マウスに高脂肪高コレステロール食を24週間摂食させ，肝線維化病態を誘導した後，餌を通常食に切り換え，経時的に観察した．その結果，肝臓における各種線維化パラメーターは抑制され，肝線維化の顕著な回復が認められた．次に，肝線維化回復期における免疫細胞動態を網羅的に解析した．その結果，病態誘導によって顕著に増加していたCD8T細胞が，回復期においても残存し続けていることが明らかとなった．CD8T細胞除去抗体を用いて本細胞の線維化回復への作用を検討したところ，CD8T細胞を除去したマウスでは，CD8T細胞を除去していないマウスと比較して，肝線維化からの回復が顕著に遷延したことから，CD8T細胞が肝線維化からの回復を促進することが示された．このCD8T細胞の性質をsingle cell transcriptome解析等の手法を用いて解析したところ，組織常在型メモリーCD8T（CD8+Trm）細胞であることが判明した．また，NASH患者の肝臓組織における検討から，CD8+ Trm細胞が，線維化の進展とともに増加していることを確認した．さらに免疫染色法を用いた検討結果から，線維化回復期のCD8T細胞は肝星細胞と非常に近接して存在していた．CD8+Trm細胞と肝星細胞の相互作用の詳細を検討したところ，CD8+Trm細胞がFasLを介して肝星細胞の細胞死を誘導しており，抗FasL抗体を用いてこのシグナルを抑制すると細胞死した星細胞の数が減少し肝線維化の回復が遷延した．以上のことから，CD8+Trm細胞が肝星細胞の細胞死を誘導し，線維化からの回復を促進することが明らかとなった．一方，モデルマウスの経時的な腸内細菌の解析の結果, NASH肝線維化により大きく変化した腸内細菌の組成は線維化の回復後も定常状態には戻らず, 線維化修復に寄与する腸内細菌の存在が示唆された.
We found that the number of intrahepatic CD8T cells remained significantly high in RES mice compared to normal diet-fed (ND) mice, while other cell subsets tended to return to the normal condition with fibrosis regression at 8 weeks following the diet switch. Single-cell transcriptome analysis revealed enrichment of intrahepatic CD8T cells with Cd44+Sell−S1pr− tissue-resident memory (Trm) phenotypes in NASH resolution. Furthermore, bulk RNA-seq analysis of isolated CD8T cells subsets revealed that both Trm subsets from HFHC mice and RES mice expressed core signature genes of Trm. Tissue residency of these cells with lower circulating potential was confirmed in parabiosis mice in which blood circulation was shared between Ly5.1 and Ly5.2 derived congenic mice. Depletion of liver CD8+ Trm cells in the resolution phase by anti-CD8α killing antibody or IL-15 neutralization prevented recovery from liver inflammation and fibrosis, while adoptive transfer of these cells protected mice from fibrosis progression, suggesting direct roles of CD8+ Trm cells in NASH resolution. Hepatic stellate cells (HSCs) demonstrated CCR5-dependent chemotaxis to RES CD8+ Trm in vitro, and HSC-specific CCR5 deficient mice generated by administrating vitamin A-CCR5 siRNA liposomes during the resolution phase showed attenuated recovery from fibrosis with less proximity between CD8+ Trm cells and HSCs. Mechanistically, CD8+ Trm cells predisposed activated HSCs to FasL-Fas-mediated apoptosis in NASH resolution both in vitro and in vivo. We finally confirmed that treatment with anti-FasL Ab during the resolution phase decreased apoptotic HSCs and partially prevented the regression from NASH. Histological assessment of patients with NASH revealed CD69+CD8+ Trm abundance in fibrotic areas, further supporting their roles in humans. Conclusion: The current study provides additional insights into the liver protective function of CD8+ Trm cells in NASH pathogenesis. Attempts to manipulate their number and/or function may serve as a potential therapeutic option for NASH in the future. Analysis of intestinal bacteria over time in the model mice revealed that the composition of intestinal bacteria that changed significantly during the development of NASH liver fibrosis did not return to the steady state even after recovery of fibrosis, suggesting that gut microbiota may contributed to liver fibrosis repair.