Single-cell RNA Research Articles

Computational Methods to Identify Cell-Fate Determinants, Identity Transcription Factors, and Niche-Induced Signaling Pathways for Stem Cell Research.

The large-scale development of high-throughput sequencing technologies has not only allowed the generation of reliable omics data related to various regulatory layers but also the development of novel computational models in the field of stem cell research. These computational approaches have enabled the disentangling of a complex interplay between these interrelated layers of regulation by interpreting large quantities of biomedical data in a systematic way. In the context of stem cell research, network modeling of complex gene-gene interactions has been successfully used for understanding the mechanisms underlying stem cell differentiation and cellular conversion. Notably, it has proven helpful for predicting cell-fate determinants and signaling molecules controlling such processes. This chapter will provide an overview of various computational approaches that rely on single-cell and/or bulk RNA sequencing data for elucidating the molecular underpinnings of cell subpopulation identities, lineage specification, and the process of cell-fate decisions. Furthermore, we discuss how these computational methods provide the right framework for computational modeling of biological systems in order to address long-standing challenges in the stem cell field by guiding experimental efforts in stem cell research and regenerative medicine.

Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases.

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4+ naive T cells with concomitant increase in CD4+ memory and CD8+ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.

Dependence on Bcl6 and Blimp1 drive distinct differentiation of murine memory and follicular helper CD4+ T cells.

During the immune response, CD4+ T cells differentiate into distinct effector subtypes, including follicular helper T (Tfh) cells that help B cells, and into memory cells. Tfh and memory cells are required for long-term immunity; both depend on the transcription factor Bcl6, raising the question whether they differentiate through similar mechanisms. Here, using single-cell RNA and ATAC sequencing, we show that virus-responding CD4+ T cells lacking both Bcl6 and Blimp1 can differentiate into cells with transcriptomic, chromatin accessibility, and functional attributes of memory cells but not of Tfh cells. Thus, Bcl6 promotes memory cell differentiation primarily through its repression of Blimp1. These findings demonstrate that distinct mechanisms underpin the differentiation of memory and Tfh CD4+ cells and define the Bcl6-Blimp1 axis as a potential target for promoting long-term memory T cell differentiation.

Abstract 14231: The Role of Altered Notch-Sox9-Hapln1 Signaling in the Pathogenesis of Bicuspid Aortic Valve

Bicuspid aortic valve (BAV) is a common congenital valve disorder with a prevalence close to 1%. Genetic studies on familial forms of BAV had previously identified NOTCH1 receptor as key mediator in the disease pathogenesis. However, the role of different Notch ligands in valve development in health and disease has remained elusive. Here we sequenced a total of 158 individuals with BAV and identified a list of 12 genes significantly enriched in the patients. Among the identified genes, there were various mutations in NOTCH1 receptor and Notch ligands. To further dissect the role of various Notch ligands in valve development, we generated knock-out models of Jag1a, Jag2b, Dll4, and Dld in Zebrafish and explored their effect on the heart outflow tract. Interestingly, Jag1a and Jag2b had opposite effects on valve diameter. Using single cell RNA and bulk RNA seq, and chip sequencing techniques on murine knock-out models of the corresponding genes, we identified a novel Notch-Sox9-Hapln1 signaling pathway dysregulated in the genetic models of Notch mutation that could underlie the changes in endocardial cells. Finally, we observed alterations in this pathway in aortic valve specimens from patients with bicuspid versus tricuspid aortic valves. In conclusion, our study identifies an association between mutations in Notch ligands and BAV and highlights the role of Notch-Sox9-Hapln1 pathway in aortic valve development and implicates its altered function as a novel pathway for the pathogenesis of BAV.

Abstract 12876: Identification of Pathogenic Immune Cell Subsets in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune related adverse events (irAE) such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T-cell-mediated mechanisms. However, the precise pathologic immune subsets and molecular changes in ICI myocarditis are unknown. Hypothesis: We hypothesize that ICI myocarditis is associated with significant quantitative and molecular changes in pathogenic immune cell subsets. Methods: To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry (CyTOF) on peripheral blood mononuclear cells from 40 patients and controls with autoimmune adverse events (irAE) on ICI including four patients with ICI myocarditis. We also profiled 15 patients/controls using single cell T-cell receptor (TCR) sequencing and RNA sequencing (scRNA-seq) with feature barcoding for surface marker expression confirmation. Results: With both methods, we found expansion of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in ICI myocarditis patients compared to controls. Using TCR sequencing, these CD8+ Temra cells were clonally expanded in myocarditis patients. Transcriptomic analysis of these Temra CD8+ clones confirmed a highly activated and cytotoxic phenotype with expression of granzyme/perforin. Longitudinal study demonstrated progression of these Temra CD8+ cells into an exhausted phenotype two months after treatment with glucocorticoids. Differential expression of proinflammatory chemokines (CCL4/CCL4L2/CCL5) was uncovered in clonally expanded Temra CD8+ cells, and ligand-receptor analysis implicated their regulation of other inflammatory cells such as monocytes and NK cells. Conclusions: Clonal cytotoxic Temra CD8+ cells are significantly increased in patients with ICI myocarditis, and have unique transcriptional changes, including upregulation of chemokines CCL4/CCL4L2/CCL5, which may serve as attractive diagnostic/therapeutic targets.

A Predictive Endothelial-Leukemia Pre-Clinical Platform to Uncover Drug Vulnerabilities for Personalized Treatments

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment.Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements.Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing.Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A).We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of “public” and “private” vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs).Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E).Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. [Display omitted] DisclosuresMelnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Expression of Immune Related Genes and Possible Regulatory Mechanisms in Alzheimer’s Disease

Immune infiltration of peripheral natural killer (NK) cells in the brain has been observed in Alzheimer’s disease (AD). Immunity-related genes (IRGs) play an essential role in immune infiltration; however, the expression of IRGs and possible regulatory mechanisms involved in AD remain unclear. The peripheral blood mononuclear cells (PBMCs) single-cell RNA (scRNA) sequencing data from patients with AD were analyzed and PBMCs obtained from the ImmPort database were screened for cluster marker genes. IRG activity was calculated using the AUCell package. A bulk sequencing dataset of AD brain tissues was analyzed to explore common IRGs between PBMCs and the brain. Relevant regulatory transcription factors (TFs) were identified from the Human TFDB database. The protein-protein interaction network of key TFs were generated using the STRING database. Eight clusters were identified, including memory CD4 T, NKT, NK, B, DC, CD8 T cells, and platelets. NK cells were significantly decreased in patients with AD, while CD4 T cells were increased. NK and DC cells exhibited the highest IRG activity. GO and KEGG analyses of the scRNA and bulk sequencing data showed that the DEGs focused on the immune response. Seventy common IRGs were found in both peripheral NK cells and the brain. Seventeen TFs were associated with IRG expression, and the PPI network indicated that STAT3, IRF1, and REL were the hub TFs. In conclusion, we propose that peripheral NK cells may infiltrate the brain and contribute to neuroinflammatory changes in AD through bioinformatic analysis of scRNA and bulk sequencing data. Moreover, STAT3 may be involved in the transcriptional regulation of IRGs in NK cells.

Identification of a Novel cGas/STING/Type I Interferon Specific Murine Cardiac Immune Landscape Following Radiation Therapy

<h3>Purpose/Objective(s)</h3> Cardiotoxicity from radiation treatment to the chest is a late toxicity that has been shown to increase with radiation dose. While clinical parameters and advances in radiation techniques have resulted in lower doses to the heart, the pathophysiologic mechanisms of radiation cardiotoxicity remains a debated topic. We have previously demonstrated that, in murine models, radiation activates a delayed, fibroblast-specific type I interferon response that is dependent on the cytosolic nucleic acid sensor, cGas and its downstream signaling protein STING. Indeed, STING knockout mice exhibit a survival advantage in comparison to wild type mice following a radiation challenge to the heart (20Gy x 1). In this study, we sought to evaluate the consequences of the type I interferon activation on cardiac tissue-specific white blood cell population and identify novel radiation-dependent changes of cardiac immune infiltrates. <h3>Materials/Methods</h3> All animal experiments were performed in accordance with IACUC guidelines. C57BL/6 wild type mice and C57BL/6 Tmem173^gt (catalytically inactive STING) were treated with either sham or 12 Gy single dose to the heart using opposing tangents. At 1 month, hearts were harvested and CD45+ positive cells were immunoprecipitated and sent for 3' single cell RNA (scRNA) sequencing using 10X genomics. ScRNA data was analyzed using Seurat 3.0 <h3>Results</h3> Our results show that radiation induces the upregulation of a subset of cardiac macrophages, interferon inducible cardiac (IFNIC) macrophages. This effect is that is abrogated in mice lacking a functional STING. Furthermore, we observed a general activation of innate immunity as evidenced by increases in the dendritic cell population as well as upregulation of non-classical class I MHC molecules in non-IFNIC macrophages. More surprisingly, we find that there is a marked increase in tissue specific B cell population following radiation, an effect that is completely abolished in mice lacking a functional STING. <h3>Conclusion</h3> We describe for the first time the immune landscape of heart tissue following radiation. We find a previously undescribed infiltration of IFNIC macrophages and a robust activation of an innate immunity. Furthermore, we find a strong effector humoral adaptive response, likely downstream. These findings open the possibility to further understand the role of immune infiltrates in mediation of normal tissue toxicity following radiation.

Single-cell transcriptome analysis yields new insights into the pathogenic mechanisms and possible genetic etiology of cardiomyopathies

Abstract Background Inherited cardiomyopathies (CM) represent a clinically heterogeneous group of primary cardiac muscle disorders with a strong genetic underpinning. Recent rapid genomic advances have led to the identification of numerous disease-causing genes for both non-syndromic (nsCM) and syndromic (sCM) cardiomyopathies. This has greatly facilitated molecular genetic testing, thus enabling accurate disease diagnosis needed for the practice of precision medicine and the optimization of patient outcome. However, many cardiomyopathies remain unexplained with the known genes and dominant genetic model of disease. Purpose To reassess the genetic features of known CM genes as a strategy to recover novel candidate CM genes. Methods Known hypertrophic CM (HCM), dilated CM (DCM) and pediatric CM genes were curated from the literature and from 23 commercial CM diagnostic panels. They were classified as non-syndromic and syndromic, and further annotated using two constraint metrics, the missense Z score and pLI score obtained from the GnomAD database. Publicly available mouse (n=6) and human (n=3) single-cell RNA (scRNA) datasets were downloaded and cardiomyocyte specific differentially expressed genes (DEGs) (fold change &amp;gt;0.1; adjusted P&amp;lt;0.0001) were recovered. Genes identified as DEGs in at least 4 mouse or 3 human scRNA datasets were recovered as nsCM candidate genes. Results Analyses of 9 scRNA datasets showed the majority of known nsCM genes are cardiomyocyte specific (Fig. 1a-b). nsCM and sCM genes have distinct expression and genetic profile. nsCM genes are associated with higher heart expression and lower loss intolerance (Fig. 1c). In contrast, syndromic CM genes mostly showed lower expression with high loss intolerance, consistent with their higher clinical impact. However, interestingly some of the nsCM genes (MYLK2, TMPO and KLF10) show low or even no detectable expression in mouse and human cardiac cells. Using the scRNA data, we assessed cellular expression of genes in the 23 commercial CM diagnostic panels. This analysis showed some of the CM genes with high cardiomyocytes expression have low coverage on the current commercial CM panels (Fig. 1d). Using human and mouse scRNA data, we recovered 224 mouse and 157 human nsCM candidate genes. MTUS2 (microtubule-associated tumor suppressor candidate 2) was identified as a strong nsCM candidate gene supported by evidence from both mouse and human studies (Fig. 1e-f). Conclusions Our analysis showed many of the nsCM genes have differential cardiomyocyte expression with low loss intolerance, while the reverse was observed for many sCM genes. We propose increasing commercial panel coverage of cardiomyocytes-specific expressed genes may help increase disease diagnostic yield. Additionally, novel candidate genes uncovered trained on cardiomyocyte expression profile may help accelerate elucidation of unsolved cardiomyopathy cases. Funding Acknowledgement Type of funding sources: None.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases

Non-small cell lung cancer (NSCLC) metastatic to the brain leptomeninges is rapidly fatal, cannot be biopsied, and cancer cells in the cerebrospinal fluid (CSF) are few; therefore, available tissue samples to develop effective treatments are severely limited. This study aimed to converge single-cell RNA-seq and cell-free RNA (cfRNA) analyses to both diagnose NSCLC leptomeningeal metastases (LM), and to use gene expression profiles to understand progression mechanisms of NSCLC in the brain leptomeninges. NSCLC patients with suspected LM underwent withdrawal of CSF via lumbar puncture. Four cytology-positive CSF samples underwent single-cell capture (n = 197 cells) by microfluidic chip. Using robust principal component analyses, NSCLC LM cell gene expression was compared to immune cells. Massively parallel qPCR (9216 simultaneous reactions) on human CSF cfRNA samples compared the relative gene expression of patients with NSCLC LM (n = 14) to non-tumor controls (n = 7). The NSCLC-associated gene, CEACAM6, underwent in vitro validation in NSCLC cell lines for involvement in pathologic behaviors characteristic of LM. NSCLC LM gene expression revealed by single-cell RNA-seq was also reflected in CSF cfRNA of cytology-positive patients. Tumor-associated cfRNA (e.g., CEACAM6, MUC1) was present in NSCLC LM patients’ CSF, but not in controls (CEACAM6 detection sensitivity 88.24% and specificity 100%). Cell migration in NSCLC cell lines was directly proportional to CEACAM6 expression, suggesting a role in disease progression. NSCLC-associated cfRNA is detectable in the CSF of patients with LM, and corresponds to the gene expression profile of NSCLC LM cells. CEACAM6 contributes significantly to NSCLC migration, a hallmark of LM pathophysiology.

Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice

BackgroundAging has long been thought to be a major risk factor for various types of cancers. However, accumulating evidence indicates increased resistance of old animals to tumor growth. An in-depth...

Doublet identification in single-cell sequencing data using scDblFinder

Doublets are prevalent in single-cell sequencing data and can lead to artifactual findings. A number of strategies have therefore been proposed to detect them. Building on the strengths of existing approaches, we developed scDblFinder, a fast, flexible and accurate Bioconductor-based doublet detection method. Here we present the method, justify its design choices, demonstrate its performance on both single-cell RNA and accessibility sequencing data, and provide some observations on doublet formation, detection, and enrichment analysis. Even in complex datasets, scDblFinder can accurately identify most heterotypic doublets, and was already found by an independent benchmark to outcompete alternatives.

Seq Your Destiny: Neural Crest Fate Determination in the Genomic Era.

Neural crest stem/progenitor cells arise early during vertebrate embryogenesis at the border of the forming central nervous system. They subsequently migrate throughout the body, eventually differentiating into diverse cell types ranging from neurons and glia of the peripheral nervous system to bones of the face, portions of the heart, and pigmentation of the skin. Along the body axis, the neural crest is heterogeneous, with different subpopulations arising in the head, neck, trunk, and tail regions, each characterized by distinct migratory patterns and developmental potential. Modern genomic approaches like single-cell RNA- and ATAC-sequencing (seq) have greatly enhanced our understanding of cell lineage trajectories and gene regulatory circuitry underlying the developmental progression of neural crest cells. Here, we discuss how genomic approaches have provided new insights into old questions in neural crest biology by elucidating transcriptional and posttranscriptional mechanisms that govern neural crest formation and the establishment of axial level identity.

A comprehensive transcriptomic analysis of alternate interferon signaling pathways in peripheral blood mononuclear cells in rheumatoid arthritis.

Interferon (IFN) signaling pathways play crucial roles in the pathogenesis of rheumatoid arthritis (RA). Prior studies have mainly studied mixed alterations in the IFN signaling pathway in RA, but these studies have not been sufficient to elucidate how imbalanced IFN signaling subtly influences immune cells. Single-cell RNA (scRNA) sequencing makes it possible to better understand the alternations in the interferon signaling pathways in RA. In the present study, we found that IFN signaling pathways were activated in natural killer (NK) cells, monocytes, T cells, B cells, and most immune cell subclasses in RA. We then explored and analyzed the connections between abnormal IFN signaling pathways and cellular functional changes in RA. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) analysis and gene regulatory network (GRN) construction were also performed to identify key transcription factors in RA. Finally, we also investigated altered IFN signaling pathways in multiple RA peripheral blood samples, which indicated that abnormal IFN signaling pathways were universally observed in RA. Our study contributes to a better understanding of the delicate and precise regulation of IFN signaling in the immune system in RA. Furthermore, common alternations in IFN signaling pathway-related transcription factors could help to identify novel therapeutic targets for RA treatment.

Single-Cell Imaging of m6 A Modified RNA Using m6 A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6 AISH-PLA).

N6 -methyladenosine (m6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m6 A-specific in situ hybridization mediated proximity ligation assay (m6 AISH-PLA) for cellular imaging of m6 A RNA, allowing to identify m6 A modification at specific location in RNAs and image m6 A RNA with single-cell and single-molecule resolution. Using m6 AISH-PLA, we investigated the m6 A level and subcellular location of HSP70 RNA103-m6 A in response to heat shock stress, and found an increased m6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6 AISH-PLA can serve in the study of m6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Single‐Cell Imaging of m6A Modified RNA Using m6A‐Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH‐PLA)

AbstractN6‐methyladenosine (m6A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m6A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m6A‐specific in situ hybridization mediated proximity ligation assay (m6AISH‐PLA) for cellular imaging of m6A RNA, allowing to identify m6A modification at specific location in RNAs and image m6A RNA with single‐cell and single‐molecule resolution. Using m6AISH‐PLA, we investigated the m6A level and subcellular location of HSP70 RNA103‐m6A in response to heat shock stress, and found an increased m6A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6AISH‐PLA can serve in the study of m6A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Clonally expanded, GPR15-expressing pathogenic effector TH2 cells are associated with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue-resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161- and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was up-regulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen-specific population with enhanced esophagus homing potential.

Zooming in on T cell clones: Are we heading to personalized treatment of allergy?

Single-cell RNA and TCR sequencing of peripheral blood and esophageal cells of human eosinophilic esophagitis uncovers antigen-restricted TH2 cells.

Integrated analysis of glycan and RNA in single cells

SummarySingle-cell sequencing has emerged as an indispensable technology to dissect cellular heterogeneity but never been applied to the simultaneous analysis of glycan and RNA. Using oligonucleotide-labeled lectins, we first established lectin-based glycan profiling of single cells by sequencing (scGlycan-seq). We then combined the scGlycan-seq with single-cell transcriptome profiling for joint analysis of glycan and RNA in single cells (scGR-seq). Using scGR-seq, we analyzed the two modalities in human induced pluripotent stem cells (hiPSCs) before and after differentiation into neural progenitor cells at the single-cell resolution. The combination of RNA and glycan separated the two cell types clearer than either one of them. Furthermore, integrative analysis of glycan and RNA modalities in single cells found known and unknown lectins that were specific to hiPSCs and coordinated with neural differentiation. Taken together, we demonstrate that scGR-seq can reveal the cellular heterogeneity and biological roles of glycans across multicellular systems.