IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.

CAR-based therapies: opportunities for immuno-medicine beyond cancer.

One of the most exciting new therapies for cancer involves the use of autologous T cells that are engineered to recognize and destroy cancerous cells. Patients with previously untreatable B cell leukaemias and lymphomas have been cured, and efforts are underway to extend this success to other tumours. Here, we discuss recent studies and emerging research aimed to extend this approach beyond oncology in areas such as cardiometabolic disorders, autoimmunity, fibrosis and senescence. We also summarize new technologies that may help to reduce the cost and increase access to related forms of immunotherapy.

CAR T cells produced in vivo to treat cardiac injury.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell­targeted lipid nanoparticles (LNPs). The efficacy of these in vivo­reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.

Immune Cells and Immunotherapy for Cardiac Injury and Repair.

Cardiac injury remains a major cause of morbidity and mortality worldwide. Despite significant advances, a full understanding of why the heart fails to fully recover function after acute injury, and why progressive heart failure frequently ensues, remains elusive. No therapeutics, short of heart transplantation, have emerged to reliably halt or reverse the inexorable progression of heart failure in the majority of patients once it has become clinically evident. To date, most pharmacological interventions have focused on modifying hemodynamics (reducing afterload, controlling blood pressure and blood volume) or on modifying cardiac myocyte function. However, important contributions of the immune system to normal cardiac function and the response to injury have recently emerged as exciting areas of investigation. Therapeutic interventions aimed at harnessing the power of immune cells hold promise for new treatment avenues for cardiac disease. Here, we review the immune response to heart injury, its contribution to cardiac fibrosis, and the potential of immune modifying therapies to affect cardiac repair.

Author Correction: Targeting cardiac fibrosis with engineered T cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting cardiac fibrosis with engineered T cells.

Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts-fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis.

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells.

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

A radial axis defined by semaphorin-to-neuropilin signaling controls pancreatic islet morphogenesis.

The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered. Here, we identify a radial signaling axis that instructs developing islet cells to disperse throughout the pancreas. A screen of pancreatic extracellular signals identified factors that stimulated islet cell development. These included semaphorin 3a, a guidance cue in neural development without known functions in the pancreas. In the fetal pancreas, peripheral mesenchymal cells expressed Sema3a, while central nascent islet cells produced the semaphorin receptor neuropilin 2 (Nrp2). Nrp2 mutant islet cells developed in proper numbers, but had defects in migration and were unresponsive to purified Sema3a. Mutant Nrp2 islets aggregated centrally and failed to disperse radially. Thus, Sema3a-Nrp2 signaling along an unrecognized pancreatic developmental axis constitutes a chemoattractant system essential for generating the hallmark morphogenetic properties of pancreatic islets. Unexpectedly, Sema3a- and Nrp2-mediated control of islet morphogenesis is strikingly homologous to mechanisms that regulate radial neuronal migration and cortical lamination in the developing mammalian brain.

Pdgfrα functions in endothelial-derived cells to regulate neural crest cells and the development of the great arteries.

Originating as a single vessel emerging from the embryonic heart, the truncus arteriosus must septate and remodel into the aorta and pulmonary artery to support postnatal life. Defective remodeling or septation leads to abnormalities collectively known as conotruncal defects, which are associated with significant mortality and morbidity. Multiple populations of cells must interact to coordinate outflow tract remodeling, and the cardiac neural crest has emerged as particularly important during this process. Abnormalities in the cardiac neural crest have been implicated in the pathogenesis of multiple conotruncal defects, including persistent truncus arteriosus, double outlet right ventricle and tetralogy of Fallot. However, the role of the neural crest in the pathogenesis of another conotruncal abnormality, transposition of the great arteries, is less well understood. In this report, we demonstrate an unexpected role of Pdgfra in endothelial cells and their derivatives during outflow tract development. Loss of Pdgfra in endothelium and endothelial-derived cells results in double outlet right ventricle and transposition of the great arteries. Our data suggest that loss of Pdgfra in endothelial-derived mesenchyme in the outflow tract endocardial cushions leads to a secondary defect in neural crest migration during development.

Epicardial YAP/TAZ orchestrate an immunosuppressive response following myocardial infarction.

Ischemic heart disease resulting from myocardial infarction (MI) is the most prevalent form of heart disease in the United States. Post-MI cardiac remodeling is a multifaceted process that includes activation of fibroblasts and a complex immune response. T-regulatory cells (Tregs), a subset of CD4+ T cells, have been shown to suppress the innate and adaptive immune response and limit deleterious remodeling following myocardial injury. However, the mechanisms by which injured myocardium recruits suppressive immune cells remain largely unknown. Here, we have shown a role for Hippo signaling in the epicardium in suppressing the post-infarct inflammatory response through recruitment of Tregs. Mice deficient in epicardial YAP and TAZ, two core Hippo pathway effectors, developed profound post-MI pericardial inflammation and myocardial fibrosis, resulting in cardiomyopathy and death. Mutant mice exhibited fewer suppressive Tregs in the injured myocardium and decreased expression of the gene encoding IFN-γ, a known Treg inducer. Furthermore, controlled local delivery of IFN-γ following MI rescued Treg infiltration into the injured myocardium of YAP/TAZ mutants and decreased fibrosis. Collectively, these results suggest that epicardial Hippo signaling plays a key role in adaptive immune regulation during the post-MI recovery phase.

Coronary vasculature patterning requires a novel endothelial ErbB2 holoreceptor.

Organogenesis and regeneration require coordination of cellular proliferation, regulated in part by secreted growth factors and cognate receptors, with tissue nutrient supply provided by expansion and patterning of blood vessels. Here we reveal unexpected combinatorial integration of a growth factor co-receptor with a heterodimeric partner and ligand known to regulate angiogenesis and vascular patterning. We show that ErbB2, which can mediate epidermal growth factor (EGF) and neuregulin signalling in multiple tissues, is unexpectedly expressed by endothelial cells where it partners with neuropilin 1 (Nrp1) to form a functional receptor for the vascular guidance molecule semaphorin 3d (Sema3d). Loss of Sema3d leads to improper patterning of the coronary veins, a phenotype recapitulated by endothelial loss of ErbB2. These findings have implications for possible cardiovascular side-effects of anti-ErbB2 therapies commonly used for cancer, and provide an example of integration at the molecular level of pathways involved in tissue growth and vascular patterning.

Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest.

Notch signaling has well-defined roles in the assembly of arterial walls and in the development of the endothelium and smooth muscle of the vasculature. Hippo signaling regulates cellular growth in many tissues, and contributes to regulation of organ size, in addition to other functions. Here, we show that the Notch and Hippo pathways converge to regulate smooth muscle differentiation of the neural crest, which is crucial for normal development of the aortic arch arteries and cranial vasculature during embryonic development. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fail to produce vascular smooth muscle, and Notch target genes such as Jagged1 fail to activate normally. We show that Yap is normally recruited to a tissue-specific Jagged1 enhancer by directly interacting with the Notch intracellular domain (NICD). The Yap-NICD complex is recruited to chromatin by the DNA-binding protein Rbp-J in a Tead-independent fashion. Thus, Hippo signaling can modulate Notch signaling outputs, and components of the Hippo and Notch pathways physically interact. Convergence of Hippo and Notch pathways by the mechanisms described here might be relevant for the function of these signaling cascades in many tissues and in diseases such as cancer.

Plasticity of Hopx(+) type I alveolar cells to regenerate type II cells in the lung.

The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging type I cells. In vivo, type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here we show that Hopx becomes restricted to type I cells during development. However, unexpectedly, lineage-labelled Hopx(+) cells both proliferate and generate type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx(+) type I cells generate organoids composed of type I and type II cells, a process modulated by TGFß signalling. These findings demonstrate unanticipated plasticity of type I cells and a bidirectional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single-cell culture.

Semaphorin signaling in cardiovascular development.

Semaphorins were originally identified as neuronal guidance molecules mediating their attractive or repulsive signals by forming complexes with plexin and neuropilin receptors. Subsequent research has identified functions for semaphorin signaling in many organs and tissues outside of the nervous system. Vital roles for semaphorin signaling in vascular patterning and cardiac morphogenesis have been demonstrated, and impaired semaphorin signaling has been associated with various human cardiovascular disorders, including persistent truncus arteriosus, sinus bradycardia and anomalous pulmonary venous connections. Here, we review the functions of semaphorins and their receptors in cardiovascular development and disease and highlight important recent discoveries in the field.

Pax3 and hippo signaling coordinate melanocyte gene expression in neural crest.

Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.

The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis.

Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineage-tracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardial-derived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways.

Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors and since then have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) has been shown previously to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. We showed recently that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e affect human umbilical vein endothelial cells similarly but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1)-blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d-mediated, but not Sema3e-mediated, inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in human umbilical vein endothelial cells, and inhibition of the PI3K/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.

Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm.

While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.