Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.

Oncology has recently undergone a revolutionary change with widespread adoption of immunotherapy for many cancers. Immunotherapy using monoclonal antibodies against checkpoint molecules, including programmed death (PD)-1, PD ligand (PD-L)1, and cytotoxic T lymphocyte-associated antigen (CTLA)-4, is effective in a significant subset of patients. However, immune-related adverse events (irAEs) have emerged as frequent complications of checkpoint blockade, likely due to the physiological role of checkpoint pathways in regulating adaptive immunity and preventing autoimmunity. As immunotherapy becomes more common, a better understanding of the etiology of irAEs and ways to limit these events is needed. At the same time, studying these new therapy-related disorders provides an opportunity to better understand naturally occurring human autoimmune and inflammatory disorders, with the potential to improve therapies for cancer and autoimmune diseases.

Negative selection, epitope mimicry and autoimmunity.

Infections often precede the onset of autoimmune disease and molecular (or epitope) mimicry is a plausible link. Cross-reacting epitopes are common between an infecting microorganism and the host because negative selection of self-reactive T-cells and B-cells is frequently incomplete. Complete eradication could lead to major voids in the immunologic repertoire. The association of an autoimmune disease with a microbial epitope may signify a causal relationship with the organism, an indirect connection through bystander effects, persistent infection or coincidence. There are well-established examples of a microbial mimic inducing a defined model of autoimmune disease in experimental animals but such instances are still relatively rare in humans. Establishing epitope mimicry as a direct cause opens opportunities for preventing the disease. Current approaches to cancer immunotherapy provides new examples of epitope mimicry between cancer antigens and normal tissue antigens.

Complete Freund's adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response.

INTRODUCTION: Complete Freund's Adjuvant (CFA) emulsified with an antigen is a widely used method to induce autoimmune disease in animal models, yet the contribution of CFA to the immune response is not well understood. We compared the effectiveness of CFA with Incomplete Freund's Adjuvant (IFA) or TiterMax Gold Adjuvant (TMax) in experimental autoimmune myocarditis (EAM) in male mice. METHODS: EAM was induced in A/J, BALB/c, and IL6KO BALB/c male mice by injection of the myocarditogenic peptide in CFA, IFA, or TMax on days 0 and 7. EAM severity was analyzed by histology on day 21. In addition, specific flow cytometry outcomes were evaluated on day 21. RESULTS: Only mice immunized with CFA and myocarditogenic peptide on both days 0 and 7 developed substantial myocarditis as measured by histology. We observed a significantly increased level of IL6 in the spleen 3 days after CFA immunization. In the spleen and heart on day 21, there was an expansion of myeloid cells in CFA-immunized mice, as compared to IFA or TMax-immunized animals. Recombinant IL-6 at the time of IFA immunization partially restored susceptibility of the mice to EAM. We also treated EAM-resistant IL-6 knockout mice with recombinant IL-6 around the time of the first immunization, on days -1 to 2, completely restoring disease susceptibility, showing that the requirement for IL-6 coincides with primary immunization. Examining APC populations in the lymph node draining the immunization site evidenced the contribution of IL-6 to the CFA-dependence of EAM was through controlling local dendritic cell (DC) trafficking. CONCLUSIONS: CFA used with myocarditogenic peptide twice is required to induce EAM in both A/J and Balb/c mice. Although IFA and TiterMax induce antibody responses, only CFA preferentially induced autoantigen-specific responses. CFA expands monocytes in the heart and in the spleen. IL-6 signaling is required during short window around primary immunization to induce EAM. In addition, IL-6 deficient mice resistance to EAM could be reversed by injecting IL-6 around first immunization. IL-6 expands dendritic cell and monocytic populations and ultimately leads to a robust T-cell driven immune response in CFA immunized mice.

Eosinophils in Autoimmune Diseases.

Eosinophils are multifunctional granulocytes that contribute to initiation and modulation of inflammation. Their role in asthma and parasitic infections has long been recognized. Growing evidence now reveals a role for eosinophils in autoimmune diseases. In this review, we summarize the function of eosinophils in inflammatory bowel diseases, neuromyelitis optica, bullous pemphigoid, autoimmune myocarditis, primary biliary cirrhosis, eosinophilic granulomatosis with polyangiitis, and other autoimmune diseases. Clinical studies, eosinophil-targeted therapies, and experimental models have contributed to our understanding of the regulation and function of eosinophils in these diseases. By examining the role of eosinophils in autoimmune diseases of different organs, we can identify common pathogenic mechanisms. These include degranulation of cytotoxic granule proteins, induction of antibody-dependent cell-mediated cytotoxicity, release of proteases degrading extracellular matrix, immune modulation through cytokines, antigen presentation, and prothrombotic functions. The association of eosinophilic diseases with autoimmune diseases is also examined, showing a possible increase in autoimmune diseases in patients with eosinophilic esophagitis, hypereosinophilic syndrome, and non-allergic asthma. Finally, we summarize key future research needs.

Natural killer cells in inflammatory heart disease.

Despite of a multitude of excellent studies, the regulatory role of natural killer (NK) cells in the pathogenesis of inflammatory cardiac disease is greatly underappreciated. Clinical abnormalities in the numbers and functions of NK cells are observed in myocarditis and inflammatory dilated cardiomyopathy (DCMi) as well as in cardiac transplant rejection [1-6]. Because treatment of these disorders remains largely symptomatic in nature, patients have little options for targeted therapies [7,8]. However, blockade of NK cells and their receptors can protect against inflammation and damage in animal models of cardiac injury and inflammation. In these models, NK cells suppress the maturation and trafficking of inflammatory cells, alter the local cytokine and chemokine environments, and induce apoptosis in nearby resident and hematopoietic cells [1,9,10]. This review will dissect each protective mechanism employed by NK cells and explore how their properties might be exploited for their therapeutic potential.

Macrophages and cardiac fibroblasts are the main producers of eotaxins and regulate eosinophil trafficking to the heart.

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ-/- IL-17A-/- mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80+ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL-4 and IL-13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil-mediated cardiac damage.

Current trends in autoimmunity and the nervous system.

In the broad field of autoimmunity and clinical immunology, experimental evidence over the past few years have demonstrated several connections between the immune system and the nervous system, both central and peripheral, leading to the definition of neuroimmunology and of an immune-brain axis. Indeed, the central nervous system as an immune-privileged site, thanks to the blood-brain barrier, is no longer a dogma as the barrier may be altered during chronic inflammation with disruptive changes of endothelial cells and tight junctions, largely mediated by adenosine receptors and the expression of CD39/CD73. The diseases that encompass the neuroimmunology field vary from primary nervous diseases such as multiple sclerosis to systemic conditions with neuropsychiatric complications, such as systemic lupus erythematosus or vasculitidies. Despite potentially similar clinical manifestations, the pathogenesis of each condition is different, but the interaction between the ultra-specialized structure that is the nervous system and inflammation mediators are crucial. Two examples come from anti-dsDNA cross-reacting with anti-N-Methyl-d-Aspartate receptor (NMDAR) antibodies in neuropsychiatric lupus or the new family of antibody-associated neuronal autoimmune diseases including classic paraneoplastic syndromes with antibodies directed to intracellular antigens (Hu, Yo, Ri) and autoimmune encephalitis. In the case of multiple sclerosis, the T cell paradigm is now complicated by the growing evidence of a B cell involvement, particularly via aquaporin antibodies, and their influence on Th1 and Th17 lineages. Inspired by a productive AARDA-sponsored colloquium among experts we provide a critical review of the literature on the pathogenesis of different immune-mediated diseases with neurologic manifestations and we discuss the basic immunology of the central nervous system and the interaction between immune cells and the peripheral nervous system.

Viral myocarditis.

PURPOSE OF REVIEW: The article traces the pathways leading from viral infection of the heart by coxsackievirus B3 to autoimmune myocarditis in its various manifestations. RECENT FINDINGS: Myocarditis can be induced by a number of different infectious agents and represents a significant cause of death especially in young individuals. Following infection, patients may develop lymphocytic, eosinophilic, or giant cell/granulomatous myocardial inflammation. It can lead to infectious dilated cardiomyopathy, a disease frequently requiring cardiac transplantation. Although acute viral myocarditis is frequently subclinical and recovery may be spontaneous, treatment of chronic myocarditis is currently unsatisfactory. Ongoing disease may be because of persistent virus in the heart or to immunopathic attack. Depending on the cause, treatment may be antiviral or immunosuppressive. Endomyocardial biopsy is proving of value in determining cause and deciding future therapy. A great deal of information about the pathogenesis of myocarditis has been gained from experimental models in rodents using heart disease induced by infection using coxsackievirus B3 or by immunization with cardiac myosin. SUMMARY: Treatment of myocarditis is still problematic and may depend on etiologic diagnosis to distinguish infectious from immune-mediated disease. Both pathogenic mechanisms may co-occur in individual patients. In the future, treatment may depend upon endomyocardial biopsy, immunohistologic testing, improved imaging, and molecular genetic analysis for providing more precise diagnoses.

Pathogenic IL-23 signaling is required to initiate GM-CSF-driven autoimmune myocarditis in mice.

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL-23 stimulation of CD4(+) T cells is required only briefly at the initiation of GM-CFS-dependent cardiac autoimmunity. IL-23 signal, acting as a switch, turns on pathogenicity of CD4(+) T cells, and becomes dispensable once autoreactivity is established. Il23a(-/-) mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL-23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4(+) T cells raised from Il23a(-/-) donors. Thus, IL-23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL-23-induced GM-CSF mediates the pathogenicity of CD4(+) T cells in EAM. The neutralization of GM-CSF abrogated cardiac inflammation. However, sustained IL-23 signaling is required to maintain IL-17A production in CD4(+) T cells. Despite inducing inflammation in Il23a(-/-) recipients comparable to wild-type (WT), autoreactive CD4(+) T cells downregulated IL-17A production without persistent IL-23 signaling. This divergence on the controls of GM-CSF-dependent pathogenicity on one side and IL-17A production on the other side may contribute to the discrepant efficacies of anti-IL-23 therapy in different autoimmune diseases.

The varying faces of IL-6: From cardiac protection to cardiac failure.

IL6 is a pleiotropic cytokine that is made in response to perturbations in homeostasis. IL6 becomes elevated in the acute response to host injury and can activate immune cells, direct immune cell trafficking, signal protective responses in local tissue, initial the acute phase response or initiate wound healing. In the short term this proinflammatory response is protective and limits host damage. It is when this acute response remains chronically activated that IL6 becomes pathogenic to the host. Chronically elevated IL6 levels lead to chronic inflammation and fibrotic disorders. The heart is a tissue where this temporal regulation of IL6 is very apparent. Studies from myocardial infarction show how short-term IL6 signaling can protect and preserve the heart tissue in response to acute damage, where long term IL6 signaling or an over-production of IL6R protein plays a causal role in cardiovascular disease. Thus, IL6 can be both protective and pathogenic, depending on the kinetics of the host response.

Natural killer cells limit cardiac inflammation and fibrosis by halting eosinophil infiltration.

Myocarditis is a leading cause of sudden cardiac failure in young adults. Natural killer (NK) cells, a subset of the innate lymphoid cell compartment, are protective in viral myocarditis. Herein, we demonstrated that these protective qualities extend to suppressing autoimmune inflammation. Experimental autoimmune myocarditis (EAM) was initiated in BALB/c mice by immunization with myocarditogenic peptide. During EAM, activated cardiac NK cells secreted interferon γ, perforin, and granzyme B, and expressed CD69, tumor necrosis factor-related apoptosis-inducing ligand treatment, and CD27 on their cell surfaces. The depletion of NK cells during EAM with anti-asialo GM1 antibody significantly increased myocarditis severity, and was accompanied by elevated fibrosis and a 10-fold increase in the percentage of cardiac-infiltrating eosinophils. The resultant influx of eosinophils to the heart was directly responsible for the increased disease severity in the absence of NK cells, because treatment with polyclonal antibody asialogangloside GM-1 did not augment myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We demonstrate that NK cells limit eosinophilic infiltration both indirectly, through altering eosinophil-related chemokine production by cardiac fibroblasts, and directly, by inducing eosinophil apoptosis in vitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.

Cardiac fibroblasts mediate IL-17A-driven inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra(-/-) mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A-fibroblast-GM-CSF-MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.

Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6.

BACKGROUND AND AIMS: Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17ß-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI. METHODS: To model DILI, we immunized BALB/c, BALB/cBy, IL-6-deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function. RESULTS: BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1ß (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17ß-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17ß-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6. CONCLUSIONS: 17ß-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.

Placenta suppresses experimental autoimmune hypophysitis through soluble TNF receptor 1.

Pregnancy modulates autoimmune diseases through diverse and still incompletely defined mechanisms, in part operating at the decidua-placenta interface. To assess the immunological contribution of placenta, we administered mouse placental proteins to a mouse model of autoimmune hypophysitis, a disease known to be strongly associated with pregnancy. Emulsified placental proteins suppressed both the cellular and humoral aspects of hypophysitis. Suppression was specific to self antigens and not seen when two foreign antigens, tetanus toxoid or tuberculin purified protein derivative, were used. Proteomic analysis revealed high levels of soluble TNF receptor 1 in placenta, suggesting that blockade of the TNF-α pathway was a mechanism of disease suppression. Placentas derived from mice deficient in TNF receptor 1 lost the ability to suppress hypophysitis. Notably, hypophysitis suppression was seen only when the TNF-α pathway was blocked locally, at the site of immunization, and not systemically. These findings provide evidence that placenta contributes to the immune tolerance of pregnancy by locally inhibiting the TNF-α pathway.

Macrophage diversity in cardiac inflammation: a review.

Cardiac inflammatory disease represents a significant public health burden, and interesting questions of immunopathologic science and clinical inquiry. Novel insights into the diverse programming and functions within the macrophage lineages in recent years have yielded a view of these cells as dynamic effectors and regulators of immunity, host defense, and inflammatory disease. In this review, we examine and discuss recent investigations into the complex participation of mononuclear phagocytic cells in the pathology of animal models of myocarditis.

Macrophages participate in IL-17-mediated inflammation.

The involvement of macrophages (MΦs) in Th17-cell responses is still poorly understood. While neutrophils are thought to be the predominant effector of Th17-cell responses, IL-17 is also known to induce myelotropic chemokines and growth factors. Other T-cell-derived cytokines induce non-classical functions, suggesting that IL-17 sigxnaling may similarly elicit unique MΦ functions. Here, we characterized the expression of subunits of the IL-17 receptor on primary murine MΦs from different anatomical compartments. The greatest expression of IL-17 receptors was observed on mucosal Ly6C(hi) "inflammatory" MΦs. We further observed upregulation of IL-17 receptors in vitro on bone marrow-derived macrophages (BMMΦs) in response to peptidoglycan or CpG oligonucleotide stimuli, and in vivo, upon CFA administration. Macrophages expressing IL-17 receptors were observed infiltrating the hearts of mice with myocarditis, and genetic ablation of IL-17RA altered MΦ recruitment. Treating primary MΦs from a wide variety of different anatomic sources (as well as cell lines) with IL-17A induced the production of unique profiles of cytokines and chemokines, including GM-CSF, IL-3, IL-9, CCL4/MIP-1ß and CCL5/RANTES. IL-17A also induced production of IL-12p70; IL-17-signaling-deficient MΦs elicited diminished IFN-γ production by responding DO11.10 CD4(+) T cells when used as APCs. These data indicate that MΦs from different anatomic locations direct IL-17-mediated responses.

Low-dose inorganic mercury increases severity and frequency of chronic coxsackievirus-induced autoimmune myocarditis in mice.

Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures. In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3 (CVB3). We examined the role of timing of iHg exposure on disease; in some experiments, mice were pretreated with iHg (200 µg/kg, every other day for 15 days) before disease induction with virus inoculation, and in others, they were treated with iHg after the acute (viral) phase of disease but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology. Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared with control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin (IL)-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease. Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.

Critical cytokine pathways to cardiac inflammation.

Infectious disease is frequently cited as a precursor of subsequent autoimmune disease in genetically susceptible hosts. However, the precise mechanisms required for the transition from infection to autoimmunity have not been well defined. We have developed a mouse model of autoimmune myocarditis initiated by infection with Coxsackievirus B3 to trace the cytokine pathways involved. We found that greater production of interleukin-1ß (IL-1ß) and tumor necrosis factor-α during the early innate response to virus infection is necessary and sufficient to induce a later heart-specific autoimmune disease. Severity of the autoimmune myocarditis is determined by the profile of a number of T helper 1 (Th1) and Th2 cytokines. Th2 responses are especially pronounced in the most severe forms of myocarditis where eosinophils are prominent. The Th1 pathway can lead to infiltration of the heart, but may be dampened by concurrent INF-γ production. Th17 cytokines also contribute to disease, but the signature Th17 cytokine, IL-17A, is not required for cardiac inflammation. Rather, IL-17A is needed for progression to dilated cardiomyopathy. These findings may provide useful markers to identify individuals prone to develop an autoimmune sequel after infection and suggest future early interventions.

The genetics of autoimmune thyroiditis: the first decade.

Most of our current understanding of the genetic predisposition to autoimmune disease can be traced to experiments performed in the decade from 1971 to 1981. Chella David was a key contributor to this research. Many of these early steps came from studies of experimental autoimmune thyroiditis. This model has been especially valuable because essentially the same disease can occur spontaneously in selected strains of animals or can be induced by deliberate immunization. From a genetic point of view, the disease has been investigated in three different species: mice, rats and chickens. The same antigen, thyroglobulin, initiates the disease in all three species. Among the main discoveries were the relationship of autoimmune disease to the major histocompatibility complex (MHC), the interplay of different subregions within the MHC in promoting or retarding development of disease, the differing roles of MHC class II and MHC I class genes in induction and effector phases, respectively, and the cumulative effect of non-MHC genes, each of which represents a small addition to overall susceptibility. Other experiments revealed that genetic differences in thyroglobulin allotypes influence susceptibility to thyroiditis. Thyroid glands differed in different strains in vulnerability to passive transfer of antibody. The first evidence of modulatory genes on the sex-related X chromosome emerged. All of these genetic findings were concurrently translated to the human disease, Hashimoto's thyroiditis, where thyroglobulin is also the initiating antigen.