Siddarth Gurajala

Synovial tissue inflammation is a hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. Here, we examine genome-wide open chromatin at single-cell resolution in 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identify 24 chromatin classes and predict their associated transcription factors, including a CD8 + GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating with an RA tissue transcriptional atlas, we propose that these chromatin classes represent 'superstates' corresponding to multiple transcriptional cell states. Finally, we demonstrate the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance, as well as the nomination of classes mediating the effects of putatively causal RA genetic variants.

Rheumatoid arthritis (RA) is a systemic autoimmune disease currently with no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in at-risk populations prior to clinical onset is crucial to establishing effective prevention strategies. Here, we applied multimodal single-cell technologies (mass cytometry and CITE-Seq) to characterize the immunophenotypes in blood from at-risk individuals (ARIs) identified through the presence of serum antibodies against citrullinated protein antigens (ACPAs) and/or first-degree relative (FDR) status, as compared with patients with established RA and people in a healthy control group. We identified significant cell expansions in ARIs compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5lo naive B cell population was expanded in ACPA-positive FDRs. Further, we uncovered the molecular phenotype of the CCR2+CD4+ T cells, expressing high levels of Th17- and Th22-related signature transcripts including CCR6, IL23R, KLRB1, CD96, and IL22. Our integrated study provides a promising approach to identify targets to improve prevention strategy development for RA.

<h3>Background</h3> Lupus nephritis (LN) is characterized by considerable variability in its clinical manifestations and histopathological findings. Understanding the cellular and molecular mechanisms underlying this heterogeneity is key for the development of personalized treatments for LN. <h3>Methods</h3> Droplet-based single-cell RNA-sequencing was applied to the analysis of dissociated kidney samples, collected from 155 LN patients with active kidney disease and 30 living donor controls as part of the Accelerating Medicines Partnership (AMP) in SLE consortium - a large- scale, multi-center study. 73,440 immune cells passing quality control were identified, spanning 134 cell subsets, representing various populations of tissue-resident and infiltrating leukocytes, as well as the activation states these cells assume as part of their disease-related activation and differentiation (figure 1). Principal component analysis (PCA) was used to characterize the variability in cell subset frequencies across the LN patients. Relationships between the resulting principal components (PCs) and the demographic, clinical and histopathological features of the patients were then assessed. <h3>Results</h3> The main source of variability in immune cell subset frequencies, as represented by the first PC (PC1), reflected the balance between lymphocytes and monocytes/macrophages. Subsequent PCs represented the balance between B cells and T cells (PC2); the levels of cytotoxic T lymphocytes and NK cells, as compared to plasma cells (PC3); and the degree of macrophage differentiation to an alternatively activated phagocytic profile (PC4). PC1 was significantly correlated with the Chronicity index, such that patients with a higher percentage of lymphocytes compared to monocytes/macrophages had a higher Chronicity score (rho = -0.422, p-value &lt; 0.001; figure 2A). A high degree of macrophage differentiation, as represented by PC4, was associated with a high Activity score (rho = 0.387, p-value &lt; 0.001; figure 2B), and, in addition, with proliferative or mixed histology class, compared to pure membranous nephritis (p-value = 0.001, Kruskal–Wallis test). The ratio of B cells to T cells, as represented by PC2, demonstrated a positive correlation with the Activity index (rho = 0.311, p-value &lt; 0.001). We further identified a significant correlation of PC1 with age; specifically, older patients had a higher relative frequency of lymphocytes compared to monocytes/macrophages (rho = -0.239, p-value = 0.003). Our analysis indicated that these relations are not driven by demographic, clinical and technical sources of variation in our data, including race, ethnicity, the mixture of different nephritic classes, and the inclusion of both first and later biopsies. <h3>Conclusion</h3> Our work identifies distinct leukocyte populations active in different LN patients and, possibly, different stages of disease, and points to potential therapeutic targets, that must be validated in mechanistic studies. This approach may pave the way to personalized treatment of LN.

ABSTRACT Objectives Lupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aim to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. Methods Single-cell RNA-sequencing (scRNA-seq) was used to profile dissociated kidney samples from 156 LN patients and 30 healthy individuals. Spatial transcriptomics (ST), utilizing a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, was applied to kidney samples acquired from 6 LN patients and 2 healthy controls. Results We generated a full catalog of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the NIH chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4 + T, GZMK + CD8 + T, B and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are indicated to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. Conclusions We suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.