Suppression of Type I Interferon Signaling in Myeloid Cells by Autoantibodies in Severe COVID-19 Patients

Abstract

Purpose: Auto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs.

Methods: We evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs.

Results: Three of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor.

Conclusion: Auto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.

Keywords: Autoantibody; BCR repertoires; COVID-19; Epitope mapping; Single-cell RNA sequencing; Type I IFNs.

Fig. 1

Detection of neutralizing auto-abs to IFN-α2, IFN-β, and IFN-ω in severe or critical COVID-19 patients. a. Measurement of auto-abs to human IFN-α2 in the plasma from mild, severe and critical COVID-19 patients using an ELISA. Black or gray circles, diamond, triangles, and rectangles represent patients carrying anti-IFN-α2 antibodies (n=7). b. Neutralizing activity of the auto-abs to IFN-α2. The phosphorylation of STAT1 in U937 cells stimulated with recombinant human IFN-α2 was evaluated in the presence of 10% plasma from patients (n=123). Black or gray circles, diamond, triangles, and rectangles represent patients carrying neutralizing anti-IFN-α2 antibodies (n=7). c. Neutralizing activity of auto-abs to IFN-β or IFN-ω. Phosphorylation of STAT1 in U937 cells stimulated with recombinant human IFN-β or IFN-ω was evaluated in the presence of 10% plasma from patients (n=123). d. Characteristics of each patient with neutralizing auto-abs to type I IFNs

Fig. 2

A longitudinal analysis of auto-abs titers and the neutralizing activity to IFN-α2 during hospitalization. a. Auto-abs to IFN-α2 titers and the neutralizing activity in four patients (Case No. 1, 5, 6, 7) who did not show improvement during hospitalization. b. Auto-abs to IFN-α2 titers and the neutralizing activity in three patients (Case No. 2, 3, 4) who recovered from COVID-19. P values were determined by Wilcoxon’s matched-pairs signed rank test

Fig. 3

A scRNA-seq analysis for leukocytes composition and ISG signaling in COVID-19 patients with auto-abs to type IFNs. a. UMAP projections of CD45⁺ cells isolated from PBMCs of 28 COVID-19 patients (Mild: n=12, Critical: n=11, Severe or critical with auto-abs: n=5 [Case no. 1, 2, 3, 4 and 5]). Nine clusters were identified, including CD4⁺ T cells (CD4), CD8⁺ T cells (CD8), NK cells (NK), B cells, plasma cells, canonical monocytes (cM), non-canonical monocytes (ncM), conventional dendritic cells (cDC), and plasmacytoid DCs (pDC). b. Bar plots depicting immune cell-type composition in COVID-19 patients showing mild and severe or critical symptoms in addition to severe or critical patients carrying auto-abs to type I IFNs. c. Heatmap showing specific type I IFN-stimulated gene (ISG), type II ISG, and shared ISG-I and ISG-II in CD45⁺ cells from the three groups. d. Single-sample gene set variant analysis (ssGSVA) scores of types I and/or II ISG in each patient group. e. Average of ssGSVA scores of types I and/or II ISG in each patient. One-way ANOVA was used for the statistical analyses. Mean ± SEM. *p <0.05, **p <0.01, ***p <0.001. f. ssGSVA scores of types I and/or II ISG in each cell type of each group

Fig. 4

Impaired SARS-CoV-2-specific responses of B cells in COVID-19 patients with auto-abs to type I IFNs. a. Clone numbers of BCR repertoires (IGH, IGK and IGL) in CD45⁺ cells from the three groups. b. Pearson’s correlation coefficient for amino acid sequences converted from the scRNA-seq data of BCR repertoire (IGH, IGK and IGL). c. Diversity indexes of the clonotypes of BCR (IGH, IGK and IGL) in mild, critical, or severe-critical patients with auto-abs. One-way ANOVA was used for the statistical analysis. Mean ± SD. ***p <0.001. d. Clonotype numbers of SARS-CoV-2-specific CDR3 of IGH, IGK and IGL in mild, critical, or severe-critical patients with auto-abs. e. The concentrations of SARS-CoV-2 S protein specific antibody in the plasma. Mann-Whitney U test was used for the statistical analyses. Mean ± SEM

Fig. 5

Identification of epitopes recognized by anti-IFN-α2 antibodies in COVID-19 patients. a. Peptide microarray against human IFN-α2 for 6 COVID-19 patients with auto-abs (Case no. 1-6), 6 COVID-19 patients without auto-abs (Case no. 8-13) and 10 healthy control subjects. The data of case no. 2 was omitted from the figure because it was not suitable for evaluation due to high background values. b. Detection of the auto-Abs recognizing the two specific peptides (IFN-α2 _80-95 and IFN-α2 _124-143) in plasma from the COVID-19 patients or healthy controls subjects by ELISA. Mean ± SD. c. Amino acid sequences of the two epitopes (IFN-α2 _80-95 and IFN-α2 _124-143) of human IFN-α2 recognized by the auto-abs (upper). The amino acids surrounded by squares indicate those that bind to the receptor. A ribbon diagram of human IFN-α2 is shown at the bottom (PDB ID: 3SE3). IFN-α2 _80-95 and IFN-α2 _124-143 are highlighted in red and blue respectively. d. Crystal structure of the human IFN-α2, IFNAR1, and IFNAR2 complex (PDB ID: 3SE3). IFN-α2, IFNAR1, and IFNAR2 are shown in light blue, pink, and yellow respectively. The red and blue colors indicate the epitopes (IFN-α2 _80-95 and _124-143) recognized by auto-abs found in the COVID-19 patients