Complement and CR2/CD21 in the Immune Pathogenesis of HIT

摘要 肝素诱导的血小板减少症(HIT)是一种危及生命的免疫介导性血栓性疾病，由 抗PF4/肝素复合体抗体。免疫反应是常见的，但人们对其了解甚少。 发生了。在上一个资助期，我们对周围的早期事件进行了基本的观察 宿主与PF4/肝素抗原复合体的相遇。具体地说，我们发现：1)优先结合 与其他白细胞或血小板相比，PF4/肝素对循环B细胞的作用，2)肝素依赖 接受肝素治疗的患者中PF4/肝素与B细胞的结合，3)PF4/肝素抗原与补体结合， 4)接受肝素治疗的患者补体介导的PF4/肝素与循环B细胞的结合，以及5)结合 补体包被抗原通过补体受体2(CR2/CD21)传递给B细胞。在以下目标中，我们将 验证PF4/肝素复合体激活补体和B细胞沉积抗原的假说 Via CD21对于HIT自身抗体的产生是必不可少的。具体目标1：补体机制 由PF4/肝素复合体激活。在初步数据中，我们发现甘露糖结合凝集素和无花果胶-2 结合PF4/肝素复合体。基于这些发现，我们将检验PF4/肝素复合体的假设 通过凝集素途径激活补体。我们将定义PF4/肝素-凝集素的结构基础 相互作用，研究凝集素与PF4/肝素复合体的功能相互作用，并研究 HIT中的补体抑制。具体目标2：CD21参与的细胞后果 PF4/肝素和补体。补体包被抗原与CD21结合增强体液免疫 103%到104%。我们假设补体包被的多价PF4/肝素与CD21结合 复合体促进抗原特异性B细胞的募集和信号传递。我们将检查下游 同源和非同源B细胞的信号、激活和增殖，在急性白血病小鼠体内进行研究 B细胞受体的固定及表达CD21的滤泡树突状细胞对抗体的作用 队形。具体目标3：检查PF4/肝素血清转换的宿主预测因素。补体涂层 在接受肝素治疗的部分患者(但不是所有患者)的B细胞上可以检测到抗原。使用一种新型的C3 捕获免疫分析，我们还显示供体对供体的显著变异在健康供体血浆孵育中 使用固定剂量的PF4/肝素。在这些观察的基础上，我们将检查宿主对抗 利用抗原阳性B细胞鉴定补体蛋白质组的PF4/肝素血清转换 作为肝素化患者血清转换的标志。通过定义启动的细胞通路 PF4/肝素抗体的形成，我们希望能揭示与其他自身抗体免疫原性相关的机制。 或外源抗原。

ABSTRACT Heparin-induced thrombocytopenia (HIT) is a life-threatening immune-mediated thrombotic disorder caused by antibodies to PF4/heparin complexes. The immune response is common and yet little is understood about its occurrence. In the last funding period, we made fundamental observations related to early events surrounding the host’s encounter with the PF4/heparin antigenic complex. Specifically, we showed: 1) preferential binding of PF4/heparin to circulating B-cells, compared with other leukocytes or platelets, 2) heparin-dependent binding of PF4/heparin to B-cells in patients receiving heparin, 3) complement fixation by PF4/heparin antigen, 4) complement-mediated PF4/heparin binding to circulating B-cells in patients receiving heparin, and 5) binding of complement-coated antigen to B-cells via complement receptor 2 (CR2/ CD21). In the following aims, we will test the hypothesis that complement activation by PF4/heparin complexes and deposition of antigen on B-cells via CD21 is essential for development of HIT autoantibodies. Specific Aim 1: Mechanism of complement activation by PF4/heparin complexes. In preliminary data, we show that mannose-binding lectin and ficolin-2 bind PF4/heparin complexes. Based on these findings, we will test the hypothesis that PF4/heparin complexes activate complement via the lectin pathway. We will define the structural basis of PF4/heparin-lectin interactions, study functional interactions of lectins with PF4/heparin complexes, and examine the role of complement inhibition in HIT. Specific Aim 2: Cellular consequences of CD21 engagement by PF4/heparin and complement. Binding of complement-coated antigen to CD21 augments humoral immunity by 103 to 104 fold. We hypothesize that binding of complement-coated multivalent PF4/heparin to the CD21 complex facilitates recruitment and signaling of antigen-specific B-cells. We will examine downstream signaling, activation and proliferation of cognate and non-cognate B-cells, perform in vivo studies in mice with a fixed B-cell receptor and characterize the contribution of CD21-expressing follicular dendritic cells to Ab formation. Specific Aim 3: Examine host predictors of PF4/heparin seroconversion. Complement-coated antigen can be detected on B-cells in some, but not all, patients receiving heparin therapy. Using a novel C3 capture immunoassay, we also show significant donor to donor variation in healthy donor plasma incubated with a fixed dose of PF4/heparin. Based on these observations, we will examine host susceptibility to anti- PF4/heparin seroconversions by characterizing the complement proteome and using antigen-positive B-cells as a marker for seroconversions in heparinized patients. By defining the cellular pathways that initiate formation of PF4/heparin Abs, we hope to uncover mechanisms relevant to the immunogenicity of other auto- or exogenous antigens.