Immune Dysregulation in HIT

说明(申请人提供)：肝素诱导的血小板减少症(HIT)是一种危及生命的血栓性疾病，由针对血小板因子4(PF4)和肝素复合体的药物依赖性抗体引起。尽管HIT的定义是一种药物引起的疾病，但它的免疫学特征与奎宁或青霉素等其他常见的药物过敏几乎没有相似之处。与大多数药物依赖性免疫反应不同，肝素的抗体反应表现出剂量依赖性，寿命短，可能缺乏免疫记忆。根据HIT对药物的抗体反应的这些非典型特征，以及已知的针对自身抗原(PF4和内源性糖胺聚糖)的抗体特异性，我们假设HIT是一种T细胞依赖性免疫疾病，其自身耐受性一过性失调。具体地说，我们假设HIT是由于免疫刺激信号(包括抗原性PF4/肝素复合体和血小板CD154)和免疫调节机制受损导致的自我耐受性的破坏。为了验证这一假设，我们建立了一种小鼠自身免疫模型，在该模型中，PF4/肝素抗体(抗MP+H)在抗原攻击后从头产生。在初步研究中，我们发现来自这些动物的抗mPF4/肝素概括了HIT免疫反应的关键方面，包括其可变发生率、血清学特异性和功能特性。我们还表明，动物产生mPF4/肝素抗体需要CD4+T辅助细胞，免疫召回在BALB/c和C57BL/6小鼠中有不同的表达。利用该动物模型，我们将：1)展示大分子PF4/肝素复合体对细胞的激活作用。为此，我们将在体外和体内进行研究，以描述淋巴细胞对抗原的反应，并证明mPF4/肝素复合体激活APC。2)阐明血小板活化和血小板CD154(Cp40L)在诱导HIT免疫应答中的作用。我们的初步研究表明，在体内，血小板激活诱导产生PF4/肝素抗体。我们假设，血小板激活对于HIT中的免疫启动是必不可少的，因为激活的血小板提供了产生抗体所需的抗原(PF4)和共刺激信号(CD154)。为了验证这一假设，我们将检测血小板活化、mPF4和血小板CD154对HIT免疫反应的增敏作用。3)阐明PF4/肝素免疫应答中受损的免疫调节机制。我们的初步研究表明，BALB/c和C57BL/6小鼠的免疫回忆模式存在显著差异，这表明动物的免疫调节功能存在差异。在这个目标中，我们将检验我们的假设，即调节性T细胞在定义HIT的免疫反应过程中发挥关键作用。预计这些研究将导致对预防或调节这种危及生命的血栓性疾病的免疫并发症的策略的深入了解。

DESCRIPTION (provided by applicant): Heparin-lnduced Thrombocytopenia (HIT) is a life-threatening thrombotic illness caused by drug-dependent antibodies directed to complexes of Platelet Factor 4 (PF4) and heparin. Although HIT, by definition, is a drug-induced disorder, its immunologic features bear little resemblance to other commonly encountered drug allergies, like quinine or penicillin. Unlike most drug-dependent immune responses, which are idiosyncratic and long-lived, the antibody response to heparin displays dose dependence, is short-lived and may lack immune memory. Based on these atypical features of the antibody response to drug, as well as known antibody specificities towards self-antigens (PF4 and endogenous glycosaminoglycans) in HIT, we posit that HIT is a T-cell dependent immune disorder in which self-tolerance is transiently dysregulated. Specifically, we hypothesize that HIT arises from a breach in self-tolerance brought about by immunostimulatory signals (involving antigenic PF4/heparin complexes and platelet CD154) and impaired immunoregulatory mechanisms. To test this hypothesis, we have developed a murine autoimmune model in which PF4/heparin antibodies (anti-mP+H) arise de novo after antigen challenge. In preliminary studies, we show that anti-mPF4/heparin from these animals recapitulate key aspects of the HIT immune response, including its variable incidence, serologic specificities and functional properties. We also show that mPF4/heparin antibody production in animals requires CD4+ T-helper cells and that immune recall is variably expressed in BALB/c and C57BL/6 mice. Using this animal model, we will: 1) Demonstrate cellular activation by macromolecular PF4/heparin complexes. In this aim, we will perform in vitro and in vivo studies to delineate lymphocyte responses to antigen and demonstrate that mPF4/heparin complexes trigger APC activation. 2) Delineate the role of platelet activation and platelet CD154 (Cp40L) in induction of the HIT immune response. Our preliminary studies indicate that in vivo platelet activation induces PF4/heparin antibody production. We hypothesize that platelet activation is essential for immune initiation in HIT, as activated platelets provide both a source of antigen (PF4) and co-stimulatory signals (CD154) needed for antibody production. To test this hypothesis, we will examine the sensitizing effects of platelet activation, mPF4 and platelet CD154 on the HIT immune response. 3) Delineate impaired immunoregulatory mechanisms in the murine immune response to PF4/heparin. Our preliminary studies show striking differences in patterns of immune recall in BALB/c and C57BL/6 mice that suggest differences in immunoregulatory function in animals. In this aim, we will test our hypothesis that regulatory T cells play a critical role in defining the course of immune response in HIT. It is expected that these studies will lead to insights into strategies that prevent or modulate immune complications from this life-threatening thrombotic disorder.