Intrarenal B cells in acute kidney allograft rejection

摘要 在一篇开创性的论文中，Sarwal等人报告了B细胞基因特征和致密簇的存在 肾活检组织中B细胞与糖皮质激素耐药T细胞介导的移植物密切相关 排斥反应(TCMR)和移植肾丢失。他们提出了一个假设，尽管护理标准 免疫抑制、浸润性B细胞在肾脏急性细胞排斥反应中起关键作用 诊所里的同种异体移植。虽然随后的一些研究支持这一假设，但另一些研究的报告则相反 结果。因此，肾内B细胞在临床TCMR中的作用目前尚不清楚，目前对其认识尚不清楚。 他们的作用是迫切需要的，以允许临床医生理性地决定B细胞耗竭是否对 逆转类固醇耐药的TCMR。我们假设肾内B细胞的其他特征的识别 需要细胞或排斥活检来更准确地预测移植的不良结果和B细胞的需求。 细胞耗竭治疗中医辨证论治。这些特征包括了解肾内B细胞的特异性和 它们的功能特性，以及活检的炎症结构。为此，我们有 开发了两种新的方法来了解人类活检组织中的原位适应性T和B细胞免疫 因为排斥异基因肾移植。首先，我们能够将单个B细胞的转录状态与 它们分泌的抗体的抗原性。从肾活检中分离的B细胞受到单细胞的影响 (SC)RNA-Seq和免疫球蛋白可变区从这些细胞中克隆并表达 相应的抗体。其次，我们开发了一种新的方法来表征空间 人体组织中不同免疫细胞群之间的结构并确定功能关系2，3。 使用一种新型的深卷积神经网络(DCNN)，与调谐神经网络(TNN)相耦合，我们可以 准确捕捉识别树突状细胞(DC)提呈抗原后T细胞形态的变化 多色共焦显微照片。因此，这种解析管道(Cell Distance Map，CDM)可以识别 并量化来自小鼠和人类的固定组织样本中的抗原呈现。我们将使用CDM来 研究T细胞与B细胞或树突状细胞(DC)的相互作用在排斥肾活检中的作用。第三，我们开发了一种 小鼠肾移植模型，以解决由观察提出的机制问题 人类肾脏活组织检查。我们将使用这三种方法来测试我们的项目假设，目标有两个： 目的1.验证假设肾内B细胞在不同的转录和功能状态下 排斥反应与B细胞受体(BCR)的特异性和排斥类型有关。 目的2：检验细胞排斥类型(T细胞介导的排斥反应与混合T细胞-和 抗体介导的排斥反应)是由T细胞和B细胞之间特有的原位细胞相互作用定义的 或可接受特定治疗干预的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.