ß-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1.

A variety of signals finely tune insulin secretion by pancreatic ß cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in ß cells is critical for insulin secretion. Mice lacking COP1 in ß cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human ß-cell pathophysiology. In normal ß cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.

Low disease risk and penetrance in Leber hereditary optic neuropathy.

The risk of Leber hereditary optic neuropathy (LHON) has largely been extrapolated from disease cohorts, which underestimate the population prevalence of pathogenic primary LHON variants as a result of incomplete disease penetrance. Understanding the true population prevalence of primary LHON variants, alongside the rate of clinical disease, provides a better understanding of disease risk and variant penetrance. We identified pathogenic primary LHON variants in whole-genome sequencing data of a well-characterized population-based control cohort and found that the prevalence is far greater than previously estimated, as it occurs in approximately 1 in 800 individuals. Accordingly, we were able to more accurately estimate population risk and disease penetrance in LHON variant carriers, validating our findings by using other large control datasets. These findings will inform accurate counseling in relation to the risk of vision loss in LHON variant carriers and disease manifestation in their family. This Matters Arising paper is in response to Lopez Sanchez et al. (2021), published in The American Journal of Human Genetics. See also the response by Mackey et al. (2022), published in this issue.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Pharmacogenomic testing: perception of clinical utility, enablers and barriers to adoption in Australian hospitals.

BACKGROUND: Despite healthcare professionals (HCP) endorsing the clinical utility of pharmacogenomics testing, use in clinical practice is limited. AIMS: To assess HCP' perceptions of pharmacogenomic testing and identify barriers to implementation. METHODS: HCP involved in prescribing decisions at three hospitals in Sydney, Australia, were invited to participate. The online survey assessed perceptions of pharmacogenomic testing, including: (i) demographic and practice variables; (ii) use, knowledge and confidence; (iii) perceived benefits; (iv) barriers to implementation; and (v) operational and/or system changes and personnel required to implement on site. RESULTS: HCP were predominantly medical practitioners (75/107) and pharmacists (25/107). HCP perceived pharmacogenomic testing was beneficial to identify reasons for drug intolerance (85/95) and risk of side-effects (86/95). Although testing was considered relevant to their practice (79/100), few HCP (23/100) reported past or intended future use (26/100). Few HCP reported confidence in their ability to identify indications for pharmacogenomic testing (14/107), order tests (19/106) and communicate results with patients (16/107). Lack of clinical practice guidelines (62/79) and knowledge (54/77) were identified as major barriers to implementation of pharmacogenomics. Comprehensive reimbursement for testing and clinical practice guidelines, alongside models-of-care involving multidisciplinary teams and local clinical champions were suggested as strategies to facilitate implementation of pharmacogenomic testing into practice. CONCLUSIONS: Pharmacogenomic testing was considered important to guide drug selection and dosing decisions. However, limited knowledge, low confidence and an absence of guidelines impede the use of pharmacogenomic testing. Establishment of local resources including multidisciplinary models-of-care was suggested to facilitate implementation of pharmacogenomics.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.

Molecular patterns in salivary duct carcinoma identify prognostic subgroups.

Salivary duct carcinoma (SDCa) is a rare cancer with high rate of metastases and poor survival despite aggressive multimodality treatment. This study analyzes the genetic changes in SDCa, their impact on cancer pathways, and evaluates whether molecular patterns can identify subgroups with distinct clinical characteristics and outcome. Clinicopathologic details and tissue samples from 66 patients (48 males, 18 females) treated between 1995 and 2018 were obtained from multiple institutions. Androgen receptor (AR) was assessed by immunohistochemistry, and the Illumina TruSight 170 gene panel was used for DNA sequencing. Male gender, lympho-vascular invasion, lymph node metastasis, and smoking were significant predictors of disease-free survival. AR was present in 79%. Frequently encountered alterations were mutations in TP53 (51%), PIK3CA (32%) and HRAS (22%), as well as amplifications of CDK4/6 (22%), ERBB2 (21%), MYC (16%), and deletions of CDKN2A (13%). TP53 mutation and MYC amplifications were associated with decreased disease-free survival. Analysis of cancer pathways revealed that the PI3K pathway was most commonly affected. Alterations in the cell cycle pathway were associated with impaired disease-free survival (HR 2.6, P = 0.038). Three subgroups based on AR and ERBB2 status were identified, which featured distinct molecular patterns and outcome. Among AR positive SDCa, HRAS mutations were restricted to AR positive tumors without ERBB2 amplification and HRAS mutations featured high co-occurrence with PIK3CA alterations, which seems specific to SDCa. AR negative SDCa were associated with poor disease-free survival in multivariate analysis (HR 4.5, P = 0.010) and none of these tumors exhibited ERBB2 amplification or HRAS mutations. AR and ERBB2 status in SDCa thus classifies tumors with distinct molecular profiles relevant to future targeted therapy. Furthermore, clinical factors such as smoking and molecular features such as MYC amplification may serve as markers of poor prognosis of SDCa.

Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.

PILRα negatively regulates mouse inflammatory arthritis.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

gCMAP: user-friendly connectivity mapping with R.

UNLABELLED: Connections between disease phenotypes and drug effects can be made by identifying commonalities in the associated patterns of differential gene expression. Searchable databases that record the impacts of chemical or genetic perturbations on the transcriptome--here referred to as 'connectivity maps'--permit discovery of such commonalities. We describe two R packages, gCMAP and gCMAPWeb, which provide a complete framework to construct and query connectivity maps assembled from user-defined collections of differential gene expression data. Microarray or RNAseq data are processed in a standardized way, and results can be interrogated using various well-established gene set enrichment methods. The packages also feature an easy-to-deploy web application that facilitates reproducible research through automatic generation of graphical and tabular reports. AVAILABILITY AND IMPLEMENTATION: The gCMAP and gCMAPWeb R packages are freely available for UNIX, Windows and Mac OS X operating systems at Bioconductor (http://www.bioconductor.org).

A census of human transcription factors: function, expression and evolution.

Transcription factors are key cellular components that control gene expression: their activities determine how cells function and respond to the environment. Currently, there is great interest in research into human transcriptional regulation. However, surprisingly little is known about these regulators themselves. For example, how many transcription factors does the human genome contain? How are they expressed in different tissues? Are they evolutionarily conserved? Here, we present an analysis of 1,391 manually curated sequence-specific DNA-binding transcription factors, their functions, genomic organization and evolutionary conservation. Much remains to be explored, but this study provides a solid foundation for future investigations to elucidate regulatory mechanisms underlying diverse mammalian biological processes.

A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Genome sequencing reanalysis increases the diagnostic yield in dystonia.

PURPOSE: We investigated the contribution of genomic data reanalysis to the diagnostic yield of dystonia patients who remained undiagnosed after prior genome sequencing. METHODS: Probands with heterogeneous dystonia phenotypes who underwent initial genome sequencing (GS) analysis in 2019 were included in the reanalysis, which was performed through gene-specific discovery collaborations and systematic genomic data reanalysis. RESULTS: Initial GS analysis in 2019 (n = 111) identified a molecular diagnosis in 11.7 % (13/111) of cases. Reanalysis between 2020 and 2023 increased the diagnostic yield by 7.2 % (8/111); 3.6 % (4/111) through focused gene-specific clinical correlation collaborative efforts [VPS16 (two probands), AOPEP and POLG], and 3.6 % (4/111) by systematic reanalysis completed in 2023 [NUS1 (two probands) and DDX3X variants, and a microdeletion encompassing VPS16]. Seven of these patients had a high phenotype-based dystonia score ≥3. Notable unverified findings in four additional cases included suspicious variants of uncertain significance in FBXL4 and EIF2AK2, and potential phenotypic expansion associated with SLC2A1 and TREX1 variants. CONCLUSION: GS data reanalysis increased the diagnostic yield from 11.7 % to 18.9 %, with potential extension up to 22.5 %. While optimal timing for diagnostic reanalysis remains to be determined, this study demonstrates that periodic re-interrogation of dystonia GS datasets can provide additional genetic diagnoses, which may have significant implications for patients and their families.

Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.

Asthma is canonically thought of as a disorder of excessive Th2-driven inflammation in the airway, although recent studies have described heterogeneity with respect to asthma pathophysiology. We have previously described distinct phenotypes of asthma based on the presence or absence of a three-gene "Th2 signature" in bronchial epithelium, which differ in terms of eosinophilic inflammation, mucin composition, subepithelial fibrosis, and corticosteroid responsiveness. In the present analysis, we sought to describe Th2 inflammation in human asthmatic airways quantitatively with respect to known mediators of inflammation and intercellular communication. Using whole-genome microarray and quantitative real-time PCR analysis of endobronchial biopsies from 27 mild-to-moderate asthmatics and 13 healthy controls with associated clinical and demographic data, we found that asthmatic Th2 inflammation is expressed over a variable continuum, correlating significantly with local and systemic measures of allergy and eosinophilia. We evaluated a composite metric describing 79 coexpressed genes associated with Th2 inflammation against the biological space comprising cytokines, chemokines, and growth factors, identifying distinctive patterns of inflammatory mediators as well as Wnt, TGF-ß, and platelet-derived growth factor family members. This integrated description of the factors regulating inflammation, cell migration, and tissue remodeling in asthmatic airways has important consequences for the pathophysiological and clinical impacts of emerging asthma therapeutics targeting Th2 inflammation.

Sticking together? Falling apart? Exploring the dynamics of the interactome.

Advances in techniques for the study of protein-protein interactions have dramatically improved our understanding of the interactome. However, we know little about the dynamics of this complex system. To better understand the dynamics of the interactome, it is important to consider what happens when single proteins are perturbed. Changes in protein abundance and post-translational modification can function as switches in the interactome, affecting protein-complex assembly and function. Changes in protein sequence or a dramatic increase in abundance might cause a promiscuous gain of interactions. These effects are not identical for all proteins and will differ depending on the number and type of interaction partners that a protein has.

Circulating Tumour DNA Biomarkers Associated with Outcomes in Metastatic Prostate Cancer Treated with Lutetium-177-PSMA-617.

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes. Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA. Design setting and participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression. Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles. Outcome measurements and statistical analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes. Results and limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5-7.7; p = 0.0036; and HR 3.3, 95% CI 1.4-7.7; p = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8-13; p = 0.0014; and HR 4.1, 95% CI 1.6-11; p = 0.004). Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC. Patient summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed.

Community Genetics screening in a pandemic: solutions for pre-test education, informed consent, and specimen collection.

A Community Genetics carrier screening program for the Jewish community has operated on-site in high schools in Sydney (Australia) for 25 years. During 2020, in response to the COVID-19 pandemic, government-mandated social-distancing, 'lock-down' public health orders, and laboratory supply-chain shortages prevented the usual operation and delivery of the annual testing program. We describe development of three responses to overcome these challenges: (1) pivoting to online education sufficient to ensure informed consent for both genetic and genomic testing; (2) development of contactless telehealth with remote training and supervision for collecting genetic samples using buccal swabs; and (3) a novel patient and specimen identification 'GeneTrustee' protocol enabling fully identified clinical-grade specimens to be collected and DNA extracted by a research laboratory while maintaining full participant confidentiality and privacy. These telehealth strategies for education, consent, specimen collection and sample processing enabled uninterrupted delivery and operation of complex genetic testing and screening programs even amid pandemic restrictions. These tools remain available for future operation and can be adapted to other programs.

Quantitative ctDNA Detection in Hepatoblastoma: Implications for Precision Medicine.

Hepatoblastoma is characterized by driver mutations in CTNNB1, making it an attractive biomarker for a liquid biopsy approach utilizing circulating tumor DNA (ctDNA). This prospective observational study sought to ascertain the feasibility of ctDNA detection in patients with hepatoblastoma and explore its associations with established clinical indicators and biomarkers, including serum Alpha-fetoprotein (AFP). We obtained 38 plasma samples and 17 tumor samples from 20 patients with hepatoblastoma. These samples were collected at various stages: 10 at initial diagnosis, 17 during neoadjuvant chemotherapy, 6 post-operatively, and 5 at disease recurrence. Utilizing a bespoke sequencing assay we developed called QUENCH, we identified single nucleotide variants and deletions in CTNNB1 ctDNA. Our study demonstrated the capability to quantitate ctDNA down to a variant allele frequency of 0.3%, achieving a sensitivity of 90% for patients at initial diagnosis, and a specificity of 100% at the patient level. Notably, ctDNA positivity correlated with tumor burden, and ctDNA levels exhibited associations with macroscopic residual disease and treatment response. Our findings provide evidence for the utility of quantitative ctDNA detection in hepatoblastoma management. Given the distinct detection targets, ctDNA and AFP-based stratification and monitoring approaches could synergize to enhance clinical decision-making. Further research is needed to elucidate the interplay between ctDNA and AFP and determine the optimal clinical applications for both methods in risk stratification and residual disease detection.

Pretreatment synovial transcriptional profile is associated with early and late clinical response in rheumatoid arthritis patients treated with rituximab.

OBJECTIVE: Personalised healthcare is contingent on the identification of biomarkers that represent disease relevant pathways and predict drug response. The authors aimed to develop a gene expression signature in synovial tissue that could enrich clinical response of rheumatoid arthritis (RA) patients to rituximab. METHODS: The authors studied synovial gene expression using high-throughput quantitative real-time-PCR in 20 RA patients who underwent arthroscopy before and after treatment with rituximab. Several objective approaches were used to explore patterns in the data and to find genes associated with changes in disease activity due to treatment. RESULTS: This analysis revealed two patient populations associated with distinct clinical, laboratory and histological features and, importantly, showed enrichment for response (60% non-responders vs 90% responders). A composite baseline gene score (GS) correlated with change in disease activity score (ΔDAS) between baseline and month 3 (r=0.74, p=0.0002), but also with ΔDAS at later time-points (month 9, r=0.54, p=0.016; month 15, r=0.45, p=0.06; month 21, r=0.72, p=0.003). Notably, the GS significantly correlated with baseline erythrocyte sedimentation rate (r=0.69, p=0.0008), but not with other DAS components. The GS genes represented T cell, macrophage, remodelling and interferon-α biology. Responders demonstrated higher expression of macrophage and T cell genes, while non-responders showed higher expression of interferon-α and remodelling genes. CONCLUSIONS: This study reveals a baseline synovial GS that correlates with early and late clinical responses to rituximab. The GS biology suggests that T cells and macrophages are important for response to B cell depleting therapy, while expression of remodelling and interferon-α genes correlates with poor response.