Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Publisher Correction: The immune cell landscape in kidneys of patients with lupus nephritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1.

Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

Cytokine induced 3-D organotypic psoriasis skin model demonstrates distinct roles for NF-κB and JAK pathways in disease pathophysiology.

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.

A multimodal and integrated approach to interrogate human kidney biopsies with rigor and reproducibility: guidelines from the Kidney Precision Medicine Project.

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate three-dimensional (3-D) molecular atlases of healthy and diseased kidney biopsies by using multiple state-of-the-art omics and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single-cell level or in 3-D space is a significant challenge that can be a futile exercise if not well controlled. We describe a "follow the tissue" pipeline for generating a reliable and authentic single-cell/region 3-D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation, and harmonization across different omics and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis, and sharing. We established benchmarks for quality control, rigor, reproducibility, and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before their being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multiomics and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.

An eQTL Landscape of Kidney Tissue in Human Nephrotic Syndrome.

Expression quantitative trait loci (eQTL) studies illuminate the genetics of gene expression and, in disease research, can be particularly illuminating when using the tissues directly impacted by the condition. In nephrology, there is a paucity of eQTL studies of human kidney. Here, we used whole-genome sequencing (WGS) and microdissected glomerular (GLOM) and tubulointerstitial (TI) transcriptomes from 187 individuals with nephrotic syndrome (NS) to describe the eQTL landscape in these functionally distinct kidney structures. Using MatrixEQTL, we performed cis-eQTL analysis on GLOM (n = 136) and TI (n = 166). We used the Bayesian "Deterministic Approximation of Posteriors" (DAP) to fine-map these signals, eQTLBMA to discover GLOM- or TI-specific eQTLs, and single-cell RNA-seq data of control kidney tissue to identify the cell type specificity of significant eQTLs. We integrated eQTL data with an IgA Nephropathy (IgAN) GWAS to perform a transcriptome-wide association study (TWAS). We discovered 894 GLOM eQTLs and 1,767 TI eQTLs at FDR < 0.05. 14% and 19% of GLOM and TI eQTLs, respectively, had >1 independent signal associated with its expression. 12% and 26% of eQTLs were GLOM specific and TI specific, respectively. GLOM eQTLs were most significantly enriched in podocyte transcripts and TI eQTLs in proximal tubules. The IgAN TWAS identified significant GLOM and TI genes, primarily at the HLA region. In this study, we discovered GLOM and TI eQTLs, identified those that were tissue specific, deconvoluted them into cell-specific signals, and used them to characterize known GWAS alleles. These data are available for browsing and download via our eQTL browser, "nephQTL."

Insights into the epidemiology and management of lupus nephritis from the US rheumatologist's perspective.

Lupus nephritis is a common and severe manifestation of systemic lupus erythematosus that disproportionately affects nonwhites and those in lower socioeconomic groups. This review discusses recent data on the incidence, prevalence, and outcomes of patients with lupus nephritis with a focus on low-income US Medicaid patients. We also review recent guidelines on diagnosis, treatment, and screening for new onset and relapses of lupus nephritis. Finally, we discuss the management of lupus nephritis from a rheumatologist's perspective, including vigilance for the common adverse events related to disease and treatment, and we review prevention and new treatment strategies.

Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1).

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

Overexpression of human SAMD9 inhibits protein translation and alters MYC signaling resulting in cell cycle arrest.

Inherited bone marrow failure syndromes often result from pathogenic mutations in genes that are important for ribosome function, namely, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, and dyskeratosis congenita. Germline mutations in SAMD9 are a frequent genetic lesion resulting in an inherited bone marrow failure syndrome with monosomy 7; some patients have severe multisystem syndromes that include myelodysplasia. The association of germline SAMD9 mutations and bone marrow failure is clear; however, to date, there is no reliable method to predict whether a novel SAMD9 mutation is pathogenic unless it is accompanied by an obvious family history and/or clinical syndrome. The difficulty with pathogenicity prediction is, in part, due to the incomplete understanding of the biological functions of SAMD9. We used a SAMD9-targeted, inducible CRISPRa system and RNA sequencing to better understand the global transcriptional changes that result from transcriptional manipulation of SAMD9. Supporting recent discoveries that SAMD9 acts as a ACNase specific for phenylalanine tRNA (tRNA-Phe), we confirmed with crosslinking and solid-phase purification that SAMD9 is an RNA binding protein and analyzed how overexpression of tRNA-Phe may reverse transcriptomic changes caused by SAMD9 activation. Our data show that overexpression of SAMD9 from the endogenous locus results in decreased cell proliferation, cell cycle progression, and global protein translation. When SAMD9 contains a gain-of-function mutation (p.E1136Q), these functional phenotypes are exacerbated but only partially rescued with tRNA-Phe overexpression, suggesting additional molecular actions of SAMD9. Additionally, we demonstrate that gene expression pathways important for ribosome biogenesis and MYC signaling are the most significantly impacted by SAMD9 overexpression.

Urine proteomic signatures of histological class, activity, chronicity, and treatment response in lupus nephritis.

Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%-40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

Topoisomerase 2ß Induces DNA Breaks To Regulate Human Papillomavirus Replication.

Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2ß is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2ß. Upon DNA cleavage, a covalent intermediate DNA-TOP2ß (TOP2ßcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2ßcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2ß proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2ß and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2ß with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2ß also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2ß. This study identifies TOP2ß as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2ß is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2ß levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2ß reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2ß through DNA break formation.

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16.

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.

Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.

Pulmonary Vascular and Right Ventricular Burden During Exercise in Interstitial Lung Disease.

BACKGROUND: Pulmonary hypertension (PH) adversely affects patient's exercise capacity in interstitial lung disease (ILD). The impact of pulmonary vascular and right ventricular (RV) dysfunction, however, has traditionally been believed to be mild and clinically relevant principally in advanced lung disease states. RESEARCH QUESTION: The aim of this study was to evaluate the relative contributions of pulmonary mechanics, pulmonary vascular function, and RV function to the ILD exercise limit. STUDY DESIGN AND METHODS: Forty-nine patients with ILD who underwent resting right heart catheterization followed by invasive exercise testing were evaluated. Patients with PH at rest (ILD + rPH) and with PH diagnosed exclusively during exercise (ILD + ePH) were contrasted with ILD patients without PH (ILD non-PH). RESULTS: Peak oxygen consumption was reduced in ILD + rPH (61 ± 10% predicted) and ILD + ePH (67 ± 13% predicted) compared with ILD non-PH (81 ± 16% predicted; P < .001 and P = .016, respectively). Each ILD hemodynamic phenotype presented distinct patterns of dynamic changes of pulmonary vascular compliance relative to pulmonary vascular resistance from rest to peak exercise. Peak RV stroke work index was increased in ILD + ePH (24.7 ± 8.2 g/m2 per beat) and ILD + rPH (30.9 ± 6.1 g/m2 per beat) compared with ILD non-PH (18.3 ± 6.4 g/m2 per beat; P = .020 and P = .014). Ventilatory reserve was reduced in ILD + rPH compared with the other groups at the anaerobic threshold, but it was similar between ILD + ePH and ILD non-PH at the anaerobic threshold (0.32 ± 0.13 vs 0.30 ± 0.11; P = .921) and at peak exercise (0.70 ± 0.17 vs 0.73 ± 0.24; P = .872). INTERPRETATION: ILD with resting and exercise PH is associated with increased exercise RV work, reduced pulmonary vascular reserve, and reduced peak oxygen consumption. The findings highlight the role of pulmonary vascular and RV burden to ILD exercise limit.

Activation of DNA damage repair factors in HPV positive oropharyngeal cancers.

The mechanisms regulating viral pathogenesis of human papillomavirus (HPV) associated oropharyngeal squamous cell cancers (OPSCC) are not well understood. In the cervix, activation of DNA damage repair pathways is critical for viral replication but little is known about their role in OPSCC. APOBEC factors have been shown to be increased in OPSCC but the significance of this is unclear. We therefore examined activation of DNA damage and APOBEC factors in HPV-induced OPSCC. Our studies show significantly increased levels of pCHK1, FANCD2, BRCA1, RAD51, pSMC1 and γH2AX foci in HPV-positive samples as compared to HPV-negative while the ATM effector kinase, pCHK2, was not increased. Similar differences were observed when the levels of proteins were examined in OPSCC cell lines. In contrast, the levels of APOBEC3B and 3A were found to be similar in both HPV-positive and -negative OPSCC. Our studies suggest members of ATR pathway and FANCD2 may be important in HPV-induced OPSCC.

Accelerating Medicines Partnership: Organizational Structure and Preliminary Data From the Phase 1 Studies of Lupus Nephritis.

The Accelerating Medicines Partnership (AMP) Lupus Network was established as a partnership between the National Institutes of Health, pharmaceutical companies, nonprofit stakeholders, and lupus investigators across multiple academic centers to apply high-throughput technologies to the analysis of renal tissue, urine, and blood from patients with lupus nephritis (LN). The AMP network provides publicly accessible data to the community with the goal of generating new scientific hypotheses and improving diagnostic and therapeutic tools so as to improve disease outcomes. We present here a description of the structure of the AMP Lupus Network and a summary of the preliminary results from the phase 1 studies. The successful completion of phase 1 sets the stage for analysis of a large cohort of LN samples in phase 2 and provides a model for establishing similar discovery cohorts.