TLR7 gain-of-function genetic variation causes human lupus

Abstract

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 RNA⁸,⁹ 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.

Main

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 blood¹, and excessive accumulation of extrafollicular helper T cells¹⁶. 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 models⁷. 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 lupus¹. 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 cells¹⁷. 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 degradation¹⁰, which synergizes with uridine-rich short RNAs binding to a second site^11,12. Here we describe the action of a de novo TLR7 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 TLR7²⁵, within the endosomal part of the receptor (Fig. 1b) and is highly conserved across species, including zebrafish (Fig. 1d). Additional analyses for rare variants in 22 genes that can cause human SLE when mutated (Supplementary Table 3) revealed a heterozygous variant in RNASEH2B, p.Ala177Thr, which, when homozygous, causes SLE²⁶.

Whole-exome sequencing (WES) analysis of additional patients with SLE identified two other variants in TLR7 (B.I.2 F507L and C.I.1 R28G; Fig. 1a–d, Extended Data Fig. 1c, Supplementary 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)….