Characterization of the antispike IgG immune response to COVID-19 vaccines in people with a wide variety of immunodeficiencies.

Research on coronavirus disease 2019 vaccination in immune-deficient/disordered people (IDP) has focused on cancer and organ transplantation populations. In a prospective cohort of 195 IDP and 35 healthy volunteers (HV), antispike immunoglobulin G (IgG) was detected in 88% of IDP after dose 2, increasing to 93% by 6 months after dose 3. Despite high seroconversion, median IgG levels for IDP never surpassed one-third that of HV. IgG binding to Omicron BA.1 was lowest among variants. Angiotensin-converting enzyme 2 pseudo-neutralization only modestly correlated with antispike IgG concentration. IgG levels were not significantly altered by receipt of different messenger RNA-based vaccines, immunomodulating treatments, and prior severe acute respiratory syndrome coronavirus 2 infections. While our data show that three doses of coronavirus disease 2019 vaccinations induce antispike IgG in most IDP, additional doses are needed to increase protection. Because of the notably reduced IgG response to Omicron BA.1, the efficacy of additional vaccinations, including bivalent vaccines, should be studied in this population.

Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling.

We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)-6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as "COVID toes," remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19.

Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy.

Time-resolved systems immunology reveals a late juncture linked to fatal COVID-19.

COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating interleukin (IL)-15. CD8+ T cell activation was apparent without signs of exhaustion. Although cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery versus fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome.

BACKGROUND: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). OBJECTIVE: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. METHODS: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. RESULTS: We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by Th1 effector T cells. CONCLUSIONS: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.

Patients with Idiopathic Pulmonary Nontuberculous Mycobacterial Disease Have Normal Th1/Th2 Cytokine Responses but Diminished Th17 Cytokine and Enhanced Granulocyte-Macrophage Colony-Stimulating Factor Production.

OBJECTIVE: Although disseminated nontuberculous mycobacterial infection is attributed to defects in the interleukin (IL)-12/interferon-γ circuit, the immunophenotype of idiopathic pulmonary nontuberculous mycobacterial (PNTM) disease is not well defined. METHOD: We phenotyped Th1, Th2, Th17, and Treg cytokines and colony-stimulating factor production from patients with idiopathic PNTM disease. Data were compared with healthy donors, cystic fibrosis (CF), and primary ciliary dyskinesia (PCD) patients with PNTM disease. Both supernatant cytokine production and intracellular cytokines expressed by various leukocyte subpopulations following mitogen and antigen stimulation were assayed by electrochemiluminescence-based multiplex immunoassay and flow cytometry, respectively. RESULTS: Regardless of antigen or mitogen stimulation, neither intracellular nor extracellular Th1, Th2, and Treg cytokine levels differed between patients and controls. Th17 cells and IL-17A levels were lower in idiopathic PNTM patients, whereas monocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in response to NTM stimulation was higher compared with healthy donors. Besides, distinct cytokine responses following stimulation by Mycobacterium abscessus and Mycobacterium avium were observed consistently within each group. CONCLUSIONS: The IL-12/IFN-γ circuit appeared intact in patients with idiopathic PNTM disease. However, idiopathic PNTM patients had reduced Th17 response and higher mycobacteria-induced monocyte GM-CSF expression.

X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by recurrent life-threatening bacterial and fungal infections and aberrant inflammation. Mutations in CYBB cause X-linked CGD and account for 65% to 70% of cases in Western countries. OBJECTIVE: We sought to understand the clinical manifestations associated with the X-linked CGD carrier state. METHODS: We undertook a comprehensive retrospective study of 162 affected female subjects. We examined dihydrorhodamine 123 (DHR) oxidation data for percentage of X-chromosome inactivation. We correlated lyonization (%DHR+) with clinical features. Where possible, we followed %DHR+ values over time. RESULTS: Clinical data were available for 93 female subjects: %DHR+ values were 46% (mean) and 47% (median; SD, 24). Using the %DHR+ value as the criterion for X inactivation, 78% of patients had levels of inactivation of 20% to 80%, suggesting random inactivation that was independent of age. In contrast, carriers with CGD-type infections had median %DHR+ values of 8% (n = 14; range, 0.06% to 48%), and those with only autoimmune or inflammatory manifestations had median %DHR+ values of 39% (n = 31; range, 7.4% to 74%). Those with both infections and autoimmunity had low %DHR+ values (n = 6; range, 3% to 14%). A %DHR+ value of less than 10% was strongly associated with infections (odds ratio, 99). Strong association persisted when %DHR+ values were less than 20% (odds ratio, 12). Autoimmunity was not associated with %DHR+ values. In 2 sets of identical twins, the %DHR+ populations tracked closely over time. Although the %DHR+ populations were very similar between sisters, those between mothers and daughters were unrelated. CONCLUSIONS: A low %DHR+ value strongly predicts infection risk in X-linked CGD carriers, and the carrier state itself is associated with autoimmunity.

Cytoskeletal abnormalities and neutrophil dysfunction in WDR1 deficiency.

Cell motility, division, and structural integrity depend on dynamic remodeling of the cellular cytoskeleton, which is regulated in part by actin polymerization and depolymerization. In 3 families, we identified 4 children with recurrent infections and varying clinical manifestations including mild neutropenia, impaired wound healing, severe stomatitis with oral stenosis, and death. All patients studied had similar distinctive neutrophil herniation of the nuclear lobes and agranular regions within the cytosol. Chemotaxis and chemokinesis were markedly impaired, but staphylococcal killing was normal, and neutrophil oxidative burst was increased both basally and on stimulation. Neutrophil spreading on glass and cell polarization were also impaired. Neutrophil F-actin was elevated fourfold, suggesting an abnormality in F-actin regulation. Two-dimensional differential in-gel electrophoresis identified abnormal actin-interacting protein 1 (Aip1), encoded by WDR1, in patient samples. Biallelic mutations in WDR1 affecting distinct antiparallel ß-strands of Aip1 were identified in all patients. It has been previously reported that Aip1 regulates cofilin-mediated actin depolymerization, which is required for normal neutrophil function. Heterozygous mutations in clinically normal relatives confirmed that WDR1 deficiency is autosomal recessive. Allogeneic stem cell transplantation corrected the immunologic defect in 1 patient. Mutations in WDR1 affect neutrophil morphology, motility, and function, causing a novel primary immunodeficiency.

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.