass spectrometry results of IRF3 K193 methylation were obtained. (I) HEK293T cells were transfected with FLAG-IRF3 wild-type or mutant plasmids, followed by transfection with either a vector or HA-KMT5A. Whole-cell lysates were collected, and IP was performed using anti-FLAG magnetic beads, followed by IB analysis. (J) Amino acid sequences at the K193 site of IRF3 were compared across different species. All immunoblotting experiments were conducted independently in triplicate, yielding consistent results.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Methylation, Transfection, Magnetic Beads, Control, shRNA, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, Mass Spectrometry, Mutagenesis, Plasmid Preparation, Western Blot

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: UNC0379 could be used as an adjuvant therapy for colorectal tumors (A) The treatment protocol of anti-PD-1 treatment combined UNC0379 in CRC. (B) C57BL/6 mice were subcutaneously injected with MC38 cells and subsequently treated with PBS, anti-mPD1, UNC0379, or a combination of anti-mPD1 and UNC0379 to establish a tumor model. (C and D) Tumor weight and volume were quantified from the samples obtained in experiment (A). (E) The concentration of IFN-β in the tumors from experiment (A) was assessed using an ELISA assay. (F–H) Flow cytometric analysis was conducted to evaluate the populations of CD8 + T cells and NK1.1+ cells in the tumors from experiment (A). For (C), (E), (G), and (H), data were analyzed using one-way ANOVA with Tukey’s post-hoc test. (D) was analyzed using two-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD, with statistical significance indicated as ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Human Interferon Beta ELISA Kit , ABclonal , RK01630.

Techniques: Adjuvant, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Human ZBP1 is a potent inducer of cell death through mechanisms divergent from mouse ZBP1

doi: 10.1038/s44319-026-00866-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant Mouse IFN-β , ABclonal , Cat#RP01076.

Techniques: Recombinant, Sequencing, Magnetic Beads, Protease Inhibitor, Software, Microscopy, Bicinchoninic Acid Protein Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Comparing CHPV propagation in cultured cells infected at different MOIs. ( a ) Schema summarizing cell-infection experiments performed in this study. ( b ) Barplot revealing progeny virus titer in the culture supernatant of MEFs at 24 h post-infection with CHPV at the indicated input MOI. ( c ) Similarly, progeny virus yield was measured at 12 h and 36 h post-infection for cells infected at MOI 2 and 20. ( d ) ELISA showing accumulation of IFNβ in a time course in the culture supernatant of cells infected with CHPV at the indicated MOI. ( e ) CHPV-mediated cell death was measured at 24 h post-infection by crystal violet staining. The abundance of viable cells at various MOI was determined relative to corresponding uninfected MEFs and presented as bargraphs. ( f ) Infection-induced cell death was similarly measured at 12 h and 36 h post-infection. Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test. ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Cell Culture, Infection, Virus, Enzyme-linked Immunosorbent Assay, Staining

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Mathematically probing CHPV multiplication and host responses at cell-saturating MOIs. ( a ) A cartoon depicting key processes involved in CHPV perpetuation at cell-saturating MOIs that were considered for mathematical analyses. Kinetic rate parameters associated with each of the reactions have been indicated. Right, a set of mathematical equations used for describing the relationship between number of infected live cells, IFN concentration, and progeny virus titer has been indicated. IFN( t ) and V ( t ) have been used to represent IFN concentration in pg/mL and viral titer in pfu/mL at time t . The fraction of live, infected cells at time t has been described as C ( t ). Other parameters have been described in . ( b – d ) Fitting mathematical equations with experimental time course data related to progeny virus titer ( b ), cell-produced IFNβ levels ( c ), and infection-inflicted cell death ( d ) observed at MOI 2 and MOI 20. Experimental data points derived from biological replicates are indicated in black circles, and solid lines represent the fitted mean values for viral titer, IFN-β levels, or fraction of live cells. Shaded regions around the solid lines indicate the corresponding 95% confidence intervals. ( e ) Bar chart comparing the values of cell death , IFN pdn , and virus pdn at MOI 2 and MOI 20 extracted from fitting exercise. Values of these rate parameters were independently determined using data from experimental replicates. Unpaired t -test was performed to determine the statistical significance. ( f ) Simulating virus yield as a function of time in the MOI 20 regime using the virus pdn , IFN pdn , or cell death rate constant values linked to MOI 2. Data represent the means of three biological replicates ± SEM. *** P ≤ 0.001; ns, not significant, ≥0.05.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Concentration Assay, Virus, Produced, Derivative Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Charting CHPV growth in Ifnar1 −/− MEFs infected at different MOI. ( a ) Barplot comparing virus yields in WT and Ifnar1 −/− MEFs determined at 24 h post-infection at the indicated MOIs. ( b ) ELISA revealing the abundance of IFNβ in the culture supernatant of Ifnar1 −/− cells infected with CHPV at the indicated MOIs. ( c ) Barplot showing cell death in WT and Ifnar1 −/− MEFs at 24 h post-infection with CHPV at the indicated MOIs. ( d – f ) Line plots depicting fitting of mathematical equations with experimental time course data on CHPV propagation in Ifnar1 −/− cells (left). Shaded regions represent 95% confidence intervals. Extracted values for various rate parameters are indicated in the accompanying bar chart (right). Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test for panels a through c and using unpaired t -test for panel d ** P ≤ 0.01; *** P ≤ 0.001; ns, not significant, ≥0.05.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Studying CHPV propagation in Nfkbia −/− MEFs infected at different MOI. ( a, b, c ) Comparing WT and Nfkbia −/− MEFs infected at various MOIs for cell death ( a ), IFNβ production ( b ), and CHPV yield ( c ). ( d – f ) Fitting experimental time course data on CHPV propagation in Nfkbia −/− cells in our mathematical model (left). Shaded regions represent 95% confidence intervals. Extracted values for various rate parameters are also indicated (right). ( g ) Line plot charting the simulated progeny virus titer estimated in the Nfkbia −/− settings at MOI 20 using altered virus pdn , cell death , and IFN pdn values that preserved the quantum of changes in these rates upon raising MOI from 2 to 20 to those determined in WT cells. Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test for panels a through c and using unpaired t -test for panel d. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Virus

Journal: Molecular Therapy Oncology

Article Title: AAV immuno-gene therapy platform delivering vectorized cytokines defines a new modality for high-grade glioma treatment

doi: 10.1016/j.omton.2026.201183

Figure Lengend Snippet: Vectorized IFNβ drives durable signaling and complete tumor regression in human glioblastoma models in vivo (A) Sustained hIFNβ secretion in human GBM6 cells treated with AAV9-hIFNβ (red, MOI = 4E5 vg/cell) or recombinant hIFNβ cytokine (r-hIFNβ, purple, 47 IU/mL, equivalent to 114 pg/mL), measured by ELISA at indicated time points. 50% media washouts every 5 h for the first 20 h in the r-hIFNβ condition mimic in vivo cytokine clearance (half-life = 4–5 h). Full media exchanges were performed at 24, 48, 72, and 96 h post-treatment. (B) Number of differentially expressed genes (DEGs, p -Adj<0.01) in GBM6 cells 24–96 h post-treatment with AAV9-hIFNβ or r-hIFNβ vs. media controls. (C) Enrichment scores for type I IFN and TNFα response pathways across treatments and time points. (D) Heatmap of the top 10 IFN and TNFα response genes (Log2FC vs. media controls) in GBM6 cells treated as in (A). (E) Schematic of orthotopic PDX (SF11411) and cell line-derived xenograft ([CDX], GBM6-FLuc) studies in athymic nu/nu mice treated intratumorally with saline, AAV9-GFP, or AAV9-hIFNβ via CED. (F) Kaplan-Meier survival curves for PDX mice treated as in (E). Saline = black, AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ (2E11 vg/brain) = red. Vertical dashed line = day of treatment (day 9). p < 0.04 by log-rank (Mantel-Cox) test. n = 30 (10 per treatment arm). (G) Longitudinal BLI of GBM6-FLuc tumor growth in CDX mice treated as in (E). Saline = black, AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ (2E11 vg/brain) = red. Thin lines = individual mice, thick lines = geometric mean. Vertical dashed line = day of treatment (day 9). ∗ p < 0.04 by Kruskal-Wallis test with Dunn’s multiple comparisons correction on day 22. n = 30 (10 per treatment arm). (G′) Representative BLI images from each treatment group 11 days post-treatment. (H) Kaplan-Meier survival curves for CDX mice. p < 0.001 by log-rank (Mantel-Cox) test. (I) Distribution of treatment responses in CDX by BLI flux (photons/second) at day 27. Tumor free = BLI flux <2.5 × 10 5 p/s, tumor reduction = ≥30% decrease from assignment on day 9, no change = between 30% decrease and 20% increase from assignment on day 9, tumor growth = ≥20% increase from assignment on day 9, death = mice that died before day 27. (J) Dose-response analysis of AAV9-hIFNβ efficacy in CDX mice. AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ hi (2E11 vg/brain) = solid red, and AAV9-hIFNβ lo (1E11 vg/brain) = dashed red. Thin lines = individual mice, thick lines = geometric mean. Vertical dashed line = day of treatment (day 9). ∗∗ p < 0.02 by Kruskal-Wallis test with Dunn’s multiple comparisons correction on day 20. n = 45 (15 per treatment arm). For data interpretation, tumor burden threshold = 2.5 × 10 5 . (J′) Representative BLI images of tumors 11 days post-treatment. (K) Kaplan-Meier survival curves from (J). p < 0.002 (AAV9-hIFNβ hi), p < 0.005 (AAV9-hIFNβ lo) by log-rank (Mantel-Cox) test compared to AAV9-GFP. (I) Distribution of treatment responses in CDX mice at day 27 by BLI flux as in (I).

Article Snippet: Cells were treated on day 2 with the following: media as a negative control, AAV9-hIFNβ (MOI 4E5), or r-hIFNβ (47 IU/mL, equivalent to 114 pg/mL,PBL Assay Science Cat#11415-1, Lot:7600).

Techniques: In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay, Saline

Journal: Molecular Therapy Oncology

Article Title: AAV immuno-gene therapy platform delivering vectorized cytokines defines a new modality for high-grade glioma treatment

doi: 10.1016/j.omton.2026.201183

Figure Lengend Snippet: The standard GL261 syngeneic GBM model fails to recapitulate interferon responses due to species-specific differences (A) Schematic of the syngeneic allograft study in immunocompetent C57BL/6J mice orthotopically engrafted with GL261-FL cells and treated intratumorally via CED with saline, AAV9-GFP (2E11 vg/brain), or AAV9-mIFNβ (2E11 vg/brain). (B) Longitudinal BLI of FLuc+ tumor growth in mice treated as in (A). Saline = black, AAV9-GFP = green, AAV9-mIFNβ = orange. Thin lines = individual mice, thick lines = geometric mean. Vertical dashed line = day of treatment (day 5). n = 45 (15 per arm). (C) Kaplan-Meier survival curves for treated mice from (B). p = 0.05 by log-rank (Mantel-Cox) test for AAV9-mIFNβ vs. AAV9-GFP. (D) UMAP of single-cell RNA-seq profiles from brains of mice treated with saline, AAV9-GFP, or AAV9-mIFNβ, colored by cell type (left) and treatment type (right). (E) Differential gene expression analysis (DESeq2) of tumor cells from AAV9-mIFNβ-treated vs. AAV9-GFP-treated mice. Red = p -Adj<0.01, gray = p -Adj>0.01. (F) Differentially expressed genes in mouse PBMCs treated with mIFNβ vs. media control (left), human PBMCs treated with hIFNβ vs. media control (middle), and mouse PBMCs treated with hIFNβ vs. media control (right). Significantly differentially expressed genes ( p -Adj<0.01) = red, with select genes labeled. (G) Venn diagrams showing minimal overlap of significantly upregulated (left) and downregulated (right) genes between mouse and human PBMCs treated with species-matched IFNβ. (H) Concordance plot comparing fold change responses to species-matched IFNβ in mouse vs. human PBMCs reveals no correlation (R 2 = 0.012), indicating that interferon responses are strongly species specific.

Article Snippet: Human PBMCs were treated with either recombinant human IFNβ (PBL Cat#11415-1) or media control and placed in an incubator (37°C, 5% CO 2 , with saturating humidity) for 24 ± 1 h. Mouse PBMCs were treated with recombinant mouse IFNβ (PBL Cat#12405-1 Lot:7573R), recombinant human IFNβ, or media control and placed in an incubator (37°C, 5% CO 2 , with saturating humidity) for 24 ± 1 h. At the time of collection, plates were spun in a swinging bucket rotor centrifuge (350 × g , 5 min).

Techniques: Saline, Single Cell, RNA Sequencing, Gene Expression, Control, Labeling