Loss of B cell Tolerance is TCR Dependent [amplicon-seq]

We tested orphan TCR autoreactivity using the peptide MHC-TCR chimeric receptor (MCR) co-culture system. In this system, cognate antigen recognition leads to TCR specific NFAT activation in MCR reporter cells expressing a mouse I-Ab MHC class II extracellular domain covalently linked to candidate peptides and an intracellular TCR signaling domain. We used mixed autoimmune bone marrow chimera spleens and kidneys as sources of cDNA to generate a transcriptome-wide library of natural autoantigen peptides . We cloned this cDNA-derived peptide (CDP) autoantigen library into the MCR retroviral backbone and transduced NFAT reporter cells to make a murine autoantigen MCR reporter library (MCR-Lib). We then used this library to screen orphan TCRs identified by scTCR-seq for autoreactivity. Amplicon NGS was performed on PCR-amplified peptide sequences from MCR-Libraries following screening with orphan TCRs

本研究采用肽-MHC-TCR嵌合受体（peptide MHC-TCR chimeric receptor, MCR）共培养体系，检测孤儿T细胞受体（orphan TCR）的自身反应性。在该体系中，表达与候选肽共价连接的小鼠I-Ab II类主要组织相容性复合体（MHC class II）胞外结构域以及细胞内T细胞受体信号结构域的MCR报告细胞，在识别同源抗原后会发生T细胞特异性的活化T细胞核因子（NFAT）激活。本研究以混合自身免疫性骨髓嵌合体的脾脏与肾脏作为互补DNA（complementary DNA, cDNA）来源，构建了全转录组天然自身抗原肽文库。将该cDNA衍生肽（cDNA-derived peptide, CDP）自身抗原文库克隆至MCR逆转录病毒骨架中，再转导至NFAT报告细胞，成功构建小鼠自身抗原MCR报告文库（MCR-Lib）。随后利用该文库对通过单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCR-seq）鉴定得到的孤儿T细胞受体开展自身反应性筛选。在完成孤儿T细胞受体的筛选后，对MCR文库中经PCR扩增得到的肽序列进行扩增子下一代测序（amplicon next-generation sequencing, Amplicon NGS）。