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

这一新的R01应用的目标是研究T细胞激活的机制和 致命性心力衰竭综合征的心脏纤维化进展的后果，目前 是美国死亡和住院的主要原因。我们是第一个报道心脏T细胞 非缺血性心力衰竭患者中与CF相关的浸润，并使用公认的实验 横断性主动脉缩窄(TAC)诱导的心衰模型，我们和其他人描述了CD4+T细胞的主要作用 细胞是非缺血性心力衰竭的主要贡献者。然而，由于T细胞机制的复杂性 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 转化为治疗心力衰竭的临床实践。我们的初步数据揭示了T细胞活化的新发现 发生在引流心脏的纵隔淋巴结(MLN)，也发生在心脏内。 梅杰提出的抗原涉及T细胞受体(TCR)参与的依赖方式 组织相容性复合体II(MHC-II)表达于抗原提呈细胞(APC)。此外，我们还 证明活化的心脏成纤维细胞(CFB)表达MHC-II，从而在心脏中发挥APC的作用 心。新的证据表明，T细胞也可以被警报、可溶性炎症所激活 在无菌炎症反应中产生的介质，通过TCR非经典途径 TCR非依赖的方式，我们的体外和体内初步数据支持这一点。基于这些 发现，我们将检验经典和非经典T细胞激活机制协同作用的中心假设 在心力衰竭的发展过程中启动和维持心力衰竭。在目标1中，我们将使用Nur77GFP小鼠，在其中T细胞 仅当通过TCR经典刺激时才表达GFP，以映射特定的位置和时间 TAC过程中典型的T细胞激活。我们还将对分选的心脏进行单细胞TCR测序 GFP+CD4+T细胞用于鉴定心力衰竭进展中的免疫优势T细胞克隆。APC负责 随着时间的推移，这种激活及其在CF和HF中的影响将在细胞特异性MHC-II-/-小鼠中进行评估。在目标2中， 我们将进行WT和ALARIN感知受损的活化的CD4+T细胞到 MHC-II-/-受体小鼠，缺乏典型的T细胞激活，通常对CF和HF具有保护作用。Cf 心功能和负责T细胞激活的警报将在体内和体外表征 化验。在目标3中，基于我们的数据表明，激活的T细胞与CFB的黏附诱导他们的 以转化生长因子β依赖的方式转化为促纤维化的肌成纤维细胞，我们将探讨其机制 转化生长因子β在经典型和非经典型活化T细胞刺激下的合成、释放及其信号转导 粘附力。我们将使用体外生化和分子检测。这些重要的研究将导致 更深入地了解如何最好地调节T细胞活化和诱导的T细胞，并提供新的 对如何预防、改善和治疗非缺血性心力衰竭的展望。

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.