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.

Associations between circulating interferon and kynurenine/tryptophan pathway metabolites: support for a novel potential mechanism for cognitive dysfunction in SLE.

OBJECTIVE: Quinolinic acid (QA), a kynurenine (KYN)/tryptophan (TRP) pathway metabolite, is an N-methyl-D-aspartate receptor agonist that can produce excitotoxic neuron damage. Type I and II interferons (IFNs) stimulate the KYN/TRP pathway, producing elevated QA/kynurenic acid (KA), a potential neurotoxic imbalance that may contribute to SLE-mediated cognitive dysfunction. We determined whether peripheral blood interferon-stimulated gene (ISG) expression associates with elevated serum KYN:TRP and QA:KA ratios in SLE. METHODS: ISG expression (whole-blood RNA sequencing) and serum metabolite ratios (high-performance liquid chromatography) were measured in 72 subjects with SLE and 73 healthy controls (HCs). ISG were identified from published gene sets and individual IFN scores were derived to analyse associations with metabolite ratios, clinical parameters and neuropsychological assessments. SLE analyses were grouped by level of ISG expression ('IFN high', 'IFN low' and 'IFN similar to HC') and level of monocyte-associated gene expression (using CIBERSORTx). RESULTS: Serum KYN:TRP and QA:KA ratios were higher in SLE than in HC (p<0.01). 933 genes were differentially expressed ≥2-fold in SLE versus HC (p<0.05). 70 of the top 100 most highly variant genes were ISG. Approximately half of overexpressed genes that correlated with KYN:TRP and QA:KA ratios (p<0.05) were ISG. In 36 IFN-high subjects with SLE, IFN scores correlated with KYN:TRP ratios (p<0.01), but not with QA:KA ratios. Of these 36 subjects, 23 had high monocyte-associated gene expression, and in this subgroup, the IFN scores correlated with both KY:NTRP and QA:KA ratios (p<0.05). CONCLUSIONS: High ISG expression correlated with elevated KYN:TRP ratios in subjects with SLE, suggesting IFN-mediated KYN/TRP pathway activation, and with QA:KA ratios in a subset with high monocyte-associated gene expression, suggesting that KYN/TRP pathway activation may be particularly important in monocytes. These results need validation, which may aid in determining which patient subset may benefit from therapeutics directed at the IFN or KYN/TRP pathways to ameliorate a potentially neurotoxic QA/KA imbalance.

Urine Single-Cell RNA Sequencing in Focal Segmental Glomerulosclerosis Reveals Inflammatory Signatures.

INTRODUCTION: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. METHODS: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). RESULTS: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. CONCLUSION: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.

Urine Proteomics and Renal Single-Cell Transcriptomics Implicate Interleukin-16 in Lupus Nephritis.

OBJECTIVE: Current lupus nephritis (LN) treatments are effective in only 30% of patients, emphasizing the need for novel therapeutic strategies. We undertook this study to develop mechanistic hypotheses and explore novel biomarkers by analyzing the longitudinal urinary proteomic profiles in LN patients undergoing treatment. METHODS: We quantified 1,000 urinary proteins in 30 patients with LN at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single-cell transcriptomics of renal biopsy sections from LN patients. RESULTS: Our analysis revealed multiple biologic pathways, including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization, which could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with LN, as compared to controls without systemic lupus erythematosus. Interleukin-16 (IL-16), CD163, and transforming growth factor ß mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction in urinary IL-16, a CD4 ligand with proinflammatory and chemotactic properties. Single-cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in LN kidneys. IL-16-producing cells were found at key sites of kidney injury. CONCLUSION: Urine proteomics may profoundly change the diagnosis and management of LN by noninvasively monitoring active intrarenal biologic pathways. These findings implicate IL-16 in LN pathogenesis, designating it as a potentially treatable target and biomarker.

The lupus nephritis management renaissance.

Over the past year, and for the first time ever, the US Food and Drug Administration approved 2 drugs specifically for the treatment of lupus nephritis (LN). As the lupus community works toward understanding how to best use these new therapies, it is also an ideal time to begin to rethink the overall management strategy of LN. In addition to new drugs, this must include how to use kidney biopsies for management and not just diagnosis, how molecular technologies can be applied to interrogate biopsies and how such data can impact management, and how to incorporate LN biomarkers into management paradigms. Herein, we will review new developments in these areas of LN and put them into perspective for disease management now and in the future.

Integrated urine proteomics and renal single-cell genomics identify an IFN-γ response gradient in lupus nephritis.

Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology, which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed in order to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by IFN-γ. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, we observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8+ T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8+ cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways could be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.

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.

Design and application of single-cell RNA sequencing to study kidney immune cells in lupus nephritis.

The immune mechanisms that cause tissue injury in lupus nephritis have been challenging to define. The advent of high-dimensional cellular analyses, such as single-cell RNA sequencing, has enabled detailed characterization of the cell populations present in small biopsy samples of kidney tissue. In parallel, the development of methods that cryopreserve kidney biopsy specimens in a manner that preserves intact, viable cells, has enabled the uniform analysis of tissue samples collected at multiple sites and across many geographic areas and demographic cohorts with high-dimensional platforms. The application of these methods to kidney biopsy samples from patients with lupus nephritis has begun to define the phenotypes of both infiltrating and resident immune cells, as well as parenchymal cells, present in nephritic kidneys. The detection of similar immune cell populations in urine suggests that it might be possible to non-invasively monitor immune activation in kidneys. Once applied to large patient cohorts, these high-dimensional studies might enable patient stratification according to patterns of immune cell activation in the kidney or identify disease features that can be used as surrogate measures of efficacy in clinical trials. Applied broadly across multiple inflammatory kidney diseases, these studies promise to enormously expand our understanding of renal inflammation in the next decade.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

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.

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.

A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.

ß Cells that Resist Immunological Attack Develop during Progression of Autoimmune Diabetes in NOD Mice.

Type 1 diabetes (T1D) is a chronic autoimmune disease that involves immune-mediated destruction of ß cells. How ß cells respond to immune attack is unknown. We identified a population of ß cells during the progression of T1D in non-obese diabetic (NOD) mice that survives immune attack. This population develops from normal ß cells confronted with islet infiltrates. Pathways involving cell movement, growth and proliferation, immune responses, and cell death and survival are activated in these cells. There is reduced expression of ß cell identity genes and diabetes antigens and increased immune inhibitory markers and stemness genes. This new subpopulation is resistant to killing when diabetes is precipitated with cyclophosphamide. Human ß cells show similar changes when cultured with immune cells. These changes may account for the chronicity of the disease and the long-term survival of ß cells in some patients.

Human systems immunology: hypothesis-based modeling and unbiased data-driven approaches.

Systems immunology is an emerging paradigm that aims at a more systematic and quantitative understanding of the immune system. Two major approaches have been utilized to date in this field: unbiased data-driven modeling to comprehensively identify molecular and cellular components of a system and their interactions; and hypothesis-based quantitative modeling to understand the operating principles of a system by extracting a minimal set of variables and rules underlying them. In this review, we describe applications of the two approaches to the study of viral infections and autoimmune diseases in humans, and discuss possible ways by which these two approaches can synergize when applied to human immunology.

In vivo dynamical interactions between CD4 Tregs, CD8 Tregs and CD4+ CD25- cells in mice.

BACKGROUND: Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1--a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4(+)CD25(-) cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity. CONCLUSIONS/SIGNIFICANCE: In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.