Simultaneous antigen receptor repertoire profiling and single-cell transcriptomics in T and B lymphocytes from limited clinical samples

 说明(由申请人提供)：在绝大多数疫苗试验中，初级读数是经验性的，并不收集有关疫苗成败的免疫决定因素的信息。近年来，几项疫苗研究将高通量转录数据与抗原性测量相结合。这些“系统疫苗学”研究已经证明了对效力和抗原性决定因素的宝贵洞察力，并证明了在人类疫苗接种的机制研究中的巨大价值。然而，一个特别未被研究的领域是在成功和不成功的疫苗接种过程中出现的抗原特异性受体的组成。在临床疫苗研究中收集抗原特异性克隆型信息将提供有价值的数据，可用于加速疫苗的开发。由于几个因素(I)基于克隆的分析的高成本和低通量(Ii)大多数高通量分析只提供关于单个基因的信息(通常是免疫球蛋白的H链或TCRs的B链)，或者(Iii)不将agR序列与免疫表型或转录数据联系起来，目前用于免疫学研究的谱系测序方法的实用性受到了限制。在初步工作中，我们开发了一种方案，使用下一代测序同时查询来自流感疫苗接种后的人骨髓B淋巴细胞的转录组和配对抗原受体序列。这项建议的目标是完成B细胞和T细胞的“-Seq”联合检测的发展，包括建立其局限性和与当代全套测序技术的基准。

 DESCRIPTION (provided by applicant): In the vast majority of vaccine trials, the primary readouts are empirical do not collect information about the immunological determinants of a vaccine's success or failure. In recent years, several vaccine studies have combined high-throughput transcriptomic data with measures of antigenicity. These "systems vaccinology" studies have proven invaluable insight into the determinants of efficacy and antigenicity, and have demonstrated the enormous value in mechanistic studies of human vaccination. However, one area that is particularly understudied is the composition of antigen specific receptors that arise during successful and unsuccessful vaccinations. Collection of antigen-specific clonotype information in clinical vaccine studies would provide valuable data that could be used to accelerate vaccine development. The utility of current methodology for repertoire sequencing for immunological studies has been limited due to several factors (i) high-cost and low-throughput of cloning based assays (ii) most high-throughput assays only provide information on a single gene(typically the H-chain of immunoglobulin or ß-chain for TCRs), or (iii) do not link ag-receptor sequences with immunophenotypic or transcriptomic data. In preliminary work, we have developed a protocol that simultaneously queries the transcriptome and paired antigen receptor sequences in B lymphocytes derived from human bone marrow after flu vaccination using next generation sequencing. The goal of this proposal is to complete development of this combined "-Seq" assay for both B and T cells, including establishment of its limitations and benchmark against contemporary repertoire sequencing techniques.