Tissue-specific enhancer-gene maps from multimodal single-cell data identify causal disease alleles.

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.

Causes and Consequences of Varying Transposable Element Activity: An Evolutionary Perspective.

Transposable elements (TEs) are genomic parasites found in nearly all eukaryotes, including humans. This evolutionary success of TEs is due to their replicative activity, involving insertion into new genomic locations. TE activity varies at multiple levels, from between taxa to within individuals. The rapidly accumulating evidence of the influence of TE activity on human health, as well as the rapid growth of new tools to study it, motivated an evaluation of what we know about TE activity thus far. Here, we discuss why TE activity varies, and the consequences of this variation, from an evolutionary perspective. By studying TE activity in nonhuman organisms in the context of evolutionary theories, we can shed light on the factors that affect TE activity. While the consequences of TE activity are usually deleterious, some have lasting evolutionary impacts by conferring benefits on the host or affecting other evolutionary processes.

Single-cell RNA-seq of Drosophila miranda testis reveals the evolution and trajectory of germline sex chromosome regulation.

Although sex chromosomes have evolved from autosomes, they often have unusual regulatory regimes that are sex- and cell-type-specific such as dosage compensation (DC) and meiotic sex chromosome inactivation (MSCI). The molecular mechanisms and evolutionary forces driving these unique transcriptional programs are critical for genome evolution but have been, in the case of MSCI in Drosophila, subject to continuous debate. Here, we take advantage of the younger sex chromosomes in D. miranda (XR and the neo-X) to infer how former autosomes acquire sex-chromosome-specific regulatory programs using single-cell and bulk RNA sequencing and ribosome profiling, in a comparative evolutionary context. We show that contrary to mammals and worms, the X down-regulation through germline progression is most consistent with the shutdown of DC instead of MSCI, resulting in half gene dosage at the end of meiosis for all 3 X's. Moreover, lowly expressed germline and meiotic genes on the neo-X are ancestrally lowly expressed, instead of acquired suppression after sex linkage. For the young neo-X, DC is incomplete across all tissue and cell types and this dosage imbalance is rescued by contributions from Y-linked gametologs which produce transcripts that are translated to compensate both gene and protein dosage. We find an excess of previously autosomal testis genes becoming Y-specific, showing that the neo-Y and its masculinization likely resolve sexual antagonism. Multicopy neo-sex genes are predominantly expressed during meiotic stages of spermatogenesis, consistent with their amplification being driven to interfere with mendelian segregation. Altogether, this study reveals germline regulation of evolving sex chromosomes and elucidates the consequences these unique regulatory mechanisms have on the evolution of sex chromosome architecture.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Understanding Spondyloarthritis Pathogenesis: The Promise of Single-Cell Profiling.

PURPOSE OF REVIEW: Single-cell profiling, either in suspension or within the tissue context, is a rapidly evolving field. The purpose of this review is to outline recent advancements and emerging trends with a specific focus on studies in spondyloarthritis. RECENT FINDINGS: The introduction of sequencing-based approaches for the quantification of RNA, protein, or epigenetic modifications at single-cell resolution has provided a major boost to discovery-driven research. Fluorescent flow cytometry, mass cytometry, and image-based cytometry continue to evolve. Spatial transcriptomics and imaging mass cytometry have extended high-dimensional analysis to cells in tissues. Applications in spondyloarthritis include the indexing and functional characterization of cells, discovery of disease-associated cell states, and identification of signatures associated with therapeutic responses. Single-cell TCR-seq has provided evidence for clonal expansion of CD8+ T cells in spondyloarthritis. The use of single-cell profiling approaches in spondyloarthritis research is still in its early stages. Challenges include high cost and limited availability of diseased tissue samples. To harness the full potential of the rapidly expanding technical capabilities, large-scale collaborative efforts are imperative.

Associations Between Rheumatoid Arthritis Clinical Factors and Synovial Cell Types and States.

OBJECTIVE: Recent studies have uncovered diverse cell types and states in the rheumatoid arthritis (RA) synovium; however, limited data exist correlating these findings with patient-level clinical information. Using the largest cohort to date with clinical and multicell data, we determined associations between RA clinical factors with cell types and states in the RA synovium. METHODS: The Accelerated Medicines Partnership Rheumatoid Arthritis study recruited patients with active RA who were not receiving disease-modifying antirheumatic drugs (DMARDs) or who had an inadequate response to methotrexate (MTX) or tumor necrosis factor inhibitors. RA clinical factors were systematically collected. Biopsies were performed on an inflamed joint, and tissue were disaggregated and processed with a cellular indexing of transcriptomes and epitopes sequencing pipeline from which the following cell type percentages and cell type abundance phenotypes (CTAPs) were derived: endothelial, fibroblast, and myeloid (EFM); fibroblasts; myeloid; T and B cells; T cells and fibroblasts (TF); and T and myeloid cells. Correlations were measured between RA clinical factors, cell type percentage, and CTAPs. RESULTS: We studied 72 patients (mean age 57 years, 75% women, 83% seropositive, mean RA duration 6.6 years, mean Disease Activity Score-28 C-reactive Protein 3 [DAS28-CRP3] score 4.8). Higher DAS28-CRP3 correlated with a higher T cell percentage (P < 0.01). Those receiving MTX and not a biologic DMARD (bDMARD) had a higher percentage of B cells versus those receiving no DMARDs (P < 0.01). Most of those receiving bDMARDs were categorized as EFM (57%), whereas none were TF. No significant difference was observed across CTAPs for age, sex, RA disease duration, or DAS28-CRP3. CONCLUSION: In this comprehensive screen of clinical factors, we observed differential associations between DMARDs and cell phenotypes, suggesting that RA therapies, more than other clinical factors, may impact cell type/state in the synovium and ultimately influence response to subsequent therapies.

Maternal dominance contributes to subgenome differentiation in allopolyploid fishes.

Teleost fishes, which are the largest and most diverse group of living vertebrates, have a rich history of ancient and recent polyploidy. Previous studies of allotetraploid common carp and goldfish (cyprinids) reported a dominant subgenome, which is more expressed and exhibits biased gene retention. However, the underlying mechanisms contributing to observed 'subgenome dominance' remains poorly understood. Here we report high-quality genomes of twenty-one cyprinids to investigate the origin and subsequent subgenome evolution patterns following three independent allopolyploidy events. We identify the closest extant relatives of the diploid progenitor species, investigate genetic and epigenetic differences among subgenomes, and conclude that observed subgenome dominance patterns are likely due to a combination of maternal dominance and transposable element densities in each polyploid. These findings provide an important foundation to understanding subgenome dominance patterns observed in teleost fishes, and ultimately the role of polyploidy in contributing to evolutionary innovations.

Systematic benchmarking of imaging spatial transcriptomics platforms in FFPE tissues.

Emerging imaging spatial transcriptomics (iST) platforms and coupled analytical methods can recover cell-to-cell interactions, groups of spatially covarying genes, and gene signatures associated with pathological features, and are thus particularly well-suited for applications in formalin fixed paraffin embedded (FFPE) tissues. Here, we benchmarked the performance of three commercial iST platforms on serial sections from tissue microarrays (TMAs) containing 23 tumor and normal tissue types for both relative technical and biological performance. On matched genes, we found that 10x Xenium shows higher transcript counts per gene without sacrificing specificity, but that all three platforms concord to orthogonal RNA-seq datasets and can perform spatially resolved cell typing, albeit with different false discovery rates, cell segmentation error frequencies, and with varying degrees of sub-clustering for downstream biological analyses. Taken together, our analyses provide a comprehensive benchmark to guide the choice of iST method as researchers design studies with precious samples in this rapidly evolving field.

Dynamic regulatory elements in single-cell multimodal data implicate key immune cell states enriched for autoimmune disease heritability.

In autoimmune diseases such as rheumatoid arthritis, the immune system attacks the body's own cells. Developing a precise understanding of the cell states where noncoding autoimmune risk variants impart causal mechanisms is critical to developing curative therapies. Here, to identify noncoding regions with accessible chromatin that associate with cell-state-defining gene expression patterns, we leveraged multimodal single-nucleus RNA and assay for transposase-accessible chromatin (ATAC) sequencing data across 28,674 cells from the inflamed synovial tissue of 12 donors. Specifically, we used a multivariate Poisson model to predict peak accessibility from single-nucleus RNA sequencing principal components. For 14 autoimmune diseases, we discovered that cell-state-dependent ('dynamic') chromatin accessibility peaks in immune cell types were enriched for heritability, compared with cell-state-invariant ('cs-invariant') peaks. These dynamic peaks marked regulatory elements associated with T peripheral helper, regulatory T, dendritic and STAT1+CXCL10+ myeloid cell states. We argue that dynamic regulatory elements can help identify precise cell states enriched for disease-critical genetic variation.

Drug Dissolution in Oral Drug Absorption: Workshop Report.

The in-person workshop "Drug Dissolution in Oral Drug Absorption" was held on May 23-24, 2023, in Baltimore, MD, USA. The workshop was organized into lectures and breakout sessions. Three common topics that were re-visited by various lecturers were amorphous solid dispersions (ASDs), dissolution/permeation interplay, and in vitro methods to predict in vivo biopharmaceutics performance and risk. Topics that repeatedly surfaced across breakout sessions were the following: (1) meaning and assessment of "dissolved drug," particularly of poorly water soluble drug in colloidal environments (e.g., fed conditions, ASDs); (2) potential limitations of a test that employs sink conditions for a poorly water soluble drug; (3) non-compendial methods (e.g., two-stage or multi-stage method, dissolution/permeation methods); (4) non-compendial conditions (e.g., apex vessels, non-sink conditions); and (5) potential benefit of having both a quality control method for batch release and a biopredictive/biorelevant method for biowaiver or bridging scenarios. An identified obstacle to non-compendial methods is the uncertainty of global regulatory acceptance of such methods.

Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes.

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Type I interferon signature and cycling lymphocytes in macrophage activation syndrome.

BACKGROUNDMacrophage activation syndrome (MAS) is a life-threatening complication of Still's disease (SD) characterized by overt immune cell activation and cytokine storm. We aimed to further understand the immunologic landscape of SD and MAS.METHODWe profiled PBMCs from people in a healthy control group and patients with SD with or without MAS using bulk RNA-Seq and single-cell RNA-Seq (scRNA-Seq). We validated and expanded the findings by mass cytometry, flow cytometry, and in vitro studies.RESULTSBulk RNA-Seq of PBMCs from patients with SD-associated MAS revealed strong expression of genes associated with type I interferon (IFN-I) signaling and cell proliferation, in addition to the expected IFN-γ signal, compared with people in the healthy control group and patients with SD without MAS. scRNA-Seq analysis of more than 65,000 total PBMCs confirmed IFN-I and IFN-γ signatures and localized the cell proliferation signature to cycling CD38+HLA-DR+ cells within CD4+ T cell, CD8+ T cell, and NK cell populations. CD38+HLA-DR+ lymphocytes exhibited prominent IFN-γ production, glycolysis, and mTOR signaling. Cell-cell interaction modeling suggested a network linking CD38+HLA-DR+ lymphocytes with monocytes through IFN-γ signaling. Notably, the expansion of CD38+HLA-DR+ lymphocytes in MAS was greater than in other systemic inflammatory conditions in children. In vitro stimulation of PBMCs demonstrated that IFN-I and IL-15 - both elevated in MAS patients - synergistically augmented the generation of CD38+HLA-DR+ lymphocytes, while Janus kinase inhibition mitigated this response.CONCLUSIONMAS associated with SD is characterized by overproduction of IFN-I, which may act in synergy with IL-15 to generate CD38+HLA-DR+ cycling lymphocytes that produce IFN-γ.

Testing a candidate meiotic drive locus identified by pool sequencing.

Meiotic drive biases the transmission of alleles in heterozygous individuals, such that Mendel's law of equal segregation is violated. Most examples of meiotic drive have been discovered over the past century based on causing sex ratio distortion or the biased transmission of easily scoreable genetic markers that were linked to drive alleles. More recently, several approaches have been developed that attempt to identify distortions of Mendelian segregation genome wide. Here, we test a candidate female meiotic drive locus in Drosophila melanogaster, identified previously as causing a ∼54:46 distortion ratio using sequencing of large pools of backcross progeny. We inserted fluorescent visible markers near the candidate locus and scored transmission in thousands of individual progeny. We observed a small but significant deviation from the Mendelian expectation; however, it was in the opposite direction to that predicted based on the original experiments. We discuss several possible causes of the discrepancy between the 2 approaches, noting that subtle viability effects are particularly challenging to disentangle from potential small-effect meiotic drive loci. We conclude that pool sequencing approaches remain a powerful method to identify candidate meiotic drive loci but that genotyping of individual progeny at early developmental stages may be required for robust confirmation.

Cardiac allograft vasculopathy diagnosed by vasodilator myocardial contrast echocardiography perfusion imaging.

Cardiac allograft vasculopathy (CAV) remains a common long-term complication of cardiac transplantation. While invasive coronary angiography is considered the gold standard, it is also invasive and lacks sensitivity to detect early, distal CAV. Although vasodilator stress myocardial contrast echocardiography perfusion imaging (MCE) is used in the detection of microvascular disease in non-transplant patients, there is little data guiding its use in transplant recipients. Herein is a case series of four heart transplant recipients that had vasodilator stress MCE performed in addition to invasive coronary angiography for CAV surveillance. MCE at rest and after regadenason was performed using a continuous infusion of lipid-shelled microbubbles. We describe a case of normal microvascular function, diffuse microvascular dysfunction, patchy sub-endocardial perfusion defects and a focal sub-endocardial perfusion defect. Cardiac allograft vasculopathy can be heralded by several different perfusion patterns on MCE in patients after orthotopic heart transplant. The varying prognoses and potential interventions for these different patterns require further investigation.

Adipocytes regulate fibroblast function, and their loss contributes to fibroblast dysfunction in inflammatory diseases.

Fibroblasts play critical roles in tissue homeostasis, but in pathologic states can drive fibrosis, inflammation, and tissue destruction. In the joint synovium, fibroblasts provide homeostatic maintenance and lubrication. Little is known about what regulates the homeostatic functions of fibroblasts in healthy conditions. We performed RNA sequencing of healthy human synovial tissue and identified a fibroblast gene expression program characterized by enhanced fatty acid metabolism and lipid transport. We found that fat-conditioned media reproduces key aspects of the lipid-related gene signature in cultured fibroblasts. Fractionation and mass spectrometry identified cortisol in driving the healthy fibroblast phenotype, confirmed using glucocorticoid receptor gene ( NR3C1 ) deleted cells. Depletion of synovial adipocytes in mice resulted in loss of the healthy fibroblast phenotype and revealed adipocytes as a major contributor to active cortisol generation via Hsd11 ß 1 expression. Cortisol signaling in fibroblasts mitigated matrix remodeling induced by TNFα- and TGFß, while stimulation with these cytokines repressed cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

The Chromatin Landscape of Pathogenic Transcriptional Cell States in Rheumatoid Arthritis.

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.

mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion.

Identifying the mechanisms underlying the regulation of immune checkpoint molecules and the therapeutic impact of targeting them in cancer is critical. Here we show that high expression of the immune checkpoint B7-H3 (CD276) and high mTORC1 activity correlate with immunosuppressive phenotypes and worse clinical outcomes in 11,060 TCGA human tumors. We find that mTORC1 upregulates B7-H3 expression via direct phosphorylation of the transcription factor YY2 by p70 S6 kinase. Inhibition of B7-H3 suppresses mTORC1-hyperactive tumor growth via an immune-mediated mechanism involving increased T-cell activity and IFN-γ responses coupled with increased tumor cell expression of MHC-II. CITE-seq reveals strikingly increased cytotoxic CD38+CD39+CD4+ T cells in B7-H3-deficient tumors. In pan-human cancers, a high cytotoxic CD38+CD39+CD4+ T-cell gene signature correlates with better clinical prognosis. These results show that mTORC1-hyperactivity, present in many human tumors including tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), drives B7-H3 expression leading to suppression of cytotoxic CD4+ T cells.

Clonal associations of lymphocyte subsets and functional states revealed by single cell antigen receptor profiling of T and B cells in rheumatoid arthritis synovium.

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.