Nakagawa Ryo, Muroyama Ryosuke, Saeki Chisato, Oikawa Tsunekazu, Kaise Yoshimi, Koike Kazuhiko, Arai Jun, Nakano Masanori, Matsubara Yasuo, Takano Keiko, Hirata Yoshihiro, Saruta Masayuki, Zeniya Mikio, Kato Naoya
Division of Advanced Genome Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Department of Gastroenterology and Hepatology, Jikei University School of Medicine, Tokyo, Japan.
Hepatol Res. 2019 Jun;49(6):653-662. doi: 10.1111/hepr.13318. Epub 2019 Feb 26.
Primary biliary cholangitis (PBC) is an autoimmune liver disease with unknown pathogenesis. In PBC, activation of T-cell receptor (TCR) signaling is associated with inflammatory cytokine production through N-Ras upregulation. Although the CD4 T cell TCR repertoire could be associated with PBC pathogenesis, it has not been evaluated. Thus, we analyzed the PBC-CD4 T cell TCR repertoire using next generation sequencing (NGS).
Four PBC patients (one treatment-naïve and three receiving ursodeoxycholic acid) and three healthy individuals were enrolled. NRAS expression in CD4 T cells was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). N-Ras dynamics in CD4 T cells were assessed by qRT-PCR and GTP-N-Ras activation assay. The TCR α- (TRA) and β-chain (TRB) repertoires on CD4 T cells were analyzed by NGS and profiled using hierarchical analysis. Motif analysis was undertaken to elucidate the structure of PBC-specific TCRs.
NRAS was upregulated in PBC relative to control CD4 T cells (P < 0.05), and N-Ras enhanced T cell activation in CD4 T cells. Among 2668 TRAs and 841 TRBs, 20 and 11, respectively, were differentially expressed in PBC compared to that in controls (P < 0.05, fold-change >2). Among them, TRAV29/J22, TRBV6-5/J2-6, and TRBV10-1/J2-1 were expressed in PBC but the expression was negligible in the controls, with more mature and longer forms observed in PBC-CD4 T cells.
N-Ras was upregulated in PBC-CD4 T cells, and it enhanced TCR activation, indicating that PBC-CD4 T cells were activated by N-Ras upregulation with differentially expressed TCR repertoires on their surfaces.
原发性胆汁性胆管炎（PBC）是一种发病机制不明的自身免疫性肝病。在PBC中，T细胞受体（TCR）信号的激活与通过N-Ras上调产生炎性细胞因子有关。尽管CD4 T细胞TCR库可能与PBC发病机制有关，但尚未进行评估。因此，我们使用下一代测序（NGS）分析了PBC-CD4 T细胞TCR库。
纳入4例PBC患者（1例未经治疗，3例接受熊去氧胆酸治疗）和3名健康个体。通过定量逆转录-聚合酶链反应（qRT-PCR）评估CD4 T细胞中NRAS的表达。通过qRT-PCR和GTP-N-Ras激活试验评估CD4 T细胞中N-Ras的动态变化。通过NGS分析CD4 T细胞上的TCR α链（TRA）和β链（TRB）库，并使用层次分析进行分析。进行基序分析以阐明PBC特异性TCR的结构。
与对照CD4 T细胞相比，PBC中NRAS上调（P < 0.05），并且N-Ras增强了CD4 T细胞中的T细胞活化。在2668条TRA和841条TRB中，与对照相比，PBC中分别有20条和11条差异表达（P < 0.05，变化倍数> 2）。其中，TRAV29/J22、TRBV6-5/J2-6和TRBV10-1/J2-1在PBC中表达，但在对照中表达可忽略不计，在PBC-CD4 T细胞中观察到更成熟和更长的形式。
PBC-CD4 T细胞中N-Ras上调，并且它增强了TCR激活，表明PBC-CD4 T细胞通过N-Ras上调而被激活，其表面TCR库差异表达。
