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

项目摘要 自身免疫性干燥症可以自发发生，如干燥综合征（SjS），也可以急性发生，如免疫相关性干燥症。 免疫检查点抑制剂（ICI）治疗癌症后的不良事件（irAE）。的大约 每年有500，000名患者接受ICI治疗，估计有3-24%的患者出现新发干燥症症状;这些 随着FDA批准ICI使用范围的扩大，数量将增加。我们定义ICI诱导的自身免疫性干燥症（ICIA- 如Ro 52所示，ICI-sicca患者中有10-25%发生了可证实的B细胞自身免疫 或Ro 60自身抗体。需要新的、特异性的生物标志物来预测谁将发生哪种类型的irAE。 ICIA-sicca中的“人的实验”能够比较Ro自身免疫前后的B细胞 发展，与SjS“自然实验”相反，其中自身免疫的起始点不是 知道的B细胞克隆在SjS中通过T细胞选择和B细胞受体（BCR）亲和力成熟而产生，但它是 尚不清楚这种高度集中的B细胞应答是否是由ICIA-干燥症中的ICI使用引起的，或者是哪种功能性B细胞应答 扩展单元子集（例如，存储器）。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发生后从患者中收集的外周血样本中 与医学博士道格·约翰逊合作，M.S.C.I.范德比尔特黑色素瘤研究项目主任和irAE 专家高通量人杂交瘤技术将用于Aim 1以鉴定分子特征 Ro 52和Ro 60结合B细胞表达的BCR。我们会发现突变和选择 在自身抗体产生之前的自身反应性B细胞扩增中起作用。外周血扩张 在SjS患者中观察到CD 21 lo B细胞（一种倾向于自身反应的亚群），并预测ICI治疗的irAE 患者因此，我们将通过比较单克隆抗体，研究Aim 2中这种和其他B细胞亚群的扩增。 ICIA-sicca中的细胞库（BCRseq）、表型（CITEseq）和转录组（RNAseq）B细胞特征 ICI治疗前和ICIA-sicca发展后的患者。我们将使用这些数据来识别 ICIA-sicca中扩增的B细胞克隆和亚群的标志。我们将进一步整合Aim 1和Aim 2数据 确定Ro 52/Ro 60特异性V基因身份、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起源， 干燥识别ICIA-sicca中其他自身抗原的自身反应性B细胞将通过无偏倚的 目标2的方法。这些研究将揭示特定的序列和表型生物标志物， 在未来预测即将发生的ICIA-Sicca和评估ICIA-Sicca的免疫抑制效果。人类 Ro 52和Ro 60单克隆抗体，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irAE， 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设置 未来研究的阶段，以研究ICI治疗后其他irAE的起源。

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.