Intrarenal B cells in acute kidney allograft rejection

摘要 在一篇开创性的论文中，Sarwal等1报道了B细胞基因特征和密集簇的存在， 肾活检组织中B细胞的数量与糖皮质激素抵抗性T细胞介导的移植物 排斥反应（TCMR）和肾移植物丢失。他们提出了一个假设，尽管标准的护理， 免疫抑制、浸润性B细胞在肾急性细胞排斥反应中起关键作用 临床上的异体移植虽然随后的一些研究支持这一假设，但另一些研究则相反。 成果。因此，肾内B细胞在临床TCMR中的作用目前尚不清楚， 迫切需要了解它们的作用，以使临床医生能够合理地决定B细胞耗竭是否对 逆转类固醇耐药TCMR。我们假设，肾内B的其他特征的识别 细胞或排斥活检的结果，以更准确地预测不良移植物的结果和B的需要 细胞耗竭以治疗TCMR。这些特征包括了解肾内B细胞的特异性， 它们的功能特性，以及活检的炎症结构。为此我们 开发了两种创新方法来了解人体活检中的原位适应性T和B细胞免疫 移植肾排斥反应首先，我们能够将单个B细胞中的转录状态与 它们分泌的抗体的抗原特异性。对来自肾活检的分选的B细胞进行单细胞免疫分析。 (sc)从这些相同的细胞克隆并表达免疫球蛋白可变区 相应的抗体。其次，我们开发了一种新的方法来描述空间 人体组织中不同免疫细胞群体之间的结构，并确定功能关系2，3. 使用一种新的深度卷积神经网络（DCNN），再加上一个调谐神经网络（TNN），我们可以 在树突状细胞（DC）识别抗原后，准确捕获T细胞形状的变化， 共聚焦显微照片。因此，该分析管道（小区距离映射，CDM）可以识别 并定量来自小鼠和人的固定组织样品中的抗原呈递。我们将使用CDM 研究T细胞与B细胞或树突状细胞（DC）在排斥肾活检中的相互作用。第三，我们开发了一个 小鼠肾移植模型，以解决由以下观察提出的机制问题： 人类肾脏活检我们将应用这三种方法来测试我们的项目假设在两个目标： 目标1。检验肾内B细胞的不同转录和功能状态在肾移植过程中的假设。 排斥与B细胞受体（BCR）特异性和排斥类型有关。 目的2：检验细胞排斥的类型（T细胞介导的排斥与混合T细胞-和 抗体介导的排斥）由T细胞和B细胞之间的特征性原位细胞相互作用定义 或适合于特定治疗干预的DC。

ABSTRACT In a seminal paper, Sarwal et al.1 reported that the presence of a B cell gene signature and dense clusters of B cells in the renal biopsies was strongly associated with glucocorticoid-resistant T cell-mediated graft rejection (TCMR) and kidney allograft loss. They proposed a hypothesis that, despite standard of care immunosuppression, infiltrating B cells play a pivotal role in a subset of acute cellular rejection of kidney allografts in the clinic. While some subsequent studies supported this hypothesis, others reported opposite outcomes. As a result, the role of intrarenal B cells in clinical TCMR is currently unclear, and an understanding of their role is urgently required to allow clinicians to rationally decide if B cell depletion might be useful for reversing steroid-resistant TCMR. We hypothesize that the identification of additional features of intrarenal B cells or of the rejecting biopsy are required to more accurately predict poor graft outcome and the need for B cell depletion to treat TCMR. These features include an understanding the specificity of intrarenal B cells and their functional properties, as well as the inflammation architecture of the biopsy. To this end, we have developed two innovative approaches to understand in situ adaptive T and B cell immunity in human biopsies from rejecting kidney allografts. First, we are able to pair the transcriptional state in single B cells with the antigenic specificity of the antibody they secrete. Sorted B cells from renal biopsies are subjected to single cell (sc) RNA-Seq, and the immunoglobulin variable regions are cloned from these same cells and expressed corresponding antibodies. Second, we have developed a novel approach to characterize the spatial architecture between different immune cell populations in human tissue and identify functional relationships2,3. Using a novel deep convolutional neural network (DCNN), coupled to a tuned neural network (TNN), we can accurately capture T cell shape change upon recognition of antigen presented by dendritic cells (DCs) in multicolor confocal micrographs. Therefore, this analytic pipeline (Cell Distance Mapping, CDM) can identify and quantify antigen presentation in fixed tissue samples from both mice and humans. We will use CDM to study T cell interactions with B cells or dendritic cells (DCs) in rejecting kidney biopsies. Third, we developed a mouse model of kidney transplantation to address mechanistic questions raised by observations made from the human renal biopsies. We will apply these three approaches to test our project hypothesis in two Aims: Aim 1. Test the hypothesis that the different transcriptional and functional states of intrarenal B cells during rejection are associated with B cell receptor (BCR) specificity and rejection type. Aim 2: Test the hypothesis that the type of cellular rejection (T cell mediated rejection vs. mixed T cell- and antibody-mediated rejection) is defined by characteristic in situ cellular interactions between T cells and B cells or DCs that are amenable to specific therapeutic intervention.