Leveraging pleiotropy identifies common-variant associations with selective IgA deficiency.

Selective IgA deficiency (SIgAD) is the most common inborn error of immunity (IEI). Unlike many IEIs, evidence of a role for highly penetrant rare variants in SIgAD is lacking. Previous SIgAD studies have had limited power to identify common variants due to their small sample size. We overcame this problem first through meta-analysis of two existing GWAS. This identified four novel common-variant associations and enrichment of SIgAD-associated variants in genes linked to Mendelian IEIs. SIgAD showed evidence of shared genetic architecture with serum IgA and a number of immune-mediated diseases. We leveraged this pleiotropy through the conditional false discovery rate procedure, conditioning our SIgAD meta-analysis on large GWAS of asthma and rheumatoid arthritis, and our own meta-analysis of serum IgA. This identified an additional 18 variants, increasing the number of known SIgAD-associated variants to 27 and strengthening the evidence for a polygenic, common-variant aetiology for SIgAD.

sMR and PTX3 levels associate with COVID-19 outcome and survival but not with Long COVID.

Biomarkers for monitoring COVID-19 disease course are lacking. Study aim was to identify biomarkers associated with disease severity, survival, long-term outcome, and Long COVID. As excessive macrophages activation is a hallmark of COVID-19 and complement activation is key in this, we selected the following proteins involved in these processes: PTX3, C1q, C1-INH, C1s/C1-INH, and sMR. EDTA-plasma concentrations were measured in 215 patients and 47 controls using ELISA. PTX3, sMR, C1-INH, and C1s/C1-INH levels were associated with disease severity. PTX3 and sMR were also associated with survival and long-term immune recovery. Lastly, sMR levels associate with ICU admittance. sMR (AUC 0.85) and PTX3 (AUC 0.78) are good markers for disease severity, especially when used in combination (AUC 0.88). No association between biomarker levels and Long COVID was observed. sMR has not previously been associated with COVID-19 disease severity, ICU admittance or survival and may serve as marker for disease course.

COVID-19 related myocardial injury is associated with immune dysregulation in symptomatic patients with cardiac MRI abnormalities.

AIMS: While acute cardiovascular complications of COVID-19 are well-described, less is known about longer-term cardiac sequelae. For many individuals with cardiac signs or symptoms arising after COVID-19 infection, the aetiology remains unclear. We examined immune profiles associated with magnetic resonance imaging (MRI) abnormalities in patients with unexplained cardiac injury after COVID-19. METHODS AND RESULTS: Twenty-one participants (mean age 47 [SD 13] years, 71% female) with long COVID (n=17), raised troponin (n=2), or unexplained new-onset heart failure (n=2), who did not have pre-existing heart conditions or recent steroid/immunosuppression treatment were enrolled a mean 346 (SD 191) days after COVID-19 infection in a prospective observational study. Cardiac MRI and blood sampling for deep immunophenotyping using mass cytometry by time of flight and measurement of proteomic inflammatory markers was performed. Nine of 21 (43%) participants had MRI abnormalities (MRI(+)), including non-ischaemic patterns of late gadolinium enhancement and/or visually overt myocardial oedema in 8 people. One patient had mildly impaired biventricular function without fibrosis or oedema, and 2 had severe left ventricular impairment. MRI(+) individuals had higher blood CCL3, CCL7, FGF-23 and CD4 Th2 cells, and lower CD8 T effector memory (TEM) cells, than MRI(-). Cluster analysis revealed lower expression of inhibitory receptors PD1 and TIM3 in CD8 TEM cells from MRI(+) patients than MRI(-) patients, and functional studies of CD8 T αß cells showed higher proportions of cytotoxic granzyme B+ secreting cells upon stimulation. CD8 TEM cells and CCL7 were the strongest predictors of MRI abnormalities in a LASSO regression model (composite AUC 0.96, 95%CI 0.88-1.0). CCL7 was correlated with diffuse myocardial fibrosis/oedema detected by quantitative T1 mapping (r=0.47, p=0.04). CONCLUSION: COVID-19 related cardiac injury in symptomatic patients with non-ischaemic myocarditis-like MRI abnormalities is associated with immune dysregulation, including decreased peripheral CD8 TEM cells and increased CCL7, persisting long after the initial infection.

Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19.

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

A biomarker-stratified comparison of top-down versus accelerated step-up treatment strategies for patients with newly diagnosed Crohn's disease (PROFILE): a multicentre, open-label randomised controlled trial.

BACKGROUND: Management strategies and clinical outcomes vary substantially in patients newly diagnosed with Crohn's disease. We evaluated the use of a putative prognostic biomarker to guide therapy by assessing outcomes in patients randomised to either top-down (ie, early combined immunosuppression with infliximab and immunomodulator) or accelerated step-up (conventional) treatment strategies. METHODS: PROFILE (PRedicting Outcomes For Crohn's disease using a moLecular biomarker) was a multicentre, open-label, biomarker-stratified, randomised controlled trial that enrolled adults with newly diagnosed active Crohn's disease (Harvey-Bradshaw Index ≥7, either elevated C-reactive protein or faecal calprotectin or both, and endoscopic evidence of active inflammation). Potential participants had blood drawn to be tested for a prognostic biomarker derived from T-cell transcriptional signatures (PredictSURE-IBD assay). Following testing, patients were randomly assigned, via a secure online platform, to top-down or accelerated step-up treatment stratified by biomarker subgroup (IBDhi or IBDlo), endoscopic inflammation (mild, moderate, or severe), and extent (colonic or other). Blinding to biomarker status was maintained throughout the trial. The primary endpoint was sustained steroid-free and surgery-free remission to week 48. Remission was defined by a composite of symptoms and inflammatory markers at all visits. Flare required active symptoms (HBI ≥5) plus raised inflammatory markers (CRP >upper limit of normal or faecal calprotectin ≥200 µg/g, or both), while remission was the converse-ie, quiescent symptoms (HBI <5) or resolved inflammatory markers (both CRP ≤ the upper limit of normal and calprotectin <200 µg/g) or both. Analyses were done in the full analysis (intention-to-treat) population. The trial has completed and is registered (ISRCTN11808228). FINDINGS: Between Dec 29, 2017, and Jan 5, 2022, 386 patients (mean age 33·6 years [SD 13·2]; 179 [46%] female, 207 [54%] male) were randomised: 193 to the top-down group and 193 to the accelerated step-up group. Median time from diagnosis to trial enrolment was 12 days (range 0-191). Primary outcome data were available for 379 participants (189 in the top-down group; 190 in the accelerated step-up group). There was no biomarker-treatment interaction effect (absolute difference 1 percentage points, 95% CI -15 to 15; p=0·944). Sustained steroid-free and surgery-free remission was significantly more frequent in the top-down group than in the accelerated step-up group (149 [79%] of 189 patients vs 29 [15%] of 190 patients, absolute difference 64 percentage points, 95% CI 57 to 72; p<0·0001). There were fewer adverse events (including disease flares) and serious adverse events in the top-down group than in the accelerated step-up group (adverse events: 168 vs 315; serious adverse events: 15 vs 42), with fewer complications requiring abdominal surgery (one vs ten) and no difference in serious infections (three vs eight). INTERPRETATION: Top-down treatment with combination infliximab plus immunomodulator achieved substantially better outcomes at 1 year than accelerated step-up treatment. The biomarker did not show clinical utility. Top-down treatment should be considered standard of care for patients with newly diagnosed active Crohn's disease. FUNDING: Wellcome and PredictImmune Ltd.

Spontaneous, persistent, T cell-dependent IFN-γ release in patients who progress to Long Covid.

After acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a proportion of patients experience persistent symptoms beyond 12 weeks, termed Long Covid. Understanding the mechanisms that cause this debilitating disease and identifying biomarkers for diagnostic, therapeutic, and monitoring purposes are urgently required. We detected persistently high levels of interferon-γ (IFN-γ) from peripheral blood mononuclear cells of patients with Long Covid using highly sensitive FluoroSpot assays. This IFN-γ release was seen in the absence of ex vivo peptide stimulation and remains persistently elevated in patients with Long Covid, unlike the resolution seen in patients recovering from acute SARS-CoV-2 infection. The IFN-γ release was CD8+ T cell-mediated and dependent on antigen presentation by CD14+ cells. Longitudinal follow-up of our study cohort showed that symptom improvement and resolution correlated with a decrease in IFN-γ production to baseline levels. Our study highlights a potential mechanism underlying Long Covid, enabling the search for biomarkers and therapeutics in patients with Long Covid.

Loss of T follicular regulatory cell-derived IL-1R2 augments germinal center reactions via increased IL-1.

Inappropriate immune activity is key in the pathogenesis of multiple diseases, and it is typically driven by excess inflammation and/or autoimmunity. IL-1 is often the effector owing to its powerful role in both innate and adaptive immunity, and, thus, it is tightly controlled at multiple levels. IL-1R2 antagonizes IL-1, but effects of losing this regulation are unknown. We found that IL-1R2 resolves inflammation by rapidly scavenging free IL-1. Specific IL-1R2 loss in germinal center (GC) T follicular regulatory (Tfr) cells increased the GC response after a first, but not booster, immunization, with an increase in T follicular helper (Tfh) cells, GC B cells, and antigen-specific antibodies, which was reversed upon IL-1 blockade. However, IL-1 signaling is not obligate for GC reactions, as WT and Il1r1-/- mice showed equivalent phenotypes, suggesting that GC IL-1 is normally restrained by IL-1R2. Fascinatingly, germline Il1r2-/- mice did not show this phenotype, but conditional Il1r2 deletion in adulthood recapitulated it, implying that compensation during development counteracts IL-1R2 loss. Finally, patients with ulcerative colitis or Crohn's disease had lower serum IL-1R2. All together, we show that IL-1R2 controls important aspects of innate and adaptive immunity and that IL-1R2 level may contribute to human disease propensity and/or progression.

Proinflammatory cytokines driving cardiotoxicity in COVID-19.

AIMS: Cardiac involvement is common in patients hospitalized with COVID-19 and correlates with an adverse disease trajectory. While cardiac injury has been attributed to direct viral cytotoxicity, serum-induced cardiotoxicity secondary to serological hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. METHODS AND RESULTS: To investigate serological drivers of cardiotoxicity in COVID-19 we have established a robust bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell (hESC)-derived cardiomyocytes. We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability independent of viral transduction, an effect that was also seen in non-COVID-19 acute respiratory distress syndrome (ARDS). Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-ß, IL-10, CRP, and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partly rescued the phenotype and preserved cardiomyocyte viability and function. Bulk RNA sequencing of serum-treated cardiomyocytes elucidated specific pathways involved in the COVID-19 response impacting cardiomyocyte viability, structure, and function. The observed effects of serum-induced cytotoxicity were cell-type selective as serum exposure did not adversely affect microvascular endothelial cell viability but resulted in endothelial activation and a procoagulant state. CONCLUSION: These results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and characterise the downstream activated pathways in human cardiomyocytes. The serum signature of patients with severe disease indicates possible targets for therapeutic intervention.

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-ß (IKKß, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis.

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

Genotyping and population characteristics of the China Kadoorie Biobank.

The China Kadoorie Biobank (CKB) is a population-based prospective cohort of >512,000 adults recruited from 2004 to 2008 from 10 geographically diverse regions across China. Detailed data from questionnaires and physical measurements were collected at baseline, with additional measurements at three resurveys involving ∼5% of surviving participants. Analyses of genome-wide genotyping, for >100,000 participants using custom-designed Axiom arrays, reveal extensive relatedness, recent consanguinity, and signatures reflecting large-scale population movements from recent Chinese history. Systematic genome-wide association studies of incident disease, captured through electronic linkage to death and disease registries and to the national health insurance system, replicate established disease loci and identify 14 novel disease associations. Together with studies of candidate drug targets and disease risk factors and contributions to international genetics consortia, these demonstrate the breadth, depth, and quality of the CKB data. Ongoing high-throughput omics assays of collected biosamples and planned whole-genome sequencing will further enhance the scientific value of this biobank.

Mapping interindividual dynamics of innate immune response at single-cell resolution.

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

Efficacy of cannabidiol in convulsive and nonconvulsive seizure types associated with treatment-resistant epilepsies in the Expanded Access Program.

The cannabidiol (CBD) Expanded Access Program (EAP), initiated in 2014, provided CBD (Epidiolex) to patients with treatment-resistant epilepsy (TRE). In the final pooled analysis of 892 patients treated through January 2019 (median exposure = 694 days), CBD treatment was associated with a 46%-66% reduction in median monthly total (convulsive plus nonconvulsive) seizure frequency. CBD was well tolerated, and adverse events were consistent with previous findings. We used pooled EAP data to investigate the effectiveness of add-on CBD therapy for individual convulsive seizure types (clonic, tonic, tonic-clonic, atonic, focal to bilateral tonic-clonic), nonconvulsive seizure types (focal with and without impaired consciousness, absence [typical and atypical], myoclonic, myoclonic absence), and epileptic spasms. CBD treatment was associated with a reduction in the frequency of convulsive seizure types (median percentage reduction = 47%-100%), and nonconvulsive seizure types and epileptic spasms (median percentage reduction = 50%-100%) across visit intervals through 144 weeks of treatment. Approximately 50% of patients had ≥50% reduction in convulsive and nonconvulsive seizure types and epileptic spasms at nearly all intervals. These results show a favorable effect of long-term CBD use in patients with TRE, who may experience various convulsive and nonconvulsive seizure types. Future controlled trials are needed to confirm these findings.

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups.

Introduction: While complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood. Methods: We therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome. Results: We show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID. Conclusion: In conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted.

A randomised study of rituximab and belimumab sequential therapy in PR3 ANCA-associated vasculitis (COMBIVAS): design of the study protocol.

BACKGROUND: Sequential B cell-targeted immunotherapy with BAFF antagonism (belimumab) and B cell depletion (rituximab) may enhance B cell targeting in ANCA-associated vasculitis (AAV) through several mechanisms. METHODS: Study design: COMBIVAS is a randomised, double-blind, placebo-controlled trial designed to assess the mechanistic effects of sequential therapy of belimumab and rituximab in patients with active PR3 AAV. The recruitment target is 30 patients who meet the criteria for inclusion in the per-protocol analysis. Thirty-six participants have been randomised to one of the two treatment groups in a 1:1 ratio: either rituximab plus belimumab or rituximab plus placebo (both groups with the same tapering corticosteroid regimen), and recruitment is now closed (final patient enrolled April 2021). For each patient, the trial will last for 2 years comprising a 12-month treatment period followed by a 12-month follow-up period. PARTICIPANTS: Participants have been recruited from five of seven UK trial sites. Eligibility criteria were age ≥ 18 years and a diagnosis of AAV with active disease (newly diagnosed or relapsing disease), along with a concurrent positive test for PR3 ANCA by ELISA. INTERVENTIONS: Rituximab 1000 mg was administered by intravenous infusions on day 8 and day 22. Weekly subcutaneous injections of 200 mg belimumab or placebo were initiated a week before rituximab on day 1 and then weekly through to week 51. All participants received a relatively low prednisolone (20 mg/day) starting dose from day 1 followed by a protocol-specified corticosteroid taper aiming for complete cessation by 3 months. OUTCOMES: The primary endpoint of this study is time to PR3 ANCA negativity. Key secondary outcomes include change from baseline in naïve, transitional, memory, plasmablast B cell subsets (by flow cytometry) in the blood at months 3, 12, 18 and 24; time to clinical remission; time to relapse; and incidence of serious adverse events. Exploratory biomarker assessments include assessment of B cell receptor clonality, B cell and T cell functional assays, whole blood transcriptomic analysis and urinary lymphocyte and proteomic analysis. Inguinal lymph node and nasal mucosal biopsies have been performed on a subgroup of patients at baseline and month 3. DISCUSSION: This experimental medicine study provides a unique opportunity to gain detailed insights into the immunological mechanisms of belimumab-rituximab sequential therapy across multiple body compartments in the setting of AAV. TRIAL REGISTRATION: ClinicalTrials.gov NCT03967925. Registered on May 30, 2019.