表現無唾液酸神經節苷脂之肝臟駐留記憶性T細胞對原發性膽汁性膽管炎參與角色之研究

Abstract

原發性膽汁性膽管炎(Primary Biliary Cholangitis, PBC)是一種自體免疫肝臟疾病，其特徵為在病患體內產生針對粒線體自體抗原的自體抗體。誘發PBC的機制有非常多因素，然而肝臟內T細胞所誘發的免疫反應在此疾病下仍然未知。在所有T細胞族群之中，組織駐留記憶性T細胞(Tissue Resident Memory, TRM)是一群具備停留與維持於特定組織中的能力而得名的細胞。在本實驗室的先前研究中發現有一群特定表現無唾液酸神經節苷脂(ASGM1)的CD8 T細胞存在於小鼠肝臟中，並與其他文獻記載之肝臟駐留記憶性T細胞具備相似特性。我們發現這群細胞在B型肝炎高壓注射轉染模式中對於病毒清除非常重要。此外，在刀豆蛋白A引發之急性肝炎模式中也發現這群細胞會於早期快速產生IFN-γ並引發急性肝炎。為了更進一步剖析這群細胞，於此論文中我們利用2-辛炔酸-卵白蛋白(2-octynoic acid-ovalbumin, 2-OA-OVA)所誘發的自體免疫膽管炎動物模式用以模擬人體的原發性膽汁性膽管炎來探討這群細胞的角色。我們發現在誘發自體免疫膽管炎的野生型小鼠與缺乏先天性淋巴細胞的NFIL3-/-小鼠給予anti-ASGM1剔除性抗體都可以觀察到膽管炎有效被抑制，證明其為透過非自然殺手細胞(nature killer cell, NK cell)誘導的機制。我們以另一種得以剔除TRM的anti-CXCR3抗體以剔除細胞，亦可以觀察到小鼠的膽管炎被抑制。我們進一步以質譜流式細胞術進行分析並發現ASGM1陽性CD8 T細胞為主要干擾素γ (IFN-γ)來源並可能導致自體免疫膽管炎的產生。透過免疫組織化學染色可以觀察到給予anti-ASGM1和anti-CXCR3抗體都能抑制肝臟中IFN-γ的堆積。此外，在以anti-ASGM1消耗細胞後利用α-半乳糖神經醯胺(α-galactosylceramide, α-GalCer)刺激NKT細胞後，血清中IFN-γ濃度明顯降低。將α-Galcer刺激活化之NKT細胞與ASGM1陽性肝臟駐留CD8 T細胞共培養後觀察到產生IFN-γ之細胞數明顯較多。綜上所述，ASGM1陽性肝臟駐留CD8 T細胞在自體免疫膽管炎模式中是IFN-γ的主要來源，並對於膽管炎的發展扮演重要的角色。

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by specific anti­mitochondrial antibodies (AMAs) targeted at mitochondrial autoantigens. The pathophysiology of PBC is multifactorial, whereas the detailed immune response triggered by the intrahepatic T lymphocytes still remains unknown. Among all T cell subsets, tissue-resident memory T cell (TRM) is a specific lineage of lymphocytes, given its name by the ability to reside and maintain in different tissues. In our previous study, we identified a distinct Asialo-GM1-positive (ASGM1+) CD8 T cell population in intrahepatic lymphocytes (IHLs) and exhibited similar properties with the previously reported liver-resident memory T cells. In the hepatitis B virus (HBV) hydrodynamic transfection model, we found that this population is crucial for the eradication of HBV. Moreover, this population was also identified as an early IFN-γ producer and critical for the initiation of the ConA-induced acute hepatitis model. To further dissect this specific population within this work, the role of ASGM1+ liver TRM cells was investigated in autoimmune cholangitis with 2-octynoic acid-ovalbumin (2-OA-OVA) immunization mouse model, comparing to the PBC disease in human bodies. We found that autoimmune cholangitis was suppressed by α-ASGM1 treatment through an NK cell-independent mechanism, with similar results shown by utilizing NFIL3-/- mice. We also applied an alternative way for liver TRM depletion by the α-CXCR3 treatment, which was also found capable of suppressing autoimmune cholangitis. Moreover, through Mass Cytometry (CyTOF) analysis, we found that ASGM1+ CD8 T cells were the main source of IFN-γ and might be responsible for the pathogenesis of autoimmune cholangitis. By immunohistochemical staining, we demonstrated that α-ASGM1 and α-CXCR3 treatment suppressed IFN-γ deposition in portal tracts. When mice were pre-treated with α-ASGM1 followed by α-galactosylceramide (α-GalCer) exposure, the serum level of IFN-γ was significantly suppressed but not IL-4. Further, when co-cultured with α-GalCer-stimulated NKT cells, we identified that ASGM1+ liver TRM cells were activated upon iNKT activation and contributed to IFN-γ production. Taken together, it was suggested that the ASGM1+ CD8 liver TRM cells were crucial for the development of 2-OA-OVA immunized autoimmune cholangitis and served as a source of IFN-γ deposition.