Mechanisms of T cell activation in cardiac fibrosis and non-ischemic heart failure

这项新的R 01应用的目标是研究T细胞活化的机制， 心脏纤维化（CF）的进展在致命的心力衰竭（HF）综合征的后果，目前， 是美国死亡和住院的主要原因。我们是第一个报告心脏T细胞 在非缺血性HF患者中与CF相关的浸润，并使用已建立的实验 在主动脉缩窄（TAC）诱导的HF模型中，我们和其他人描述了CD 4 + T细胞在HF中的主要作用。 细胞作为非缺血性HF的主要贡献者。然而，由于T细胞免疫调节机制的复杂性， 由于纤维化激活导致炎症、CF和HF，目前还没有免疫调节或抗纤维化治疗， 转化为治疗HF的临床实践。我们的初步数据揭示了T细胞活化 发生在引流心脏的纵隔淋巴结（mLN）中，也发生在心脏内， 依赖性方式，涉及T细胞受体（TCR）通过主要抗原呈递的抗原接合 组织相容性复合物II（MHC-II）在抗原呈递细胞（APC）上表达。我们另外 证明活化的心脏成纤维细胞（CFB）表达MHC-II，因此可以在心脏中起APC的作用。 心新出现的证据表明，T细胞也可以被alarmins、可溶性炎症因子激活。 无菌炎症反应中产生的介质，通过TCR非依赖性途径，在“非经典” 我们的体外和体内初步数据支持这一点.基于这些 研究结果，我们将测试经典和非经典T细胞激活机制合作的中心假设 在HF进展过程中启动和维持CF。在目标1中，我们将使用Nur 77 GFP小鼠，其中T细胞 只有当通过TCR经典刺激时才表达GFP，以绘制GFP的特异性位置和时间。 TAC期间典型的T细胞活化。我们还将对心脏分选细胞进行单细胞TCR测序， GFP+ CD 4 + T细胞以鉴定HF进展中的免疫显性T细胞克隆。APC负责 将在细胞特异性MHC-II-/-小鼠中评价这种激活及其在CF和HF中随时间的作用。在目标2中， 我们将进行WT和alarmin敏感受损的活化的CD 4 + T细胞的过继转移实验， MHC-II-/-受体小鼠，其缺乏经典的T细胞活化并且通常受到保护免于CF和HF。CF、 心脏功能和负责T细胞活化的alarmin将在体内和体外表征 测定。在目标3中，基于我们的数据表明活化的T细胞粘附到CFB诱导其活化。 转化为促纤维化肌成纤维细胞在TGFβ依赖的方式，我们将探讨其机制 经典和非经典活化T细胞对CFB中TGFβ合成、释放和信号转导的影响 粘连我们将使用体外生物化学和分子测定。这些重要的研究将导致 更深入地了解如何最好地调节T细胞活化和T细胞诱导的HF，并提供新的 如何预防、改善和治疗非缺血性HF的前景。

The goal of this new R01 application is to investigate the mechanisms of T cell activation and the consequences in the progression of cardiac fibrosis (CF) in the deadly syndrome of heart failure (HF), currently the leading cause of mortality and hospitalizations in the USA. We were the first to report cardiac T cell infiltration associated with CF in patients with non-ischemic HF, and using the well- established experimental model of HF induced by transverse aortic constriction (TAC), we and others described a major role for CD4+ T cells as major contributors to non-ischemic HF. However, due to the complexity of the mechanisms of T cell activation resulting in inflammation, CF and HF, no immunomodulatory or anti-fibrotic therapies have yet translated to clinical practice to treat HF. Our preliminary data reveal the novel finding that T cell activation occurs in the mediastinal lymph nodes that drain the heart (mLN), and also within the heart in a classic dependent manner that involves T cell receptor (TCR) engagement by antigens presented by Major histocompatibility complex II (MHC-II) expressed on antigen presenting cells (APC). We additionally demonstrate that activated cardiac fibroblasts (CFB) express MHC-II and thus could function as APC in the heart. Emerging evidence suggests that T cells can also be activated by alarmins, soluble inflammatory mediators produced in response to sterile inflammation, through TCR independent pathways in a “non classic" TCR- independent manner, and our in vitro and in vivo preliminary data is in support of this. Based on these findings, we will test the central hypothesis that classic and non-classic T cell activation mechanisms cooperate to initiate and sustain CF during the progression of HF. In aim 1, we will use Nur77GFP mice, in which T cells express GFP only when stimulated classically through the TCR, to map the specific location and timing of classic T cell activation during TAC. We will additionally perform single cell TCR sequencing on heart sorted GFP+CD4+ T cells to identify the immunodominant T cell clones in HF progression. The APC responsible for such activation and its effects in CF and HF over time will be evaluated in cell specific MHC-II-/- mice. In aim 2, we will perform adoptive transfer experiments of WT and alarmin sensing-impaired activated CD4+ T cells into MHC-II-/- recipient mice, which lack classic T cell activation and are normally protected from CF and HF. CF, cardiac function and the alarmins responsible for T cell activation will be characterized in in vivo and in vitro assays. In aim 3, based on our data indicating that activated T cell adhesion to CFB induces their transformation to pro-fibrotic myofibroblast in a TGFβ dependent manner, we will investigate the mechanisms of TGFβ synthesis, release and signaling in CFB in response to classically and non classically activated T cell adhesion. We will use in vitro biochemical and molecular assays. These important studies will result in a deeper understanding of how best to regulate T cell activation and T cell induced in HF and provide new perspectives on how to prevent, ameliorate and treat non-ischemic HF.