Immune Dysregulation in HIT

描述（由申请人提供）：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，由靶向血小板因子4 （PF4）和肝素复合物的药物依赖性抗体引起。虽然从定义上讲，HIT是一种药物引起的疾病，但其免疫学特征与其他常见的药物过敏（如奎宁或青霉素）几乎没有相似之处。与大多数药物依赖性免疫反应不同，肝素抗体反应具有剂量依赖性，是短暂的，并且可能缺乏免疫记忆。基于这些抗体对药物反应的非典型特征，以及HIT中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，其中自我耐受性短暂失调。具体来说，我们假设HIT是由免疫刺激信号（包括抗原PF4/肝素复合物和血小板CD154）和免疫调节机制受损引起的自我耐受性破坏引起的。为了验证这一假设，我们开发了一种小鼠自身免疫模型，其中PF4/肝素抗体（抗mp +H）在抗原攻击后重新产生。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。我们还发现动物体内mPF4/肝素抗体的产生需要CD4+ t辅助细胞，并且免疫召回在BALB/c和C57BL/6小鼠中表达不同。利用这种动物模型，我们将：1)证明大分子PF4/肝素复合物对细胞的激活作用。为此，我们将进行体外和体内研究，以描绘淋巴细胞对抗原的反应，并证明mPF4/肝素复合物触发APC激活。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，体内血小板活化可诱导PF4/肝素抗体的产生。我们假设血小板激活对于HIT的免疫启动至关重要，因为活化的血小板提供了抗体产生所需的抗原（PF4）和共刺激信号（CD154）的来源。为了验证这一假设，我们将研究血小板活化、mPF4和血小板CD154对HIT免疫应答的致敏作用。3)描述PF4/肝素在小鼠免疫应答中的免疫调节机制受损。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，这表明动物免疫调节功能存在差异。为此，我们将验证我们的假设，即调节性T细胞在确定HIT的免疫反应过程中起关键作用。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见解。

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.