Defining the antigenic determinants of the adaptive immune response in IgG4-related disease

这个指导研究计划的目的是发展佩鲁吉诺博士成为一个独立的研究者， 纤维化的免疫机制。研究目标包括识别特定的自我蛋白质驱动 在IgG 4相关疾病（IgG 4-RD）的背景下的适应性免疫应答，最近描述的免疫- 介导的纤维化疾病。候选人：佩鲁吉诺博士是哈佛大学流变学系的一名初级教员。 马萨诸塞州总医院，自2015年以来一直在进行基于实验室的研究。他的短期 职业目标是在分子生物学方面打下更坚实的基础，提高他在统计分析方面的技能， 熟练掌握单细胞测序，获得创建和筛选肽-MHC酵母的专业知识 展示图书馆，并培养他独立调查的专业技能。这些目标将是 通过哈佛大学和哈佛临床和翻译中心的一系列课程的支持 科学中心。他最近发表了四篇第一作者论文，第一篇将半乳糖凝集素-3确定为新颖的B IgG 4-RD中的细胞自身抗原（变态反应和临床免疫学杂志，2019），第二次将 IgG 4-RD中的自身抗体反应与疾病严重程度（关节炎和风湿病学，2019），第三次建立 具有细胞毒性特征的CD 4 + T细胞作为系统性硬化症中可能的疾病驱动因素（临床医学杂志 Investigation，2020）和第4次在IgG 4-RD中建立细胞毒性CD 4 + T细胞的效应子表型（Journal 过敏和临床免疫学，2020）。这项工作构成了K 08提案的基础。导师制， 培训活动和环境：Perugino博士自2009年以来一直接受Shiv Pillai博士的直接指导。 2015年和博士约翰·斯通自2014年以来担任导师在调查和转化研究，分别。 培训计划以在这一指导小组下学到的技能为基础。在他的顾问的指导下， Perugino博士将熟练掌握（1）单细胞RNA测序（Alex Shalek博士），（2）IG/TCR 库分析（Dr. MarkDavis），（3）肽-MHC酵母展示文库开发（Dr. MichaelBirnbaum）， 免疫细胞相互作用（Michael Brenner博士）和生物统计分析（Musie Ghebremichael博士）。研究 项目：该提案的总体假设是IgG 4-RD中的免疫应答由免疫应答驱动。 B细胞和T细胞之间针对源自相同蛋白抗原的特异性表位的串扰。利用 鉴定IgG 4-RD中B和T细胞的克隆扩增，该建议需要确定 显性B和T细胞克隆，其随后将用于单细胞克隆可溶性抗原受体， 用它们作为探针拉下同源抗原这些互补的方法，一个侧重于B 细胞（目标1）和T细胞（目标2），需要验证B和T细胞反应中最大的 美国IgG 4-RD生物样本的单中心队列。这些研究将作为基础， 博士Perugino的未来目标是鉴定区分临床肿瘤的HLA和抗原决定簇， 不同免疫介导的纤维化疾病的表型，如IgG 4-RD，作为独立的研究者。

This mentored research program aims to develop Dr. Perugino into an independent investigator studying the immunologic mechanisms of fibrosis. The research goals entail the identification of specific self-proteins driving adaptive immune responses in the context of IgG4-related disease (IgG4-RD), a recently described immune- mediated fibrotic disease. Candidate: Dr. Perugino is a junior faculty member in the Rheumatology Unit at Massachusetts General Hospital and has been conducting bench-based research since 2015. His short-term career goals are to build a stronger foundation in molecular biology, advance his skills in statistical analysis, develop proficiency in single-cell sequencing, gain expertise in creating and screening peptide-MHC yeast display libraries, and build his professional skills towards independent investigation. These goals will be supported by a series of coursework through Harvard University and the Harvard Clinical and Translational Science Center. He has recently published four first-author papers, the first identifying galectin-3 as a novel B cell self-antigen in IgG4-RD (Journal of Allergy and Clinical Immunology, 2019), the 2nd linking the diversity of auto-antibody responses with disease severity in IgG4-RD (Arthritis & Rheumatology, 2019), a 3rd establishing CD4+ T cells with cytotoxic features as likely disease drivers in systemic sclerosis (Journal of Clinical Investigation, 2020) and a 4th establishing the effector phenotype of cytotoxic CD4+ T cells in IgG4-RD (Journal of Allergy and Clinical Immunology, 2020). This work forms the foundation for this K08 proposal. Mentorship, Training Activities, and Environment: Dr. Perugino has been under the direct mentorship of Dr. Shiv Pillai since 2015 and Dr. John Stone since 2014 acting as mentors in investigation and translational research, respectively. The training plan builds upon the skills learned under this mentorship team. Guided by his advisors and collaborators, Dr. Perugino will gain proficiency in (1) single cell RNA sequencing (Dr. Alex Shalek), (2) Ig/TCR repertoire analyses (Dr. Mark Davis), (3) peptide-MHC yeast display library development (Dr. Michael Birnbaum), immune cell interactions (Dr. Michael Brenner) and biostatistical analysis (Dr. Musie Ghebremichael). Research Program: The overarching hypothesis of this proposal is that the immune response in IgG4-RD is driven by the crosstalk between B and T cells directed at specific epitopes derived from the same protein antigen. Leveraging the identification of clonal expansions of B and T cells in IgG4-RD, the proposal entails the determination of dominant B and T cell clones, which will subsequently be used to single cell clone soluble antigen receptors and use those as probes to pull down their cognate antigens. These complementary approaches, one focused on B cells (Aim 1) and the other on T cells (Aim 2), entail the validation of B and T cell responses among the largest single-center cohort of IgG4-RD bio-samples in the United States. These studies will serve as a foundation for Dr. Perugino's future goals of identifying the HLA and antigenic determinants that distinguish the clinical phenotypes of different immune-mediated fibrotic diseases, such as IgG4-RD, as an independent investigator.