Synovial tissue inflammation is the 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. We measured genome-wide open chromatin at single cell resolution from 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identified 24 chromatin classes and predicted their associated transcription factors, including a CD8+ GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating an RA tissue transcriptional atlas, we found that the chromatin classes represented 'superstates' corresponding to multiple transcriptional cell states. Finally, we demonstrated 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.

Technologies such as Cellular Indexing of Transcriptomes and Epitopes sequencing (CITE-seq) and RNA Expression and Protein sequencing (REAP-seq) augment unimodal single-cell RNA sequencing (scRNA-seq) by simultaneously measuring expression of cell-surface proteins using antibody derived oligonucleotide tags (ADT). These protocols have been increasingly used to resolve cellular populations that are difficult to infer from gene expression alone, and to interrogate the relationship between gene and protein expression at a single-cell level. However, the ADT-based protein expression component of these assays remains widely underutilized as a primary tool to discover and annotate cell populations, in contrast to flow cytometry which has used surface protein expression in this fashion for decades. Therefore, we hypothesized that computational tools used for flow cytometry data analysis could be harnessed and scaled to analyze ADT data. Here we apply Ozette Discovery™, a recently-developed method for flow cytometry analysis, to re-analyze a large (>400,000 cells) published COVID-19 CITE-seq dataset. Using the protein expression data alone, Ozette Discovery is able to identify granular, robust, and interpretable cellular phenotypes in a high-throughput manner. In particular, we identify a population of CLEC12A+CD11b+CD14- myeloid cells that are specifically expanded in patients with critical COVID-19, and can only be resolved by their protein expression profiles. Using the longitudinal gene expression data from this dataset, we find that early expression of interferon response genes precedes the expansion of this subset, and that early expression of PRF1 and GZMB within specific Ozette Discovery phenotypes provides a RNA biomarker of critical COVID-19. In summary, Ozette Discovery demonstrates that taking a protein-centric approach to cell phenotype annotation in CITE-seq data can achieve the potential that dual RNA/protein assays provide in mixed samples: instantaneous in silico flow sorting, and unbiased RNA-seq profiling.### Competing Interest StatementDA, MW, SP, FL, MJ and AM are employees of and hold stock and/or stock options in Ozette Technologies. GF and EG are employees and founders of and hold stock and/or stock options in Ozette Technologies.

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA. Activated B cells and T cells accumulate within joints of patients with rheumatoid arthritis. Here, the authors use single-cell transcriptome and repertoire profiling to identify clonally expanded synovial B cells and T cells and define their phenotypes and predicted cell-cell interactions.

Translating genome-wide association study (GWAS) loci into causal variants and genes requires accurate cell-type-specific enhancer-gene maps from disease-relevant tissues. Building enhancer-gene maps is essential but challenging with current experimental methods in primary human tissues. Here we developed a nonparametric statistical method, SCENT (single-cell enhancer target gene mapping), that models association between enhancer chromatin accessibility and gene expression in single-cell or nucleus multimodal RNA sequencing and ATAC sequencing data. We applied SCENT to 9 multimodal datasets including >120,000 single cells or nuclei and created 23 cell-type-specific enhancer-gene maps. These maps were highly enriched for causal variants in expression quantitative loci and GWAS for 1,143 diseases and traits. We identified likely causal genes for both common and rare diseases and linked somatic mutation hotspots to target genes. We demonstrate that application of SCENT to multimodal data from disease-relevant human tissue enables the scalable construction of accurate cell-type-specific enhancer-gene maps, essential for defining noncoding variant function.

ABSTRACTLimited ancestral diversity has impaired our ability to detect risk variants more prevalent in non-European ancestry groups in genome-wide association studies (GWAS). We constructed and analyzed a multi-ancestry GWAS dataset in the Alzheimer’s Disease (AD) Genetics Consortium (ADGC) to test for novel shared and ancestry-specific AD susceptibility loci and evaluate underlying genetic architecture in 37,382 non-Hispanic White (NHW), 6,728 African American, 8,899 Hispanic (HIS), and 3,232 East Asian individuals, performing within-ancestry fixed-effects meta-analysis followed by a cross-ancestry random-effects meta-analysis. We identified 13 loci with cross-ancestry associations including known loci at/nearCR1,BIN1,TREM2,CD2AP,PTK2B,CLU,SHARPIN,MS4A6A,PICALM,ABCA7,APOEand two novel loci not previously reported at 11p12 (LRRC4C) and 12q24.13 (LHX5-AS1). Reflecting the power of diverse ancestry in GWAS, we observed theSHARPINlocus using 7.1% the sample size of the original discovering single-ancestry GWAS (n=788,989). We additionally identified three GWS ancestry-specific loci at/near (PTPRK(P=2.4×10-8) andGRB14(P=1.7×10-8) in HIS), andKIAA0825(P=2.9×10-8in NHW). Pathway analysis implicated multiple amyloid regulation pathways (strongest withPadjusted=1.6×10-4) and the classical complement pathway (Padjusted=1.3×10-3). Genes at/near our novel loci have known roles in neuronal development (LRRC4C, LHX5-AS1, andPTPRK) and insulin receptor activity regulation (GRB14). These findings provide compelling support for using traditionally-underrepresented populations for gene discovery, even with smaller sample sizes.

ObjectiveThe synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF‐C) and its receptor, VEGFR‐3, are crucial for lymphangiogenesis. However, their involvement in age‐related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF‐C/VEGFR‐3 pathway contribute to the development and progression of age‐related OA, using a murine model of naturally occurring joint disease.MethodsSLS function was assessed in the knees of young (3‐month‐old) and aged (19–24‐month‐old) male and female C57BL/6J mice via a newly established in vivo IVIS‐dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF‐C treatment on SLS function and OA pathology in aged mice. RNA‐sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints.ResultsResults showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[μW/cm2]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA‐sequencing of the synovial tissue showed that Vegf‐c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF‐C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF‐C–treated aged mice versus 52 ± 15% in vehicle‐treated aged mice; P = 0.012), increased total articular cartilage cross‐sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF‐C–treated aged mice versus 0.26 ± 0.07 mm2 in vehicle‐treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13–positive staining area within total synovial area in 22‐month‐old VEGF‐C–treated mice versus 22‐month‐old vehicle‐treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004).ConclusionSLS function is reduced in the knee joints of aged mice due to decreased VEGF‐C/VEGFR‐3 signaling. VEGF‐C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF‐C/VEGFR‐3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age‐related OA.

Cellular senescence plays important roles in age-related diseases, including musculoskeletal disorders. Senescent cells (SCs) exert a senescence-associated secretory phenotype (SASP) by producing SASP factors, some of which overlap with factors produced by inflammatory cells (Inf-Cs). However, the differences between SCs and Inf-Cs and how they interact with each other during fracture repair have not been well studied. Here, we analyzed single cell RNA sequencing data of aged mouse fracture callus stromal cells. We defined Inf-Cs as cells that express NF-κB Rela/Relb, SCs as cells that express the senescence genes, Cdkn1a, Cdkn2a or Cdkn2c, and inflammatory SCs (Inf-SCs) as cells that express both NF-κB and senescence genes. Differentially expressed genes and pathway analyses revealed that Inf-SCs and SCs had a similar gene expression profile and upregulated pathways that are related to DNA damage/oxidation-reduction and cellular senescence, while Inf-Cs expressed different gene signatures and pathways from SCs and Inf-SCs, mainly related to inflammation. Cellchat software analysis indicated that SCs and Inf-SCs are potential ligand-producing cells that affect Inf-Cs as target cells. Cell culture experiments demonstrated that SC conditioned medium promoted inflammatory gene expression by callus-derived mesenchymal progenitor cells, and Inf-Cs had reduced osteoblast differentiation capacity. In summary, we have identified three cell subclusters associated with inflammation and senescence in callus stromal cells, predicted potential effects of Inf-SCs and SCs on Inf-Cs by production of active ligands, and demonstrated that when mesenchymal progenitors acquire inflammatory phenotypes their osteogenic potential is reduced.

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK + cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

Background: It remains unclear whether the variable biologic features of B-lymphoblastic leukemia (B-ALL) reflect distinct cells of origin in the hierarchy of early B cell development. Recombination activating genes (RAG) rearrange the immunoglobulin heavy chain (IgH) locus in B lymphoid progenitors, resulting in unique variable (Vh), diversity (D), and joining (Jh) gene rearrangements. In B-ALL, dominant IgH sequences are used for clone detection and surveillance. Further, subclone diversity resulting from RAG-mediated Vh gene switching is a prevalent feature of many B-ALLs, but its biologic basis has not been determined. To test whether discrete properties of each B-ALL reflect derivation from distinct B cell stages, we assessed the relationship between IgH variable gene (IGHV) characteristics and gene expression. Methods: We used targeted IgH sequencing (Illumina MiSeq) to identify dominant IgH clones and to assess the extent of IgH subclone diversification in pre-treatment specimens from 22 patients with B-ALL. We compared the gene expression profile (GEP) of cases with Vh-switch-mediated subclone diversity to those without. We then used single-cell RNA sequencing (scRNAseq) of 5 patients' B-ALL to test whether the capacity to undergo IgH diversification is patient-specific or clone-intrinsic, and we characterized variation in GEP between intrapatient IgH clones. Results: We discovered that each B-ALL with at least 1 dominant IgH clone demonstrates either minimal or extensive RAG-mediated subclone evolution. Gene set enrichment analysis (GSEA) revealed that genes associated with distinct B cell developmental stages distinguish these 2 groups. We detected dominant IgH clones in 19 of 22 patients (86.4%). In patients with 'diverse' B-ALL characterized by RAG-mediated intra-clonal diversification (N=9; 40.9%), we observed extensive subclone evolution arising from Vh recombination (median: 482 subclones; range: 2-2600) and enriched expression of genes associated with regulation of a hematopoietic stem cell state. In those without diversity (N=10; 45.5%) - the 'homogeneous' cohort - leukemia cells showed enriched expression of genes involving cell fate commitment. GSEA further revealed biologically distinct phenotypes between groups, as reflected in enrichment in the diverse cohort of Hallmark gene sets involving key cellular metabolic pathways including oxidative phosphorylation and glycolysis. Furthermore, distinct dominant IgH clones from a single patient could be individually diverse or homogenous with respect to Vh-switch-mediated subclone evolution. scRNAseq revealed that dominant IgH clones inconsistently express IGHV mRNA and differ from one another in their gene expression. In 4 of 5 patients, we detected IgH complementarity-determining region 3 (CDR3) mRNA expression from at least 1 clone. The GEP of IGHV-expressing leukemia cells was clearly resolved from other B-ALL cells in 3 of these 4 cases, with distinctive gene expression characterized by RAG1 pathway up-expression. Conclusions: Tumor evolution requires genetic diversity and is a fundamental property of cancer. Using primary B-ALL specimens, we assessed RAG-mediated intraclonal IgH variation to characterize the extent of diversity in B-ALL and to discover how it relates to fundamental biologic features of this common pediatric malignancy. We found that B-ALL may be divided into 2 groups: leukemias with RAG-mediated intra-clonal IGHV sequence diversification ('diverse') and those lacking diversity ('homogeneous'). GEP suggests that the difference in diversity of the IGHV repertoire reflects the B cell stage at leukemic initiation: B-ALL specimens with subclone diversity had a GEP suggestive of an earlier differentiation state compared to specimens with a homogenous IGHV repertoire. Furthermore, we discovered that a single leukemia can be simultaneously composed of both genetically stable and genetically diverse clonal populations. Our single-cell GEP data reveal that: 1) even within a single patient's leukemia, genetically-distinct clonal populations demonstrate a distinct profile of mRNA expression, and 2) B-ALL cells which express IGHV mRNA are characterized by greater expression of RAG pathway-related genes. Our findings suggest that variability in IGHV diversification in B-ALL is a useful marker of biological properties related to the B cell stage of origin.

Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identify a synovial B cell population characterized by co-expression of a family of orphan nuclear receptors (NR4A1-3), which is highly enriched in RA synovial tissue. A transcriptomic profile of NR4A synovial B cells significantly overlaps with germinal center light zone B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell state. NR4A B cells co-express lymphotoxins α and β and IL-6, supporting functions in ELS promotion. Expanded and shared clones between synovial NR4A B cells and plasma cells and the rapid upregulation with BCR stimulation point to in situ differentiation. Together, we identify a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in local adaptive immune responses.

T cell-derived pro-inflammatory cytokines are a major driver of rheumatoid arthritis (RA) pathogenesis. Although these cytokines have traditionally been attributed to CD4 T cells, we have found that CD8 T cells are notably abundant in synovium and make more interferon (IFN)-γ and nearly as much tumor necrosis factor (TNF) as their CD4 T cell counterparts. Furthermore, using unbiased high-dimensional single-cell RNA-seq and flow cytometric data, we found that the vast majority of synovial tissue and synovial fluid CD8 T cells belong to an effector CD8 T cell population characterized by high expression of granzyme K (GzmK) and low expression of granzyme B (GzmB) and perforin. Functional experiments demonstrate that these GzmK+ GzmB+ CD8 T cells are major cytokine producers with low cytotoxic potential. Using T cell receptor repertoire data, we found that CD8 GzmK+ GzmB+ T cells are clonally expanded in synovial tissues and maintain their granzyme expression and overall cell state in blood, suggesting that they are enriched in tissue but also circulate. Using GzmK and GzmB signatures, we found that GzmK-expressing CD8 T cells were also the major CD8 T cell population in the gut, kidney, and coronavirus disease 2019 (COVID-19) bronchoalveolar lavage fluid, suggesting that they form a core population of tissue-associated T cells across diseases and human tissues. We term this population tissue-enriched expressing GzmK or TteK CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 TteK cells have the potential to drive inflammation.

SummaryRheumatoid arthritis (RA) is a prototypical autoimmune disease that causes destructive tissue inflammation in joints and elsewhere. Clinical challenges in RA include the empirical selection of drugs to treat patients, inadequate responders with incomplete disease remission, and lack of a cure. We profiled the full spectrum of cells in inflamed synovium from patients with RA with the goal of deconstructing the cell states and pathways characterizing pathogenic heterogeneity in RA. Our multicenter consortium effort used multi-modal CITE-seq, RNA-seq, and histology of synovial tissue from 79 donors to build a >314,000 single-cell RA synovial cell atlas with 77 cell states from T, B/plasma, natural killer, myeloid, stromal, and endothelial cells. We stratified tissue samples into six distinct cell type abundance phenotypes (CTAPs) individually enriched for specific cell states. These CTAPs demonstrate the striking diversity of RA synovial inflammation, ranging from marked enrichment of T and B cells (CTAP-TB) to a congregation of specific myeloid, fibroblast, and endothelial cells largely lacking lymphocytes (CTAP-EFM). Disease-relevant cytokines, histology, and serology metrics are associated with certain CTAPs. This comprehensive RA synovial atlas and molecular, tissue-based CTAP stratification reveal new insights into RA pathology and heterogeneity, which could lead to novel targeted-treatment approaches in RA.