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NUDT1 promotes the accumulation and longevity of CD103+ TRM cells in primary biliary cholangitis

  • Author Footnotes
    # BH, ZL and QQ joint first co-authorship.
    Bingyuan Huang
    Footnotes
    # BH, ZL and QQ joint first co-authorship.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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    # BH, ZL and QQ joint first co-authorship.
    Zhuwan Lyu
    Footnotes
    # BH, ZL and QQ joint first co-authorship.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Author Footnotes
    # BH, ZL and QQ joint first co-authorship.
    Qiwei Qian
    Footnotes
    # BH, ZL and QQ joint first co-authorship.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Yong Chen
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Jun Zhang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Bo Li
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Yikang Li
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Jubo Liang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Qiaoyan Liu
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • You Li
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Ruiling Chen
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Min Lian
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Xiao Xiao
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Qi Miao
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Qixia Wang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Jingyuan Fang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Zhexiong Lian
    Affiliations
    Chronic Disease Laboratory, Institutes for Life Sciences and School of Medicine, South China University of Technology, Guangzhou 510006, China
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  • Yanmei Li
    Affiliations
    Department of Clinical Immunology and Rheumatology, Tianjin Medical University General Hospital, Tianjin, China
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  • Ruqi Tang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Thomas Helleday
    Affiliations
    Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institute, 171 76 Stockholm, Sweden

    Weston Park Cancer Centre, Department of Oncology and Metabolism, University of Sheffield, Sheffield S10 2RX, United Kingdom
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  • Author Footnotes
    † MEG, ZY and XM joint last co-authorship.
    M. Eric Gershwin
    Correspondence
    Division of Rheumatology, Department of Medicine, Allergy and Clinical Immunology, University of California at Davis, Davis, CA, USA.
    Footnotes
    † MEG, ZY and XM joint last co-authorship.
    Affiliations
    Division of Rheumatology, Department of Medicine, Allergy and Clinical Immunology, University of California at Davis, Davis, CA, USA
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  • Author Footnotes
    † MEG, ZY and XM joint last co-authorship.
    Zhengrui You
    Correspondence
    Corresponding authors. Addresses: Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China.
    Footnotes
    † MEG, ZY and XM joint last co-authorship.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Author Footnotes
    † MEG, ZY and XM joint last co-authorship.
    Xiong Ma
    Correspondence
    Corresponding authors. Addresses: Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China.
    Footnotes
    † MEG, ZY and XM joint last co-authorship.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China
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  • Author Footnotes
    # BH, ZL and QQ joint first co-authorship.
    † MEG, ZY and XM joint last co-authorship.
Open AccessPublished:June 23, 2022DOI:https://doi.org/10.1016/j.jhep.2022.06.014

      Highlights

      • CD103+ TRM cells are the dominant population of PDC-E2-specific autoreactive cytotoxic T cells in livers of patients with PBC.
      • CD103+ TRM cells exhibit cytotoxicity against autologous cholangiocytes in PBC.
      • Upon antigenic stimulation, metabolic reprogramming and DNA damage response of CD103+ TRM cells is orchestrated by NUDT1.
      • NUDT1-dependent DNA damage resistance promotes long-term survival of CD103+ TRM cells via PARP1-TGFβ-Smad axis.

      Background & Aims

      Pyruvate dehydrogenase (PDC)-E2 specific CD8+ T cells play a leading role in biliary destruction in PBC. However, there are limited data on the characterization of these autoantigen-specific CD8+ T cells, particularly in the liver. Herein, we aimed to identify pathogenic intrahepatic CD8+ T-cell subpopulations and investigate their immunobiology in PBC.

      Methods

      Phenotypic and functional analysis of intrahepatic T-cell subsets were performed by flow cytometry. CD103+ TRM cell frequency was evaluated by histological staining. The transcriptome and metabolome were analyzed by RNA-seq and liquid chromatography-mass spectrometry, respectively. Cytotoxicity of TRM cells against cholangiocytes was assayed in a 3D organoid co-culture system. Moreover, the longevity (long-term survival) of TRM cells in vivo was studied by 2-octynoic acid-BSA (2OA-BSA) immunization, Nudt1 conditional knock-out and adoptive co-transfer in a murine model.

      Results

      Intrahepatic CD103+ TRM (CD69+CD103+CD8+) cells were significantly expanded, hyperactivated, and potentially specifically reactive to PDC-E2 in patients with PBC. CD103+ TRM cell frequencies correlated with clinical and histological indices of PBC and predicted poor ursodeoxycholic acid response. NUDT1 blockade suppressed the cytotoxic effector functions of CD103+ TRM cells upon PDC-E2 re-stimulation. NUDT1 overexpression in CD8+ T cells promoted tissue-residence programming in vitro; inhibition or knockdown of NUDT1 had the opposite effect. Pharmacological blockade or genetic deletion of NUDT1 eliminated CD103+ TRM cells and alleviated cholangitis in mice immunized with 2OA-BSA. Significantly, NUDT1-dependent DNA damage resistance potentiates CD8+ T-cell tissue-residency via the PARP1-TGFβR axis in vitro. Consistently, PARP1 inhibition restored NUDT1-deficient CD103+ TRM cell durable survival and TGFβ-Smad signaling.

      Conclusions

      CD103+ TRM cells are the dominant population of PDC-E2-specific CD8+ T lymphocytes in the livers of patients with PBC. The role of NUDT1 in promoting pathogenic CD103+ TRM cell accumulation and longevity represents a novel therapeutic target in PBC.

      Lay summary

      Primary biliary cholangitis (PBC) is a rare inflammatory condition of the bile ducts. It can be treated with ursodeoxycholic acid, but a large percentage of patients respond poorly to this treatment. Liver-infiltrating memory CD8+ T cells recognizing the PDC-E2 immunodominant epitope are critical in the pathogenesis of PBC. We identifed the key pathogenic CD8+ T cell subset, and worked out the mechanisms of its hyperactivation and longevity, which could be exploited therapeutically.

      Graphical abstract

      Keywords

      Introduction

      Patients with primary biliary cholangitis (PBC) develop a multilineage response to a highly restricted peptide of the E2 component of pyruvate dehydrogenase (PDC-E2) including anti-mitochondrial antibodies and autoreactive T-cell responses.
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      The clinical usage and definition of autoantibodies in immune-mediated liver disease: a comprehensive overview.
      CD8+ cytotoxic T lymphocytes (CTLs) are thought to be directly involved in the apoptosis of biliary epithelial cells (BECs) during PBC progression.
      • Wakabayashi K.
      • Lian Z.X.
      • Leung P.S.
      • Moritoki Y.
      • Tsuneyama K.
      • Kurth M.J.
      • et al.
      Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease.
      Memory CD8+ T cells recognizing the PDC-E2 immunodominant epitope are found only in patients with PBC and are significantly enriched in the liver compared to peripheral blood, highlighting their disease- and organ-specificity.
      • Kita H.
      • Matsumura S.
      • He X.S.
      • Ansari A.A.
      • Lian Z.X.
      • Van De Water J.
      • et al.
      Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis.
      Treatment of PBC has been dominated by routine ursodeoxycholic acid (UDCA) therapy, which is ineffective in 30-40% of patients.
      • Lleo A.
      • Wang G.Q.
      • Gershwin M.E.
      • Hirschfield G.M.
      Primary biliary cholangitis.
      High-risk patients would probably benefit from early combined treatment with UDCA and immunotherapies.
      • Hirschfield G.M.
      • Beuers U.
      • Corpechot C.
      • Invernizzi P.
      • Jones D.
      • Marzioni M.
      • et al.
      EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      • Vierling J.M.
      • Adams D.
      • Alpini G.
      • et al.
      The challenges of primary biliary cholangitis: what is new and what needs to be done.
      Despite the lack of uniform success with targeted therapies based on autoantigen-reactive CTLs in PBC to date, this approach remains promising.
      • Zhu C.
      • Boucheron N.
      • Müller A.C.
      • Májek P.
      • Claudel T.
      • Halilbasic E.
      • et al.
      24-Norursodeoxycholic acid reshapes immunometabolism in CD8(+) T cells and alleviates hepatic inflammation.
      Therefore, phenotypic and functional characterization of the pathogenic cytotoxic T-cell subsets is urgently needed.
      • Kita H.
      • Matsumura S.
      • He X.S.
      • Ansari A.A.
      • Lian Z.X.
      • Van De Water J.
      • et al.
      Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis.
      ,
      • Tsuda M.
      • Ambrosini Y.M.
      • Zhang W.
      • Yang G.-X.
      • Ando Y.
      • Rong G.
      • et al.
      Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis.
      Increasing evidence support that relapsed/refractory autoimmune diseases are partially driven by tissue-resident memory CD8+ T (TRM) cells, a recently identified T-cell subset that lodges permanently in non-lymphoid tissues without recirculating.
      • Beura L.K.
      • Mitchell J.S.
      • Thompson E.A.
      • Schenkel J.M.
      • Mohammed J.
      • Wijeyesinghe S.
      • et al.
      Intravital mucosal imaging of CD8 + resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory article.
      ,
      • Park C.O.
      • Kupper T.S.
      The emerging role of resident memory T cells in protective immunity and inflammatory disease.
      TRM cells often express CD69, integrins CD49a and CD103 that mediate long-term retention within tissues.
      • Sun H.
      • Sun C.
      • Xiao W.
      • Sun R.
      Tissue-resident lymphocytes: from adaptive to innate immunity.
      ,
      • Topham D.J.
      • Reilly E.C.
      Tissue-resident memory CD8+ T cells: from phenotype to function.
      TRM cells within the human liver can be further divided by CD103 expression into 2 subpopulations exhibiting substantial functional differences: CD103- TRM cells (CD69+CD103-CD8+ T cells) and CD103+ TRM cells (CD69+CD103+CD8+ T cells).
      • Pallett L.J.
      • Davies J.
      • Colbeck E.J.
      • Robertson F.
      • Hansi N.
      • Easom N.J.W.
      • et al.
      IL-2high tissue-resident T cells in the human liver: sentinels for hepatotropic infection.
      ,
      • Swadling L.
      • Pallett L.J.
      • Maini M.K.
      Liver-resident CD8+ T cells: learning lessons from the local experts.
      Upon antigen or cytokine stimulation, CD103+ TRM cells are stronger effectors than CD103- TRM cells, exhibiting robust inflammatory sentinel activity and long-term survival (longevity) in diseased livers.
      • Pallett L.J.
      • Davies J.
      • Colbeck E.J.
      • Robertson F.
      • Hansi N.
      • Easom N.J.W.
      • et al.
      IL-2high tissue-resident T cells in the human liver: sentinels for hepatotropic infection.
      ,
      • Kim J.H.
      • Han J.W.
      • Choi Y.J.
      • Rha M.-S.
      • Koh J.Y.
      • Kim K.H.
      • et al.
      Functions of human liver CD69+CD103-CD8+ T cells depend on HIF-2α activity in healthy and pathologic livers.
      Interestingly, in patients with chronic HBV infection, CD103- TRM cells have specificity for both hepatotropic and non-hepatotropic viruses, whereas CD103+ TRM subsets are only HBV-specific cells. Moreover, antigen-nonspecific activation of liver-resident CD8+ T cells contributes to inflammation in HDV.
      • Kefalakes H.
      • Horgan X.J.
      • Jung M.K.
      • Amanakis G.
      • Kapuria D.
      • Bolte F.J.
      • et al.
      Liver-resident bystander CD8(+) T cells contribute to liver disease pathogenesis in chronic hepatitis D virus infection.
      Beyond the antigen specificity, CD103+ TRM cells also exhibit distinct functional heterogeneity and plasticity. In cholangiocarcinoma, CD103+ TRM cells express prominent exhaustion-related checkpoint receptors, but still retain a T-bethigh Eomeslow population with reinvigoration potential.
      • Kim H.-D.
      • Jeong S.
      • Park S.
      • Lee Y.J.
      • Ju Y.S.
      • Kim D.
      • et al.
      Implication of CD69+CD103+ tissue-resident-like CD8+ T cells as a potential immunotherapeutic target for cholangiocarcinoma.
      The balance between CD103+ TRM and exhausted T cells is also predictive of outcomes in hepatocellular carcinoma.
      • Barsch M.
      • Salié H.
      • Schlaak A.E.
      • Zhang Z.
      • Hess M.
      • Mayer L.S.
      • et al.
      T cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma.
      Herein, we report that in PBC, expanded CD103+ TRM cells are the dominant intrahepatic population of PDC-E2-specific autoreactive CTLs. Upon antigenic stimulation, CD103+ TRM cells require nucleoside diphosphate X hydrolase 1 (NUDT1), a nucleotide pool sanitizing enzyme, to improve tolerance to oxidative stress derived from metabolic reprogramming. Genetic deletion or pharmacological blockade of NUDT1 eliminates CD103+ TRM cells in our PBC murine model and is accompanied by reduced portal inflammation. Significantly, NUDT1-dependent oxidative DNA damage resistance imprints CD8+ T-cell tissue-residency via the PARP1-TGFβR axis in vitro. Consistently, PARP1 inhibition restores NUDT1-deficient CD103+ TRM cell survival and TGFβ-Smad signaling both in vitro and in vivo.

      Materials and methods

      Patients and study samples

      Demographic and clinical features of study participants are included in Table S1 and Table S2. All individuals were enrolled in Shanghai Renji Hospital and provided written, informed consent. The study was carried out under the principles of the Declaration of Helsinki and approved by the research ethics boards of Renji Hospital, Shanghai Jiao Tong University.

      Animal studies

      We generated Nudt1flox/flox mice using a standard CRISPR/Cas9-mediated gene editing strategy. To specifically delete the NUDT1 from T cells, Nudt1flox/flox mice were bred with a CD4-Cre transgenic mouse strain to generate the Nudt1△CD4 mice on a C57BL/6 background. Procedure details of 2OA immunization and T-cell adoptive transfer/co-transfer can be found in the supplementary methods.
      Additional information on the materials and methods used can be found in the supplementary methods and supplementary CTAT table.

      Results

      CD103+ TRM cells are significantly expanded and hyperactivated in PBC

      A significantly increased frequency of CD103+ TRM cells (CD69+CD103+CD8+ T cells) among liver-infiltrating CD8+ T cells was observed in patients with PBC compared to healthy controls (HCs) (Fig. 1A). CD103+ TRM cells from patients with PBC demonstrated a higher expression of PD-1 and CXCR3 (Fig. 1B) and secreted significantly more cytokines including granzyme-B, IFN-γ, IL-2 and IL-17 (Fig. 1C) than those of HCs, indicating CD103+ TRM cells were hyperactivated with enhanced effector functions in PBC. CD103+ TRM cells also exhibited significantly higher proliferation/self-renewal related transcription factors, including Ki-67 and TCF-1, but simultaneously expressed higher exhaustion-associated TOX (Fig. 1C). The seemingly contradictory transcriptional program revealed a substantial internal heterogeneity of CD103+ TRM cell phenotypes.
      Figure thumbnail gr1
      Fig. 1Phenotype and autoantigen-specificity of CD103- and CD103+ TRM cells in PBC.
      (A) Frequency of liver-infiltrating CD103+ TRM cells in HCs (n = 15) and patients with PBC (n = 18). (B and C) Expression of surface markers (B), intracellular cytokines and transcription factors (C) in liver-infiltrating CD103+ TRM cells from HCs (n = 15) and patients with PBC (n = 18). (D) Representative flow cytometry plots of ex vivo expansion of PDC-E2159-167-specific cells in paired intrahepatic CD103- and CD103+ TRM cells from HLA-A∗0201+ patients with PBC, stained with CFSE and PDC-E2159-167 tetramer. (E) Precursor frequency of PDC-E2-specific CD8+ T cells in paired intrahepatic CD103- and CD103+ TRM cell subsets (n = 10). (F) Relative proportions of CD103- and CD103+ cells in total PDC-E2–specific reactive CD69+CD8+ T cells (n = 10). (G) CD103- and CD103+ TRM cells from patients with PBC were stimulated with a mix of PDC-E2 peptides for 6 h. The percentage of IFN-γ+ or granzyme-B+ cells in each subpopulation was analyzed (n = 10). (H) IFN-γ concentration in culture supernatants upon PDC-E2 peptides stimulation. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Mann-Whitney U test (A-C), Wilcoxon matched-pairs test (E-H). CFSE, carboxyfluorescein succinimidyl ester; E2 component of pyruvate dehydrogenase; HCs, healthy controls; HLA, human leukocyte antigen; IFN, interferon; MFI, mean fluorescence intensity; PBC, primary biliary cholangitis; PDC-E2, E2 component of pyruvate dehydrogenase; TRM cells, tissue-resident memory T cells.

      Autoreactivity of CD103+ TRM cells is potentially specific to PDC-E2 in PBC

      We sorted liver-infiltrating CD103+ TRM (CD69+CD103+CD8+) cells, CD103- TRM (CD69+CD103-CD8+) cells and “non-resident” CD69-CD103-CD8+ T cells from liver explants of HLA-A∗0201+ patients with PBC to expand peptide-specific CTL lines with PDC-E2159-167 peptide. Distinct populations of the PDC-E2159-167 tetramer+ CD8+ T cells were detected after a 12–14-day expansion of paired CD103+ and CD103- TRM cells (Fig. 1D), while PDC-E2159-167 tetramer+ CD8+ T cells derived from the CD69-CD103-CD8+ counterpart were still undetectable.
      The frequency of PDC-E2 peptide-specific T-cell precursors before expansion was estimated by the number of cell divisions. The average frequency of PDC-E2159-167-specific reactive cells among liver-infiltrating CD103+ TRM cells was significantly higher (over 3-fold) than that among CD103- TRM cells (Fig. 1E). Combining with their respective proportions in liver-infiltrating CD8+ T cells, more than 60% of the PDC-E2 reactive CD69+CD8+ T cells were CD103+ TRM cells (Fig. 1F). Moreover, CD103+ TRM cells sorted from all HLA-A∗0201+ HCs and disease controls failed to expand tetramer+ cells at detectable and comparable levels (Fig. S1B). Upon PDC-E2 peptide stimulation, CD103+ TRM cells produced more IFN-γ and granzyme-B compared to paired CD103- TRM cells (Fig. 1G). The prevalence of autoantigen-reactive T cells in CD103- and CD103+ TRM cell subsets and their differential antigen responsiveness was also confirmed by measurement of IFN-γ in supernatants (Fig. 1H; Fig. S1C). As expected, CD103+ TRM cells from HCs and disease controls did not respond to the stimulation (Fig. S1D). Overall, these findings reflect that CD103+ TRM cells are the major population of PDC-E2-specific autoreactive CD8+ T lymphocytes in the livers of patients with PBC.

      CD103+ TRM cells are clinically relevant in PBC

      We confirmed that CD103+ TRM cells accumulate around damaged interlobular bile ducts in the peri-portal areas by immunofluorescence co-staining of CD8, CD69, CD103 and CK7 (Fig. 2A). The numbers of CD103+CD8+ cells in the portal area in PBC were significantly higher than in HCs and other chronic liver diseases (Fig. 2B). In treatment-naive patients with PBC, either absolute numbers (Fig. 2C) or percentages among CD8+ cells (Fig. S2A) of portal area-infiltrating CD103+CD8+ cells positively correlated with biochemical parameters, including serum levels of alkaline phosphatase (ALP), gamma-glutamyltransferase and total bilirubin. No statistical correlation was observed between CD103+CD8+ cell numbers or frequencies and serum alanine aminotransferase, aspartate aminotransferase, IgM and IgG (Fig. S2B-C). For histological indices, portal-infiltrating CD103+CD8+ cell numbers (Fig. 2D) and frequencies (Fig. S2D) were both positively correlated with degree of hepatic inflammation and fibrosis stage. Interestingly, patients who responded to UDCA had significantly lower numbers and percentages of baseline CD103+CD8+ cells compared to those who were non-responders (Fig. 2E).
      Figure thumbnail gr2
      Fig. 2Correlation of CD103+ TRM cells with the clinical and histological indices in PBC.
      (A) Representative multiplex co-staining of CD8, CD69, CD103 and CK7(×200) in PBC liver. (B) Quantification of portal area-infiltrating CD103+CD8+ cells in the livers of HCs (n = 15) and patients with PBC (n = 66), AIH (n = 38), CHB (n = 36) and NAFLD (n = 32). (C and D) Numbers of CD103+CD8+ cells positively correlated with serum ALP, GGT, TBIL levels (C) or hepatic inflammation degree and fibrosis stage (D) in patients with PBC (n = 66). (E) UDCA responders (n = 42) exhibited lower absolute numbers and percentages of CD103+CD8+ cells than non-responders (n = 24). (F) Representative multiplex co-staining (above) and its phenotype-mapped image (below) with CK7+ (red) and TRM cells (black dots, CD69+CD103-CD8+ cells; blue dots, CD69+CD103+CD8+ cells). (G) The average distance to the nearest BECs from CD103+ TRM cells was significantly lower than that from CD103- TRM cells. (H) Spatial localization of CD103- TRM cells and CD103+ TRM cells within different distances from nearest CK7+ BECs. Mean ± SEM. ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Student’s t test (B, E), Spearman correlation test (C-D), paired t test (E-H). AIH, autoimmune hepatitis; ALP, alkaline phosphatase; BECs, biliary epithelial cells; CHB, chronic hepatitis B; GGT, Gamma-glutamyl transferase; HPF, high-power field; NAFLD, non-alcholic fatty liver disease; PBC, primary biliary cholangitis; TBIL, total bilirubin; UDCA, ursodeoxycholic acid.

      CD103+ TRM cells localize near biliary epithelial cells and are cytotoxic in PBC

      To examine the spatial relationship between BECs and TRM cells, we conducted nearest-neighbor analysis of CD103- TRM (CD69+CD103-CD8+) cells or CD103+ TRM (CD69+CD103+CD8+) cells with respect to CK7+ BECs (Fig. 2F). The average distance to the nearest BECs from CD103+ TRM cells was significantly lower than that from CD103- TRM cells (Fig. 2G), and was also lower than CD103+ TRM cells from HCs or other liver diseases (Fig. S2E-G). Within close proximity (0–25 μm) to BECs, the frequency of CD103+ TRM cells was higher than that of CD103- TRM cells. In contrast, CD8+ T cells localized further from BECs were more enriched with CD103- TRM cells (Fig. 2H).
      These results led us to further confirm the direct cytotoxicity of TRM cells against BECs, we established the 3D organoid co-culture system containing intrahepatic cholangiocytes and T cells. CD103+ TRM cells derived from patients with PBC produced significantly higher degranulation when co-cultured with autologous cholangiocytes (Fig. 3A), which was not observed in HCs or disease controls (Fig. S3A). Blocking assays demonstrated that cholangiocyte growth arrest and apoptosis caused by TRM cell killing were mediated by both Fas/FasL-dependent and perforin/granzymeB-dependent pathways, with the latter being dominant (Fig. 3B-D). Confocal 3D reconstructions showed granzyme-B/perforin-positive TRM cells accumulate around the damaged cholangiocyte organoids in the co-culture system (supplementary movie 1). Consistently, in portal areas of PBC livers, most CD103+ TRM cells close to the BECs were positive for granzyme-B and/or perforin (Fig. 3E; larger fields of view are shown in Fig. S3C). In addition to 3D organoid co-culture, similar results were also observed in planar co-culture of TRM cells and autologous primary cholangiocytes (Fig. S3D-E).
      Figure thumbnail gr3
      Fig. 3CD103+ TRM cells present cytotoxicity against cholangiocytes in PBC.
      (A) Representative flow cytometry plots showing degranulation (CD107a and granzyme-B measured by flow cytometry) of CD103+ TRM cells (derived from patients with PBC) cultured with and without autologous cholangiocytes as target cells. (B) Representative bright-field microscopy images showing morphology of cholangiocyte organoid-T cell co-culture systems (Top row); Representative CK7/DAPI immunofluorescence co-staining (Middle row) and apoptosis measured by Annexin V/PI of cholangiocytes (Bottom row). (C) Size of cholangiocyte organoids during co-culture in the presence of specific inhibitors/antibodies of cytotoxic pathways. (D) Residual cytotoxicity against autologous cholangiocytes of CD103+ TRM cells in the presence of specific inhibitors/antibodies of cytotoxic pathways. (E) Representative histological multiplex co-staining of CD103, granzyme-B, Perforin and CK7(×400) in liver portal area of PBC. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Wilcoxon matched-pairs test (A). Mann-Whitney U test (C-D). DAPI, 4,6-Diamidine-2-phenylindole; PBC, primary biliary cholangitis; PI, propidium iodide.

      CD103+ TRM cells within PBC livers accumulate high levels of ROS, but exhibit decreased dNTP pool oxidation and subsequent DNA damage

      Persistent antigenic stimulation impairs mitochondrial oxidative phosphorylation (OXPHOS) and results in functional exhaustion or replicative senescence in T cells.
      • Vardhana S.A.
      • Hwee M.A.
      • Berisa M.
      • Wells D.K.
      • Yost K.E.
      • King B.
      • et al.
      Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.
      ,
      • Acerbi G.
      • Montali I.
      • Ferrigno G.D.
      • Barili V.
      • Schivazappa S.
      • Alfieri A.
      • et al.
      Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.
      While TRM cells within different tissues are reported to utilize increased mitochondrial β-oxidation to support their longevity.
      • Pan Y.
      • Tian T.
      • Park C.O.
      • Lofftus S.Y.
      • Mei S.
      • Liu X.
      • et al.
      Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.
      ,
      • Varanasi S.K.
      • Kumar S.V.
      • Rouse B.T.
      Determinants of tissue-specific metabolic adaptation of T cells.
      CD103+ TRM cells demonstrated more fatty acid internalization (Fig. 4A) and higher mitochondrial respiratory activity (Fig. 4B) compared with the CD103- TRM subset in PBC. Oxygen consumption rate analysis showed mitochondrial respiratory capacity was also higher in CD103+ TRM cells (Fig. S4A). Consistently, higher reactive oxygen species (ROS) production was observed in CD103+ TRM cells in PBC (Fig. 4C), which was also higher than CD103+ TRM cells isolated from controls (Fig. S4B).
      Figure thumbnail gr4
      Fig. 4Mitochondrial metabolic reprogramming and oxidative DNA damage response of CD103+ TRM cells is orchestrated by NUDT1 in PBC.
      (A-C) Exogenous fatty acid uptake, active mitochondria and intracellular ROS levels in CD103- and CD103+ TRM cells from patients with PBC (n = 16) monitored by Bodipy-FL-C16, Mt-DR and Cell-ROX Reagent, respectively. (D) Relative quantification of nucleotide metabolites in paired CD103- and CD103+ TRM cells from patients with PBC (n = 5). (E and F) DNA double strand breaks assessed by phosphorylated activation of γ-H2AX (E) and CHK2 (F) in CD103- and CD103+ TRM cells from patients with PBC (n = 16). (G) NUDT1 expression among different intrahepatic CD8+ T-cell subsets from patients with PBC (n = 16). (H) Representative multiplex co-staining of CD8, CD103, NUDT1 and CK7(400×) showing the juxtaposition of NUDT1-expressing CD103+ TRM cells and the damaged interlobular bile ducts. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Wilcoxon matched-pairs test (A-G). Bodipy, boron dipyrromethene dyes; CHK2, checkpoint kinase 2; MFI, mean fluorescence intensity; Mt-DR, Mito-Tracker Deep Red; NUDT1, nucleoside diphosphate X hydrolase 1; PBC, primary biliary cholangitis.
      Liquid chromatography-mass spectrometry analysis revealed a significant difference in their nucleotide metabolism. Strikingly, oxidized dNTPs, especially 8-hydroxy-dGTP and 2-hydroxy-dATP, were substantially lower in CD103+ TRM cells (Fig. 4D). Insertion of oxidized dNTPs into the genome leads to a DNA damage response (DDR). Consistently, indicators of DNA double-strand breaks, including γ-H2AX and pCHK2, showed that CD103+ TRM cells had a lower DDR level (Fig. 4E-F). Accordingly, we detected the expression of NUDT1, the key enzyme degrading oxidized purine dNTPs in mammalian cells; flow cytometry analysis demonstrated that NUDT1 was selectively upregulated in CD103+ TRM cells, rather than other counterparts in PBC (Fig. 3G). Furthermore, CD103+ TRM cells expressed higher NUDT1 than CD103+ TRM cells isolated from HCs or disease controls (Fig. S4C). Immunofluorescence co-staining of NUDT1, CD8, CD103 and CK7 in PBC livers confirmed high NUDT1 expression of CD103+ TRM cells in close proximity to or even within the damaged interlobular bile ducts (Fig. 3H).

      NUDT1 regulates the function and intrahepatic accumulation of CD103+ TRM cells in PBC

      Next, we investigated whether NUDT1 regulates the effector function of CD103+ TRM cells in PBC. Intracellular co-staining was performed for the comparison of NUDT1low with NUDT1high CD103+ TRM cell subpopulations. NUDT1high CD103+ TRM cells expressed increased granzyme-B, IFN-γ, IL-2, and IL-17 (Fig. 5A), as well as exhibiting significantly higher Ki-67, TCF-1 (Fig. S4D). Upon re-stimulation with PDC-E2 peptides or anti-CD3/28, Karonudib (NUDT1 inhibitor) suppressed cytokine production from the CD103+ subset (Fig. 5B; Fig. S5A), but not from the CD103- subset (Fig. S4E). In stark contrast, such effects were not observed in IL-15 or IL-12+IL-18 stimulated bystander-activated CD103+ TRM cells (Fig. S5B-C).
      Figure thumbnail gr5
      Fig. 5NUDT1 promotes the function and phenotype of CD103+ TRM cells in PBC.
      (A) Cytokine-producing cell percentages in NUDT1low and NUDT1high CD103+ TRM subpopulations from patients with PBC (n = 10). (B) CD103+ TRM cells were stimulated with a mix of PDC-E2 peptides for 6 h in the presence of Karonudib (NUDT1 inhibitor) or vehicle control (n = 8). The percentage of IFN-γ+ or granzyme-B+ cells was analyzed. (C-E) Peripheral CD8+ T cells were sequentially exposed with 3-day TCR engagement (0.25 μg/ml αCD3/28) followed by 3-day TGFβ (50 ng/ml) to induce tissue-resident phenotype in vitro. Induced CD69+CD103+ cell frequencies in CD8+ T cells were increased by NUDT1 overexpression (C), and decreased by NUDT1 knockdown (D) or Karonudib treatment (E). (F) NUDT1 expression in paired peripheral CD8+ MPECs and SLECs from patients with PBC and HCs. (G) Elevated NUDT1 expression in peripheral MPECs of patients with PBC compared with HCs. (H) NUDT1 expression in paired peripheral CXCR3-/CXCR3+ or CXCR6-/CXCR6+ MPECs from patients with PBC. (I) Frequency of liver-infiltrating MPECs from HCs (n = 15) and patients with PBC (n = 18). (J) NUDT1 expression in peripheral MPECs were positively correlated with the absolute numbers and frequencies of CD103+CD8+ cells in PBC liver tissues. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Wilcoxon matched-pairs test (A, B, F, H), Mann-Whitney U test (C-E, G, I), Spearman correlation test (J). IFN, interferon; MFI, mean fluorescence intensity; MPECs, memory precursor effector cells; NUDT1, nucleoside diphosphate X hydrolase 1; PBC, primary biliary cholangitis; PDC-E2, E2 component of pyruvate dehydrogenase; SLECs, short-lived effector cells.
      We next utilized a well-established TRM cell de novo induction system to examine if the tissue-residency imprinted on peripheral CD8+ T cells is regulated by NUDT1
      • Pallett L.J.
      • Davies J.
      • Colbeck E.J.
      • Robertson F.
      • Hansi N.
      • Easom N.J.W.
      • et al.
      IL-2high tissue-resident T cells in the human liver: sentinels for hepatotropic infection.
      ,
      • Swadling L.
      • Pallett L.J.
      • Diniz M.O.
      • Baker J.M.
      • Amin O.E.
      • Stegmann K.A.
      • et al.
      Human liver memory CD8+ T cells use autophagy for tissue residence.
      ,
      • You Z.
      • Li Y.
      • Wang Q.
      • Zhao Z.
      • Li Y.
      • Qian Q.
      • et al.
      The clinical significance of hepatic CD69+CD103+CD8+ resident-memory T cells in autoimmune hepatitis.
      (Fig. 5C). Lentivirus-mediated NUDT1 overexpression significantly increased the frequency (Fig. 5C) and cytokine production (Fig. S5D) of de novo induced CD69+CD103+CD8+ cells, whereas NUDT1 knockdown (Fig. 4D; Fig. S5E) or Karonudib treatment (Fig. 5E; Fig. S5F) resulted in the opposite effect. Consistently, Karonudib rescued the cholangiocytes from growth defects and apoptosis induced by co-cultured autologous TRM cells (Fig. S5G-H). In addition, NUDT1 overexpression increased the oxygen consumption rate of CD8+ T cells after tissue-residency induction (Fig. S5I), either NUDT1 knockdown (Fig. S5J) or Karonudib (Fig. S5K) decreased their mitochondrial respiratory capacity.
      Based on differential KLRG1 and CD127 expression, we distinguish peripheral CD8+ short-lived effector cells (CD127KLRG1+) from memory precursor effector cells (MPECs, CD127+KLRG1) (Fig. 5F), the latter has been reported to be the dominant precursor cell source of TRM cell replenishment within different tissues.
      • Kurd N.S.
      • He Z.
      • Louis T.L.
      • Milner J.J.
      • Omilusik K.D.
      • Jin W.
      • et al.
      Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing.
      • Kok L.
      • Dijkgraaf F.E.
      • Urbanus J.
      • Bresser K.
      • Vredevoogd D.W.
      • Cardoso R.F.
      • et al.
      A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool.
      • Mani V.
      • Bromley S.K.
      • Äijö T.
      • Mora-Buch R.
      • Carrizosa E.
      • Warner R.D.
      • et al.
      Migratory DCs activate TGF-b to precondition naïve CD8+T cells for tissue-resident memory fate.
      Significantly higher NUDT1 expression in peripheral MPECs than short-lived effector cells was observed in patients with PBC, but not in HCs (Fig. 5F). MPECs in patients with PBC exhibited higher NUDT1 expression than those in HCs (Fig. 5G). In addition, upregulated NUDT1 was observed in circulating MPECs expressing CXCR3 and CXCR6, which mediate hepatic T-cell recruitment and residence (Fig. 5H). As expected, the frequencies of intrahepatic MPECs (potential TRM cell precursors) in PBC were significantly higher than those in HCs (Fig. 5I). Of note, the mean fluorescence intensity of NUDT1 in circulating MPECs positively correlated with the percentage of CD103+ TRM cells in paired liver tissue specimens (Fig. 5J).
      In summary, NUDT1 maintains the effector functions of CD103+ TRM cells during autoantigen recall response in PBC, and also promotes their hepatic accumulation by imprinting a tissue-residency phenotype on their circulating precursor counterparts.

      NUDT1-dependent DNA damage resistance potentiates CD8+ T-cell residency via the PARP1-TGFβ-Smad axis in vitro

      To further understand the mechanisms whereby NUDT1 promotes CD103+ TRM cell programming, we compared transcriptomes between NUDT1-overexpressing and control de novo induced TRM cells using RNA-sequencing analysis. A gene ontology enrichment analysis revealed that the genes associated with DDR were downregulated in the NUDT1-overexpressing group (Fig. 6A), consistent with the known functions of NUDT1 in modulating resistance to oxidative DNA damage
      • Karsten S.
      • Fiskesund R.
      • Zhang X.-M.
      • Marttila P.
      • Sanjiv K.
      • Pham T.
      • et al.
      MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.
      ,
      • Huber K.V.
      • Salah E.
      • Radic B.
      • Gridling M.
      • Elkins J.M.
      • Stukalov A.
      • et al.
      Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy.
      ; while the most critical events in TGFβ-Smad signaling including R-smad binding and smad protein-complex assembly were substantially upregulated by NUDT1 overexpression (Fig. 6B). Gene set variation analysis based on the KEGG pathway database also highlighted the increased levels of TGFβ signaling in the NUDT1-overexpressing group (Fig. 6C; Fig. S6A). Thus, we hypothesized that NUDT1-dependent DNA damage resistance would contribute to CD103+ TRM cell development or maintenance via upregulation of TGFβ-Smad signaling.
      Figure thumbnail gr6
      Fig. 6NUDT1-dependent DNA damage resistance potentiates CD8+ T-cell residency upon activation via PARP1-TGFβ signaling in vitro.
      (A-C) RNA was extracted from NUDT1-overexpressing and control CD8+ T cells on the 6th day of tissue-residency de novo induction and subjected to RNA-seq (n = 3). Gene ontology enrichment analysis (A and B) and gene set variation analysis based on KEGG pathway database (C) indicating the DEGs. (D-F) Levels of cleaved PARP-1 in CD8+ T cells were decreased by NUDT1 overexpression (D) and increased by NUDT1 knockdown (E) or Karonudib (NUDT1 inhibitor) treatment (F). (G-H) 1 μM Karonudib or vehicle control was added to the culture media concomitantly with TCR engagement, with or without pretreatment of 0.5 μM Rucaparib (PARP1 inhibitor) 6 h before TCR engagement. Induced CD69+CD103+ cell frequency (G), MFI of TGFβRII and pSmad2/3 (H) in CD8+ T cells in different groups were analyzed. (I) Cleaved PARP-1 in CD103- and CD103+ TRM cells from patients with PBC (n = 18). (J) MFI of TGFβRII and pSmad2/3 in cleaved PARP-1- and cleaved PARP-1+ CD8+ T cells from patients with PBC (n = 18). (K) TGFβRII expression was negatively correlated with cleaved PARP-1+ proportions in CD8+ T cells. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Mann-Whitney U test (C-E, G, I), Wilcoxon matched-pairs test (I-J), Spearman correlation test (K). DEGs, differentially expressed genes; KEGG, kyoto encyclopedia of genes and genomes; NUDT1, nucleoside diphosphate X hydrolase 1; PARP1, poly (ADP-ribose) polymerase-1.
      Recent studies showed that poly (ADP-ribose) polymerase-1 (PARP1), a central player in several different DDR pathways, can regulate the expression of TGFβ receptors and downstream smad signaling in T cells.
      • Lönn P.
      • van der Heide L.P.
      • Dahl M.
      • Hellman U.
      • Heldin C.-H.
      • Moustakas A.
      PARP-1 attenuates smad-mediated transcription.
      ,
      • Zhang P.
      • Nakatsukasa H.
      • Tu E.
      • Kasagi S.
      • Cui K.
      • Ishikawa M.
      • et al.
      PARP-1 regulates expression of TGF-β receptors in T cells.
      As expected, the percentage of cleaved PARP1 (the active fragment cleavage of PARP1)-positive cells was reduced in NUDT1-overexpressing CD8+ T cells compared to controls (Fig. 6D). In contrast, either NUDT1 knockdown (Fig. 6E) or NUDT1 inhibitor treatment (Fig. 6F) increased the level of cleaved PARP1. Pretreatment with an inhibitor (Rucaparib) of PARP1 reversed the reduction in CD103+ TRM cell frequency induced by Karonudib (Fig. 6G); all other tested inhibitors of different DDR signaling proteins (including DNA-dependent protein kinase catalytic subunit, ATM, ATR, CHK1 and Wee1) fail to rescue it (Fig. S6D-H). Furthermore, Rucaparib pretreatment restored the inhibitory effect of Karonudib on TGFβRII and phospho-Smad2/3 expression upon TCR/TGFβ sequential exposure (Fig. 6H), but not under IL15/TGFβ sequential induction (Fig. S6C). Finally, we validated this PARP1-TGFβ-Smad axis in liver-infiltrating CD8+ T cells from patients with PBC; CD103+ TRM cells showed a lower level of cleaved PARP1 than the CD103- TRM subset (Fig. 6I) and cleaved PARP1+ CD8+ T cells displayed lower expression of TGFβRII and phospho-Smad2/3 (Fig. 6J). Moreover, either TGFβRII (Fig. 6K) or phospho-Smad2/3 expression (Fig. S6I) was negatively correlated with cleaved PARP1+ proportions in CD8+ T cells.
      Overall, NUDT1-dependent DNA damage resistance upregulates TGFβ-Smad signaling by preventing PARP1 hyperactivation, thus promoting CD103+ TRM cell programming in PBC.

      Inhibition of NUDT1 eliminates CD103+ TRM cells and attenuates 2OA-BSA-induced cholangitis in vivo

      To examine the potential impact of NUDT1 on CD103+ TRM cells and their roles in PBC development in vivo, we induced cholangitis in mice immunized with BSA-conjugated 2OA as described.
      • Wakabayashi K.
      • Lian Z.X.
      • Leung P.S.
      • Moritoki Y.
      • Tsuneyama K.
      • Kurth M.J.
      • et al.
      Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease.
      Liver-infiltrating CD69+CD103+CD8+ TRM cell frequency was significantly increased in 2OA-immunized mice, and was specifically eliminated by Karonudib oral gavage (Fig. 7A). Liver inflammation (Fig. 7B) and serum ALP levels (Fig. 7C) in 2OA-immunized mice were significantly decreased by Karonudib. After being immunized with 2OA for 12 weeks, T cell-specific NUDT1-deficient Nudt1△CD4 mice developed a considerably lower CD103+ TRM cell frequency compared with NUDT1flfl control littermates (Fig. 7D), accompanied by the alleviation of portal inflammation (Fig. 7E) and decrease in serum ALP (Fig. 7F). Splenic pan-T cells from 2OA-immunized Nudt1△CD4 or Nudt1flfl mice were purified and adoptively transferred into Rag1−/− mice (Fig. 7G); HE staining revealed that NUDT1 knockdown in T cells led to decreased cholangitis in a cell-autonomous fashion (Fig. 7H).
      Figure thumbnail gr7
      Fig. 7Pharmacological blockade or genetic deletion of NUDT1 eliminates CD103+ TRM cells in vivo.
      (A-F) Liver tissues and liver-infiltrating lymphocytes were collected 12 weeks after primary 2OA-BSA immunization. (A) Liver-infiltrating CD69+CD103+ cell frequency in CD8+ T cells from mice immunized with 2OA or control under Karonudib or vehicle control treatment. (B) Representative H&E staining of liver portal areas and the mHAI score of the liver from indicated mice. (C) Serum ALP levels of indicated mice. (D) Liver-infiltrating CD69+CD103+ cell frequency from NUDT1△CD4 mice and NUDT1flfl control littermates immunized with 2OA or control. (E) Representative H&E staining of liver portal areas and the mHAI score of the liver from indicated mice. (F) Serum ALP levels of indicated mice. (G-H) T cells from NUDT1△CD4 or NUDT1flfl mice were adoptively transferred into 6-week-old Rag1-/- recipient mice; liver tissues were collected for histological evaluation 8-weeks post-transfer. (G) Workflow of the experimental procedure. (H) Representative H&E staining of liver sections from different recipients. Mean ± SEM. ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Mann-Whitney U test (A-H). ALP, alkaline phosphatase; mHAI, modified hepatitis activity index; NUDT1, nucleoside diphosphate X hydrolase 1; 2OA, 2-octynoic acid.

      NUDT1 promoted longevity of CD103+ TRM cells during cholangitis in a PARP1-dependent manner

      To further confirm if NUDT1 promotes the long-term residency phenotype of CD8+ T cells in vivo, an adoptive cell co-transfer experiment was performed. Pan-T cells were isolated from the spleens of wild-type CD45.1 mice and Nudt1△CD4-CD45.2 mice after 4-week 2OA-immunization, reactivated in vitro and then adoptively co-transferred into 6-week-old Rag1-/- recipient mice. Recipients were sacrificed on the indicated date and liver-infiltrating lymphocytes were collected for flow cytometry analysis (Fig. 8A). NUDT1 deficiency led to a progressive loss of CD103+ TRM cells over time (Fig. 8B), but only had a slight effect on total liver-infiltrating CD8+ cell frequency (Fig. 8C).
      Figure thumbnail gr8
      Fig. 8NUDT1 promotes long-term survival of CD103+ TRM cells in a PARP1-dependent manner in vivo.
      (A-C) Pan-T cells were isolated from WT-CD45.1 mice and NUDT1△CD4-CD45.2 mice after 4-week 2OA-immunization, then mixed (1:1 ratio) and adoptively co-transferred into 6-week-old Rag1-/- recipients. Workflow of the experimental procedure is shown (A). Mean fold-change (±SEM; n = 5) in frequencies of CD45.1- or CD45.2-expressing CD103+ TRM cells (B) or total CD8+ T cells (C) from recipients 12/16/20-weeks post-transfer. (D) Liver-infiltrating CD69+CD103+ cell frequency in CD8+ T cells from 12-week 2OA-immunized Parp1-/- or WT littermates under Karonudib (NUDT1 inhibitor) or vehicle treatment. (E) Liver-infiltrating CD69+CD103+ cell frequency in CD8+ T cells, MFI of TGFβRII and MFI of pSmad2/3 in CD103+ TRM cells from 12-week 2OA-immunized Nudt1△CD4 mice or Nudt1flfl littermates under Rucaparib or vehicle treatment. Mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. Mann-Whitney U test (B-E). 2OA, 2-octynoic acid; MFI, mean fluorescence intensity; NUDT1, nucleoside diphosphate X hydrolase 1.
      Next, to investigate if the mechanism by which NUDT1 promotes CD103+ TRM cell longevity also relies on PARP1, Karonudib or vehicle oral gavage were performed in 2OA-immunized Parp1-/- mice and wild-type littermates. Karonudib failed to abrogate CD103+ TRM cells in Parp1-/- mice after immunization with 2OA-CFA for 12 weeks (Fig. 8D). Pharmacological inhibition of PARP1 activity by Rucaparib also restored the frequency of CD103+ TRM cells, as well as their TGFβRII and phosphor-Smad2/3 expression (Fig. 8E). In addition, Rucaparib treatment also partially restored the frequency of KLRG1lowCD127high MPECs in NUDT1-deficient T cells (Fig. S7A-B).
      Taken together, genetic ablation or pharmacological blockade of PARP1 restored NUDT1-deficient CD103+ TRM cell longevity during 2OA-induced cholangitis and skewed the TRM cells towards a long-lived MPEC phenotype.

      Discussion

      Liver-infiltrating PDC-E2-specific memory CD8+ T cells play a leading role in biliary destruction in PBC progression. However, phenotypic and functional characterization of effector CD8+ T cells in PBC have been primarily confined to peripheral blood. In this study, we have comprehensively studied intrahepatic CD103+ TRM cells, with respect to their autoantigen-specificity, near-BECs localization and cytotoxicity against BECs. We also suggest that their antigen-dependent hyperactivation, and the longevity phenotype are based upon NUDT1.
      We previously proposed that residual CD103+ TRM cells may trigger a robust and rapid recall response, resulting in recurrence of liver inflammation in patients with autoimmune hepatitis discontinuing glucocorticoid treatment.
      • You Z.
      • Li Y.
      • Wang Q.
      • Zhao Z.
      • Li Y.
      • Qian Q.
      • et al.
      The clinical significance of hepatic CD69+CD103+CD8+ resident-memory T cells in autoimmune hepatitis.
      In the present study, we found an apparent link between baseline CD103+ TRM cell numbers and response to UDCA in PBC. Hence, CD103+ TRM cells may reflect the underlying disease state, severity or prognosis, since UDCA response is regarded as a disease-specific indicator for risk stratification and prognosis in PBC.
      • Hirschfield G.M.
      • Beuers U.
      • Corpechot C.
      • Invernizzi P.
      • Jones D.
      • Marzioni M.
      • et al.
      EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      • Vierling J.M.
      • Adams D.
      • Alpini G.
      • et al.
      The challenges of primary biliary cholangitis: what is new and what needs to be done.
      We also demonstrated that CD103+ TRM cells account for the majority of PDC-E2-specific CD8+ T lymphocytes and present direct cytotoxicity against BECs in PBC livers. This provides a plausible explanation for the organ specificity and chronicity of PBC, since the pathogenic CD103+ TRM cells exhibit a longevity phenotype and rarely dislodge from their residence sites once established. The data also suggests that future immune-based therapies of relapsed/refractory PBC could focus on the elimination of CD103+ TRM cells.
      Persistent antigen stimulation limits the mitochondrial oxidative capacity of T cells.
      • Vardhana S.A.
      • Hwee M.A.
      • Berisa M.
      • Wells D.K.
      • Yost K.E.
      • King B.
      • et al.
      Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.
      ,
      • Scharping N.E.
      • Rivadeneira D.B.
      • Menk A.V.
      • Vignali P.D.A.
      • Ford B.R.
      • Rittenhouse N.L.
      • et al.
      Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.
      Paradoxically, CD103+ TRM cells within different tissues utilize increased mitochondrial OXPHOS to support their longevity.
      • Pan Y.
      • Tian T.
      • Park C.O.
      • Lofftus S.Y.
      • Mei S.
      • Liu X.
      • et al.
      Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.
      One of the detrimental effects of excess ROS derived from OXPHOS is oxidative DNA damage. We revealed CD103+ TRM cells within PBC livers accumulate high levels of ROS, but show decreased dNTP pool oxidation and subsequent DDR, in line with previous reports that liver CD103+ TRM cells have longevity phenotypes.
      • Kim J.H.
      • Han J.W.
      • Choi Y.J.
      • Rha M.-S.
      • Koh J.Y.
      • Kim K.H.
      • et al.
      Functions of human liver CD69+CD103-CD8+ T cells depend on HIF-2α activity in healthy and pathologic livers.
      ,
      • Pallett L.J.
      • Burton A.R.
      • Amin O.E.
      • Rodriguez-Tajes S.
      • Patel A.A.
      • Zakeri N.
      • et al.
      Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.
      We noted that CD103+ TRM cells within PBC livers have higher expression of the enzyme NUDT1. By comparing the different functions of NUDT1low and NUDT1high CD103+ TRM subpopulations, together with a series of further in vitro experiments, we also identified that NUDT1 potentiates CD103+ TRM cell programming via PARP1-TGFβ-Smad signaling. Adoptive co-transfer experiments revealed that NUDT1 not only promotes intrahepatic CD103+ TRM cell survival, but also skews TRM cells towards a long-lived MPEC phenotype in a PARP1-dependent manner in vivo.
      There remain limitations to our findings, including the fact that animal experiments cannot perfectly mirror the pathophysiology of human disease, especially the PBC-specific antigenic effects. Even so, we suppose that NUDT1, as the central hub of metabolic reprogramming and DDR, could be key to deciphering the heterogeneity of TRM cells from an immunometabolism perspective.
      The relationship between metabolic reprogramming, ROS production, NUDT1 activity and DDR signaling in T cell-driven autoimmunity remain controversial.
      • Karsten S.
      • Fiskesund R.
      • Zhang X.-M.
      • Marttila P.
      • Sanjiv K.
      • Pham T.
      • et al.
      MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.
      Meanwhile, cellular senescence characterized by morphological changes or DDR factor expression is an intrinsic feature of cholangiocytes and hepatocytes in PBC,
      • Manco R.
      • Clerbaux L.A.
      • Verhulst S.
      • Bou Nader M.
      • Sempoux C.
      • Ambroise J.
      • et al.
      Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury.
      ,
      • Ferreira-Gonzalez S.
      • Lu W.Y.
      • Raven A.
      • Dwyer B.
      • Man T.Y.
      • O'Duibhir E.
      • et al.
      Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
      and has a detrimental role in biliary injury and impairment of liver regeneration.
      • Manco R.
      • Clerbaux L.A.
      • Verhulst S.
      • Bou Nader M.
      • Sempoux C.
      • Ambroise J.
      • et al.
      Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury.
      ,
      • Ferreira-Gonzalez S.
      • Lu W.Y.
      • Raven A.
      • Dwyer B.
      • Man T.Y.
      • O'Duibhir E.
      • et al.
      Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
      Therefore, using broad-spectrum DNA-damaging targeted drugs may inflict further biliary damage in PBC. However, NUDT1 expression is extremely low in the hepatic parenchyma, suggesting reduced toxicity if it is inhibited.
      In summary, we identified CD103+ TRM cells as the major fraction of PDC-E2-specific autoreactive CD8+ T cells within PBC livers, while their inappropriate expansion, antigen-dependent hyperactivation and longevity phenotype were dependent on NUDT1 activity. On the basis of these investigations, we propose a new paradigm for therapeutic immune modulation in PBC, exploiting targeted manipulation of PARP1-centered DDRs, that regulate TGFβ signaling in CD103+ TRM cells. This approach allows for highly selective suppression of autoantigen-specific reactive T cells, displays clear therapeutic benefits, and has less off-target toxicity.

      Abbreviations

      2OA, 2-octynoic acid; ALP, alkaline phosphatase; BECs, biliary epithelial cells; CFSE, carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocyte; DDR, DNA damage response; HCs, healthy controls; OXPHOS, oxidative phosphorylation; MPECs, memory precursor effector cells; NUDT1, nucleoside diphosphate X hydrolase 1; PARP1, poly (ADP-ribose) polymerase-1; PBC, primary biliary cholangitis; PDC-E2, E2 component of pyruvate dehydrogenase; ROS, reactive oxygen species; TRM cells, tissue-resident memory T cells; UDCA, ursodeoxycholic acid.

      Financial support

      This work was supported by the National Natural Science Foundation of China grants (#82130017 and 81830016 to XM, #81922010 and 81873561 to RT, #81900523 to YL, #81790634 and 82070581 to QW, #81800504 to ML, #81421001 to JF), Shanghai Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission (#21ZR1438700 to WC) and the Innovative research team of high-level local universities in Shanghai (#SHSMU-ZLCX20211600).

      Authors’ contributions

      XM, MEG and ZY designed and supervised the study. XM and RT acquired funding. BH, ZY, ZL and QQ performed the experiments. BH, ZY, ZL, QQ, YC, YL, JL, YL, BL, JZ and RC collected samples and clinical information. BH, ZL, QQ, ZY, ZL, TH analyzed the data. BH drafted the manuscript. XM, MEG and ZY reviewed the manuscript.

      Data availability statement

      Data are available upon request from the corresponding author Dr. Xiong Ma.

      Conflict of interest

      The authors have no conflicting financial interests.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgement

      We are grateful to all the participants who contributed to this study.

      Supplementary data

      The following are the supplementary data to this article:

      References

        • Terziroli Beretta-Piccoli B.
        • Mieli-Vergani G.
        • Vergani D.
        The clinical usage and definition of autoantibodies in immune-mediated liver disease: a comprehensive overview.
        J Autoimmun. 2018; 95: 144-158
        • Wakabayashi K.
        • Lian Z.X.
        • Leung P.S.
        • Moritoki Y.
        • Tsuneyama K.
        • Kurth M.J.
        • et al.
        Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease.
        Hepatology. 2008; 48: 531-540
        • Kita H.
        • Matsumura S.
        • He X.S.
        • Ansari A.A.
        • Lian Z.X.
        • Van De Water J.
        • et al.
        Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis.
        J Clin Invest. 2002; 109: 1231-1240
        • Lleo A.
        • Wang G.Q.
        • Gershwin M.E.
        • Hirschfield G.M.
        Primary biliary cholangitis.
        Lancet. 2020; 396: 1915-1926
        • Hirschfield G.M.
        • Beuers U.
        • Corpechot C.
        • Invernizzi P.
        • Jones D.
        • Marzioni M.
        • et al.
        EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
        J Hepatol. 2017; 67: 145-172
        • Terziroli Beretta-Piccoli B.
        • Mieli-Vergani G.
        • Vergani D.
        • Vierling J.M.
        • Adams D.
        • Alpini G.
        • et al.
        The challenges of primary biliary cholangitis: what is new and what needs to be done.
        J Autoimmun. 2019; 105102328
        • Zhu C.
        • Boucheron N.
        • Müller A.C.
        • Májek P.
        • Claudel T.
        • Halilbasic E.
        • et al.
        24-Norursodeoxycholic acid reshapes immunometabolism in CD8(+) T cells and alleviates hepatic inflammation.
        J Hepatol. 2021; 75: 1164-1176
        • Tsuda M.
        • Ambrosini Y.M.
        • Zhang W.
        • Yang G.-X.
        • Ando Y.
        • Rong G.
        • et al.
        Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis.
        Hepatol (Baltimore, Md.). 2011; 54: 1293-1302
        • Beura L.K.
        • Mitchell J.S.
        • Thompson E.A.
        • Schenkel J.M.
        • Mohammed J.
        • Wijeyesinghe S.
        • et al.
        Intravital mucosal imaging of CD8 + resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory article.
        Nat Immunol. 2018; 19: 173-182
        • Park C.O.
        • Kupper T.S.
        The emerging role of resident memory T cells in protective immunity and inflammatory disease.
        Nat Med. 2015; 21: 688-697
        • Sun H.
        • Sun C.
        • Xiao W.
        • Sun R.
        Tissue-resident lymphocytes: from adaptive to innate immunity.
        Cell Mol Immunol. 2019; 16: 205-215
        • Topham D.J.
        • Reilly E.C.
        Tissue-resident memory CD8+ T cells: from phenotype to function.
        Front Immunol. 2018; 9
        • Pallett L.J.
        • Davies J.
        • Colbeck E.J.
        • Robertson F.
        • Hansi N.
        • Easom N.J.W.
        • et al.
        IL-2high tissue-resident T cells in the human liver: sentinels for hepatotropic infection.
        J Exp Med. 2017; 214: 1567-1580
        • Swadling L.
        • Pallett L.J.
        • Maini M.K.
        Liver-resident CD8+ T cells: learning lessons from the local experts.
        J Hepatol. 2020; 72: 1049-1051
        • Kim J.H.
        • Han J.W.
        • Choi Y.J.
        • Rha M.-S.
        • Koh J.Y.
        • Kim K.H.
        • et al.
        Functions of human liver CD69+CD103-CD8+ T cells depend on HIF-2α activity in healthy and pathologic livers.
        J Hepatol. 2020; 72: 1170-1181
        • Kefalakes H.
        • Horgan X.J.
        • Jung M.K.
        • Amanakis G.
        • Kapuria D.
        • Bolte F.J.
        • et al.
        Liver-resident bystander CD8(+) T cells contribute to liver disease pathogenesis in chronic hepatitis D virus infection.
        Gastroenterology. 2021;
        • Kim H.-D.
        • Jeong S.
        • Park S.
        • Lee Y.J.
        • Ju Y.S.
        • Kim D.
        • et al.
        Implication of CD69+CD103+ tissue-resident-like CD8+ T cells as a potential immunotherapeutic target for cholangiocarcinoma.
        Liver Int. 2021; 41: 764-776
        • Barsch M.
        • Salié H.
        • Schlaak A.E.
        • Zhang Z.
        • Hess M.
        • Mayer L.S.
        • et al.
        T cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma.
        J Hepatol. 2022;
        • Vardhana S.A.
        • Hwee M.A.
        • Berisa M.
        • Wells D.K.
        • Yost K.E.
        • King B.
        • et al.
        Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.
        Nat Immunol. 2020; 21: 1022-1033
        • Acerbi G.
        • Montali I.
        • Ferrigno G.D.
        • Barili V.
        • Schivazappa S.
        • Alfieri A.
        • et al.
        Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.
        J Hepatol. 2021; 74: 783-793
        • Pan Y.
        • Tian T.
        • Park C.O.
        • Lofftus S.Y.
        • Mei S.
        • Liu X.
        • et al.
        Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.
        Nature. 2017; 543: 252-256
        • Varanasi S.K.
        • Kumar S.V.
        • Rouse B.T.
        Determinants of tissue-specific metabolic adaptation of T cells.
        Cell Metab. 2020; 32: 908-919
        • Swadling L.
        • Pallett L.J.
        • Diniz M.O.
        • Baker J.M.
        • Amin O.E.
        • Stegmann K.A.
        • et al.
        Human liver memory CD8+ T cells use autophagy for tissue residence.
        Cell Rep. 2020; 30: 687-698.e6
        • You Z.
        • Li Y.
        • Wang Q.
        • Zhao Z.
        • Li Y.
        • Qian Q.
        • et al.
        The clinical significance of hepatic CD69+CD103+CD8+ resident-memory T cells in autoimmune hepatitis.
        Hepatology. 2021; 74: 847-863
        • Kurd N.S.
        • He Z.
        • Louis T.L.
        • Milner J.J.
        • Omilusik K.D.
        • Jin W.
        • et al.
        Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing.
        Sci Immunol. 2020; 5
        • Kok L.
        • Dijkgraaf F.E.
        • Urbanus J.
        • Bresser K.
        • Vredevoogd D.W.
        • Cardoso R.F.
        • et al.
        A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool.
        J Exp Med. 2020; 217
        • Mani V.
        • Bromley S.K.
        • Äijö T.
        • Mora-Buch R.
        • Carrizosa E.
        • Warner R.D.
        • et al.
        Migratory DCs activate TGF-b to precondition naïve CD8+T cells for tissue-resident memory fate.
        Science. 2019; 366
        • Karsten S.
        • Fiskesund R.
        • Zhang X.-M.
        • Marttila P.
        • Sanjiv K.
        • Pham T.
        • et al.
        MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.
        Cell Death Differ. 2021;
        • Huber K.V.
        • Salah E.
        • Radic B.
        • Gridling M.
        • Elkins J.M.
        • Stukalov A.
        • et al.
        Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy.
        Nature. 2014; 508: 222-227
        • Lönn P.
        • van der Heide L.P.
        • Dahl M.
        • Hellman U.
        • Heldin C.-H.
        • Moustakas A.
        PARP-1 attenuates smad-mediated transcription.
        Mol Cell. 2010; 40: 521-532
        • Zhang P.
        • Nakatsukasa H.
        • Tu E.
        • Kasagi S.
        • Cui K.
        • Ishikawa M.
        • et al.
        PARP-1 regulates expression of TGF-β receptors in T cells.
        Blood. 2013; 122: 2224-2232
        • Scharping N.E.
        • Rivadeneira D.B.
        • Menk A.V.
        • Vignali P.D.A.
        • Ford B.R.
        • Rittenhouse N.L.
        • et al.
        Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.
        Nat Immunol. 2021; 22: 205-215
        • Pallett L.J.
        • Burton A.R.
        • Amin O.E.
        • Rodriguez-Tajes S.
        • Patel A.A.
        • Zakeri N.
        • et al.
        Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.
        J Exp Med. 2020; 217e20200050
        • Manco R.
        • Clerbaux L.A.
        • Verhulst S.
        • Bou Nader M.
        • Sempoux C.
        • Ambroise J.
        • et al.
        Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury.
        J Hepatol. 2019; 70: 1180-1191
        • Ferreira-Gonzalez S.
        • Lu W.Y.
        • Raven A.
        • Dwyer B.
        • Man T.Y.
        • O'Duibhir E.
        • et al.
        Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
        Nat Commun. 2018; 9: 1020