SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens

We developed a modular, high-throughput discovery platform to simultaneously test whether Mut PIK3Ca is immunogenic and to retrieve paired a/b TCR gene sequences that confer specificity to this NeoAg. This method, termed Stimulation Induced Functional TCR sequencing (SIFT-seq), combines single-cell (sc) TCR V(D)J and transcriptome sequencing. Here, microwell cultures of in vitro stimulated T cells with confirmed neoantigen-specific recognition are selected for SIFT-seq. Matched aliquots of selected wells are acutely stimulated with autologous antigen-presenting cells presenting WT or Mut PI3Ka and the transcriptomic profile of individual clonotypes is assessed to identify neoantigen-specific T cells and retrieve their TCR gene sequences. Naïve CD8+ T cells from healthy donor samples were stimulated in vitro with mutated PIK3CA. Following a qPCR screen to identify mutation-specific upregulation of inflammatory transcript, positive wells were selcted for SIFT-seq. Matched aliquots were restimulated acutely with antigen-presenting wells expressing WT or mutated PIK3CA and subject to single-cell RNA-seq and V(D)J immunoprofiling using the 10x genomics platform. All clonotypes associated with a mutation-specific activation signature were identified and their TCR gene sequences were retrieved. Candidate TCR gene sequences were retrovirally transferred to open-repertoire T cell to confirm mutation-specific recognition. Note from submitter: The FASTQ files are not supplied because the data was generated from human patients/donors (patient privacy).

我们开发了一款模块化高通量发现平台，可同时检测突变型PI3KCA（Mut PIK3Ca）是否具有免疫原性，并获取赋予该新抗原（NeoAg）特异性的配对α/β T细胞受体（TCR）基因序列。该方法被命名为刺激诱导功能性TCR测序（Stimulation Induced Functional TCR sequencing, SIFT-seq），整合了单细胞（sc）TCR V(D)J测序与转录组测序。本研究中，我们选取经体外刺激且已证实具有新抗原特异性识别能力的T细胞微孔培养物，用于SIFT-seq实验。将筛选得到的微孔的匹配等分试样，用呈递野生型（WT）或突变型PI3KCA的自体抗原呈递细胞进行急性刺激，并评估单个克隆型的转录组特征，以识别新抗原特异性T细胞并获取其TCR基因序列。从健康供体样本中分离的初始CD8+ T细胞，经突变型PI3KCA进行体外刺激。通过实时定量聚合酶链反应（qPCR）筛选以鉴定炎症转录本的突变特异性上调表达，将阳性微孔选取用于SIFT-seq实验。将阳性微孔的匹配等分试样，用表达野生型或突变型PI3KCA的自体抗原呈递细胞进行急性再次刺激，并采用10x基因组学（10x Genomics）平台开展单细胞RNA测序与V(D)J免疫分型。所有与突变特异性激活特征相关的克隆型均被成功鉴定，并获取其对应的TCR基因序列。候选TCR基因序列通过逆转录病毒转导至开放受体库T细胞中，以验证其突变特异性识别能力。提交者备注：由于本数据来源于人类患者/供体，为保护患者隐私，未提供FASTQ文件。