IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Loss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation.

Affinity-independent plasma cell differentiation in germinal centers.

The role of antibody affinity in plasma cell (PC) differentiation from germinal centers (GCs) remains contested. Parallel studies by Sprumont et al. and Sutton and Gao et al. show that PCs emerging from GCs produce antibodies with a diverse range of affinities and lack signatures of affinity-based selection. Therefore, commitment to the PC lineage is affinity independent.

Monogenic lupus: insights into disease pathogenesis and therapeutic opportunities.

PURPOSE OF REVIEW: This review aims to provide an overview of the genes and molecular pathways involved in monogenic lupus, the implications for genome diagnosis, and the potential therapies targeting these molecular mechanisms. RECENT FINDINGS: To date, more than 30 genes have been identified as contributors to monogenic lupus. These genes are primarily related to complement deficiency, activation of the type I interferon (IFN) pathway, disruption of B-cell and T-cell tolerance and metabolic pathways, which reveal the multifaceted nature of systemic lupus erythematosus (SLE) pathogenesis. SUMMARY: In-depth study of the causes of monogenic lupus can provide valuable insights into of pathogenic mechanisms of SLE, facilitate the identification of effective biomarkers, and aid in developing therapeutic strategies.

Rare SH2B3 coding variants in lupus patients impair B cell tolerance and predispose to autoimmunity.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.

The transcription factor ZEB2 drives the formation of age-associated B cells.

Age-associated B cells (ABCs) accumulate during infection, aging, and autoimmunity, contributing to lupus pathogenesis. In this study, we screened for transcription factors driving ABC formation and found that zinc finger E-box binding homeobox 2 (ZEB2) is required for human and mouse ABC differentiation in vitro. ABCs are reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with toll-like receptor 7 (TLR7)-driven lupus, ZEB2 is essential for ABC formation and autoimmune pathology. ZEB2 binds to +20-kb myocyte enhancer factor 2b (Mef2b)'s intronic enhancer, repressing MEF2B-mediated germinal center B cell differentiation and promoting ABC formation. ZEB2 also targets genes important for ABC specification and function, including Itgax. ZEB2-driven ABC differentiation requires JAK-STAT (Janus kinase-signal transducer and activator of transcription), and treatment with JAK1/3 inhibitor reduces ABC accumulation in autoimmune mice and patients. Thus, ZEB2 emerges as a driver of B cell autoimmunity.

DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.

Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the ß-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αß and γδ T cells, even in the absence of transforming growth factor-ß. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.

Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection.

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

The Role of Genetic Risk Factors in Pathogenesis of Childhood-Onset Systemic Lupus Erythematosus.

The pathogenesis of childhood-onset systemic lupus erythematosus (cSLE) is complex and not fully understood. It involves three key factors: genetic risk factors, epigenetic mechanisms, and environmental triggers. Genetic factors play a significant role in the development of the disease, particularly in younger individuals. While cSLE has traditionally been considered a polygenic disease, it is now recognized that in rare cases, a single gene mutation can lead to the disease. Although these cases are uncommon, they provide valuable insights into the disease mechanism, enhance our understanding of pathogenesis and immune tolerance, and facilitate the development of targeted treatment strategies. This review aims to provide a comprehensive overview of both monogenic and polygenic SLE, emphasizing the implications of specific genes in disease pathogenesis. By conducting a thorough analysis of the genetic factors involved in SLE, we can improve our understanding of the underlying mechanisms of the disease. Furthermore, this knowledge may contribute to the identification of effective biomarkers and the selection of appropriate therapies for individuals with SLE.

Genetics of SLE: mechanistic insights from monogenic disease and disease-associated variants.

The past few years have provided important insights into the genetic architecture of systemic autoimmunity through aggregation of findings from genome-wide association studies (GWAS) and whole-exome or whole-genome sequencing studies. In the prototypic systemic autoimmune disease systemic lupus erythematosus (SLE), monogenic disease accounts for a small fraction of cases but has been instrumental in the elucidation of disease mechanisms. Defects in the clearance or digestion of extracellular or intracellular DNA or RNA lead to increased sensing of nucleic acids, which can break B cell tolerance and induce the production of type I interferons leading to tissue damage. Current data suggest that multiple GWAS SLE risk alleles act in concert with rare functional variants to promote SLE development. Moreover, introduction of orthologous variant alleles into mice has revealed that pathogenic X-linked dominant and recessive SLE can be caused by novel variants in TLR7 and SAT1, respectively. Such bespoke models of disease help to unravel pathogenic pathways and can be used to test targeted therapies. Cell type-specific expression data revealed that most GWAS SLE risk genes are highly expressed in age-associated B cells (ABCs), which supports the view that ABCs produce lupus autoantibodies and contribute to end-organ damage by persisting in inflamed tissues, including the kidneys. ABCs have thus emerged as key targets of promising precision therapeutics.

Innate virus-sensing pathways in B cell systemic autoimmunity.

Although all multicellular organisms have germ line-encoded innate receptors to sense pathogen-associated molecular patterns, vertebrates also evolved adaptive immunity based on somatically generated antigen receptors on B and T cells. Because randomly generated antigen receptors may also react with self-antigens, tolerance checkpoints operate to limit but not completely prevent autoimmunity. These two systems are intricately linked, with innate immunity playing an instrumental role in the induction of adaptive antiviral immunity. In this work, we review how inborn errors of innate immunity can instigate B cell autoimmunity. Increased nucleic acid sensing, often resulting from defects in metabolizing pathways or retroelement control, can break B cell tolerance and converge into TLR7-, cGAS-STING-, or MAVS-dominant signaling pathways. The resulting syndromes span a spectrum that ranges from chilblain and systemic lupus to severe interferonopathies.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

De Novo PACSIN1 Gene Variant Found in Childhood Lupus and a Role for PACSIN1/TRAF4 Complex in Toll-like Receptor 7 Activation.

OBJECTIVE: Increased Toll-like receptor 7 (TLR-7) signaling leading to the production of type I interferon (IFN) is an important contributor to human systemic lupus erythematosus (SLE). Protein kinase C and casein kinase substrate in neurons 1 (PACSIN1), a molecule that regulates synaptic vesicle recycling, has been linked to TLR-7/TLR-9-mediated type I IFN production in humans and mice, but the underlying mechanism is unknown. We undertook this study to explore the pathogenicity and underlying mechanism of a de novo PACSIN1 missense variant identified in a child with SLE. METHODS: PACSIN1 Q59K de novo and null variants were introduced into a human plasmacytoid dendritic cell line and into mice using CRISPR/Cas9 editing. The effects of the variants on TLR-7/TLR-9 signaling in human and mouse cells, as well as PACSIN1 messenger RNA and IFN signature in SLE patients, were assessed using real-time polymerase chain reaction and flow cytometry. Mechanisms were investigated using luciferase reporter assays, RNA interference, coimmunoprecipitation, and immunofluorescence. RESULTS: We established that PACSIN1 forms a trimolecular complex with tumor necrosis factor receptor-associated factor 4 (TRAF4) and TRAF6 that is important for the regulation of type I IFN. The Q59K mutation in PACSIN1 augments binding to neural Wiskott-Aldrich syndrome protein while it decreases binding to TRAF4, leading to unrestrained TRAF6-mediated activation of type I IFN. Intriguingly, PACSIN1 Q59K increased TLR-7 but not TLR-9 signaling in human cells, leading to elevated expression of IFNß and IFN-inducible genes. Untreated SLE patients had high PACSIN1 expression in peripheral blood cells that correlated positively with IFN-related genes. Introduction of the Pacsin1 Q59K mutation into mice caused increased surface TLR-7 and TRAIL expression in B cells. CONCLUSION: PACSIN1 Q59K increases IFNß activity through the impairment of TRAF4-mediated inhibition of TLR-7 signaling, possibly contributing to SLE risk.

TFR Cells Express Functional CCR6 But It Is Dispensable for Their Development and Localization During Splenic Humoral Immune Responses.

Follicular T cells including T follicular helper (TFH) and T follicular regulatory (TFR) cells are essential in supporting and regulating the quality of antibody responses that develop in the germinal centre (GC). Follicular T cell migration during the propagation of antibody responses is largely attributed to the chemokine receptor CXCR5, however CXCR5 is reportedly redundant in migratory events prior to formation of the GC, and CXCR5-deficient TFH and TFR cells are still capable of localizing to GCs. Here we comprehensively assess chemokine receptor expression by follicular T cells during a model humoral immune response in the spleen. In addition to the known follicular T cell chemokine receptors Cxcr5 and Cxcr4, we show that follicular T cells express high levels of Ccr6, Ccr2 and Cxcr3 transcripts and we identify functional expression of CCR6 protein by both TFH and TFR cells. Notably, a greater proportion of TFR cells expressed CCR6 compared to TFH cells and gating on CCR6+CXCR5hiPD-1hi T cells strongly enriched for TFR cells. Examination of Ccr6-/- mice revealed that CCR6 is not essential for development of the GC response in the spleen, and mixed bone marrow chimera experiments found no evidence for an intrinsic requirement for CCR6 in TFR cell development or localisation during splenic humoral responses. These findings point towards multiple functionally redundant chemotactic signals regulating T cell localisation in the GC.

Extrafollicular Plasmablasts Present in the Acute Phase of Infections Express High Levels of PD-L1 and Are Able to Limit T Cell Response.

During infections with protozoan parasites or some viruses, T cell immunosuppression is generated simultaneously with a high B cell activation. It has been described that, as well as producing antibodies, plasmablasts, the differentiation product of activated B cells, can condition the development of protective immunity in infections. Here, we show that, in T. cruzi infection, all the plasmablasts detected during the acute phase of the infection had higher surface expression of PD-L1 than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in a BCR-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection expressed PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. In vitro experiments showed that PD-L1hi plasmablasts suppressed the T cell response, partially via PD-L1. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, whose peaks of response precede the peak of germinal center response, may have a modulatory function in infections, thus influencing T cell response.

TLR7 gain-of-function genetic variation causes human lupus.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Monocytes asphyxiate germinal centers.

Some bacteria and parasites, such as Salmonella, actively disrupt germinal centers and elicit only low affinity antibodies, but the mechanisms by which microbes alter these responses is poorly understood. In this issue of Immunity, Biram et al. (2022) uncover a mechanism by which Salmonella recruits Sca-1+ monocytes to germinal centers and impairs metabolic adaptation.

P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance.

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.

Structural and functional analysis of target recognition by the lymphocyte adaptor protein LNK.

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.