Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-toxin

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor kappa B signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

Impaired IL-23-dependent induction of IFN-gamma underlies mycobacterial disease in patients with inherited TYK2 deficiency

Human cells homozygous for rare loss-of-expression (LOE) TYK2 alleles have impaired, but not abolished, cellular responses to IFN-alpha/beta (underlying viral diseases in the patients) and to IL-12 and IL-23 (underlying mycobacterial diseases). Cells homozygous for the common P1104A TYK2 allele have selectively impaired responses to IL-23 (underlying isolated mycobacterial disease). We report three new forms of TYK2 deficiency in six patients from five families homozygous for rare TYK2 alleles (R864C, G996R, G634E, or G1010D) or compound heterozygous for P1104A and a rare allele (A928V). All these missense alleles encode detectable proteins. The R864C and G1010D alleles are hypomorphic and loss-of-function (LOF), respectively, across signaling pathways. By contrast, hypomorphic G996R, G634E, and A928V mutations selectively impair responses to IL-23, like P1104A. Impairment of the IL-23-dependent induction of IFN-gamma is the only mechanism of mycobacterial disease common to patients with complete TYK2 deficiency with or without TYK2 expression, partial TYK2 deficiency across signaling pathways, or rare or common partial TYK2 deficiency specific for IL-23 signaling.ANRS Nord-Sud ; CIBSS ; CODI ; Comité para el Desarrollo de la Investigación ; Fulbright Future Scholarshi

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19.

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-w (IFN-w) (13 patients), against the 13 types of IFN-a (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men

Inherited PD-1 deficiency: single-cell RNA sequencing (scRNASeq) of PBMCs

To identify transcriptional modules perturbed by inherited PD-1 deficiency, single-cell RNA sequencing was performed on PBMCs obtained from the ten-year-old PD-1-deficient patient and his healthy six-year-old brother (wild-type for the PDCD1 allele). The preparation was enriched in viable cells by resuspending 1×10^6 cells in 200 μL of 2% FBS and 1 mM calcium chloride in PBS and passing the suspension through a strainer with 40 µm pores, before removing dead cells with the EasySep Dead Cell Removal Kit (StemCell Technologies, Cat: 17899). The initial viability of ~50% was increased to 79% and 75% for the samples of the patient and his brother, respectively. The samples were loaded onto a 10X Genomics Chromium chip for single-cell capture. Libraries were prepared with the Chromium Single Cell 3’ Reagent Kit (v3.1). Library quality was assessed with a Bioanalyzer DNA chip. The libraries were sequenced on one lane (S4 flowcell) of an Illumina NovaSeq 6000 sequencer. Sequences were processed with Cell Ranger 3.1.0. The filtered feature-cell matrix was used for subsequent analyses. Separately, PBMCs from four healthy controls were processed and sequenced as described above (i.e., historical controls), and the data were analyzed simultaneously. Publicly available control PBMC datasets were downloaded from the 10X Genomics website (https://support.10xgenomics.com/single-cell-gene-expression/datasets). The filtered feature-cell matrix was retrieved for the following datasets: frozen PBMCs (Donors A to C) from Zheng et al.64, 33,000 PBMCs from a healthy donor from the Chromium demonstration (v1 Chemistry, processed by Cell Ranger 1.1.0), 8,000 PBMCs from a healthy donor from the Chromium demonstration (v2 Chemistry, processed by Cell Ranger 2.1.0), 10,000 PBMCs from a healthy donor from the Chromium demonstration (v3 Chemistry, processed by Cell Ranger 3.0.0) with or without cell surface protein levels determined by TotalSeq, 5,000 PBMCs from a healthy donor from the Chromium Next GEM demonstration (v3.1 Chemistry, processed by Cell Ranger 3.0.2) with or without cell surface protein levels determined by TotalSeq, and a total of 66,000 PBMCs from an aggregate of eight Chromium connect channels and eight manual channels (v3.1 Chemistry, processed by Cell Ranger 3.1.0). These 10 datasets were used as the baseline for subsequent analyses

Repitope Performance Check Data

Performance test results of the Repitope v3.0.1.(https://github.com/masato-ogishi/Repitope)The suggested cutoffs are 0.36 and 0.54 for MHC-I and MHC-II, respectively

Repitope Performance Check Data

Performance test results of the Repitope v3.0.1.(https://github.com/masato-ogishi/Repitope)The suggested cutoffs are 0.36 and 0.54 for MHC-I and MHC-II, respectively.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Inherited PD-1 deficiency: single-cell RNA sequencing (scRNASeq) of PBMCs

To identify transcriptional modules perturbed by inherited PD-1 deficiency, single-cell RNA sequencing was performed on PBMCs obtained from the ten-year-old PD-1-deficient patient and his healthy six-year-old brother (wild-type for the PDCD1 allele). The preparation was enriched in viable cells by resuspending 1×10^6 cells in 200 μL of 2% FBS and 1 mM calcium chloride in PBS and passing the suspension through a strainer with 40 µm pores, before removing dead cells with the EasySep Dead Cell Removal Kit (StemCell Technologies, Cat: 17899). The initial viability of ~50% was increased to 79% and 75% for the samples of the patient and his brother, respectively. The samples were loaded onto a 10X Genomics Chromium chip for single-cell capture. Libraries were prepared with the Chromium Single Cell 3’ Reagent Kit (v3.1). Library quality was assessed with a Bioanalyzer DNA chip. The libraries were sequenced on one lane (S4 flowcell) of an Illumina NovaSeq 6000 sequencer. Sequences were processed with Cell Ranger 3.1.0. The filtered feature-cell matrix was used for subsequent analyses. Separately, PBMCs from four healthy controls were processed and sequenced as described above (i.e., historical controls), and the data were analyzed simultaneously. Publicly available control PBMC datasets were downloaded from the 10X Genomics website (https://support.10xgenomics.com/single-cell-gene-expression/datasets). The filtered feature-cell matrix was retrieved for the following datasets: frozen PBMCs (Donors A to C) from Zheng et al.64, 33,000 PBMCs from a healthy donor from the Chromium demonstration (v1 Chemistry, processed by Cell Ranger 1.1.0), 8,000 PBMCs from a healthy donor from the Chromium demonstration (v2 Chemistry, processed by Cell Ranger 2.1.0), 10,000 PBMCs from a healthy donor from the Chromium demonstration (v3 Chemistry, processed by Cell Ranger 3.0.0) with or without cell surface protein levels determined by TotalSeq, 5,000 PBMCs from a healthy donor from the Chromium Next GEM demonstration (v3.1 Chemistry, processed by Cell Ranger 3.0.2) with or without cell surface protein levels determined by TotalSeq, and a total of 66,000 PBMCs from an aggregate of eight Chromium connect channels and eight manual channels (v3.1 Chemistry, processed by Cell Ranger 3.1.0). These 10 datasets were used as the baseline for subsequent analyses

Inherited IL-23R deficiency: single-cell RNA sequencing (scRNASeq) of cryopreserved PBMCs

Single-cell RNA sequencing analysis was performed on cryopreserved PBMCs from patients with inherited IL-23R deficiency, together with healthy controls, patients with IL-12Rb1 and IL-12Rb2 deficiencies, and a patient with a heterozygous STAT1 gain-of-function mutation. PBMCs were analyzed either directly after thawing (Baseline) or incubated for 6 hours in vitro with or without recombinant IL-23 (IL23Stim; 100 ng/mL).THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV