Journal: The Journal of Experimental Medicine
Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design
doi: 10.1084/jem.20242039
Figure Lengend Snippet: Development of a simIFNα to counteract anti-IFNα autoAbs. (A) IFNα2 amino acid sequence with residues targeted by autoAbs in bold, and residues selected for substitution that are bound by IFNAR1/IFNAR2 underlined. Numbering refers to the mature form of IFNα2. (B) Immunostimulatory activity of the indicated IFNα2 mutants compared with IFNα2 WT on AIR cells at 16 h after stimulation. Input IFNα2 amounts were first normalized to 10 7 HiBiT luciferase units before the stimulatory activity of each mutant was titrated out using fourfold serial dilutions. The dilutions at which each protein induced AIR cell activity 10-fold over baseline were calculated by nonlinear regression curve fitting using GraphPad Prism 10. Data represent mean values from n = 2 replicates. ND: not detectable. Blue bars indicate mutants studied further. (C) Immunostimulatory activity of IFNα2 R33A and IFNα2 R120E mutants using HEK293T cell supernatants at the indicated dilutions. Data are normalized to the luciferase signal from unstimulated cells. Data represent mean values from n = 3 replicates. (D) Western blot analysis of Avi-tagged IFNα2 R33A/R120E protein, as compared to IFNα2 WT , in cell and supernatant fractions from transfected HEK293T cells. β-Actin was used as a loading/specificity control. (E) Immunostimulatory activity of IFNα2 R33A/R120E , as compared to IFNα2 WT , on AIR cells at the indicated dilution and at 16 h after stimulation. Data are normalized to the luciferase signal obtained from the unstimulated control. Data represent mean values from n = 3 replicates. (F) Comparison of anti-IFNα IgG autoAb reactivity with IFNα2 WT and IFNα2 R33A/R120E proteins using the HiBiT-based qIP assay for six plasmas. Data represent mean values from n = 3 replicates. (G) Neutralization of 1 ng/ml IFNα2 activity on AIR cells by six plasmas, and inhibition of neutralization by preincubation of plasmas with simIFNα. Data are normalized to the luciferase signals from the mock plasma–treated conditions. (H) Relative levels of anti-IFNα IgG autoAbs and virus-specific IgG antibodies (HIV: BG505 SOSIP; COVID: S2) before (pre-) and after plasma depletion using control microparticle beads, or microparticle beads coupled to simIFNα. Data are expressed as MFI FC values made relative to the values derived from six negative control (healthy donor) plasma samples without anti-IFN-I or anti-virus IgG. (I) IFNα2 neutralization activities of the indicated anti-IFNα IgG autoAb–positive plasma samples before and after depletion as described in H. Data are normalized to the luciferase signal from a healthy control plasma–treated condition. Dashed lines in G and I indicate neutralization thresholds, set at 25% activity relative to the IFN-only condition (G) or healthy donor control (I). For all data panels, results shown are representative of at least n = 2 similar experiments. Statistical significance between groups was determined using unpaired t tests (F, H, and I): ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also . FC, fold change. Source data are available for this figure: .
Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).
Techniques: Sequencing, Activity Assay, Luciferase, Mutagenesis, Western Blot, Transfection, Control, Comparison, Neutralization, Inhibition, Clinical Proteomics, Virus, Derivative Assay, Negative Control
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
