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.

UNCovering new causes of monogenic systemic lupus erythematosus.

UNC93B1 is essential for the stability and endosomal trafficking of nucleic-acid sensing Toll-like receptors (TLRs) including TLR7 and TLR8. Increased TLR7 responses are associated with lupus autoimmunity in both mice and humans. In a recent article, Al-Azab et al. demonstrate the role of a variant of UNC93B1 (p.V117L) in the induction of pediatric systemic lupus erythematosus in patients and in mice through TLR7/8 hyperresponsiveness. They also highlight a potential role for the pharmacological inhibition of interleukin-1 receptor-associated kinase (IRAK) 1 and/or 4 in ameliorating disease.

A BATF-ling connection between B cells and follicular helper T cells.

The transcription factor BATF directly regulates key components of the formation and function of follicular helper T cells and antibody class switching in B cells.

Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2'-O-methyl gapmer oligonucleotides.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.

The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.

Follicular helper T (Tfh) cells provide selection signals to germinal center B cells, which is essential for long-lived antibody responses. High CXCR5 and low CCR7 expression facilitates their homing to B cell follicles and distinguishes them from T helper 1 (Th1), Th2, and Th17 cells. Here, we showed that Bcl-6 directs Tfh cell differentiation: Bcl-6-deficient T cells failed to develop into Tfh cells and could not sustain germinal center responses, whereas forced expression of Bcl-6 in CD4(+) T cells promoted expression of the hallmark Tfh cell molecules CXCR5, CXCR4, and PD-1. Bcl-6 bound to the promoters of the Th1 and Th17 cell transcriptional regulators T-bet and RORgammat and repressed IFN-gamma and IL-17 production. Bcl-6 also repressed expression of many microRNAs (miRNAs) predicted to control the Tfh cell signature, including miR-17-92, which repressed CXCR5 expression. Thus, Bcl-6 positively directs Tfh cell differentiation, through combined repression of miRNAs and transcription factors.

Heterozygosity for Roquinsan leads to angioimmunoblastic T-cell lymphoma-like tumors in mice.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (T(FH)) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the "san" allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of T(FH) cells preceded tumor development, and clonal rearrangements in the TCR-ß genes were present in most tumors. Furthermore, T(FH) cells exhibited increased clonality compared with non-T(FH) cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent T(FH) development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a T(FH)-derived origin. Roquin(san/+) mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated T(FH) cells or their consequences.

Zeb2 drives the formation of CD11c+ atypical B cells to sustain germinal centers that control persistent infection.

CD11c+ atypical B cells (ABCs) are an alternative memory B cell lineage associated with immunization, infection, and autoimmunity. However, the factors that drive the transcriptional program of ABCs have not been identified, and the function of this population remains incompletely understood. Here, we identified candidate transcription factors associated with the ABC population based on a human tonsillar B cell single-cell dataset. We identified CD11c+ B cells in mice with a similar transcriptomic signature to human ABCs, and using an optimized CRISPR-Cas9 knockdown screen, we observed that loss of zinc finger E-box binding homeobox 2 (Zeb2) impaired ABC formation. Furthermore, ZEB2 haplo-insufficient Mowat-Wilson syndrome (MWS) patients have decreased circulating ABCs in the blood. In Cd23Cre/+Zeb2fl/fl mice with impaired ABC formation, ABCs were dispensable for efficient humoral responses after Plasmodium sporozoite immunization but were required to control recrudescent blood-stage malaria. Immune phenotyping revealed that ABCs drive optimal T follicular helper (TFH) cell formation and germinal center (GC) responses and they reside at the red/white pulp border, likely permitting better access to pathogen antigens for presentation. Collectively, our study shows that ABC formation is dependent on Zeb2, and these cells can limit recrudescent infection by sustaining GC reactions.

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.

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.