透過高維度分析揭示肝臟內不同ASGM1+ CD8 T細胞群的表型特徵

Abstract

肝臟是人體最大的內臟器官，具有嚴格的免疫調節特性，在面對無害的抗原時能夠維持免疫耐受性，同時也保持對病原體入侵的免疫監控。在肝臟免疫中，組織駐留型記憶T細胞被認為是重要的發炎反應驅動者。在我們過去的研究中，發現一群表現無唾液酸神經節苷脂的肝臟CD8 T細胞群，這群細胞對於乙型肝炎病毒感染中的病毒清除以及ConA所誘導的急性肝炎模型都扮演重要的角色，並且這群細胞展現與其他文獻所記載之肝臟駐留型記憶T細胞相似的特徵，包括表現LFA-1以及CD69等肝臟駐留記憶T細胞的表面標誌，以及在細胞過繼實驗中展現回到肝臟的能力。然而ASGM1並未有過被用作肝臟駐留型記憶T細胞表面標誌的先例，因此我們認為這群肝臟ASGM1+ CD8 T細胞是由複雜的細胞群所組成，其中可能包含一群肝臟駐留型記憶T細胞，並且這群細胞對於引發肝炎扮演重要的角色。因此本研究旨在透過高維度的免疫特徵分析，以單細胞RNA定序針對這群肝臟ASGM1+ CD8 T細胞的基因表達進行分析，以揭示不同的細胞次群，並確認其中是否包含肝臟駐留型記憶T細胞。研究結果顯示，根據基因的表現情況可以將這群肝臟ASGM1+ CD8 T細胞細分為十個次群，其中有一群與我們過去所發現的細胞特徵最為相似，包括表現組織駐留型T細胞的表面標誌以及干擾素γ等的基因。我們也進一步透過流式細胞術初步分析並且確認這群ASGM1+ CD8 T細胞群當中存在部分細胞會表現肝臟駐留記憶T細胞的表面標誌。除此之外，我們也觀察到在ConA誘導急性肝炎模型中，這群具有肝臟駐留型記憶T細胞表型的ASGM1+ CD8 T細胞會快速被活化並且開始製造干擾素γ。綜合上述，我們結合高維度單細胞RNA定序與流式細胞術，揭示肝臟ASGM1⁺ CD8 T細胞族群中的表型異質性與潛在功能分化狀態，並鑑定出一群具有肝臟駐留表型特徵且可迅速活化的效應性T細胞，為後續深入探討其在肝臟免疫反應中的功能奠定基礎。

The liver is the largest internal organ in the human body, with distinct immune regulatory characteristics. When encountering harmless antigens, the liver maintains immune tolerance toward these antigens while simultaneously preserving immune surveillance against invading pathogens. Within the liver immune system, liver-resident memory T cells (liver TRM) serve as critical drivers of inflammation. In our previous study, we identified a distinct population of ASGM1+ CD8 T cells present within the intrahepatic lymphocyte population. These ASGM1+ CD8 T cells play a critical role in both the clearance of hepatitis B virus (HBV) and the pathogenesis of ConA-induced hepatitis mouse model. In addition, ASGM1+ CD8 T cells exhibit reported characteristics of liver TRM, including the expression of TRM surface markers such as LFA-1 and CD69, and the ability to home back to the liver in adoptive transfer experiments. However, ASGM1 itself is not considered a canonical surface marker for distinguishing liver TRM. Therefore, we hypothesize that intrahepatic ASGM1+ CD8 T cells consist of multiple subsets, including a subset of liver TRM, which may serve as key initiators of hepatitis. This study aims to investigate intrahepatic ASGM1+ CD8 T cells through high-dimensional immune profiling by analyzing their gene expression using single-cell RNA sequencing (scRNA-seq). We also aim to investigate whether liver TRM cells exist within the ASGM1+ CD8 T cell population. In our results, scRNA-seq analysis revealed that ASGM1+ CD8 T cells can be divided into ten distinct clusters based on differential gene expression. One of these clusters exhibits characteristics most similar to those described in our previous study, including the expression of genes encoding TRM surface markers and interferon-gamma. Given that gene expression does not always correspond directly to protein expression, we further conducted flow cytometry analysis to confirm that a subset of ASGM1+ CD8 T cells expresses well-established or recently identified surface markers characteristic of liver TRM. In addition, we observed that, in the ConA-induced hepatitis model, the ASGM1+ CD8 T cells with a liver TRM-like phenotype were rapidly activated and began producing interferon-γ (IFN-γ). Taken together, by integrating high-dimensional single-cell RNA sequencing with flow cytometry, we revealed the phenotypic heterogeneity and potential functional states within the intrahepatic ASGM1+ CD8 T cell population. This analysis led to the identification of a TRM-like T cell subset capable of rapid activation, laying a foundation for future studies to further investigate their role in liver immune responses.