T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

A New Mouse Model of Chronic Myocarditis Induced by Recombinant Bacille Calmette-Guèrin Expressing a T-Cell Epitope of Cardiac Myosin Heavy Chain-α.

Dilated cardiomyopathy (DCM) is a potentially lethal disorder characterized by progressive impairment of cardiac function. Chronic myocarditis has long been hypothesized to be one of the causes of DCM. However, owing to the lack of suitable animal models of chronic myocarditis, its pathophysiology remains unclear. Here, we report a novel mouse model of chronic myocarditis induced by recombinant bacille Calmette-Guérin (rBCG) expressing a CD4+ T-cell epitope of cardiac myosin heavy chain-α (rBCG-MyHCα). Mice immunized with rBCG-MyHCα developed chronic myocarditis, and echocardiography revealed dilation and impaired contraction of ventricles, similar to those observed in human DCM. In the heart, CD62L-CD4+ T cells were increased and produced significant amounts of IFN-γ and IL-17 in response to cardiac myosin. Adoptive transfer of CD62L-CD4+ T cells induced myocarditis in the recipient mice, which indicated that CD62L-CD4+ T cells were the effector cells in this model. rBCG-MyHCα-infected dendritic cells produced proinflammatory cytokines and induced MyHCα-specific T-cell proliferation and Th1 and Th17 polarization. This novel chronic myocarditis mouse model may allow the identification of the central pathophysiological and immunological processes involved in the progression to DCM.

Phosphoinositide 3-kinase inhibitor LY294002 ameliorates the severity of myosin-induced myocarditis in mice.

BACKGROUND: Myocarditis, characterized by myocyte necrosis, fibrosis, and degeneration with mononuclear cell infiltration, always causes heart failure in patients. Phosphoinositide 3-kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in cardiac diseases. Although previous research has suggested that PI3K was involved in cardiac diseases such as myocardial infarction, it is still unclear whether the inhibition of PI3K is essential for the treatment of myosin-induced myocarditis. The aim of this study was to explore whether pharmacological blockade of PI3K is able to protect mice against experimental autoimmune myocarditis (EAM). MATERIALS AND METHODS: We used the cardiac myosin-induced murine EAM model to investigate the therapeutic effect of PI3K inhibitor LY294002 on autoimmune myocarditis in mice. RESULTS: LY294002 significantly alleviated EAM injury in mice, as indicated by the reduction of cardiac necrosis, inflammatory infiltrates, and CD3(+) T cells. LY294002 also decreased the expression of p-Akt upon cardiac myosin treatment in the cardiac tissue of the mice. In the present study, LY294002 resulted in a moderate reduction in absolute CD4(+) cell numbers and a significant decrease in the absolute numbers of CD8(+) cells. Consequently, LY294002 increased the CD4(+)/CD8(+) ratio compared with peptide treatment alone. CONCLUSION: This report provides evidence that PI3K inhibitor LY294002 has potent effects against cardiac injury caused by EAM, suggesting that it has therapeutic value for the treatment of myocarditis.

Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis.

BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.

Insulin promotes T cell recovery in a murine model of autoimmune myocarditis.

Glucose-insulin-potassium (GIK) is a useful adjunct to myocarditis. Besides its essential action in energy metabolism, insulin also exerts an anti-inflammatory effect. This study investigated the effect of insulin on myocardial inflammation in experimental autoimmune myocarditis (EAM) in mice and its potential role in T cell regulation. Mice were divided randomly into a normal control group, a saline-treated EAM group and an insulin-treated EAM group. The histopathological changes of myocardium, α-myosin heavy chain (MyHCα)(614-629) antigen-specific autoantibody titre, the serum level of cardiac troponin I (cTnI), mitogen-activated protein kinase (MAPK) family members' activity and content were measured. Furthermore, the phenotype of T lymphocyte subsets in splenocytes was analysed to evaluate the immune status of mice. Insulin reduced serum cTnI of EAM mice on days 14 and 21 (P < 0·05) after immunization, with no changes in blood glucose and autoantibody production. Western blot revealed that extracellular signal-regulated protein kinase (ERK1/2) may be a determining factor in this process. Total ERK1/2 and phospho-ERK1/2 (p-ERK1/2) were both up-regulated in insulin-treated mice after immunization. We also found that insulin treatment promoted T cell recovery without changing the naive-to-memory T-cell ratio; in particular, CD3(+) T cells in insulin-treated mice proliferated more vigorously than in control mice (P < 0·05). We report here for the first time that insulin alleviates myocarditis in the EAM model. These data show that insulin has a direct effect on T cell proliferation in EAM. It is possible that GIK or insulin may assist T cell recovery towards normal in myocarditis, especially for diabetic or hyperglycaemic patients.

Role of impaired central tolerance to α-myosin in inflammatory heart disease.

For more than a half century, autoimmunity has been linked to a diverse array of heart diseases, including rheumatic carditis, myocarditis, Chagas' cardiomyopathy, post-myocardial infarction (Dressler's) syndrome, and idiopathic dilated cardiomyopathy. Why the heart is targeted by autoimmunity in these seemingly unrelated conditions has remained enigmatic. Here, we discuss our recent studies indicating that this susceptibility is mediated by impaired negative selection of autoreactive α-myosin heavy-chain-specific CD4(+) T cells in the thymus of both mice and humans. We describe how this process may place the heart at increased risk for autoimmune attack following ischemic or infectious injury, providing a rationale for the development of antigen-specific tolerogenic therapies.

Myocarditis: a defect in central immune tolerance?

Myocarditis, or inflammation of the heart, is a potentially devastating disease that can result from both viral infection and autoimmune attack of self antigens in the heart. In the current issue of the JCI, Lv and colleagues use a genetically susceptible mouse model to show that myocarditis is a T cell-mediated autoimmune disease that occurs due to insufficient thymic negative selection of α-myosin-reactive T cells.

Impaired thymic tolerance to α-myosin directs autoimmunity to the heart in mice and humans.

Autoimmunity has long been linked to myocarditis and its sequela, dilated cardiomyopathy, the leading causes of heart failure in young patients. However, the underlying mechanisms are poorly defined, with most clinical investigations focused on humoral autoimmunity as the target for intervention. Here, we show that the α-isoform of myosin heavy chain (α-MyHC, which is encoded by the gene Myh6) is the pathogenic autoantigen for CD4+ T cells in a spontaneous mouse model of myocarditis. Further, we found that Myh6 transcripts were absent in mouse medullary thymic epithelial cells (mTECs) and peripheral lymphoid stromal cells, which have been implicated in mediating central and peripheral T cell tolerance, respectively. Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis, demonstrating that α-MyHC is a primary autoantigen in this disease process. Remarkably, we found that humans also lacked α-MyHC in mTECs and had high frequencies of α-MyHC-specific T cells in peripheral blood, with markedly augmented T cell responses to α-MyHC in patients with myocarditis. Since α-MyHC constitutes a small fraction of MyHC in human heart, these findings challenge the longstanding notion that autoimmune targeting of MyHC is due to its cardiac abundance and instead suggest that it is targeted as a result of impaired T cell tolerance mechanisms. These results thus support a role for T cell-specific therapies for myocarditis.

Discovering novel targets for autoantibodies in dilated cardiomyopathy.

There is increasing evidence that a large proportion of dilated cardiomyopathy (DCM) cases are mediated by autoimmune processes. Since DCM is a fatal disorder with rapid aggravation and is the leading cause of heart transplantation, further insights into disease pathogenesis are needed. Recent studies have separated the pathogenic capacity of autoantibodies and initial clinical trials removing such autoantibodies via immunoadsorption have been promising. In order to elucidate the full autoantibody repertoire involved in DCM, we applied an autoantibody screening test using ventricular and atrial proteomes as autoantigenic sources and subsequently tested the autoantibody-binding patterns of sera from dogs with spontaneous DCM. With this method, we detected five potentially DCM-related autoantigens which were identified by MS as being: myosin heavy chain cardiac muscle alpha isoform, alpha cardiac actin, mitochondrial aconitate hydratase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and brain glycogen phosphorylase (GPBB). The recovery of two known DCM autoantigens (myosin heavy chain and alpha cardiac actin) and the discovery of three novel autoantigens (mitochondrial aconitate hydratase, GADPH, and GPBB) underscore the efficacy of this experimental method and the significance of the spontaneous canine DCM model.

MyD88 signaling controls autoimmune myocarditis induction.

BACKGROUND: Experimental autoimmune myocarditis (EAM) is a CD4+ T-cell-mediated mouse model of postviral cardiomyopathy. Activation of interleukin-1 type 1 and Toll-like receptors that share the common downstream adaptor molecule MyD88 is required for disease induction. The specific role of MyD88 in myocarditis, however, is not known. METHODS AND RESULTS: In contrast to control littermates, MyD88(-/-) mice were protected from myocarditis after immunization with alpha-myosin heavy chain-derived peptide (MyHC-alpha) and complete Freund's adjuvant. Disease resistance of MyD88(-/-) mice resulted from impaired expansion of heart-specific CD4+ T cells after immunization. Intrinsic defects of MyD88(-/-) CD4+ T cells were excluded. In contrast, MyD88(-/-) but not MyD88(+/+) primary antigen presenting dendritic cells (DCs) were defective in their capacity to prime CD4+ T cells. This defect mainly resulted from the inability of MyD88(-/-) DCs to release tumor necrosis factor-alpha. The critical role of MyD88 signaling in DCs in the peripheral lymphatic compartments was finally proven by repetitive injection of activated, MyHC-alpha-loaded MyD88(+/+) DCs that fully restored T-cell expansion and myocarditis in MyD88(-/-) mice. CONCLUSIONS: Autoimmune myocarditis induction depends on MyD88 signaling in self-antigen presenting cells in the peripheral compartments. We conclude that MyD88 might become a target for prevention of heart-specific autoimmunity and cardiomyopathy.

The osteopontin - CD44 pathway is superfluous for the development of autoimmune myocarditis.

Osteopontin (OPN) and CD44 have been implicated in the development of autoimmune diseases, including arthritis and multiple sclerosis, as well as chronic inflammatory diseases, such as atherosclerosis and colitis. To investigate their roles in autoimmune myocarditis induced by immunization with heart alpha-myosin (MyHC-alpha), a mouse model of human cardiomyopathy, we analyzed mice lacking OPN or CD44v6/v7, a CD44 isoform that binds OPN. Both, OPN(-/-) and CD44v6/v7(-/-) mice developed myocarditis with the same prevalence and severity as BALB/c wild-type controls. Furthermore, treatment of BALB/c mice with a pan-neutralizing anti-CD44 antibody did not affect the disease outcome. Consistently, expansion of MyHC-alpha-specific autoimmune CD4(+) T cells and MyHC-alpha autoantibody responses from either CD44v6/v7(-/-) mice or OPN(-/-) mice was indistinguishable from their wild-type controls. Thus, OPN and CD44v6/v7 are merely spectators rather than protagonists in autoimmune myocarditis.

Dendritic cell-induced autoimmune heart failure requires cooperation between adaptive and innate immunity.

Genetic susceptibility and autoimmunity triggered by microbial infections are factors implicated in the pathogenesis of dilated cardiomyopathy, the most common cause of heart failure in young patients. Here we show that dendritic cells (DCs) loaded with a heart-specific self peptide induce CD4+ T-cell-mediated myocarditis in nontransgenic mice. Toll-like receptor (TLR) stimulation, in concert with CD40 triggering of self peptide-loaded dendritic cells, was shown to be required for disease induction. After resolution of acute myocarditis, DC-immunized mice developed heart failure, and TLR stimulation of these mice resulted in relapse of inflammatory infiltrates. Injection of damaged, syngeneic cardiomyocytes also induced myocarditis in mice if TLRs were activated in vivo. DC-induced myocarditis provides a unifying theory as to how tissue damage and activation of TLRs during infection can induce autoimmunity, relapses and cardiomyopathy.

Activation of dendritic cells through the interleukin 1 receptor 1 is critical for the induction of autoimmune myocarditis.

Dilated cardiomyopathy, resulting from myocarditis, is the most common cause of heart failure in young patients. We here show that interleukin (IL)-1 receptor type 1-deficient (IL-1R1(-/-)) mice are protected from development of autoimmune myocarditis after immunization with alpha-myosin-peptide(614-629). CD4(+) T cells from immunized IL-1R1(-/-) mice proliferated poorly and failed to transfer disease after injection into naive severe combined immunodeficiency (SCID) mice. In vitro stimulation experiments suggested that the function of IL-1R1(-/-)CD4(+) T cells was not intrinsically defect, but their activation by dendritic cells was impaired in IL-1R1(-/-) mice. Accordingly, production of tumor necrosis factor (TNF)-alpha, IL-1, IL-6, and IL-12p70 was reduced in dendritic cells lacking the IL-1 receptor type 1. In fact, injection of immature, antigen-loaded IL-1R1(+/+) but not IL-1R1(-/-) dendritic cells into IL-1R1(-/-) mice fully restored disease susceptibility by rendering IL-1R1(-/-) CD4(+) T cells pathogenic. Thus, IL-1R1 triggering is required for efficient activation of dendritic cells, which is in turn a prerequisite for induction of autoreactive CD4(+) T cells and autoimmunity.

Interleukin-6-deficient mice resist development of autoimmune myocarditis associated with impaired upregulation of complement C3.

BACKGROUND: Interleukin (IL)-6 regulates various aspects of the immune response. In the context of heart diseases, it has been recognized as a prognostic factor for dilated cardiomyopathy, which often results from myocarditis. METHODS AND RESULTS: Using IL-6-deficient mice, we studied the role of IL-6 in a model of autoimmune myocarditis resulting from immunization with a peptide derived from cardiac alpha-myosin. Prevalence and severity of myocarditis were markedly reduced in the absence of IL-6. CD4+ T cells from immunized IL-6-deficient mice proliferated poorly on restimulation with specific antigen in vitro and did not mediate disease on adoptive transfer into IL-6-competent RAG-2-deficient mice, which otherwise lack B cells and T cells. Production of complement C3, a crucial factor for the development of myocarditis, was strongly upregulated in IL-6+/+ but not in IL-6-deficient mice after immunization. CONCLUSIONS: Our results demonstrate that IL-6 is required for the expansion of autoimmune CD4+ T cells and the pathogenesis of autoimmune myocarditis, possibly by upregulation of complement C3.

A novel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ, SJL/J, and SWR/J mice.

Idiopathic dilated cardiomyopathy (DCM) is responsible for approximately 25% of all cases of congestive heart failure. We have recently shown that immunization of autoimmune-susceptible SWXJ mice with whole cardiac myosin leads to T cell-mediated experimental autoimmune myocarditis (EAMC) and DCM. We have now identified two disease-inducing peptides from cardiac alpha-myosin heavy chain (CAMHC). Our approach involved the use of a novel MHC class II-binding motif contained in several peptides known to be immunogenic in SWXJ (H-2(q,s)) mice or in the parental SJL/J (H-2(s)) or SWR/J (H-2(q)) mouse strains. Two of four CAMHC peptides containing the -KXXS- peptide motif were found to be immunogenic. Immunization of SWXJ or parental SJL/J and SWR/J mice with CAMHC peptides palpha406-425 or palpha1631-1650 resulted in EAMC and DCM, characterized by inflammation, fibrosis, and decompensated right-sided ventricular dilatation. Despite mediating high incidences of severe disease, both peptides were found to be cryptic determinants, thereby providing further evidence for the importance and perhaps predominance of self crypticity in autoimmunity. Both peptides showed dual parental I-A(q) and I-A(s) restriction and mediated passive transfer of disease with activated CD4(+) T cells. An intact motif was necessary for antigenicity because loss of activity occurred in peptides containing nonconservative substitutions at the motif's terminal lysine and serine residues. Our studies provide a new model for EAMC and DCM in strains of mice widely used in autoimmune studies. Moreover, the -KXXS- motif may be particularly useful in implicating previously overlooked proteins as autoimmune targets and in facilitating the development of new organ-specific autoimmune mouse models for human diseases.

Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: pathogenetic and clinical significance.

Dilated cardiomyopathy (DCM) is a relevant cause of heart failure and a common indication for heart transplantation. It may be idiopathic, familial/genetic, viral, autoimmune or immune-mediated associated with a viral infection. Myocarditis is an inflammatory disease of the myocardium; it may be idiopathic, infectious or autoimmune and may heal or lead to DCM. Thus, in a patient subset, myocarditis and DCM are thought to represent the acute and chronic stages of an organ-specific autoimmune disease of the myocardium. In keeping with this hypothesis, autoimmune features in patients with myocarditis/DCM include: familial aggregation; a weak association with HLA-DR4; abnormal expression of HLA class II on cardiac endothelium on endomyocardial biopsy; and detection of organ- and disease-specific cardiac autoantibodies of the IgG class in the sera of affected patients and symptom-free relatives. The cardiac autoantibodies detected by immunofluorescence are directed against multiple antigens. Two of these, first identified using immunoblotting and confirmed by ELISA, are the atrial-specific alpha- and the ventricular and skeletal muscle beta-heavy chain isoform. The alpha-myosin isoform fulfils the expected criteria for organ-specific autoimmunity, in that immunization with cardiac, but not skeletal myosin reproduces, in susceptible mouse strains, the human disease phenotype of myocarditis/DCM; in addition, alpha-myosin is entirely cardiac-specific. Additional antigenic targets of heart-reactive autoantibodies include unknown sarcolemmal proteins, mitochondrial enzymes, beta-adrenergic and muscarinic receptors. For some of these antibodies, there is in vitro evidence for a functional role. The organ-specific cardiac autoantibodies detected by immunofluorescence in symptom-free relatives were associated with echocardiographic features suggestive of early disease. Mid-term follow-up suggests that these antibodies are predictive markers of progression to DCM among symptom-free relatives with or without abnormal echocardiographic findings.