Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Targeting immune-fibroblast cell communication in heart failure.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction1,2. However, the molecular mechanisms driving immune-fibroblast cell communication in human cardiac disease remain unexplored and there are at present no approved treatments that directly target cardiac fibrosis3,4. Here we performed multiomic single-cell gene expression, epitope mapping and chromatin accessibility profiling in 45 healthy donor, acutely infarcted and chronically failing human hearts. We identified a disease-associated fibroblast trajectory that diverged into distinct populations reminiscent of myofibroblasts and matrifibrocytes, the latter expressing fibroblast activator protein (FAP) and periostin (POSTN). Genetic lineage tracing of FAP+ fibroblasts in vivo showed that they contribute to the POSTN lineage but not the myofibroblast lineage. We assessed the applicability of experimental systems to model cardiac fibroblasts and demonstrated that three different in vivo mouse models of cardiac injury were superior compared with cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin-1ß (IL-1ß) signalling drove the emergence of FAP/POSTN fibroblasts within spatially defined niches. In vivo, we deleted the IL-1 receptor on fibroblasts and the IL-1ß ligand in CCR2+ monocytes and macrophages, and inhibited IL-1ß signalling using a monoclonal antibody, and showed reduced FAP/POSTN fibroblasts, diminished myocardial fibrosis and improved cardiac function. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and preserve organ function.

Adaptive exchange sustains cullin-RING ubiquitin ligase networks and proper licensing of DNA replication.

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.

Blood transcriptomic signature in type-2 biomarker-low severe asthma and asthma control.

BACKGROUND: Patients with type-2 (T2) cytokine-low severe asthma often have persistent symptoms despite suppression of T2 inflammation with corticosteroids. OBJECTIVES: We sought to analyze whole blood transcriptome from 738 samples in T2-biomarker-high/-low patients with severe asthma to relate transcriptomic signatures to T2 biomarkers and asthma symptom scores. METHODS: Bulk RNA-seq data were generated for blood samples (baseline, week 24, week 48) from 301 participants recruited to a randomized clinical trial of corticosteroid optimization in severe asthma. Unsupervised clustering, differential gene expression analysis, and pathway analysis were performed. Patients were grouped by T2-biomarker status and symptoms. Associations between clinical characteristics and differentially expressed genes (DEGs) associated with biomarker and symptom levels were investigated. RESULTS: Unsupervised clustering identified 2 clusters; cluster 2 patients were blood eosinophil-low/symptom-high and more likely to be receiving oral corticosteroids (OCSs). Differential gene expression analysis of these clusters, with and without stratification for OCSs, identified 2960 and 4162 DEGs, respectively. Six hundred twenty-seven of 2960 genes remained after adjusting for OCSs by subtracting OCS signature genes. Pathway analysis identified dolichyl-diphosphooligosaccharide biosynthesis and assembly of RNA polymerase I complex as significantly enriched pathways. No stable DEGs were associated with high symptoms in T2-biomarker-low patients, but numerous associated with elevated T2 biomarkers, including 15 that were upregulated at all time points irrespective of symptom level. CONCLUSIONS: OCSs have a considerable effect on whole blood transcriptome. Differential gene expression analysis demonstrates a clear T2-biomarker transcriptomic signature, but no signature was found in association with T2-biomarker-low patients, including those with a high symptom burden.

Blinatumomab-induced T cell activation at single cell transcriptome resolution.

BACKGROUND: Bi-specific T-cell engager (BiTE) antibody is a class of bispecific antibodies designed for cancer immunotherapy. Blinatumomab is the first approved BiTE to treat acute B cell lymphoblastic leukemia (B-ALL). It brings killer T and target B cells into close proximity, activating patient's autologous T cells to kill malignant B cells via mechanisms such as cytolytic immune synapse formation and inflammatory cytokine production. However, the activated T-cell subtypes and the target cell-dependent T cell responses induced by blinatumomab, as well as the mechanisms of resistance to blinatumomab therapy are largely unknown. RESULTS: In this study, we performed single-cell sequencing analysis to identify transcriptional changes in T cells following blinatumomab-induced T cell activation using single cells from both, a human cell line model and a patient-derived model of blinatumomab-mediated cytotoxicity. In total, the transcriptome of 17,920 single T cells from the cell line model and 2271 single T cells from patient samples were analyzed. We found that CD8+ effector memory T cells, CD4+ central memory T cells, naïve T cells, and regulatory T cells were activated after blinatumomab treatment. Here, blinatumomab-induced transcriptional changes reflected the functional immune activity of the blinatumomab-activated T cells, including the upregulation of pathways such as the immune system, glycolysis, IFNA signaling, gap junctions, and IFNG signaling. Co-stimulatory (TNFRSF4 and TNFRSF18) and co-inhibitory (LAG3) receptors were similarly upregulated in blinatumomab-activated T cells, indicating ligand-dependent T cell functions. Particularly, B-ALL cell expression of TNFSF4, which encodes the ligand of T cell co-stimulatory receptor TNFRSF4, was found positively correlated with the response to blinatumomab treatment. Furthermore, recombinant human TNFSF4 protein enhanced the cytotoxic activity of blinatumomab against B-ALL cells. CONCLUSION: These results reveal a target cell-dependent mechanism of T-cell activation by blinatumomab and suggest that TNFSF4 may be responsible for the resistant mechanism and a potential target for combination therapy with blinatumomab, to treat B-ALL or other B-cell malignancies.

Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling.

BACKGROUND: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. RESULTS: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5' v1 and 3' v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. CONCLUSION: Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

Recent Advances in Single-Cell Profiling and Multispecific Therapeutics: Paving the Way for a New Era of Precision Medicine Targeting Cardiac Fibroblasts.

PURPOSE OF REVIEW: Cardiac fibroblast activation contributes to fibrosis, maladaptive remodeling and heart failure progression. This review summarizes the latest findings on cardiac fibroblast activation dynamics derived from single-cell transcriptomic analyses and discusses how this information may aid the development of new multispecific medicines. RECENT FINDINGS: Advances in single-cell gene expression technologies have led to the discovery of distinct fibroblast subsets, some of which are more prevalent in diseased tissue and exhibit temporal changes in response to injury. In parallel to the rapid development of single-cell platforms, the advent of multispecific therapeutics is beginning to transform the biopharmaceutical landscape, paving the way for the selective targeting of diseased fibroblast subpopulations. Insights gained from single-cell technologies reveal critical cardiac fibroblast subsets that play a pathogenic role in the progression of heart failure. Combined with the development of multispecific therapeutic agents that have enabled access to previously "undruggable" targets, we are entering a new era of precision medicine.

TREM2-activating antibodies abrogate the negative pleiotropic effects of the Alzheimer's disease variant Trem2R47H on murine myeloid cell function.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an orphan immune receptor expressed on cells of myeloid lineage such as macrophages and microglia. The rare variant R47H TREM2 is associated with an increased risk for Alzheimer's disease, supporting the hypothesis that TREM2 loss of function may exacerbate disease progression. However, a complete knockout of the TREM2 gene in different genetic models of neurodegenerative diseases has been reported to result in both protective and deleterious effects on disease-related end points and myeloid cell function. Here, we describe a Trem2R47H transgenic mouse model and report that even in the absence of additional genetic perturbations, this variant clearly confers a loss of function on myeloid cells. The Trem2R47H variant-containing myeloid cells exhibited subtle defects in survival and migration and displayed an unexpected dysregulation of cytokine responses in a lipopolysaccharide challenge environment. These subtle phenotypic defects with a gradation in severity across genotypes were confirmed in whole-genome RNA-Seq analyses of WT, Trem2-/-, and Trem2R47H myeloid cells under challenge conditions. Of note, TREM2-activating antibodies that boost proximal signaling abrogated survival defects conferred by the variant and also modulated migration and cytokine responses in an antibody-, ligand-, and challenge-dependent manner. In some instances, these antibodies also boosted WT myeloid cell function. Our studies provide a first glimpse into the boost in myeloid cell function that can be achieved by pharmacological modulation of TREM2 activity that can potentially be ameliorative in neurodegenerative diseases such as Alzheimer's disease.

Voltage-gated sodium channel function and expression in injured and uninjured rat dorsal root ganglia neurons.

The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.

A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

BACKGROUND: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype. METHODS: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues. RESULTS: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and ß-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy. CONCLUSION: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.

Single Cell Multi-Omics of an iPSC Model of Human Sinoatrial Node Development Reveals Genetic Determinants of Heart Rate and Arrhythmia Susceptibility.

Cellular heterogeneity within the sinoatrial node (SAN) is functionally important but has been difficult to model in vitro , presenting a major obstacle to studies of heart rate regulation and arrhythmias. Here we describe a scalable method to derive sinoatrial node pacemaker cardiomyocytes (PCs) from human induced pluripotent stem cells that recapitulates differentiation into distinct PC subtypes, including SAN Head, SAN Tail, transitional zone cells, and sinus venosus myocardium. Single cell (sc) RNA-sequencing, sc-ATAC-sequencing, and trajectory analyses were used to define epigenetic and transcriptomic signatures of each cell type, and to identify novel transcriptional pathways important for PC subtype differentiation. Integration of our multi-omics datasets with genome wide association studies uncovered cell type-specific regulatory elements that associated with heart rate regulation and susceptibility to atrial fibrillation. Taken together, these datasets validate a novel, robust, and realistic in vitro platform that will enable deeper mechanistic exploration of human cardiac automaticity and arrhythmia.

Attenuated IL-2 muteins leverage the TCR signal to enhance regulatory T cell homeostasis and response in vivo.

Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs' sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.

Phagocytosis increases an oxidative metabolic and immune suppressive signature in tumor macrophages.

Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.

Autonomous IL-36R signaling in neutrophils activates potent antitumor effector functions.

While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.

Targeting Immune-Fibroblast Crosstalk in Myocardial Infarction and Cardiac Fibrosis.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1ß) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1ß signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1ß and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.

RAB18 is a key regulator of GalNAc-conjugated siRNA-induced silencing in Hep3B cells.

Small interfering RNA (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown, and the entire siRNA delivery process is very inefficient, likely because of steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a genome-wide pooled knockout screen on the basis of delivery of GalNAc-conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary genome-wide pooled knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of RAB18 improved the efficacy of siRNA delivered by GalNAc, cholesterol, or antibodies, but not siRNA delivered by Lipofectamine transfection, suggesting a role for RAB18 in siRNA delivery and intracellular trafficking.

In vitro assessment of an engineered tBID-based safety switch system in human T lymphocytes.

BACKGROUND: Cell therapy as a promising therapeutic modality to treat cancer has been intensively studied for decades. However, the clinical trials have indicated that patients under T cell therapy may develop severe cytokine release syndrome resulting in hospitalization or even death. Furthermore, genetic modifications to promote proliferation and persistence of T cells could result in high numbers of long-lived engineered cells in patients after treatment. METHODS: We incorporated the pro-apoptotic truncated BH3 interacting-domain death agonist (tBID) with the mutant ecDHFR destabilizing domain to form a novel recombinant protein as the major component of an engineered tBID-based safety switch system, which would be unstable and quickly degraded in the absence of trimethoprim (TMP) but, upon TMP treatment, should become stabilized and allow tBID to induce cell death experimentally. RESULTS: The novel tBID-based safety switch could be regulated through a small molecule inducer, TMP, to control undesired toxicity or ablate the engineered cells as needed. We systematically compared and assessed several tBID-based safety switch constructs with the clinically validated safety switches, including human herpes simplex virus thymidine kinase (HSV-TK) and inducible Caspase 9 (iCasp9). With optimization, we were able to achieve significant killing potency in vitro in Jurkat or human primary T cells. CONCLUSIONS: We demonstrated that our engineered tBID-based safety switch was able to eliminate up to ~90% of transduced human primary T cells within 72 h after activation, providing an alternative switch system to manage safety concerns for cell therapy.

Chamber-enriched gene expression profiles in failing human hearts with reduced ejection fraction.

Heart failure with reduced ejection fraction (HFrEF) constitutes 50% of HF hospitalizations and is characterized by high rates of mortality. To explore the underlying mechanisms of HFrEF etiology and progression, we studied the molecular and cellular differences in four chambers of non-failing (NF, n = 10) and HFrEF (n = 12) human hearts. We identified 333 genes enriched within NF heart subregions and often associated with cardiovascular disease GWAS variants. Expression analysis of HFrEF tissues revealed extensive disease-associated transcriptional and signaling alterations in left atrium (LA) and left ventricle (LV). Common left heart HFrEF pathologies included mitochondrial dysfunction, cardiac hypertrophy and fibrosis. Oxidative stress and cardiac necrosis pathways were prominent within LV, whereas TGF-beta signaling was evident within LA. Cell type composition was estimated by deconvolution and revealed that HFrEF samples had smaller percentage of cardiomyocytes within the left heart, higher representation of fibroblasts within LA and perivascular cells within the left heart relative to NF samples. We identified essential modules associated with HFrEF pathology and linked transcriptome discoveries with human genetics findings. This study contributes to a growing body of knowledge describing chamber-specific transcriptomics and revealed genes and pathways that are associated with heart failure pathophysiology, which may aid in therapeutic target discovery.

Phytosterol accumulation results in ventricular arrhythmia, impaired cardiac function and death in mice.

Heart failure (HF) and cardiac arrhythmias share overlapping pathological mechanisms that act cooperatively to accelerate disease pathogenesis. Cardiac fibrosis is associated with both pathological conditions. Our previous work identified a link between phytosterol accumulation and cardiac injury in a mouse model of phytosterolemia, a rare disorder characterized by elevated circulating phytosterols and increased cardiovascular disease risk. Here, we uncover a previously unknown pathological link between phytosterols and cardiac arrhythmias in the same animal model. Phytosterolemia resulted in inflammatory pathway induction, premature ventricular contractions (PVC) and ventricular tachycardia (VT). Blockade of phytosterol absorption either by therapeutic inhibition or by genetic inactivation of NPC1L1 prevented the induction of inflammation and arrhythmogenesis. Inhibition of phytosterol absorption reduced inflammation and cardiac fibrosis, improved cardiac function, reduced the incidence of arrhythmias and increased survival in a mouse model of phytosterolemia. Collectively, this work identified a pathological mechanism whereby elevated phytosterols result in inflammation and cardiac fibrosis leading to impaired cardiac function, arrhythmias and sudden death. These comorbidities provide insight into the underlying pathophysiological mechanism for phytosterolemia-associated risk of sudden cardiac death.