Immunization against Atherosclerosis

描述(由申请人提供)：动脉粥样硬化的部分原因是致病的CD4+效应T细胞(TEF)对病变中上调的抗原(“动脉粥样硬化抗原”)有反应。重要的是，CD4+CD25+Foxp3+调节性T细胞(Treg)是T细胞的一个亚群，其功能是抑制TJeff的反应，已被证明在控制动脉粥样硬化中发挥作用。在动脉粥样硬化的背景下，致病的Tref和抑制性Treg之间可能存在失衡，以响应动脉粥样硬化抗原，导致促进动脉粥样硬化的发生。这些想法促使我们设计了一种抗动脉粥样硬化的策略，旨在通过在体内增加动脉粥样硬化特异性的Treg来重置Treg和TJeff之间的平衡。此前，我们借鉴传统佐剂的免疫增强作用，探索了“耐受佐剂”的概念，设计了一种新的免疫策略。在迟发性超敏反应(DTH)和自发性I型糖尿病小鼠模型中，我们发现，在免疫抑制剂地塞米松(DEX)作为耐受性佐剂的影响下进行多肽抗原免疫时，分别导致迟发性超敏反应(DTH)和自身免疫性糖尿病的长期脱敏和阻断。我们发现，这种保护作用与抗原特异性Treg的选择性扩增有关。在这些结果的鼓舞下，我们将在这里检验我们的假设，即使用DEX和动脉粥样硬化抗原抑制免疫(SI)可以选择性地扩大动脉粥样硬化特异性Treg并减轻动脉粥样硬化。在特定的目标#1下，我们将确定SI是否增加了治疗小鼠病变部位和外周血中Treg/Tef的比率。为此，雄性ApoE-/-/Foxp3-GFP+/+小鼠(我们最近产生的)将接受SI治疗，联合使用DEX和HSP60衍生多肽HP1。作为对照，小鼠将单独接受PBS或HP1治疗。治疗小鼠的无名动脉将被冷冻切除，并将进行免疫组织化学分析以量化TJeff(CD4+Foxp3-)和Treg(CD4+Foxp3-GFP+)。Treg的抗原特异性将通过从主动脉弓制备的单个细胞通过ELISPOT进行分析。此外，还将通过免疫染色和流式细胞术对外周血中的TJeff和Treg进行定量，并通过免疫多肽HP1对Treg的增殖反应来评估Treg的抗原特异性。在特定目标#2下，我们将评估SI在治疗动物中的抗动脉粥样硬化效果。为此，已建立动脉粥样硬化的雄性载脂蛋白E-/-小鼠将被分成3组(n=6)。其中一组将在治疗前进行解剖，以确定病变范围的基线。两个治疗组各给予HP1单独治疗(对照组)或SI治疗。随后，无名动脉的病变将通过组织化学/形态计量学分析进行量化。此外，将对无名动脉切片进行巨噬细胞、平滑肌细胞(SMC)和胶原含量的分析。将对获得的数据进行统计分析。在特定的目标#3下，我们将利用采用转移来确定SI诱导的Treg是否有助于SI的抗动脉粥样硬化作用。为此，ApoE-/-/Foxp3-GFP+小鼠将接受SI治疗；4周后，将从脾中分离Treg(Foxp3-GFP+)并用作供者Treg(测试Treg)。从未经治疗的小鼠中分离出的Treg将用作对照Treg。从发展为动脉粥样硬化晚期的ApoE-/-小鼠中分离出的CD4+/Foxp3-细胞将被用作致病因子。随后，致病的TREF将被单独(对照)过继转移到受体ApoE-/-/RAG-2-/-小鼠(T细胞和B细胞缺乏)中，或与测试Treg或对照Treg结合(以不同的比例)。6周后，对受者动脉粥样硬化病变程度进行分析。这些特定目标的完成可能会产生一种安全、有效和实用的抗动脉粥样硬化疗法。此外，这项研究将进一步确定动脉粥样硬化特异性Treg在控制动脉粥样硬化中的作用，并为最佳靶向这些Treg提供有用的见解。

DESCRIPTION (provided by applicant): Atherosclerosis is driven partially by pathogenic CD4+ effector T cells (Teff) reactive to antigens upregulated in the lesion ("athero-antigens"). Importantly, CD4+CD25+Foxp3+ regulatory T cells (Treg), a subset of T cells that function to suppress the response of Teff, have been shown to play a role in controlling atherosclerosis. Within the context of atherosclerosis an imbalance may exist between pathogenic Teff and suppressive Treg in response to athero-antigens, leading to the promotion of atherogenesis. These notions prompted us to design an anti-atherosclerosis strategy aimed at resetting the balance between Treg and Teff via increasing athero-specific Treg in vivo. Previously, mirroring the use of the conventional adjuvants for enhancing immunity, we explored the concept of "tolerogenic adjuvant" and designed a novel immunization strategy. In mouse models of delayed-type hypersensitivity (DTH) and spontaneous type I diabetes, we showed that immunization with peptide antigen, when performed under the influence of the immunosuppressant dexamethasone (DEX) serving as tolerogenic adjuvant (a strategy we termed "suppressed immunization"), caused long-term desensitization of DTH and blockade of autoimmune diabetes, respectively. We showed that such protective efficacy was associated with selective expansion of antigen-specific Treg. Encouraged by these results, we here will test our hypothesis that suppressed immunization (SI) using DEX and athero-antigens may selectively expand athero-specific Treg and reduce atherosclerosis. Under Specific Aim #1, we will determine whether SI increases the ratio of Treg to Teff at the lesion site and in peripheral blood in treated mice. To that end, male ApoE-/-/Foxp3-GFP+/+ mice (we have recently generated) will be treated with SI combining DEX with HSP60-derived peptide HP1. As controls, mice will be treated with PBS or HP1 alone. Innominate arteries from treated mice will be cryosectioned, and immunohistochemical analysis will be performed to quantify Teff (CD4+Foxp3-) and Treg (CD4+Foxp3-GFP+). Antigen specificity of Treg will be analyzed via ELISPOT with single cells prepared from the aortic arch. In addition, Teff and Treg in peripheral blood will be quantified via immunostaining and flow cytometry, and antigen specificity of the Treg will be assessed by their proliferation in response to the immunizing peptide HP1. Under Specific Aim #2, we will assess anti-atherogenic efficacy of SI in treated animals. To that end, male ApoE-/- mice with established atherosclerosis will be divided into 3 groups (n e 6). One group will be dissected before treatment to establish the baseline for lesion extent. Each of the 2 treatment groups will be treated with HP1 alone (control) or SI. Subsequently, lesions in the innominate artery will be quantified by histochemical/morphometric analysis. In addition, sections of the innominate artery will be analyzed for contents of macrophage, smooth muscle cell (SMC), and collagen. Statistical analyses will be performed on obtained data. Under Specific Aim #3, we will exploit adoptive transfer to determine whether SI-induced Treg contribute to the anti-atherogenic efficacy of SI. To that end, ApoE-/-/Foxp3-GFP+ mice will be treated with SI; 4 weeks later, Treg (Foxp3-GFP+) will be isolated from the spleen and used as donor Treg (test Treg). Treg isolated from non-treated mice will be used as control Treg. CD4+/Foxp3- cells isolated from ApoE-/- mice that have developed advanced atherosclerosis will be used as pathogenic Teff. Subsequently, pathogenic Teff will be adoptively transferred alone (control), or in combination (in various ratios) with test Treg or control Treg, into recipient ApoE-/-/Rag-2-/- mice (which are T cell- and B cell-deficient). Six weeks later, atherosclerosis lesion extent in recipients will be analyzed. Completion of these specific aims is likely to generate a safe, effective, and practical anti-atherosclerosis therapy. In addition, this study will further define the role of athero-specific Treg in controlling atherosclerosis and provide useful insights pertaining to targeting these Treg optimally.