Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

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 of RAG1/RAG2 on 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 a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

RAG Represents a Widespread Threat to the Lymphocyte Genome.

The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer.

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

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 system1-20. Blocking either viral replication with remdesivir21-23 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.

FOXP3 deficiency, from the mechanisms of the disease to curative strategies.

FOXP3 gene is a key transcription factor driving immune tolerance and its deficiency causes immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX), a prototypic primary immune regulatory disorder (PIRD) with defective regulatory T (Treg) cells. Although life-threatening, the increased awareness and early diagnosis have contributed to improved control of the disease. IPEX currently comprises a broad spectrum of clinical autoimmune manifestations from severe early onset organ involvement to moderate, recurrent manifestations. This review focuses on the mechanistic advancements that, since the IPEX discovery in early 2000, have informed the role of the human FOXP3+ Treg cells in controlling peripheral tolerance and shaping the overall immune landscape of IPEX patients and carrier mothers, contributing to defining new treatments.

Nuclear-penetrating scleroderma autoantibody inhibits topoisomerase 1 cleavage complex formation.

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.

Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Normal B cells express ZAP70 in chronic lymphocytic leukemia: A link between autoimmunity and lymphoproliferation?

ZAP70 has a prognostic value in chronic lymphocytic leukemia (CLL), through altered B-cell receptor signaling, which is important in CLL pathogenesis. A good correlation between ZAP70 expression in CLL cells and the occurrence of autoimmune phenomena has been reported. Yet, the great majority of CLL-associated autoimmune cytopenia is due to polyclonal immunoglobulin (Ig) G synthesized by nonmalignant B cells, and this phenomenon is poorly understood. Here, we show, using flow cytometry, that a substantial percentage of CD5- nonmalignant B cells from CLL patients expresses ZAP70 compared with CD5- B cells from healthy subjects. This ZAP70 expression in normal B cells from CLL patients was also evidenced by the detection of ZAP70 mRNA at single-cell level with polyclonal Ig heavy- and light-chain gene transcripts. ZAP70+ normal B cells belong to various B-cell subsets and their presence in the naïve B-cell subset suggests that ZAP70 expression may occur during early B-cell development in CLL patients and potentially before malignant transformation. The presence of ZAP70+ normal B cells is associated with autoimmune cytopenia in CLL patients in our cohort of patients, and recombinant antibodies produced from these ZAP70+ nonmalignant B cells were frequently autoreactive including anti-platelet reactivity. These results provide a better understanding of the implication of ZAP70 in CLL leukemogenesis and the mechanisms of autoimmune complications of CLL.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Impaired B-cell tolerance checkpoints promote the development of autoimmune diseases and pathogenic autoantibodies.

A role for B cells in autoimmune diseases is now clearly established both in mouse models and humans by successful treatment of multiple sclerosis and rheumatoid arthritis with anti-CD20 monoclonal antibodies that eliminate B cells. However, the underlying mechanisms by which B cells promote the development of autoimmune diseases remain poorly understood. Here, we review evidence that patients with autoimmune disease suffer from defects in early B-cell tolerance checkpoints and therefore fail to counterselect developing autoreactive B cells. These B-cell tolerance defects are primary to autoimmune diseases and may result from altered B-cell receptor signaling and dysregulated T-cell/regulatory T-cell compartment. As a consequence, large numbers of autoreactive naive B cells accumulate in the blood of patients with autoimmune diseases and may promote autoimmunity through the presentation of self-antigen to T cells. In addition, new evidence suggests that this reservoir of autoreactive naive B cells contains clones that may develop into CD27- CD21-/lo B cells associated with increased disease severity and plasma cells secreting potentially pathogenic autoantibodies after the acquisition of somatic hypermutations that improve affinity for self-antigens.

A Novel AICDA Splice-Site Mutation in Two Siblings with HIGM2 Permits Somatic Hypermutation but Abrogates Mutational Targeting.

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.

The transmembrane activator TACI triggers immunoglobulin class switching by activating B cells through the adaptor MyD88.

BAFF and APRIL are innate immune mediators that trigger immunoglobulin G (IgG) and IgA class-switch recombination (CSR) in B cells by engaging the receptor TACI. The mechanism that underlies CSR signaling by TACI remains unknown. Here we found that the cytoplasmic domain of TACI encompasses a conserved motif that bound MyD88, an adaptor that activates transcription factor NF-kappaB signaling pathways via a Toll-interleukin 1 (IL-1) receptor (TIR) domain. TACI lacks a TIR domain, yet triggered CSR via the DNA-editing enzyme AID by activating NF-kappaB through a Toll-like receptor (TLR)-like MyD88-IRAK1-IRAK4-TRAF6-TAK1 pathway. TACI-induced CSR was impaired in mice and humans lacking MyD88 or the kinase IRAK4, which indicates that MyD88 controls a B cell-intrinsic, TIR-independent, TACI-dependent pathway for immunoglobulin diversification.

Early B cell tolerance defects in neuromyelitis optica favour anti-AQP4 autoantibody production.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients.

Patients with common variable immunodeficiency with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses.

BACKGROUND: The lack of pathogen-protective, isotype-switched antibodies in patients with common variable immunodeficiency (CVID) suggests germinal center (GC) hypoplasia, yet a subset of patients with CVID is paradoxically affected by autoantibody-mediated autoimmune cytopenias (AICs) and lymphadenopathy. OBJECTIVE: We sought to compare the physical characteristics and immunologic output of GC responses in patients with CVID with AIC (CVID+AIC) and without AIC (CVID-AIC). METHODS: We analyzed GC size and shape in excisional lymph node biopsy specimens from 14 patients with CVID+AIC and 4 patients with CVID-AIC. Using paired peripheral blood samples, we determined how AICs specifically affected B-and T-cell compartments and antibody responses in patients with CVID. RESULTS: We found that patients with CVID+AIC displayed irregularly shaped hyperplastic GCs, whereas GCs were scarce and small in patients with CVID-AIC. GC hyperplasia was also evidenced by an increase in numbers of circulating follicular helper T cells, which correlated with decreased regulatory T-cell frequencies and function. In addition, patients with CVID+AIC had serum endotoxemia associated with a dearth of isotype-switched memory B cells that displayed significantly lower somatic hypermutation frequencies than their counterparts with CVID-AIC. Moreover, IgG+ B cells from patients with CVID+AIC expressed VH4-34-encoded antibodies with unmutated Ala-Val-Tyr and Asn-His-Ser motifs, which recognize both erythrocyte I/i self-antigens and commensal bacteria. CONCLUSIONS: Patients with CVID+AIC do not contain mucosal microbiota and exhibit hyperplastic yet inefficient GC responses that favor the production of untolerized IgG+ B-cell clones that recognize both commensal bacteria and hematopoietic I/i self-antigens.

A novel ATM mutation associated with elevated atypical lymphocyte populations, hyper-IgM, and cutaneous granulomas.

Ataxia-Telangiectasia (AT) is an immunodeficiency most often associated with T cell abnormalities. We describe a patient with a hyper-IgM phenotype and immune cell abnormalities that suggest a distinct clinical phenotype. Significant B cell abnormalities with increased unswitched memory B cells, decreased naive transitional B cells, and an elevated frequency of CD19+CD38loCD27-CD10-CD21-/low B cells expressing high levels of T-bet and Fas were demonstrated. The B cells were hyporesponsive to in vitro stimulation through the B cell receptor, Toll like receptors (TLR) 7 and 9, and CD40. T cell homeostasis was also disturbed with a significant increase in γδ T cells, circulating T follicular helper cells (Tfh), and decreased numbers of T regulatory cells. The ATM mutations in this patient are posited to have resulted in the perturbations in the frequencies and distributions of B and T cell subsets, resulting in the phenotype in this patient. KEY MESSAGES: A novel mutation creating a premature stop codon and a nonsense mutation in the ATM gene are postulated to have resulted in the unique clinical picture characterized by abnormal B and T cell populations, lymphocyte subset dysfunction, granuloma formation, and a hyper-IgM phenotype. CAPSULE SUMMARY: A patient presented with ataxia-telangiectasia, cutaneous granulomas, and a hyper-IgM phenotype; a novel combination of mutations in the ATM gene was associated with abnormal distributions, frequencies, and function of T and B lymphocyte subsets.

Transitional B cells in quiescent SLE: An early checkpoint imprinted by IFN.

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells, as the most immature naïve B cell subset upstream of pathogenic B cells, display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated, mostly Caucasian, SLE patients, we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition, early transitional B cells from the bone marrow displayed the highest interferon score, reflecting a B cell interferon burden of central origin. Hence, the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment, CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass, ultimately leading in periphery to the expansion of autoantibodies-secreting cells.

A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production.

Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B- and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans.