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.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease [1][2][3][4][5][6][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 RNA 8,9 and binds to guanosine [10][11][12] . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP [10][11][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 Tlr7 Y264H 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.
Although systemic lupus erythematosus (SLE) is generally a polygenic autoimmune disease, the discovery of monogenic lupus cases and rare pathogenic variants has provided important insights into disease mechanisms, including important roles of complement, type I interferons and B cell survival [13][14][15] . There is accumulating evidence that patients with SLE display phenotypes that are consistent with increased TLR7 signalling associated with elevated IgD − CD27 − double-negative B cells and, more specifically, the CXCR5 − CD11c + subset (also known as DN2 B cells or age-associated B cells (ABCs)) in the peripheral blood 1 , and excessive accumulation of extrafollicular helper T cells 16 . Genome-wide association studies have identified common polymorphisms in or near TLR7 that segregate with SLE [2][3][4] . In mice, increased TLR7 signalling due to the duplication of the TLR7-encoding Yaa locus or to transgenic TLR7 expression exacerbates autoimmunity 5,6 and deletion of TLR7 prevents or ameliorates disease in other lupus models 7 . Despite this mounting link between TLR7 and the pathogenesis of lupus, no human SLE cases due to TLR7 variants have been reported to date. There is also conflicting evidence as to how TLR7 overexpression causes autoimmunity, particularly, the relative roles of TLR7-driven spontaneous germinal centres (GCs) versus the role of TLR7-driven double-negative B cells; the latter have been proposed to originate extrafollicularly and be pathogenic in lupus 1 . Most mouse lupus models in which TLR7 has a role in pathogenicity display increased formation of GCs and T follicular helper (T FH ) cells 5,6 and it has been proposed that TLR7 drives GCs enriched in self-reactive B cells 17 . However, recent reports have demonstrated that lupus can develop independently of GCs in mouse models in which disease is dependent on MyD88 signalling 18,19 .
TLR7 and TLR8 selectively detect a subset of RNA sequences [20][21][22] . On the basis of recent knowledge of how TLR8 senses RNA degradation products to trigger downstream signalling 23,24 , it is thought that one ligand-recognition site in TLR7 binds to guanosine or 2′,3′-cyclophosphate guanosine monophosphate (cGMP), derived from GTP degradation 10 , which synergizes with uridine-rich short RNAs binding to a second site 11,12 . Here we describe the action of a de novo TLR7 Article TLR7 Y264H causes autoimmunity in mice Our modelling suggested TLR7 Y264H would increase affinity to endogenous ligands. To investigate whether TLR7 Y264H could cause SLE, we introduced the orthologous allele into C57BL/6 mice using CRISPR-Cas9 editing. WES analysis confirmed that Tlr7 Y264H was the only relevant CRISPR-induced coding variant segregating with the phenotype. The resulting strain was named kika and Tlr7 Y264H is hereafter named the kik allele. A CRISPR-generated line lacking TLR7 protein owing to a 1-bp deletion was included as a control (Extended Data Fig. 2a, b). Male or female mice (aged 12 weeks) carrying one or two kik alleles displayed splenomegaly with increased cellularity (Fig. 1j), decreased survival (Fig. 1k) and ANAs with nuclear, cytoplasmic, cell-cycle-dependent and Golgi staining (Fig. 1l, Extended Data Fig. 3a). These were detected from 6 weeks of age in female kika mice (Extended Data Fig. 3d, e). Male Tlr7 kik/Y and female Tlr7 kik/+ kika mice also developed antibodies to the TLR7 ligands ssRNA and Smith protein (Sm) and ribonucleoprotein (RNP), an RNA-containing nuclear self antigen 7 (Fig. 1m), and 10-20% had weak double-stranded DNA reactivity (Extended Data Fig. 3b, c). These results suggest TLR7 Y264H is an X chromosome-linked dominant GOF allele.

H264 increases the response to guanosine
To validate the predicted increased affinity of H264 for guanosine, we tested the response of Tlr7 kik/Y kika bone-marrow-derived macrophages (BMDMs) to increasing doses of guanosine and R848. Although no difference was observed with R848 ( Fig. 1n and data not shown), we found an increased responsiveness to guanosine from kika BMDMs compared with wild-type BMDMs ( Fig. 1o and Extended Data Fig. 3g). Both the first (guanosine-binding) and second (uridine-binding) sites of TLR7 are necessary for ssRNA-induced TLR7 signalling 8,10,12 . We tested the responsiveness of wild-type ands mutant BMDMs to ssRNAs lacking uridine (ss41-L) or containing 6 to 10 uridines. Interestingly, whereas ssRNA sensing by wild-type BMDMs correlated with uridine content, ssRNA sensing by kika BMDMs was independent of uridine content, and ssRNA lacking uridine induced TLR7 activity in kika cells (Extended Data Fig. 3h). These results collectively indicate that the kika mutation selectively increases sensing of guanosine to the first site independently of activity at the uridine-selective second site, therefore raising the sensitivity to otherwise non-TLR7-stimulating ssRNAs.

Tissue damage caused by TLR7 Y264H
In-depth phenotyping of kika mice revealed marked thrombocytopenia as seen in the proband (Fig. 2a) and a slightly lower white blood cell count (Extended Data Fig. 5a). Proliferative glomerulonephritis was evident in kidneys ( Fig. 2b), as well as expanded mesangial matrix with electron-dense deposits and increased mesangial cellularity (Fig. 2c). Lymphoid infiltrates were seen in the liver, salivary glands and pancreas (Fig. 2d) where they occasionally formed peri-islet follicular structures. Exocrine pancreatic tissue was often replaced by fat, particularly in Tlr7 kik/kik mice (Fig. 2d). Other findings included subpleural, perivascular and interstitial infiltrates in the lungs; myocyte degeneration and necrosis in skin panniculus muscle; focal myocardial fibrosis, splenic lymphomas (4 out of 6 mice); chronic lymphadenitis in the lymph nodes and gut; and hyperplasia of Peyer's patches (Supplementary Table 4, Extended Data Fig. 4). Serum levels of IFNγ, IL-6, IL-10 and TNF were increased (Fig. 2e, Extended Data Fig. 3f).
Flow cytometry analysis of kika spleens revealed a reduced T:B cell ratio with an increase in total B cells, spontaneous GCs, and increased plasma cells and ABCs; ABCs were also expanded in the blood and kidneys ( Fig. 2f-i, Extended Data Fig. 5b, c). The percentages of splenic marginal zone B cells were decreased, but the total numbers were not decreased (Extended Data Fig. 5d). Effector or memory CD4 + CD44 high cells, including T FH cells and CXCR3 + extrafollicular helper CD4 + T cells, were increased in kika mice ( Fig. 2j-l) as well as circulating single-residue gain-of-function (GOF) variant that increases the affinity of TLR7 for guanosine and cGMP, causing enhanced TLR7 activation and childhood-onset SLE.

TLR7 variants in patients with SLE
We undertook whole-genome sequencing of a Spanish girl who was diagnosed with SLE at the age of 7 (Supplementary Table 1). She first presented with refractory autoimmune thrombocytopenia and had elevated anti-nuclear antibodies (ANAs) and hypocomplementaemia. She went on to develop inflammatory arthralgias, constitutional symptoms, intermittent episodes of hemichorea, and had mild mitral insufficiency and renal involvement after admission with a hypertensive crisis. Bioinformatics analysis revealed a de novo, TLR7 p.Tyr264His (Y264H) missense variant that was predicted to be damaging by SIFT and CADD ( Fig. 1a-c (family A) and Supplementary Table 2). This variant was not present in the databases of normal human genome variation (gnomAD, ExAC, dbSNP). Examination of the BAM files together with paternity analysis confirmed that the mutation occurred de novo (Extended Data Fig. 1a, b, d). The mutated tyrosine residue lies in the eighth leucine-rich repeat of TLR7 25 , within the endosomal part of the receptor (Fig. 1b) and is highly conserved across species, including zebrafish ( Fig. 1d) Tables 1, 2). Notably, in family B the mother had SLE from her mid-twenties and the daughter was diagnosed with neuromyelitis optica in the presence of ANAs and antibodies to aquaporin-4 (AQP4-Ab, also known as NMO-IgG) in the serum and cerebrospinal fluid. Both carried the TLR7 F507L variant, which was also highly conserved (Fig. 1d). No additional rare variants in the 22 SLE-causing genes were identified in these families (Supplementary Table 3).

TLR7 Y264H increases guanosine sensing
To test whether pathogenic variants enhance TLR7 signalling and downstream NF-κB activation, we transfected RAW264.7 cells with plasmids encoding the different TLR7 mutants. Compared with wild-type cells, overexpression of TLR7 Y264H and TLR7 F507L constructs showed enhanced NF-κB activation for the mutant variants after 2′,3′-cGMP stimulation, whereas overexpression of TLR7 R28G showed enhanced NF-κB activation after stimulation with guanosine and single-stranded RNA (ssRNA) (Fig. 1e, f). Using a published TLR7 structure, we mapped the mutated Tyr264 residue to TLR7 ligand-binding site 1, where R837/R848 and guanosine or its analogues (such as 2′,3′-cGMP) bind 10 . The Tyr264 side-chain OH has been shown to form a hydrogen bond with 2′,3′-cGMP 10 . To further understand whether the Y264H mutation could enhance TLR7 sensing or activation, we used the method of thermodynamic integration within molecular dynamics simulations [27][28][29] to determine the relative binding affinity of the ligands to dimeric TLR7 (Extended Data Fig. 1e, f). Guanosine, but not R848, appeared to have increased affinity to its binding site with both singly (H264) and doubly protonated (H + 264) protonated variants (Fig. 1g). The site of the mutation, Tyr264, was close (albeit not bound) to the guanosine, which formed a hydrogen bond to Thr586, limiting the access of water to this end of the ligand (Fig. 1h). By contrast, the mutants H264 and H + 264, appeared to allow solvent to access this region, helping to stabilize a cluster of water molecules that provide a favourable environment for the polar ribose ring of guanosine (Fig. 1i). In these simulations, Y264H + also displayed stronger attractive electrostatic interactions with guanosine, providing a plausible explanation for the much larger predicted affinity to this variant. The electrostatic attraction of H + 264 is predicted to be even greater for the natural ligand 2′,3′-cGMP that carries a negative charge. CXCR5 high PD-1 high GC T FH cells that are usually only seen in secondary lymphoid tissues (Extended Data Fig. 5e). Plasmacytoid dendritic cells (pDCs) appeared to be activated with increased MHC-II and reduced siglec-H expression 30 (Extended Data Fig. 5f). TLR7-deficient mice lacked spontaneous GC B cells, T FH cells and ABCs, and had reduced plasma cells (Extended Data Fig. 5g), supporting that the expansion of these subsets in kika mice is driven by the TLR7 GOF.

B-cell-intrinsic effects of TLR7 Y264H
To establish which phenotypes were cell autonomous, mixed bone marrow chimeras were generated by adoptively transferring 100% wild-type CD45.2 or kika CD45.2 bone marrow, or 50:50 mixes of either wild-type CD45.1:kika CD45.2 or wild-type CD45.1:wild-type CD45.2 bone marrow into sublethally irradiated Rag1 −/− mice (Fig. 3a, b, Extended Data Fig. 5h). Autoantibodies were present in chimeric mice receiving either 100% or 50% kika bone marrow cells (Fig. 3a, Extended Data Fig. 5j). Expansion of GCs, ABCs and plasma cells (Fig. 3b) was cell-intrinsic whereas all T cell phenotypes and reduction in marginal zone B cells were largely cell-extrinsic (Fig. 3b, Extended Data Fig. 5h, i). The ABCs of kika mice expressed more TLR7 although not to the extent seen in Yaa mice that express two copies of Tlr7 (Fig. 3c). By contrast, the functional consequences of the Y264H mutation were more severe than the Yaa allele, as seen by higher levels of anti-RNA and smRNP autoantibodies and ABCs in kika mice (Fig. 3d, Extended Data Fig. 5k).    [R848] (μg ml -1 )

Article
We investigated whether the Y264H variant leads to spontaneous TLR7 cleavage and activation in the absence of stimulation 12 . Western blot analysis of splenocyte lysates from kika and wild-type littermates using two different antibodies against both the C and N termini revealed the presence of the approximately 75-kDa C-terminal-cleaved and 65-kDa N-terminal cleaved TLR7 product in splenocytes from unimmunized kika mice (Fig. 3e). Such cleaved fragments have been reported to be indicative of the active form of TLR7 31 . MyD88 was also increased in kika splenocytes ( Fig. 3f), consistent with enhanced TLR signalling 32 .
Proband A.II.1 was also heterozygous for RNASEH2B p.Ala177Thr, which, when homozygous, causes SLE and Aicardi-Goutières syndrome 26 . RNASHE2B activates cGAS-STING, which increases type 1 IFN production and TLR7 signalling 33 . To test whether this allele might act in functional epistasis with TLR7 Y264H , we generated mice carrying an Rnaseh2b 1 bp deletion, leading to a premature stop codon at amino acid 175 (Extended Data Fig. 6a). Heterozygous mice were viable and had no immunological phenotypes (Extended Data Fig. 6b), whereas the mutation was embryonically lethal in homozygous mice, as previously reported for Rnaseh2b-knockout mice 34 (Extended Data Fig. 6e-h). Besides a mild enhancement in the expression of type I IFN signature genes (Extended Data Fig. 6i), Rnaseh2b hemizygosity did not exacerbate the cellular or serological phenotypes of Tlr7 kik/+ female mice (Extended Data Fig. 6c, d). However, it is possible that there may be functional epistatic effects of these two RNA-interacting proteins in the presence of environmental triggers.

Enhanced survival of BCR-activated cells
We next examined the stage at which TLR7 Y264H breaks B cell tolerance. We hypothesized that constitutive TLR7 signalling may provide an aberrant signal 2 to self-reactive B cells that have bound to self-antigen through their BCR (signal 1) and would otherwise die within 72 h, as   occurs in anergic B cells 35 and in immature CD93 + B cells stimulated with anti-IgM 36 . We could not use CD93 to purify immature splenic B cells because we found that agonistic TLR7 treatment of mature B cells upregulated CD93 (Extended Data Fig. 7a). We therefore activated either total splenic B cells or CD93 + B220 low immature bone marrow cells from kika, wild-type and Yaa mice carrying a Tlr7 duplication 6,7 , stimulated them with R837 or anti-IgM and performed live cell counts 72 h later. We observed that anti-IgM, but not R837, enhanced the survival of total, mature and immature kika B cells compared with control cells (Fig. 3g, Extended Data Fig. 7b-d). This differs from reports using TLR7 transgenic B cells, which displayed increased survival only when activated with a TLR ligand and not with anti-IgM 37 , again suggesting sustained activation of TLR7(Y264H) by endogenous ligands. RNA-sequencing (RNA-seq) analysis of kika and control splenocytes cultured for 20 h with anti-IgM revealed that 203 and 34 transcripts were upregulated or downregulated, respectively, by more than twofold (P < 0.05) in kika cells (Fig. 3i). Upregulated transcripts included the anti-apoptotic genes Mxd3 38 and Serpina3g 39 , as well as IL28ra (also known as Ifnlr1), which encodes the common IFNλ-1/2/3 receptor 40 . Levels of the transcription factor SOX5, which decreases B cell proliferative capacity while allowing plasmablast differentiation, were also 41 . Other upregulated transcripts included Cxcr3, which promotes lupus nephritis 42 . We confirmed a decreased tendency for apoptosis in kika ABCs with decreased expression of active caspase-3 and also a small decrease in proliferation (Fig. 3h). Overall, these results suggest that hypersensitive TLR7 signalling enables the survival of B cells that bind to self-antigen through their surface BCR.

MyD88 dependence and GC independence
To confirm that the observed aberrant B cell survival after IgM stimulation was due to enhanced TLR7 signalling, we crossed kika mice with Myd88-knockout mice. MyD88 deficiency completely rescued kika phenotypes, including splenomegaly (Fig. 4a), accumulation of ABCs, GC B cells, plasma cells, eT H cells (Fig. 4b, Extended Data Fig. 8) and autoantibody formation (Fig. 4c). The aberrant survival of B cells    a-e, k-o), independent experiments. Experiments in a-e were performed once with n > 25 mice, and experiments in j-n were performed once with n = 4 HC-teens, n = 3 HC-adults, n = 1 patient. Statistical analysis was performed using one-way ANOVA with Tukey multiple-comparison test (a-c); Mann-Whitney U-tests (g); and two-way ANOVA with Tukey multiple-comparison test (d, n). Exact P values are shown. receiving only signal 1 was completely abrogated in anti-IgM-activated kika B cells lacking MyD88 (Fig. 4d) without changes in proliferation (Fig. 4e). It remains controversial whether the spontaneous GCs of lupus-prone mice contribute to the autoimmune phenotype, with some suggesting that TLR7 promotes the appearance of self-reactive GC B cells that produce autoantibodies 17 and others proposing that the pathogenic B cells are ABCs of extrafollicular origin 1 . To resolve this question, we crossed kika mice with Bcl6 flox/flox ;Cd23 cre mice that cannot form GCs. In the F 2 -intercross offspring, we enumerated GC B cells and confirmed that kika Bcl6 flox/flox ;Cd23 cre mice had a substantial reduction in GC B cells (Fig. 4f). Despite the paucity of GC B cells, kika Bcl6 flox/flox ;Cd23 cre mice developed autoantibodies and an even more pronounced expansion of ABCs and plasma cells than that observed in their GC-forming kika littermates (Fig. 4g, h). These results support the idea that TLR7-driven autoimmunity is GC independent.
We finally analysed the PBMCs from proband A.II.1 carrying the Y264H variant to confirm the phenotypes observed in the kika mice. Flow cytometry analysis revealed that ABCs and the parental IgD − CD27 − B cells were substantially increased compared with gender-and age-matched controls (Fig. 4j, k). As seen in kika mice, TLR7 and MyD88 protein expression were increased in the pDCs and B cells of the proband (Fig. 4l, m), as was the cleaved TLR7 product (Fig. 4i). Increased levels of ABCs, TLR7 and MyD88 were not seen in the most unrelated patients with SLE (Extended Data Fig. 9a, b). PBMCs in proband A.II.1 also revealed upregulation of the NF-κB activation marker CD25 in unstimulated monocytes (Fig. 4n), increasing its expression after TLR7 stimulation (Extended Data Fig. 9c). The proband with the TLR7 Y264H allele, but not the mother (A.1.2), carrying the RNASEH2B A177T variant allele (Fig. 4o), had an increased type I IFN signature.
We conclude that TLR7 GOF can cause B-cell-driven autoimmunity including SLE due to increased affinity to guanosine, leading to a lowered threshold for TLR7 activation. Although the human TLR7 Y264H variant is sufficient to induce lupus in mice with no clear additive effects of Rnaseh2b hemizygosity apart from increased type I IFN gene transcripts, an exacerbating role of this variant in humans may occur in the presence of environmental stimuli, including ssRNA viruses such as SARS-CoV-2 that are dependent on TLR7 immunity 43 . The TLR7 GOF promotes the survival of BCR-activated immature B cells, which are known to be enriched in self-reactivity 44 . Activation of self-reactive B cells by self-antigen in the presence of a constitutive signal 2 is likely to promote differentiation and autoantibody production of B cells that would otherwise be destined to die in the absence of T cell help.
Notably, despite enhanced TLR7 signalling causing the cellautonomous accumulation of ABCs and GC B cells, GC B cells are dispensable for the autoimmune phenotype and rather protect against it. Extrafollicular ABCs are therefore the most likely source of pathogenicity. It will be important to determine whether this is true for all cases of SLE, or only for patients in whom excessive TLR7 signalling is the dominant pathogenic pathway. Although highly damaging TLR7 GOF mutations are rare, our data, together with evidence of increased TLR7 signalling in a large fraction of patients with SLE 1 , suggest that TLR7 is a key upstream driver of human SLE. Therapies blocking TLR7 itself or MyD88 may be more effective than therapies blocking GCs in patients with SLE due to increased TLR7 signalling.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-022-04642-z.

Mice
Mice were bred and maintained in specific-pathogen-free conditions at the Australian National University (ANU), Canberra, Australia. Experimentation was performed according to the regulations approved by the local institution ethics committee, including the Australian National University's Animal and human Experimentation Ethics Committee. Estimations of the expected change between experimental and control groups allowed the use of power analysis to estimate the group size that would enable detection of statistically significant differences. For in vitro experiments, randomization was not required given that there were no relevant covariates. Blinding was used for microscopy: histological analysis, electron microscopy imaging. Mice were used from 6-12 weeks, except for survival curves and tissue assessment (12-26 weeks). Both male and female mice were used and their genders are indicated in most figures (the Y chromosome is indicated in the genotype, that is, male mice).

Human WES and variant identification
Written informed consent was obtained as part of the Centre for Personalised Immunology Program. The study was approved by and complies with all relevant ethical regulations of the Australian National University and ACT Health Human Ethics Committees, the University Hospitals Institutional Review Board, or by the Renji Hospital Ethics Committee of Shanghai Jiaotong University School of Medicine. For WES analysis, DNA samples were enriched using the Human SureSelect XT2 All Exon V4 Kit and sequenced using the Illumina HiSeq 2000 (Illumina) system. Bioinformatics analysis was performed at JCSMR, ANU as previously described 45 . A search for 'de novo', coding, novel or ultrarare (MAF < 0.0005) variants among 100 SLE trios identified a proband with a de novo, novel variant in TLR7 (Y64H) (family A). A further search for rare variants (MAF < 0.005) in TLR7 across our three systemic autoimmune cohorts (Australia, Europe and China) that have undergone WES at our Centre for Personalised Immunology (~500 probands) identified 2 additional probands (families B-C). A further proband was identified at Baylor-Hopkins Center for Mendelian Genomics, where the family was recruited as a part of a study investigating monogenic causes of neuroimmune disorders in families with early disease onset (≤10 years). All family members provided written informed consent under Baylor College of Medicine Institutional Review Board (IRB) protocol H-29697. A detailed description of the exome sequencing approach, data processing, filtration and analysis for that particular family can be found in the supplementary information of ref. 46 . All probands were subsequently analysed for rare variants in 22 genes proven to cause human SLE (Supplementary Table 1).
Fc receptors were blocked using purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences) and then stained for 30 min at 4 °C, in the dark, with primary and secondary antibodies. Intracellular staining was performed using the FOXP3 Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Samples were acquired on the Fortessa or Fortessa X-20 cytometer with FACSDiva (BD, Biosciences) and analysed using FlowJo v.10 (FlowJo). All fluorescence-activated cell sorting (FACS) and microscopy analysis was carried out at the Microscopy and Cytometry Facility, Australian National University.

Sanger sequencing
Primers for human TLR7 DNA sequencing were used at 10 µM (primer sequences available on request). PCR amplification was carried out using Phusion Hot Start II DNA Polymerase II (Thermo Fisher Scientific) and under the conditions recommended by the manufacturer. PCR amplicons were electrophoresed and excised bands were purified using the QIAquick Gel Extraction Kit (Qiagen). Sanger sequencing was completed using Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems) using the same primers used for PCR amplification. Sequencing reactions were run on the 3730 DNA Analyze (Applied Biosystems) system at the ACRF Biomolecular Resource Facility, Australian National University.

Immunohistochemistry
Liver, pancreas and kidneys were fixed in 10% neutral buffer formalin solution, embedded in paraffin and stained with H&E.

Bone marrow chimera experimentation
For competitive bone marrow chimeras, Rag1 −/− mice were irradiated and injected intravenously with equal numbers of bone marrow cells from either wild-type or kika CD45.2 and wild-type CD45.1 mice. Mice were given Bactrim in their drinking water for 48 h before injection and for 6 weeks after injection, and housed in sterile cages. After 22 weeks of reconstitution, mice were taken down for phenotyping by flow cytometry.

ADVIA blood analysis
Orbital bleeds were performed on mice and blood samples were run on the ADVIA system (Siemens Advia 1200).

Statistics
Statistical analysis was carried out using R software v.3.6.1 (The R Foundation for Statistical Computing) and the Emmeans package. Mouse spleen mass data were analysed using two experiments as a blocking factor and one-way ANOVA, followed by a pairwise estimated marginal means comparison of genotypes. Mouse cellular phenotyping, ELISAs, white blood cell and platelet count analyses were performed using a log linear regression model and one-way ANOVA, followed by a pairwise estimated marginal means comparison of genotypes. Purified B cell cultures were analysed using a linear regression model and one-way ANOVA, followed by a pairwise estimated marginal means comparison of genotypes and stimulatory effect. Luciferase assay statistics were analysed using one-way ANOVA with Bonferroni multiple-comparison test (Prism, GraphPad). All data were filed using Microsoft Excel 2016 and graphed using PRISM.

DNA, RNA and nRNP ELISAs
Plates were coated with poly-l-lysine (Sigma-Aldrich) before addition of 2.5 µg of either DNA (D7290, Sigma-Aldrich), RNA (AM7120G, Thermo Fisher Scientific) or nRNP (SRC-1000, Immunovision). Plates were then blocked in ELISA blocking buffer (PBS and 1% BSA) for 2 h at room temperature. Mouse serum was diluted 1:40 with ELISA coating buffer (0.05 M sodium carbonate anhydrous/sodium hydrogen carbonate, pH 9.6), and incubated in the ELISA plates overnight at 4 °C. The plates were washed and goat anti-mouse IgG-AP antibodies (Alkaline Phosphatase, Southern Biotech) were added for 1 h at 37 °C. Phosphatase substrate (Sigma-Aldrich, S0942) was used as described by the manufacturer. The samples were read using the Infinite 200 PRO Tecan Microplate Reader (Tecan Group) at an absorbance of 405 nm and normalized to background absorbance at 605 nm.

Hep-2/C. luciliae immunofluorescence
ANAs and dsDNA were determined using Hep-2 and Crithidia luciliae slides (both from NOVA Lite), respectively. Serum was diluted 1:40 for Hep-2 slides and 1:20 for Crithidia slides and stained as described by the manufacturer using donkey anti-mouse IgG Alexa-488 (Molecular Probes) as the secondary antibody. The slides were imaged using an Olympus IX71 inverted fluorescence microscope.

RNA-seq analysis
Total B cells were obtained from wild-type or kika mouse spleens and purified using the Mouse B Cell Isolation Kit (Miltenyi Biotec) and stimulated with anti-mouse IgM (10 µg ml −1 ) for 20 h. Total RNA was extracted using RNeasy Mini Kits (74104, Qiagen). Sequencing was performed using the NextSeq500 platform and analysis was conducted using the following R packages: limma, edgeR and enhanced volcano 49 . For the patient, type I IFN single-cell RNA-seq analysis was performed. PBMCs were isolated from frozen human samples as previously described 50 . Live cells were next purified by FACS using 7AAD and labelled with TotalSeq anti-human hashtags (BioLegend). The number of cells was determined and 10,000 cells per sample were run on the 10x Chromium platform (10x Genomics). Library preparation and sequencing were performed by The Biomedical Research Facility according to the manufacturer's instructions for the Chromium Next GEM Single Cell 5′ Kit v2. The samples were sequenced using the NovaSeq 6000 (Illumina) system. The FASTQ files were aligned to the human GRCh38 reference genome using 10x Genomics Cell Ranger pipeline v.6.0.1. Statistical analysis, clustering and visualization were conducted using Seurat v.4.0.1 in the R environment.

Molecular dynamics simulations
Details of the computational modelling are provided in the Supplementary Methods.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
For the genomic data relating to the families in Fig. 1, family A has been deposited in SRA, under BioProject accession number PRJNA798834; family B has been deposited at the Baylor-Hopkins Center for Mendelian Genomics, AnVIL repository under the participant IDs BH14453-1 (proband), BH14453-2 (mother) and BH14453-3 (father); family C has been submitted to the EGA (EGAS00001005965). Sequencing data in Fig. 3i have been deposited at the Gene Expression Omnibus under accession number GSE196316 and Fig.4o has been deposited at Open Science Framework (OFS) under accession number pw62n.