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IFNγ-secreting T cells are central for the maintenance of immune surveillance within the central nervous system (CNS). It was previously reported in healthy donors that the T-cell environment in the CNS induces distinct signatures related to cytotoxic capacity, CNS trafficking, tissue adaptation, and lipid homeostasis. These findings suggested that the CNS milieu consisting predominantly of lipids mediated the metabolic conditions leading to IFNγ-secreting brain CD4 T cells. Here, we demonstrate that the supplementation of CD4 + CD45RO + CXCR3 + cells with cholesterol modulates their function and increases IFNG expression. The heightened IFNG expression was mediated by the activation of the serum/glucocorticoid-regulated kinase (SGK1). Inhibition of SGK1 by a specific enzymatic inhibitor significantly reduces the expression of IFNG Our results confirm the crucial role of lipids in maintaining T-cell homeostasis and demonstrate a putative role of environmental factors to induce effector responses in CD4 + effector/memory cells. These findings offer potential avenues for further research targeting lipid pathways to modulate inflammatory conditions., (© 2024 Hanin et al.)

Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research., Competing Interests: COMPETING INTERESTS The authors declare that there is no conflict of interest.

Autoimmune diseases, among the most common disorders of young adults, are mediated by genetic and environmental factors. Although CD4 + FOXP3 + regulatory T cells (T regs ) play a central role in preventing autoimmunity, the molecular mechanism underlying their dysfunction is unknown. Here, we performed comprehensive transcriptomic and epigenomic profiling of T regs in the autoimmune disease multiple sclerosis (MS) to identify critical transcriptional programs regulating human autoimmunity. We found that up-regulation of a primate-specific short isoform of PR domain zinc finger protein 1 (PRDM1-S) induces expression of serum and glucocorticoid-regulated kinase 1 (SGK1) independent from the evolutionarily conserved long PRDM1 , which led to destabilization of forkhead box P3 (FOXP3) and T reg dysfunction. This aberrant PRDM1-S/SGK1 axis is shared among other autoimmune diseases. Furthermore, the chromatin landscape profiling in T regs from individuals with MS revealed enriched activating protein-1 (AP-1)/interferon regulatory factor (IRF) transcription factor binding as candidate upstream regulators of PRDM1-S expression and T reg dysfunction. Our study uncovers a mechanistic model where the evolutionary emergence of PRDM1-S and epigenetic priming of AP-1/IRF may be key drivers of dysfunctional T regs in autoimmune diseases.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P  < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P  = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P  = 0.007), although not different than controls, and may be associated with decreased survival ( P  = 0.009 in Kaplan-Meier analysis; and P  = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Probabilistic topic modelling has become essential in many types of single-cell data analysis. Based on probabilistic topic assignments in each cell, we identify the latent representation of cellular states. A dictionary matrix, consisting of topic-specific gene frequency vectors, provides interpretable bases to be compared with known cell type-specific marker genes and other pathway annotations. However, fitting a topic model on a large number of cells would require heavy computational resources-specialized computing units, computing time and memory. Here, we present a scalable approximation method customized for single-cell RNA-seq data analysis, termed ASAP, short for Annotating a Single-cell data matrix by Approximate Pseudobulk estimation. Our approach is more accurate than existing methods but requires orders of magnitude less computing time, leaving much lower memory consumption. We also show that our approach is widely applicable for atlas-scale data analysis; our method seamlessly integrates single-cell and bulk data in joint analysis, not requiring additional preprocessing or feature selection steps., (© 2024 Subedi et al.)

The discovery of FOXP3 + regulatory T (T reg ) cells as a distinct cell lineage with a central role in regulating immune responses provided a deeper understanding of self-tolerance. The transcription factor FOXP3 serves a key role in T reg cell lineage determination and maintenance, but is not sufficient to enable the full potential of T reg cell suppression, indicating that other factors orchestrate the fine-tuning of T reg cell function. Moreover, FOXP3-independent mechanisms have recently been shown to contribute to T reg cell dysfunction. FOXP3 mutations in humans cause lethal fulminant systemic autoinflammation (IPEX syndrome). However, it remains unclear to what degree T reg cell dysfunction is contributing to the pathophysiology of common autoimmune diseases. In this Review, we discuss the origins of T reg cells in the periphery and the multilayered mechanisms by which T reg cells are induced, as well as the FOXP3-dependent and FOXP3-independent cellular programmes that maintain the suppressive function of T reg cells in humans and mice. Further, we examine evidence for T reg cell dysfunction in the context of common autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis., (© 2024. Springer Nature Limited.)

Rationale : Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives : To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods : Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results : Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3 hi /CCL4 hi and S100A hi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100A hi classical monocytes differentiate into SPP1 hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZM hi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFβ and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions : Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZM hi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100A hi and CCL3 hi /CCL4 hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Rare immune-mediated cardiac tissue inflammation can occur after vaccination, including after SARS-CoV-2 mRNA vaccines. However, the underlying immune cellular and molecular mechanisms driving this pathology remain poorly understood. Here, we investigated a cohort of patients who developed myocarditis and/or pericarditis with elevated troponin, B-type natriuretic peptide, and C-reactive protein levels as well as cardiac imaging abnormalities shortly after SARS-CoV-2 mRNA vaccination. Contrary to early hypotheses, patients did not demonstrate features of hypersensitivity myocarditis, nor did they have exaggerated SARS-CoV-2-specific or neutralizing antibody responses consistent with a hyperimmune humoral mechanism. We additionally found no evidence of cardiac-targeted autoantibodies. Instead, unbiased systematic immune serum profiling revealed elevations in circulating interleukins (IL-1β, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix metalloproteases (MMP1, MMP8, MMP9, and TIMP1). Subsequent deep immune profiling using single-cell RNA and repertoire sequencing of peripheral blood mononuclear cells during acute disease revealed expansion of activated CXCR3 + cytotoxic T cells and NK cells, both phenotypically resembling cytokine-driven killer cells. In addition, patients displayed signatures of inflammatory and profibrotic CCR2 + CD163 + monocytes, coupled with elevated serum-soluble CD163, that may be linked to the late gadolinium enhancement on cardiac MRI, which can persist for months after vaccination. Together, our results demonstrate up-regulation in inflammatory cytokines and corresponding lymphocytes with tissue-damaging capabilities, suggesting a cytokine-dependent pathology, which may further be accompanied by myeloid cell-associated cardiac fibrosis. These findings likely rule out some previously proposed mechanisms of mRNA vaccine--associated myopericarditis and point to new ones with relevance to vaccine development and clinical care.

Maintenance of peripheral tolerance by CD4 + Foxp3 + regulatory T cells (Tregs) is essential for regulating autoreactive T cells. The loss of function of Foxp3 leads to autoimmune disease in both animals and humans. An example is the rare, X-linked recessive disorder known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy X-linked) syndrome. In more common human autoimmune diseases, defects in Treg function are accompanied with aberrant effector cytokines such as IFNγ. It has recently become appreciated that Tregs plays an important role in not only maintaining immune homeostasis but also in establishing the tissue microenvironment and homeostasis of non-lymphoid tissues. Tissue resident Tregs show profiles that are unique to their local environments which are composed of both immune and non-immune cells. Core tissue-residence gene signatures are shared across different tissue Tregs and are crucial to homeostatic regulation and maintaining the tissue Treg pool in a steady state. Through interaction with immunocytes and non-immunocytes, tissue Tregs exert a suppressive function via conventional ways involving contact dependent and independent processes. In addition, tissue resident Tregs communicate with other tissue resident cells which allows Tregs to adopt to their local microenvironment. These bidirectional interactions are dependent on the specific tissue environment. Here, we summarize the recent advancements of tissue Treg studies in both human and mice, and discuss the molecular mechanisms that maintain tissue homeostasis and prevent pathogenesis., Competing Interests: DH has received funding for his laboratory from Bristol Myers Squibb and Genentech. Further information regarding funding is available at: https://openpaymentsdata.cms.gov/physician/166753. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cheru, Hafler and Sumida.)

Hyperosmotic stress triggers an evolutionally preserved, fundamental cellular response. A growing body of evidence has highlighted the role of extra-renal, interstitial hyperosmolality in maintaining local tissue immune homeostasis and potentially driving tissue inflammation in human diseases. The hyperosmotic stress response initiates cellular shrinkage, oxidative stress, metabolic remodeling and cell cycle arrest, all of which are adjusted by a counteractive adaptative response that includes osmolyte synthesis, upregulation of ion transporters and induction of heat shock proteins. Recent studies have revealed that high osmolality can impact immune cell differentiation and activation pathways in a cell type specific manner. The fine-tuning of the immune response depends on the tissue microenvironment. Accordingly, novel therapeutic approaches that target hyperosmolality-mediated inflammation may be identified by furthering our understanding of hyperosmotic response in the context of disease. In this review, we discuss the cellular and molecular mechanisms by which hyperosmotic stress response regulates interstitial homeostasis and pathogenic inflammation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4 + T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1 high CXCR5 - CD4 + T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection., Competing Interests: Declaration of interests D.A.H. has received research funding from Bristol-Myers Squibb, Novartis, Sanofi, and Genentech. He has been a consultant for Bayer Pharmaceuticals, Bristol Myers Squibb, Compass Therapeutics, EMD Serono, Genentech, Juno Therapeutics, Novartis Pharmaceuticals, Proclara Biosciences, Sage Therapeutics, and Sanofi Genzyme. Further information regarding funding is available at https://openpaymentsdata.cms.gov/physician/166753/general-payments. N.K. reports personal fees from Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Indalo, Theravance, LifeMax, Three Lake Partners, and RohBar in the last 36 months and Equity in Pliant. N.K. is also a recipient of a grant from Veracyte and non-financial support from Miragen. In addition, N.K. has patents on New Therapies in Pulmonary Fibrosis and ARDS (unlicensed) and Peripheral Blood Gene Expression as biomarkers in IPF (licensed to biotech), all outside the submitted work. S.H.K. receives consulting fees from Northrop Grumman. K.B.H. receives consulting fees from Prellis Biologics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies identifying hundreds of susceptibility loci for autoimmune diseases indicate that genes active in immune cells predominantly mediate risk. However, identification and functional characterization of causal variants remain challenging. Here, we focused on the immunomodulatory role of a protective variant of histone deacetylase 7 (HDAC7). This variant (rs148755202, HDAC7.p.R166H) was identified in a study of low-frequency coding variation in multiple sclerosis (MS). Through transcriptomic analyses, we demonstrate that wild-type HDAC7 regulates genes essential for the function of Foxp3 + regulatory T cells (T regs ), an immunosuppressive subset of CD4 T cells that is generally dysfunctional in patients with MS. Moreover, T reg -specific conditional hemizygous deletion of HDAC7 increased the severity of experimental autoimmune encephalitis (EAE), a mouse model of neuroinflammation. In contrast, T regs transduced with the protective HDAC7 R166H variant exhibited higher suppressive capacity in an in vitro functional assay, mirroring phenotypes previously observed in patient samples. In vivo modeling of the human HDAC7 R166H variant by generation of a knock-in mouse model bearing an orthologous R150H substitution demonstrated decreased EAE severity linked to transcriptomic alterations of brain-infiltrating T regs , as assessed by single-cell RNA sequencing. Our data suggest that dysregulation of epigenetic modifiers, a distinct molecular class associated with disease risk, may influence disease onset. Last, our approach provides a template for the translation of genetic susceptibility loci to detailed functional characterization, using in vitro and in vivo modeling.

B cell depletion in patients with relapsing-remitting multiple sclerosis (RRMS) markedly prevents new MRI-detected lesions and disease activity, suggesting the hypothesis that altered B cell function leads to the activation of T cells driving disease pathogenesis. Here, we performed comprehensive analyses of CD40 ligand- (CD40L-) and IL-21-stimulated memory B cells from patients with MS and healthy age-matched controls, modeling the help of follicular helper T cells (Tfh cells), and found a differential gene expression signature in multiple B cell pathways. Most striking was the impaired TIGIT expression on MS-derived B cells mediated by dysregulation of the transcription factor TCF4. Activated circulating Tfh cells (cTfh cells) expressed CD155, the ligand of TIGIT, and TIGIT on B cells revealed their capacity to suppress the proliferation of IL-17-producing cTfh cells via the TIGIT/CD155 axis. Finally, CCR6+ cTfh cells were significantly increased in patients with MS, and their frequency was inversely correlated with that of TIGIT+ B cells. Together, these data suggest that the dysregulation of negative feedback loops between TIGIT+ memory B cells and cTfh cells in MS drives the activated immune system in this disease.

T cells can acquire CD20 from B cells via trogocytosis, then contribute to autoimmune neurological disease while becoming targets for therapeutically effective B cell depletion.

Single-cell technology can be used to understand the genetic basis of human diseases.

Although inhibition of T cell coinhibitory receptors has revolutionized cancer therapy, the mechanisms governing their expression on human T cells have not been elucidated. In the present study, we show that type 1 interferon (IFN-I) regulates coinhibitory receptor expression on human T cells, inducing PD-1/TIM-3/LAG-3 while inhibiting TIGIT expression. High-temporal-resolution mRNA profiling of IFN-I responses established the dynamic regulatory networks uncovering three temporal transcriptional waves. Perturbation of key transcription factors (TFs) and TF footprint analysis revealed two regulator modules with different temporal kinetics that control expression of coinhibitory receptors and IFN-I response genes, with SP140 highlighted as one of the key regulators that differentiates LAG-3 and TIGIT expression. Finally, we found that the dynamic IFN-I response in vitro closely mirrored T cell features in acute SARS-CoV-2 infection. The identification of unique TFs controlling coinhibitory receptor expression under IFN-I response may provide targets for enhancement of immunotherapy in cancer, infectious diseases and autoimmunity., (© 2022. Springer Nature America, Inc.)

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100A hi /HLA-DR lo classical monocytes and activated LAG-3 hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8 + clones, unmutated IGHG + B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19., (© 2022. The Author(s).)

Protective immunity against COVID-19 likely depends on the production of SARS-CoV-2-specific plasma cells and memory B cells postinfection or postvaccination. Previous work has found that germinal center reactions are disrupted in severe COVID-19. This may adversely affect long-term immunity against reinfection. Consistent with an extrafollicular B cell response, patients with severe COVID-19 have elevated frequencies of clonally expanded, class-switched, unmutated plasmablasts. However, it is unclear whether B cell populations in individuals with mild COVID-19 are similarly skewed. In this study, we use single-cell RNA sequencing of B cells to show that in contrast to patients with severe COVID-19, subjects with mildly symptomatic COVID-19 have B cell repertoires enriched for clonally diverse, somatically hypermutated memory B cells ∼30 d after the onset of symptoms. This provides evidence that B cell responses are less disrupted in mild COVID-19 and result in the production of memory B cells., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The increasing availability of single-cell data revolutionizes the understanding of biological mechanisms at cellular resolution. For differential expression analysis in multi-subject single-cell data, negative binomial mixed models account for both subject-level and cell-level overdispersions, but are computationally demanding. Here, we propose an efficient NEgative Binomial mixed model Using a Large-sample Approximation (NEBULA). The speed gain is achieved by analytically solving high-dimensional integrals instead of using the Laplace approximation. We demonstrate that NEBULA is orders of magnitude faster than existing tools and controls false-positive errors in marker gene identification and co-expression analysis. Using NEBULA in Alzheimer's disease cohort data sets, we found that the cell-level expression of APOE correlated with that of other genetic risk factors (including CLU, CST3, TREM2, C1q, and ITM2B) in a cell-type-specific pattern and an isoform-dependent manner in microglia. NEBULA opens up a new avenue for the broad application of mixed models to large-scale multi-subject single-cell data.

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8 + T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C., Competing Interests: Declaration of interests D.A.H. has received research funding from Bristol-Myers Squibb, Novartis, Sanofi, and Genentech. He has been a consultant for Bayer Pharmaceuticals, Bristol Myers Squibb, Compass Therapeutics, EMD Serono, Genentech, Juno Therapeutics, Novartis Pharmaceuticals, Proclara Biosciences, Sage Therapeutics, and Sanofi Genzyme. Further information regarding funding is available on: https://openpaymentsdata.cms.gov/physician/166753/general-payments. N.K. reports personal fees from Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Indalo, Theravance, LifeMax, Three Lake Partners, RohBar in the last 36 months, and Equity in Pliant. N.K. is also a recipient of a grant from Veracyte and non-financial support from Miragen. All outside the submitted work; In addition, N.K. has patents on New Therapies in Pulmonary Fibrosis and ARDS (unlicensed) and Peripheral Blood Gene Expression as biomarkers in IPF (licensed to biotech). S.H.K. receives consulting fees from Northrop Grumman. K.B.H. receives consulting fees from Prellis Biologics. B.S. is a former SomaLogic, Inc. (Boulder, CO, USA) employee and a company shareholder. All other authors declared that they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.