Eric Meffre

Gain-of-function (GOF) variants in human TLR7 have recently been reported in 11 cases, six of which were diagnosed with systemic lupus erythematosus (SLE). We have identified the X-linked L840R TLR7 variant in hemizygosity in a male patient with SLE and in heterozygosity in his clinically asymptomatic mother. The leucine 840 is located at the first amino acid of TLR7 transmembrane domain and is conserved across various species. The L840R substitution is predicted to be deleterious by various scoring algorithms and may therefore affect TLR7 function. Molecular dynamics simulations of TLR7–UNC93B1 interactions revealed that R840 alters nearby amino acids interactions, resulting in increased hydrogen bond between E834 of TLR7 with R157 of UNC93B1. Finally, the L840R TLR7 variant has increased activity compared with WT, as measured with a nuclear factor κB (NF-κB)–specific luciferase reporter upon stimulation with TLR7 agonist R848. Hence, hemizygosity for L840R confers GOF for NF-κB activation and underlies SLE by potentially increasing TLR7 binding to UNC93B1.

Summary paragraph: Multiple sclerosis (MS) is a chronic autoimmune disease targeting the central nervous system (CNS). MS develops almost exclusively in individuals previously infected with Epstein–Barr virus (EBV) ¹ , yet the mechanisms linking EBV infection to MS pathogenesis remain incompletely defined. Here we characterized EBV-infected B cells in MS and demonstrated that EBV directly infects autoreactive anti-CNS antigen B cells and reprograms them into pro-inflammatory antigen-presenting cells (APCs). EBV⁺ B cells in MS were enriched within the CD27⁺CD21 ^low memory B-cell subset and exhibited upregulated B cell activation and APC transcriptional programs. Recombinant antibodies derived from MS blood and cerebrospinal fluid (CSF) EBV⁺ B cells bound brain tissue, and several cross-bound both MS-associated autoantigens and Epstein-Barr virus nuclear antigen-1 (EBNA1). In vitro , EBV⁺ B cells functioned as APCs that stimulated T peripheral helper cells, with associated activation of EBV⁻ anti-CNS antigen B cells. Collectively, these findings support a mechanistic framework in which EBV infects and transcriptionally reprograms autoreactive anti-CNS antigen B cells into APCs that drive pathogenic anti-CNS antigen T cell and EBV ^- B cell responses in MS.

Common variable immunodeficiency (CVID) is a primary antibody defect that leads to frequent infections, but inflammatory complications appear in 30–50%, leading to increased morbidity and mortality. We have previously shown that circulating bacterial 16S ribosomal DNA (rDNA), originating from gut commensals, is significantly increased in the serum of patients with CVID with inflammatory conditions (P = 0.0007). Here we examined the relationships between serum 16S ribosomal DNA (rDNA), isotype-switched memory (SM) B cells, serum IgA, IgA+ SM B cells, serum IFN-γ, and serum B cell–activating factor (BAFF). We found a significant inverse correlation between serum 16S ribosomal DNA (rDNA) concentrations and the numbers of isotype SM B cells, IgA+ SM B cells, and serum IgA levels in our large cohort, suggesting that loss of IgA in the mucosal barrier permits bacterial transcytosis. Loss of SM B cells and lower serum IgA concentrations were both associated with increased serum IFN-γ, as well as increased CXCL9 and serum BAFF concentrations. Serum BAFF was also significantly associated with IFN-γ levels and inversely correlated with baseline serum IgA. We conclude that loss of IgA, accompanied by mucosal defects in CVID, may permit bacterial transcytosis, resulting in excessive IFN-γ and BAFF production, both of which promote autoimmune and inflammatory complications in this immune defect.

Checkpoint inhibitors targeting CTLA-4 and PD-1 revolutionized the treatment of cancer patients, but their use is limited by the emergence of immune-related adverse events (irAEs). We assessed autoreactive B cell frequencies in the blood of cancer patients before and after treatment with checkpoint inhibitors by testing the reactivity of recombinant antibodies cloned from single B cells. We found that anti–PD-1 and anti–CTLA-4 combination therapy induced the emergence of autoreactive mature naive B cells, whereas central B cell tolerance remained functional. In contrast, anti–PD-1 alone did not alter autoreactive B cell counterselection. Anti–CTLA-4 injections in humanized mice also resulted in the production of autoreactive B cells, whereas anti–PD-1 did not. We conclude that CTLA-4 but not PD-1 is required for the removal of developing autoreactive mature naive B cells and that CTLA-4 blockade broadens the peripheral B cell repertoire, which likely contains clones that promote not only irAEs but also antitumor responses.

Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.

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 β2-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.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non–SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non–SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact ofRAG1/RAG2on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an ‘experiment of nature’ to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal aRAG-dependent ‘domino effect’ that impacts stringency of tolerance and B cell fate in the periphery.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20}. Blocking either viral replication with remdesivir^21,22,23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Hyper-IgM syndrome type 2 (HIGM2) is a B cell intrinsic primary immunodeficiency caused by mutations in AICDA encoding activation-induced cytidine deaminase (AID) which impair immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM). Whereas autosomal-recessive AID-deficiency (AR-AID) affects both CSR and SHM, the autosomal-dominant form (AD-AID) due to C-terminal heterozygous variants completely abolishes CSR but only partially affects SHM. AR-AID patients display enhanced germinal center (GC) reactions and autoimmune manifestations, which are not present in AD-AID, suggesting that SHM but not CSR regulates GC reactions and peripheral B cell tolerance. Herein, we describe two siblings with HIGM2 due to a novel homozygous AICDA mutation (c.428-1G > T) which disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant that lacks 10 highly conserved amino acids encoded by exon 4 (AID-ΔE4a). AID-ΔE4a patients suffered from defective CSR and enhanced GC reactions and were therefore indistinguishable from other AR-AID patients. However, the AID-ΔE4a variant only partially affected SHM as observed in AD-AID patients. In addition, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns. Hence, qualitative defects in AID function and altered SHM rather than global decreased SHM activity may account for the disease phenotype in these patients.