Justin M. Johnson

The epitope spreading cascade during progression of experimental autoimmune encephalomyelitis and multiple sclerosis

Summary: We have made the following observations regarding self‐recognition during the development and progression of murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS); I) chronic progression of EAE is accompanied by a sequential, predictable cascade of neo‐autoreactivity, commonly referred to as epitope spreading, presumably caused by endogenous self‐priming during autoimmune‐mediated tissue damage; 2) there is an invariant relationship between the progression of EAE and the emergence of epitope spreading; 3) progression of EAE can be inhibited by the induction of antigen‐specific tolerance to spreading determinants after onset of initial neurologic symptoms; 4) CD4+ Th1 cells responding to spreading determinants are autonomously encephalitogenic; 5) epitope spreading occurs during the development of MS and in some cases involves HLA‐DP class II‐restricted self‐recognition; and 6) progression of both EAE and MS is accompanied by the decline of primary T‐cell autoreactivity associated with disease onset and by the concurrent emergence of the epitope spreading cascade. Our studies directly challenge the traditional view that EAE and MS are initiated and maintained by autoreactivity directed against a single predominant myelin protein or determinant. Our results indicate that progression of EAE and MS involves a shifting of T‐cell autoreactivity from primary initialing self‐determinants to defined cascades of secondary determinants that sustain the inflammatory self‐recognition process during disease progression.

An autoimmune-mediated strategy for prophylactic breast cancer vaccination

Although vaccination is most effective when used to prevent disease, cancer vaccine development has focused predominantly on providing therapy against established growing tumors. The difficulty in developing prophylactic cancer vaccines is primarily due to the fact that tumor antigens are variations of self proteins and would probably mediate profound autoimmune complications if used in a preventive vaccine setting. Here we use several mouse breast cancer models to define a prototypic strategy for prophylactic cancer vaccination. We selected α-lactalbumin as our target vaccine autoantigen because it is a breast-specific differentiation protein expressed in high amounts in the majority of human breast carcinomas and in mammary epithelial cells only during lactation. We found that immunoreactivity against α-lactalbumin provides substantial protection and therapy against growth of autochthonous tumors in transgenic mouse models of breast cancer and against 4T1 transplantable breast tumors in BALB/c mice. Because α-lactalbumin is conditionally expressed only during lactation, vaccination-induced prophylaxis occurs without any detectable inflammation in normal nonlactating breast tissue. Thus, α-lactalbumin vaccination may provide safe and effective protection against the development of breast cancer for women in their post–child-bearing, premenopausal years, when lactation is readily avoidable and risk for developing breast cancer is high.

Modulation of the IL-10/IL-12 cytokine circuit by interferon-β inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis

IFN-beta has been shown to be effective in the treatment of multiple sclerosis (MS). However, the primary mechanism by which IFN-beta mediates its therapeutic effect remains unclear. Recent studies indicate that under defined conditions, IFN-beta may downregulate DC expression of IL-12. We and others have shown that IFN-beta may also downregulate IL-10. In light of the recently proposed paradigm that an IL-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease, we examined the effect of IFN-beta on the development and behavior of the autoreactive T cell repertoire during experimental autoimmune encephalomyelitis (EAE), an animal model sharing many features with MS. SWXJ mice were immunized with the immunodominant p139-151 determinant of myelin proteolipid protein (PLP), and at onset of EAE were treated every other day with IFN-beta. After eight weeks of treatment, we assessed autoreactivity and observed no significant IFN-beta effect on splenocyte proliferation or splenocyte production of IFN-gamma, IL-2, IL-4, or IL-5 in response to the priming determinant used to initiate disease. However, in IFN-beta treated mice, the cytokine profile in response to the priming immunogen was significantly skewed toward an increased production of IL-10 and a concurrent decreased production of IL-12. Moreover, the in vivo modulation of the IL-10/IL-12 immunoregulatory circuit in response to the priming immunogen was accompanied by an aborted development of epitope spreading. Our results indicate that IFN-beta induces a reciprocal modulation of the IL-10/IL-12 cytokine circuit in vivo. This skewed autoreactivity establishes an inflammatory microenvironment that effectively prevents endogenous self-priming thereby inhibiting the progression of disease associated with epitope spreading.

β1-Adrenergic Receptor Autoantibodies Mediate Dilated Cardiomyopathy by Agonistically Inducing Cardiomyocyte Apoptosis

Background— Antibodies to the &bgr;1-adrenergic receptor (&bgr;1AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby &bgr;1AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM. Methods and Results— We used the &bgr;1AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the &bgr;1AR. After transfer into naive male hosts, &bgr;1AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of &bgr;1AR IgG before transfer and by selective pharmacological antagonism of host &bgr;1AR but not &bgr;2AR. We found that &bgr;1AR autoantibodies shifted the &bgr;1AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by &bgr;1AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK. Conclusions— Our data show how &bgr;1AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Murine autoimmune hearing loss mediated by CD4+ T cells specific for inner ear peptides

Autoimmune sensorineural hearing loss (ASNHL) is characterized typically by bilateral, rapidly progressive hearing loss that responds therapeutically to corticosteroid treatment. Despite its name, data implicating autoimmunity in the etiopathogenesis of ASNHL have been limited, and targeted self-antigens have not been identified. In the current study we show that the inner ear-specific proteins cochlin and beta-tectorin are capable of targeting experimental autoimmune hearing loss (EAHL) in mice. Five weeks after immunization of SWXJ mice with either Coch 131-150 or beta-tectorin 71-90, auditory brainstem responses (ABR) showed significant hearing loss at all frequencies tested. Flow cytometry analysis showed that each peptide selectively activated CD4(+) T cells with a proinflammatory Th1-like phenotype. T cell mediation of EAHL was determined by showing significantly increased ABR thresholds 6 weeks after adoptive transfer of peptide-activated CD4(+) T cells into naive SWXJ recipients. Immunocytochemical analysis showed that leukocytic infiltration of inner ear tissues coincided with onset of hearing loss. Our study provides a contemporary mouse model for clarifying our understanding of ASNHL and facilitating the development of novel effective treatments for this clinical entity. Moreover, our data provide experimental confirmation that ASNHL may be a T cell-mediated organ-specific autoimmune disorder of the inner ear.

Increased Frequencies of Cochlin-Specific T Cells in Patients with Autoimmune Sensorineural Hearing Loss

Autoimmune sensorineural hearing loss (ASNHL) is the most common cause of sudden hearing loss in adults. Although autoimmune etiopathogenic events have long been suspected in ASNHL, inner ear-specific Ags capable of targeting T cell autoreactivity have not been identified in ASNHL. In this study, we show by ELISPOT analysis that compared with normal hearing age- and sex-matched control subjects, ASNHL patients have significantly higher frequencies of circulating T cells producing either IFN-γ (p = 0.0001) or IL-5 (p = 0.03) in response to recombinant human cochlin, the most abundant inner ear protein. In some patients, cochlin responsiveness involved both CD4+ and CD8+ T cells whereas other patients showed cochlin responsiveness confined to CD8+ T cells. ASNHL patients also showed significantly elevated cochlin-specific serum Ab titers compared with both normal hearing age- and sex-matched control subjects and patients with noise- and/or age-related hearing loss (p < 0.05 at all dilutions tested through 1/2048). Our study is the first to show T cell responsiveness to an inner ear-specific protein in ASNHL patients, and implicates cochlin as a prominent target Ag for mediating autoimmune inner ear inflammation and hearing loss.

Treatment with BBB022A or rolipram stabilizes the blood-brain barrier in experimental autoimmune encephalomyelitis: an additional mechanism for the therapeutic effect of type IV phosphodiesterase inhibitors

We examined the treatment effects of two structurally distinct phosphodiesterase type IV (PDE IV) inhibitors, BBB022 and rolipram, in murine and rat models of experimental autoimmune encephalomyelitis (EAE). Based on our data, we propose a mechanism of action which may supplement immunomodulatory effects of PDE IV inhibitors. In particular, PDE inhibitors promote elevation of intracellular cAMP levels, increasing the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes. Such an effect on central nervous system (CNS) vascular endothelium has the potential to reduce disease severity in EAE, because both inflammatory cells and humoral factors readily cross a disrupted blood-brain barrier (BBB). In this report, we demonstrate the capacity of BBB022 and rolipram to decrease clinical severity of EAE. further, PDE IV inhibitors significantly reduced BBB permeability in the spinal cords of mice with EAE. These results provide evidence that PDE IV-inhibitors may exert therapeutic effects in EAE by modifying cerebrovascular endothelial permeability, reducing tissue edema as well as entry of inflammatory cells and factors.

Human and murine lymphocyte neurotrophin expression is confined to B cells.

Recent reports indicate that autoreactive T cells may produce neurotrophic factors capable of mediating repair and regeneration of damaged neurons. By using semiquantitative RT‐PCR, we examined gene expression of nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and the trkB BDNF receptor in autoreactive T cells from SWXJ mice immunized with the p104–117 encephalitogen of myelin proteolipid protein (PLP 104–117). We observed antigen‐inducible expression of NGF and BDNF, but not NT‐3 and trkB, in lymph node cells activated with PLP 104–117. To determine which leukocyte subpopulation expressed neurotrophins, CD4+, CD8+, B220+, CD11b+, and NK1.1+ cells were purified from activated primary cultures, and their mRNAs were analyzed. Neurotrophin expression was also measured in CD3+ T cells purified from mouse CNS during acute onset of experimental autoimmune encephalomyelitis as well as in resting and activated human T cells and B cells purified from peripheral blood of normal subjects. In all cases, we found that neurotrophin expression was confined exclusively to B cells (B220+) in both mouse and human. CD3+, CD4+, and CD8+ T cells as well as NK1.1+ cells and CD11b+ monocytes and macrophages did not express any detectable BDNF, NGF, NT‐3, or trkB under any conditions. Our data indicate that B cells rather than T cells are the predominant if not the only source of leukocyte‐derived neurotrophins and as such may provide “protective autoimmunity” in repair and regeneration of the injured nervous system.

Generation of autonomously pathogenic neo-autoreactive TH1 cells during the development of the determinant spreading cascade in murine autoimmune encephalomyelitis

Chronic progression of autoimmune disease is accompanied by the acquisition of autoreactivity to new self‐determinants. Recent evidence indicates that this process, commonly referred to as determinant spreading, may be pathogenic for chronicity. Our studies on experimental autoimmune encephalomyelitis (EAE), a murine model widely used in multiple sclerosis (MS) studies, have shown that determinant spreading develops as a predictable sequential cascade of neo‐autoimmunity during progression to chronic disease. By 7–8 weeks after immunization of (SWRxSJL)F1 mice with the immunodominant myelin proteolipid protein determinant (PLP 139–151), splenocytes consistently respond to the immunodominant myelin basic protein determinant (MBP 87–99). In the present study, we directly address the pathogenicity of neo‐autoimmunity resulting from endogenous self‐priming during the course of disease. Our results indicate that T cells responding to the spreading MBP 87–99 determinant produce a proinflammatory cytokine profile consistent with type 1 helper T cells (Th1) cells. In addition, splenocytes activated to the spreading MBP 87–99 determinant consistently transfer acute EAE in naive recipients even when T cells reactive to the priming PLP 139–151 immunogen are eliminated by bromodeoxyuridine (BUdR)‐mediated photolysis. Our data indicate that endogenous neo‐autoantigen priming during chronic autoimmune disease generates type 1 helper T cells (Th1) cells that are autonomously pathogenic. These results provide further evidence supporting the view that determinant spreading is a pathogenic process that leads to chronic progression of autoimmune disease.

Autoimmune Targeted Disruption of the Pituitary-Ovarian Axis Causes Premature Ovarian Failure

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women’s health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215–234 peptide derived from mouse inhibin-α activates CD4+ T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-α-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-α-targeted experimental autoimmune oophoritis is initiated by CD4+ Th1 T cells that stimulate B cells to produce inhibin-α-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-α autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.