Molecular Characterization of Autoreactive B Cells in Immune Checkpoint Inhibitor-Induced Autoimmune Sicca

项目总结 自身免疫性干燥可自发发生，如Sjӧgren‘s综合征(SjS)，也可急性发生为免疫相关的 免疫检查点抑制剂(ICI)治疗癌症的不良事件(IRAE)。在大约 每年有50万名患者接受ICIS治疗，估计有3%-24%的患者出现新的干燥症状；这些 随着FDA对ICI使用的批准范围扩大，这一数字将会增加。我们定义ICI诱导的自身免疫性干燥(ICIA- Sicca)，如Ro52所示，10-25%的ICI-SICCA患者发展为可证明的B细胞自身免疫 或者Ro60自身抗体。需要新的、特定的生物标记物来预测谁会患上哪些类型的irAEs。 ICIA-SICCA中的“人的实验”能够比较Ro自身免疫前后的B细胞 与SJS的“自然实验”不同，在SJS的“自然实验”中，自身免疫的起点不是 为人所知。在SjS中，B细胞克隆是通过T细胞选择和B细胞受体(BCR)亲和力成熟而产生的，但它是 尚不清楚这种高度集中的B细胞反应是否源于ICIA-SICCA中ICI的使用，或者是哪种功能性B细胞 单元子集(例如，存储器)被扩展。为了填补这些知识空白，我们建立了一个精心策划的生物库 在ICI治疗前和ICIA-SICCA发生后采集的患者外周血样本 与道格·约翰逊，医学博士，M.S.C.I.，范德比尔特黑色素瘤研究项目主任和IRAE合作 专家。目标1将使用高通量人类杂交瘤技术来鉴定分子特征 与Ro52和Ro60结合的B细胞表达的BCR。我们将认识到突变和选择的作用 在自身抗体产生之前的自身反应性B细胞增殖中起作用。外周血液扩张 CD21lo B细胞(一种易于发生自身反应的亚群)在SjS患者中被观察到，并预测ICI治疗中的irAEs 病人。因此，我们将通过比较单个-B细胞亚群来研究该B细胞亚群和目标2中其他B细胞亚群的扩展。 ICIA-SICCA的细胞谱(BCRseq)、表型(CITEseq)和转录(RNAseq)B细胞特征 患者在ICI治疗前和ICIA-SICCA发展后。我们将使用这些数据来确定 ICIA-SICCA中扩增的B细胞克隆和亚群的特征我们将进一步整合目标1和目标2的数据 确定Ro52/Ro60特异的V基因同一性、克隆相关性和表型信息以推断 ICIA中识别SjS相关自身抗原的B细胞的功能能力和发育来源 干酪。识别ICIA-SICCA中其他自身抗原的自身反应性B细胞将由无偏见的 目标2中的方法。这些研究将发现可以使用的特定序列和表型生物标志物 预测即将发生的ICIA-SICCA并评估ICIA-SICCA的免疫抑制效果。人类 Ro52和Ro60单抗，自身反应性BCR基序数据库，以及平行 我们将生成的BCRseq/CITEseq/RNAseq数据将公开提供，以支持RARE和 自发性自身免疫性疾病研究。我们将创建的实验和分析蓝图将 为进一步研究ICI治疗后其他irAEs的起源奠定了基础。

PROJECT SUMMARY Autoimmune sicca can arise spontaneously, as in Sjӧgren’s syndrome (SjS), or acutely, as an immune-related adverse event (irAE) following immune checkpoint inhibitor (ICI) therapy for cancer. Of the approximately 500,000 patients treated with ICIs annually, an estimated 3-24% develop new-onset sicca symptoms; these numbers will increase as FDA approvals for ICI use broaden. We define ICI-induced autoimmune sicca (ICIA- sicca) as the 10-25% of ICI-sicca patients who develop demonstrable B cell autoimmunity, as shown by Ro52 or Ro60 autoantibodies. New, specific biomarkers are needed to predict who will develop which types of irAEs. The “experiment of man” in ICIA-sicca enables comparison of B cells before and after Ro autoimmunity develops, in contrast to the SjS “experiment of nature”, in which the initiation point of autoimmunity is not known. B cell clones arise via T cell selection and B cell receptor (BCR) affinity maturation in SjS, but it is unknown whether such highly focused B cell responses result from ICI use in ICIA-sicca, or which functional B cell subsets (e.g. memory) are expanded. To fill these gaps in knowledge, we built a carefully curated biobank of peripheral blood samples collected from patients before ICI therapy and following ICIA-sicca development in collaboration with Doug Johnson, M.D., M.S.C.I., Vanderbilt Melanoma Research Program Director and irAE expert. High-throughput human hybridoma technology will be used in Aim 1 to identify the molecular features of BCRs expressed by Ro52 and Ro60-binding B cells. We will discern the role that mutation and selection plays in autoreactive B cell expansion that precedes autoantibody production. Peripheral blood expansion of CD21lo B cells (an autoreactive-prone subset) is observed in SjS patients and predicts irAEs in ICI-treated patients. We will therefore investigate expansion of this and other B cell subsets in Aim 2 by comparing single- cell repertoire (BCRseq), phenotypic (CITEseq), and transcriptomic (RNAseq) B cell signatures in ICIA-sicca patients before ICI treatment and following ICIA-sicca development. We will use these data to identify hallmarks of expanded B cell clones and subsets in ICIA-sicca. We will further integrate Aim 1 and Aim 2 data to determine Ro52/Ro60-specific V gene identity, clonal relatedness, and phenotypic information to infer the functional capacity and developmental origins of B cells that recognize SjS-associated autoantigens in ICIA- sicca. Autoreactive B cells that recognize other autoantigens in ICIA-sicca will be identified by the unbiased approach in Aim 2. These studies will uncover specific sequence and phenotypic biomarkers that can be used in the future to predict impending ICIA-sicca and assess immunosuppressive efficacy in ICIA-sicca. Human Ro52 and Ro60 monoclonal antibodies, the autoreactive BCR motif database, and the parallel BCRseq/CITEseq/RNAseq data we will generate will be made publicly available to support irAE and spontaneous autoimmune disease research. The experimental and analysis blueprints we will create will set the stage for future studies to investigate the origins of other irAEs following ICI treatment.