TCR repertoire sequencing analysis using RNA on fibrotic livers. TCR repertoire sequencing analysis using RNA on fibrotic livers

Fibrosis is the final path of nearly every form of chronic disease and accounts for up to 45% of all deaths in the developed world. However, antifibrotic therapies that target fibrogenic cells are lacking. We tested whether specific immunization against ADAM12 can elicit an antigen-specific cytotoxic T cell response to ameliorate liver fibrosis. In this study, we performed T cell receptor (TCR) alpha- and beta-chain repertoire sequencing on fibrotic livers to further characterize the T cell response and to detect potential TCR clonotypes. We observed TCR clonality of liver-infiltrating T cells from v-A12- and control-vaccinated mice with minimal overlap to v-CTRL mice in two α-chain sequences. However, the vast majority of expanded clones from v-A12-vaccinated animals showed a unique sequence pattern. Moreover, there was no overlap in the β-chain sequences between v-A12-vaccinated and control mice, suggesting a vaccination-induced expansion of antigen-specific TCR clonotypes. Overall design: Livers were collected and used for total RNA extraction. Then total RNA was used for TCR alpha- and beta-chain sequencing. Comparative gene expression analysis of TCR alpha- and beta-chain seq data for livers from these four groups (sham_ v-CTRL, sham_v-A12, CCl4_v-CTRL and CCl4_v-A12) were performed.

纤维化是几乎所有慢性疾病的最终病理转归，在发达国家中其导致的死亡占总死亡人数的45%之多。然而，当前仍缺乏针对促纤维化细胞的抗纤维化治疗手段。本研究探讨了针对ADAM12的特异性免疫是否可诱发抗原特异性细胞毒性T细胞应答，以缓解肝纤维化。
本研究对纤维化肝脏进行了T细胞受体（T cell receptor, TCR）α链与β链谱系测序，以进一步解析T细胞应答，并检测潜在的TCR克隆型。
研究观察到，v-A12疫苗接种组与对照疫苗接种组小鼠的肝浸润T细胞的TCR克隆性在两条α链序列上与v-CTRL小鼠仅存在极少量重叠。然而，v-A12疫苗接种组动物体内绝大多数扩增的克隆均呈现独特的序列模式。此外，v-A12疫苗接种组与对照组小鼠的β链序列无任何重叠，这表明疫苗接种诱导了抗原特异性TCR克隆型的扩增。
总体实验设计：收集肝脏组织用于总RNA提取，随后利用总RNA进行TCRα链与β链测序。对四组小鼠（假手术对照疫苗组sham_v-CTRL、假手术ADAM12疫苗组sham_v-A12、四氯化碳诱导对照疫苗组CCl4_v-CTRL、四氯化碳诱导ADAM12疫苗组CCl4_v-A12）的肝脏TCRα、β链测序数据进行了比较基因表达分析。