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Cited by 75Cancer Immunotherapy and BiomarkersT-cell and B-cell ImmunologyCancer, Stress, Anesthesia, and Immune Response

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma

Open access
Zheng, Chunxing, Snow, Bryan E., Elia, Andrew J., Nechanitzky, Robert, Dominguez-Brauer, Carmen, Liu, Shaofeng, Tong, Yin, Cox, Maureen A. +13 more·2023·Nature Cancer
DOI 10.1038/s43018-023-00624-wPMC PMC10597839
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Abstract

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca 2+–NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

AI snapshot

Written from the paper’s full text.

TL;DR

Cholinergic CD4+ T cells expressing choline acetyltransferase (ChAT) are induced during hepatocellular carcinoma (HCC) development and support antitumor immunosurveillance. Their cholinergic activity restrains TCR-induced Ca2+–NFAT signaling, preventing hyperactivated CD25+ Treg cells and dysfunctional PD-1+ T cells that otherwise impair HCC control.

Key findings

  • In a mouse multihit HCC model combining CRISPR-mediated Trp53/Pten deletion and Sleeping Beauty–driven Myc overexpression, tumors were visible by day 15 and substantial nodules were present by day 25; immune cells infiltrated around MYC+ preneoplastic cells and HCC.
  • ChAT-expressing CD4+ T cells accumulated in HCC, including Foxp3+ Treg cells and PD-1+ dysfunctional T cells; scRNA-seq of 15,703 hepatic CD4+ T cells identified 11 clusters, with ChAT–GFP+ cells enriched in C3 (Cxcr6+Pdcd1+), C4, C9 and C10 clusters.
  • Tumor antigens drove clonal expansion of cholinergic CD4+ T cells: among the top 30 TCR types, 25 were observed in HCC-bearing mice and were more commonly found on ChAT–GFP+ T cells; after inducible OVA expression in HCC, about 40% of OVA-specific TCR Vβ5+ CD4+ T cells expressed ChAT versus about 2% in untreated mice.
  • T cell–specific Chat ablation (Chatfl/fl; Cd4-cre) accelerated HCC, increased tumor nodules and liver weights, and increased NASH-derived HCC incidence after 15 months on a Western diet; mechanistically, Chat loss increased TCR-induced Ca2+ influx and NFAT nuclear translocation, increased CD25 on Treg cells and PD-1 on Tconv cells, and PD-1 blockade significantly reduced HCC in Chatfl/fl; Cd4-cre mice.

Methods

  • Mouse HCC was induced by hydrodynamic injection of duplex CRISPR vectors targeting Trp53 and Pten plus Sleeping Beauty transposon–mediated Myc overexpression; an alternative NASH-HCC model used 15 months of Western diet.
  • Immune profiling used flow cytometry, immunohistochemistry, scRNA-seq/CITE-seq/TCR-seq on sorted ChAT–GFP+ and ChAT–GFP− CD4+ T cells from four control and four HCC-bearing livers, plus Ca2+ flux and NFAT nuclear translocation assays.
  • Human HCC parallels were examined using published scRNA-seq datasets and TCGA expression/survival data; statistical analyses included t-tests, ANOVA, log-rank tests, Fisher’s exact test and correlation tests as indicated.

Limitations

  • The paper states that there could be compensation by other receptors or interspecies differences, particularly after Chrm3/Chrm5 knockout in mouse HCC cells did not significantly affect HCC development.
  • The paper notes that cholinergic activity of T cells was most relevant when triggered by “self” tumor antigens; OVA-immunized mice were equally protected against HCC regardless of Chat ablation, so the findings may not generalize to potently immunogenic foreign antigens.
  • Evident from the text: human evidence is largely correlative/descriptive from existing datasets, and direct functional validation of CHAT-expressing T cells in human HCC is not provided.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s43018-023-00624-w

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 47 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Antigen-stimulated CD4+ T cells were increased in the HCC model.

    “Analysis of OX40, a transient marker of T cell antigen receptor (TCR) activation, showed that antigen-stimulated CD4+ T cells were increased (Extended Data Fig. 1e).”
    Quote in the paperSubstantial claim
  • Findingown data

    Infiltrating immune cells, including CD3+ T cells and CD11b+ antigen-presenting cells, were positioned around MYC+ preneoplastic cells and in established HCC.

    “The infiltrating immune cells, including CD3+ T cells and CD11b+ antigen-presenting cells (APCs), were positioned around MYC+ preneoplastic cells and in established HCC (Fig. 1g and Extended Data Fig. 1f).”
    Quote in the paperSubstantial claim
  • Findingown data

    Combining CRISPR-mediated Trp53/Pten ablation and transposon-based Myc overexpression induces rapid HCC development in mice.

    “Synergism between Myc expression and Trp53/Pten ablation induced rapid HCC development.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The CRISPR/transposon mouse model recapitulates key features of human HCC.
  • Findingown data

    In a mouse multihit model of HCC, two populations of CD4+ T cells expressing choline acetyltransferase are induced, including regulatory T cells and dysfunctional PD-1+ T cells.

    “induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells”
    Quote in the paperSubstantial claim
    • Rests on an assumption: ChAT–GFP reporter expression faithfully marks cells expressing ChAT.
  • Findingown data

    In a mouse multihit model of hepatocellular carcinoma, the adaptive immune response is activated.

    “In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response”
    Quote in the paperSubstantial claim
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 26

  • May combine two claims.

    Findingown data

    Neoplasms were visible on the liver surface by 15 days after injection, with substantial tumor nodules present by day 25.

    “Neoplasms were visible on the liver surface by 15 d after injection, with substantial tumor nodules present by day 25 (Fig. 1b).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The CRISPR/transposon mouse model recapitulates key features of human HCC.

  • May combine two claims.

    Findingown data

    NSG mice developed more severe disease and died sooner than immunocompetent animals.

    “Compared to immunocompetent animals, NSG mice developed more severe disease and died sooner, with tumor cells diffused throughout the liver instead of confined in discrete nodules (Fig. 1h and Extended Data Fig. 1g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cluster C7 cells expressed Cxcr6 but were negative for Pdcd1.

    “C7 cells expressed Cxcr6 but were negative for Pdcd1;”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the mouse HCC model, there was a marked increase in Foxp3+ChAT-GFP+ T cells alongside expansion of Treg cells during HCC development.

    “In our model, we observed a marked increase in Foxp3+ChAT–GFP+ T cells alongside expansion of Treg cells during HCC development (Fig. 3a–c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In HCC livers, the ChAT-GFP+ compartment contained a substantial number of C3 (Cxcr6+Pdcd1+) cells but was devoid of C7 (Cxcr6+Pdcd1-) cells.

    “In HCC livers, the ChAT–GFP+ compartment contained a substantial number of C3 (Cxcr6+Pdcd1+) cells but was devoid of C7 (Cxcr6+Pdcd1–) cells (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The prevalent TCRs in control mice preferentially belonged to ChAT-GFP- T cells, while those in HCC-bearing mice were more commonly found on ChAT-GFP+ T cells.

    “Intriguingly, the prevalent TCRs in control mice preferentially belonged to ChAT–GFP– T cells, while those in HCC-bearing mice were more commonly found on ChAT–GFP+ T cells (Fig. 4a,b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    T cells bearing TCR 3 were exclusively ChAT-GFP- cells in HCC and belonged primarily to cluster C7 (Cxcr6+Pdcd1-).

    “Notably, T cells bearing TCR 3 were exclusively ChAT–GFP– cells in HCC (Fig. 4b) and belonged primarily to cluster C7 (Cxcr6+Pdcd1–; Extended Data Fig. 4c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was a marked shift from naive T cells to effector T cells in the presence of HCC and induction of the C3 cluster.

    “We observed a marked shift from naive T cells to effector T cells in the presence of HCC and, in particular, the induction of the C3 cluster.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percentages of ChAT-GFP+CD8+ T cells and NKT cells were significantly elevated in HCC but to a lesser extent than CD4+ T cells.

    “The percentages of ChAT–GFP+CD8+ T cells and NKT cells were also significantly elevated in HCC but to a lesser extent (Fig. 2b).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: ChAT–GFP reporter expression faithfully marks cells expressing ChAT.

  • May combine two claims.

    Findingown data

    ChAT-GFP+CD4+ T cells were comprised mainly of CD44+ activated T cells that significantly increased during HCC progression.

    “These ChAT–GFP+CD4+ T cells were comprised mainly of CD44+ activated T cells that significantly increased during HCC progression (Fig. 2a,b).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: ChAT–GFP reporter expression faithfully marks cells expressing ChAT.

  • May combine two claims.

    Findingown data

    ChAT-GFP+Foxp3+ Treg cells and ChAT-GFP+ Tconv cells accumulated in HCC tissue, particularly at the tumor border, and were present in immune cell clusters associated with neoplastic hepatocytes.

    “Histologically, ChAT–GFP+Foxp3+ Treg cells and ChAT–GFP+ Tconv cells accumulated in HCC tissue, in particular at the tumor border, and were also present in immune cell clusters associated with neoplastic hepatocytes (Fig. 3h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Compared to ChAT-GFP− cells, the ChAT-GFP+ population lacked the naive T cell clusters as well as C7, with C2 cells also underrepresented.

    “Compared to ChAT–GFP− cells, the ChAT–GFP+ population lacked the naive T cell clusters as well as C7, with C2 cells also underrepresented (Fig. 2d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In Chat-GFP; OT-II mice subjected to standard HCC induction without OVA expression, ChAT-GFP+ T cells did not become significantly elevated, and most were CD4+ T cells expressing a natural repertoire of TCRs that were Vβ5-.

    “First, we subjected Chat-GFP; OT-II mice to standard HCC induction, which does not involve OVA expression. We found that ChAT–GFP+ T cells did not become significantly elevated, and most were CD4+ T cells expressing a natural repertoire of TCRs that were Vβ5– (Fig. 4e–g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percentage of ChAT-GFP+ B cells did not differ between control and HCC-bearing livers, and ChAT-GFP expression by NK cells and CD11b+ myeloid cells was negligible.

    “In comparison, the percentage of ChAT–GFP+ B cells did not differ between control and HCC-bearing livers, and the expression of ChAT–GFP by NK cells and CD11b+ myeloid cells was negligible (Extended Data Fig. 2b).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: ChAT–GFP reporter expression faithfully marks cells expressing ChAT.

  • May combine two claims.

    Findingown data

    The numbers of tumor nodules and liver weights were significantly increased in Chatfl/fl; Cd4-cre mice.

    “The numbers of tumor nodules and liver weights were also significantly increased in Chatfl/fl; Cd4-cre mice (Fig. 5b–e).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cd4-cre mediates T cell-specific deletion of Chat.

  • May combine two claims.

    Findingown data

    PD-1 was highly expressed by OVA-specific Tconv cells, and ~40% of these cells were ChAT-GFP+.

    “PD-1 was highly expressed by these OVA-specific Tconv cells, and ~40% of these cells were ChAT–GFP+ (Extended Data Fig. 5a).”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: OVA expression in HCC cells mimics a tumor antigen.

  • May combine two claims.

    Findingreasoning

    HCC progression was not exacerbated in Rag1-/- mice but instead was alleviated.

    “In contrast to the significantly increased tumor burden in Chatfl/fl; Cd4-cre mice, HCC progression was not exacerbated in Rag1–/– mice but instead was alleviated (Extended Data Fig. 7f,g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PD-1 levels strongly correlated with HCC grade in Chatfl/fl; Cd4-cre mice but not in Chatfl/fl mice.

    “Notably, PD-1 levels strongly correlated with HCC grade in Chatfl/fl; Cd4-cre mice but not in Chatfl/fl mice (Fig. 7c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PD-1 blockade significantly reduced HCC development in Chatfl/fl; Cd4-cre mice, substantially eliminating the differences in tumor progression.

    “However, PD-1 blockade significantly reduced HCC development in Chatfl/fl; Cd4-cre mice, substantially eliminating the differences in tumor progression (Fig. 7e,f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The HCC model does not involve viral vectors or mutant proteins; as such, the tumor antigens involved are likely 'self' proteins.

    “It should be stressed that our HCC model does not involve viral vectors or mutant proteins; as such, the tumor antigens involved here are likely ‘self’ proteins.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using scRNA-seq and flow cytometric analysis, the study demonstrated the induction of Foxp3+ Treg cells and PD-1+ Tconv cells in the mouse HCC model and showed that ChAT-expressing T cells predominantly belong to these two populations.

    “Using scRNA-seq and flow cytometric analysis, we have demonstrated the induction of Foxp3+ Treg cells and PD-1+ Tconv cells in our mouse HCC model and have shown that ChAT-expressing T cells predominantly belong to these two populations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ChAT-expressing Tconv cells induced in HCC are primarily PD-1+ T cells that coexpress inhibitory immunoreceptors such as Tim-3, Lag-3 and CTLA-4.

    “ChAT-expressing Tconv cells induced in HCC are primarily PD-1+ T cells (Fig. 3e,f) that coexpress inhibitory immunoreceptors, such as Tim-3 (Havcr2), Lag-3, CTLA-4 and other molecules characteristic of T cell exhaustion and dysfunction (Fig. 2f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The existence and phenotypes of human ChAT-expressing T cells are consistent with the mouse scRNA-seq data, indicating that a similar induction program of ChAT-expressing T cells occurs in human HCC.

    “Thus, the existence and phenotypes of human ChAT-expressing T cells are consistent with our scRNA-seq data on T cells from mouse HCC, indicating that a similar induction program of ChAT-expressing T cells also occurs in human HCC.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IFNγ has a pivotal function in antitumor immunity.

    “IFNγ, a hallmark of the type 1 helper T cell immune response, has a pivotal function in antitumor immunity37.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Castro, F., Cardoso, A. P., Gonçalves, R. M., Serre, K. & Oliveira, M. J. Interferon-γ at the crossroads of tumor immune surveillance or evasion. Front. Immunol. 9, 847 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Innate cytotoxic NK cells play a crucial role in antitumor immune response in HCC.

    “In addition to adaptive immune responses, innate cytotoxic NK cells play a crucial role in antitumor immune response in HCC38,39.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [38] Mikulak, J., Bruni, E., Oriolo, F., Di Vito, C. & Mavilio, D. Hepatic natural killer cells: organ-specific sentinels of liver immune homeostasis and physiopathology. Front. Immunol. 10, 946 (2019). source

      Not checked in the free run.

    • cites [39] Cheng, M., Chen, Y., Xiao, W., Sun, R. & Tian, Z. NK cell-based immunotherapy for malignant diseases. Cell. Mol. Immunol. 10, 230–252 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Tumor antigens can induce ChAT-expressing Treg cells and PD-1+ Tconv cells, and the expansion of ChAT-expressing T cells in liver cancer depends on TCR activation by tumor antigens.

    “Taken together, these data confirm that tumor antigens can induce ChAT-expressing Treg cells and PD-1+ Tconv cells and demonstrate that the expansion of ChAT-expressing T cells in liver cancer depends on TCR activation by tumor antigens.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: OVA expression in HCC cells mimics a tumor antigen.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Liver Treg cells have a crucial function in the maintenance of peripheral tolerance.

    “Liver Treg cells have a crucial function in the maintenance of this peripheral tolerance state53.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Tiegs, G. & Lohse, A. W. Immune tolerance: what is unique about the liver. J. Autoimmun. 34, 1–6 (2010). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Mobilization of Ca2+ in T cells is a biphasic event divided into the initial releasing of intracellular ER Ca2+ stores and the subsequent extracellular Ca2+ influx from CRAC channels.

    “Mobilization of Ca2+ in T cells is a biphasic event divided into the initial releasing of intracellular ER Ca2+ stores and the subsequent extracellular Ca2+ influx from ‘Ca2+ release-activated Ca2+’ (CRAC) channels50.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [50] Feske, S., Skolnik, E. Y. & Prakriya, M. Ion channels and transporters in lymphocyte function and immunity. Nat. Rev. Immunol. 12, 532–547 (2012). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    CD25 expression is controlled by the transcription factor NFAT, which is regulated by Ca2+ signaling.

    “CD25 expression is controlled by the transcription factor NFAT, which is regulated by Ca2+ signaling42.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [42] Hermann-Kleiter, N. & Baier, G. NFAT pulls the strings during CD4+ T helper cell effector functions. Blood 115, 2989–2997 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Gq/11-coupled M1, M3 and M5 mAChRs elicit Ca2+ release from the endoplasmic reticulum by activating the PLC/IP3/IP3R cascade, the same pathway triggered by TCR signaling.

    “The Gq/11-coupled M1, M3 and M5 mAChRs elicit Ca2+ release from the endoplasmic reticulum (ER) by activating the downstream phospholipase C/inositol 1,4,5-trisphosphate/inositol 1,4,5-trisphosphate receptor (PLC/IP3/IP3R) cascade49, the same pathway triggered by TCR signaling.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] Wess, J., Eglen, R. M. & Gautam, D. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nat. Rev. Drug Discov. 6, 721–733 (2007). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    TCR-induced Ca2+-NFAT signaling is indispensable for T cell exhaustion and induces the expression of inhibitory surface receptors such as PD-1, Lag-3 and Tim-3.

    “TCR-induced Ca2+–NFAT signaling is indispensable for T cell exhaustion and induces the expression of inhibitory surface receptors, such as PD-1, Lag-3 and Tim-3 (ref. 43).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity 42, 265–278 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Tumor antigens induce the expansion of ChAT-expressing Treg cells and PD-1+ Tconv cells.

    “Collectively, our study supports a model (Fig. 8g) whereby tumor antigens induce the expansion of ChAT-expressing Treg cells and PD-1+ Tconv cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In the absence of Chat in T cells, an unleashing of PD-1 inhibitory activity occurs that may restrict the functions of antitumor Tconv cells, allowing HCC progression.

    “Thus, in the absence of Chat in T cells, an unleashing of PD-1 inhibitory activity occurs that may restrict the functions of antitumor Tconv cells, allowing HCC progression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The clonal expansion of Treg cells and exhausted T cells in individuals with HCC has been revealed by scRNA-seq.

    “The clonal expansion of Treg cells and exhausted T cells in individuals with HCC has been revealed by scRNA-seq34.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Zheng, C. et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169, 1342–1356 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    In the absence of such cholinergic modulation, the Ca2+-NFAT pathway becomes hyperactivated to increase immunosuppression by Treg cells and to impose dysfunction on Tconv cells, resulting in compromised antitumor immunity.

    “In the absence of such cholinergic modulation, the Ca2+–NFAT pathway becomes hyperactivated to increase immunosuppression by Treg cells and to impose dysfunction on Tconv cells, resulting in compromised antitumor immunity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Activation of mAChR depletes the IP3-sensitive Ca2+ pool and renders T cells unresponsive to further CRAC channel-dependent stimulation.

    “However, the activation of mAChR depletes the IP3-sensitive Ca2+ pool (ER Ca2+) and renders the T cells unresponsive to further CRAC channel-dependent stimulation52.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Premack, B. A., McDonald, T. V. & Gardner, P. Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca2+-ATPase inhibitors. J. Immunol. 152, 5226–5240 (1994). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In the livers of individuals with HCC, Treg cell infiltration is prominently elevated, alongside the exhaustion and impaired function of CD8+ T cells.

    “In the livers of individuals with HCC, Treg cell infiltration is prominently elevated, alongside the exhaustion and impaired function of CD8+ T cells34,54.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [34] Zheng, C. et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169, 1342–1356 (2017). source

      Not checked in the free run.

    • cites [54] Fu, J. et al. Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients. Gastroenterology 132, 2328–2339 (2007). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The collective functions of Treg cells and Tconv cells in HCC are neutral in Chatfl/fl mice, whereas Chat deletion in T cells tilts the balance toward HCC promotion.

    “This result suggested that the collective functions of Treg cells and Tconv cells in HCC are neutral in Chatfl/fl mice, whereas Chat deletion in T cells tilts the balance toward HCC promotion.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    ACh regulates intracellular Ca2+ levels by binding to nAChRs and mAChRs.

    “ACh regulates intracellular Ca2+ levels by binding to nAChRs and mAChRs48.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [48] Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A. & Hudspeth, A. J. Principles of Neural Science 5th edn (McGraw-Hill Education, 2013).

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    This altered Ca2+ status restrains the expression of CD25 by Treg cells and the expression of PD-1 and other exhaustion markers by Tconv cells.

    “This altered Ca2+ status restrains the expression of CD25 by Treg cells and the expression of PD-1 and other exhaustion markers by Tconv cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Many actions of ACh in the nervous system are mediated through Ca2+ signaling via either G-protein-coupled mAChRs or ionotropic nAChRs.

    “Many actions of ACh in the nervous system are mediated through Ca2+ signaling via either G-protein-coupled mAChRs or ionotropic nAChRs48.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A. & Hudspeth, A. J. Principles of Neural Science 5th edn (McGraw-Hill Education, 2013).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The attempts at replication were successful.

    “The attempts at replication were successful.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    For analysis of c-MYC expression in liver sections, random 20× non-tumor fields of the c-MYC IHC scans from liver sections of individual mice were output using NDP.view2 and blindly analyzed using ImageJ with the plugin 'IHC Profiler'.

    “For analysis of c-MYC expression in liver sections, random 20× non-tumor fields of the c-MYC IHC scans from liver sections of individual mice were output using NDP.view2 and blindly analyzed using ImageJ (https://imagej.nih.gov/ij/) with the plugin ‘IHC Profiler’62.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [62] Varghese, F., Bukhari, A. B., Malhotra, R. & De, A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS ONE 9, e96801 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Blinding was performed in quantification of liver sections but not surface tumor nodules.

    “Blinding was performed in quantification of liver sections but not surface tumor nodules.”
    • Also stated in: § Statistics and reproducibility · plain
      “Blinding was also performed for quantitative analyses of liver sections.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The Chrm3 and Chrm5 sgRNAs only targeted the cells receiving the pX330-p53-Pten vector.

    “The Chrm3 and Chrm5 sgRNA expression vectors were combined with pX330-p53-Pten, SB100X and pT2-Myc for HCC induction; in this way, the Chrm3 and Chrm5 sgRNAs only targeted the cells receiving the pX330-p53-Pten vector.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The M1, M3 and M5 mAChRs are coupled with Gq/11 and elicit the PLC/IP3/IP3R cascade to trigger ER Ca2+ release, whereas M2 and M4 mAChRs are generally linked via Gi/o to cAMP production.

    “The M1, M3 and M5 mAChRs are coupled with Gq/11 and elicit the PLC/IP3/IP3R cascade to trigger ER Ca2+ release, whereas M2 and M4 mAChRs are generally linked via Gi/o to cAMP production48.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A. & Hudspeth, A. J. Principles of Neural Science 5th edn (McGraw-Hill Education, 2013).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    For comparing liver weights and nodule numbers, five to ten mice per group were sufficient to achieve statistical significance.

    “For comparing liver weights and nodule numbers, five to ten mice per group were sufficient to achieve statistical significance.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The T cell immune response to HCC antigens is reportedly dysfunctional and unreactive to HCC.

    “Indeed, the T cell immune response to HCC antigens is reportedly dysfunctional and unreactive to HCC55.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [55] Schietinger, A. et al. Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 45, 389–401 (2016). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Mice from each litter were randomly divided into groups to guarantee that sex-matched and genotype-matched individuals obtained different treatments.

    “To control for the treatments (including plasmids and antibodies), mice from each litter were randomly divided into groups to guarantee that sex-matched and genotype-matched individuals obtained different treatments.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Multiplets and cells with ambiguous hashing were filtered out.

    “the multiplets and cells with ambiguous hashing were filtered out.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Early mortalities (<5 d) were removed from the analysis.

    “Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.”
    • Also stated in: § Statistics and reproducibility · plain
      “Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Early mortalities (<5 d) were due to injection-associated death.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Maximal cholinergic stimulation mediated by overexpressed M1 receptor completely emptied the ER Ca2+ store, the Ca2+ pool essential for TCR-induced Ca2+ signaling.

    “In the Premack study, the maximal cholinergic stimulation mediated by overexpressed M1 receptor completely emptied the ER Ca2+ store, the Ca2+ pool essential for TCR-induced Ca2+ signaling.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Certain mechanisms, including the inactivation of IP3 receptors, can be triggered to attenuate ER Ca2+ release.

    “Certain mechanisms, including the inactivation of IP3 receptors, can be triggered to attenuate ER Ca2+ release57,58.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [57] Yamashita, M. ‘Quantal’ Ca2+ release reassessed—a clue to oscillation and synchronization. FEBS Lett. 580, 4979–4983 (2006). source

      Not checked in the free run.

    • cites [58] Rossi, A. M. et al. Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3. Cell Rep. 37, 109932 (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Quantification of immune cell clusters in scans of H&E-stained liver sections and determination of c-MYC, p53 and PTEN expression in tumor clones present in scans of IHC-stained liver sections were performed using NDP.view2 in a blinded fashion.

    “Quantification of immune cell clusters in scans of H&E-stained liver sections and determination of c-MYC, p53 and PTEN expression in tumor clones present in scans of IHC-stained liver sections were performed using NDP.view2 (Hamamatsu) in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ER Ca2+ release mediated by cholinergic signaling occurred in a quantal manner: a submaximal concentration of cholinergic agonist rapidly released a fraction of the ER Ca2+ store, followed by a slower or terminated Ca2+ release.

    “Moreover, the ER Ca2+ release mediated by cholinergic signaling occurred in a quantal manner: a submaximal concentration of cholinergic agonist rapidly released a fraction of the ER Ca2+ store, followed by a slower or terminated Ca2+ release56,57.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [56] Muallem, S., Pandol, S. J. & Beeker, T. G. Hormone-evoked calcium release from intracellular stores is a quantal process. J. Biol. Chem. 264, 205–212 (1989). source

      Not checked in the free run.

    • cites [57] Yamashita, M. ‘Quantal’ Ca2+ release reassessed—a clue to oscillation and synchronization. FEBS Lett. 580, 4979–4983 (2006). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    After excluding low-quality cells (counts of <30,000; percentage of mitochondrial counts of <30), gene expression was normalized using the recommended counts per million method.

    “After excluding low-quality cells (counts of <30,000; percentage of mitochondrial counts of <30), gene expression was normalized using the recommended counts per million method.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    ChAT deficiency in T cells exacerbates the dysfunction of Tconv cells and reinforces the immunosuppressive function of Treg cells, significantly compromising immunosurveillance against liver cancer.

    “Our data suggest that ChAT deficiency in T cells exacerbates the dysfunction of Tconv cells and reinforces the immunosuppressive function of Treg cells, significantly compromising immunosurveillance against liver cancer.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Blinding was achieved during injection by putting littermates of different genotypes into new cages lacking mouse information.

    “Blinding was achieved during injection by putting littermates of different genotypes into new cages lacking mouse information.”
    • Also stated in: § Statistics and reproducibility · plain
      “For vector delivery by hydrodynamic injection, blinding was achieved during injection by placing littermates of different genotypes into new cages lacking mouse information.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Pilot experiments were used to estimate the sample size necessary to generate statistically significant results using the appropriate statistical tests.

    “Pilot experiments were used to estimate the sample size necessary to generate statistically significant results using the appropriate statistical tests.”
    Quote in the paperNot a substantial claim
59 of 59

  • The paper relies on this without support.

    ChAT–GFP reporter expression faithfully marks cells expressing ChAT.

    “We analyzed the expression pattern of ChAT in liver tissues of reporter mice expressing green fluorescent protein (GFP) under the control of transcriptional regulatory elements for ChAT (Chat-GFP reporter mice).”

  • The paper relies on this without support.

    Cd4-cre mediates T cell-specific deletion of Chat.

    “we deleted Chat specifically in T cells by crossing mice carrying the conditional Chatfl allele with mice expressing the Cd4-cre transgene, thereby obtaining Chatfl/fl; Cd4-cre progeny.”

  • The paper relies on this without support.

    ChAT expression in T cells confers cholinergic activity (ACh synthesis).

    “subpopulations of CD4+ T cells expressing choline acetyltransferase (ChAT), the rate-limiting enzyme governing ACh synthesis, are induced during the development of liver cancer in mice”

  • The authors state this as an assumption.

    OVA expression in HCC cells mimics a tumor antigen.

    “Next, we used OVA to mimic a tumor antigen and devised a vector allowing the inducible expression of cytosolic OVA with constitutive expression of Myc.”

  • The paper relies on this without support.

    The CRISPR/transposon mouse model recapitulates key features of human HCC.

    “To mimic the multihit process of human liver carcinogenesis, we combined these two approaches by ablating Trp53 and Pten using duplex CRISPR and overexpressing Myc using the Sleeping Beauty transposon (Fig. 1a).”

  • The paper relies on this without support.

    Tumor antigens in this HCC model are weakly immunogenic self proteins.

    “Such proteins are postulated to be weakly immunogenic, particularly within the tolerogenic immune milieu in the liver.”

  • The paper relies on this without support.

    The immune milieu of the liver at steady state favors tolerance over immune responses.

    “The immune milieu of the liver at steady state favors tolerance over immune responses to prevent overreaction to innocuous antigens from food, microbial substances or by-products of metabolism.”

  • The paper relies on this without support.

    Early mortalities (<5 d) were due to injection-associated death.

    “Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.”
    “Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Lymphocytes are a critical source of ACh in various tissues, including the liver.

    “Recent evidence has identified lymphocytes as a critical source of ACh in various tissues, including the liver.15,16”

    From B cell-derived acetylcholine promotes liver regeneration by regulating Kupffer cell and hepatic CD8+ T cell function (2025) · cites this paper as [16]

    The Results section (‘ChAT-expressing T cells are induced during HCC development’; page unavailable) identifies lymphocytes as the dominant cholinergic cells in healthy and HCC-bearing livers. The paper does not establish that lymphocytes are a critical source of ACh across various other tissues, so the broader tissue claim is not supported by this source. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 6 gives two sample sizes for the same treatment group

    Minor

    The legend assigns the Chatfl/fl + anti-CD25 group both 7 and 9 mice. It does not identify which count applies to that group.

  • Figure 7 gives conflicting anti-PD-1 group sizes

    Minor

    The legend assigns 10 mice to the Chatfl/fl + anti-PD-1 group, then assigns 11 to the same group with "anti" misspelled as "anit."

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A. & Hudspeth, A. J. Principles of Neural Science 5th edn (McGraw-Hill Education, 2013).

Cited by

Sorted by citations

Related

Authors
Zheng, C., Snow, B. E., Elia, A. J., Nechanitzky, R., Dominguez-Brauer, C., Liu, S., Tong, Y., Cox, M. A., Focaccia, E., Wakeham, A. C., Haight, J., Tobin, C., Hodgson, K., Gill, K. T., Ma, W., Berger, T., Heikenwälder, M., Saunders, M. E., Fortin, J., Leung, S. Y., & Mak, T. W.
Affiliations
  • University Health Network (CA)
  • Queen Mary Hospital (CN)
  • German Cancer Research Center (DE)
Corresponding author
Mak, Tak W.
ORCID
Venue
Nature Cancer
Publisher
Springer Science and Business Media LLC
Published
2023-08-28
Volume
4
Issue
10
Pages
1437-1454
ISSN
2662-1347 (ISSN-L)
DOI
10.1038/s43018-023-00624-w
PMC
PMC10597839
OpenAlex
W4386225610
Cited by
75
References
61
Citations by year
2026: 29 · 2025: 27 · 2024: 17 · 2023: 2
FWCI
9.09
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Cancer (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • The HKU Scholars Hub (University of Hong Kong) (submittedVersion, cc-by-nc-nd)
Funders
  • Gouvernement du Canada | Canadian Institutes of Health Research (143268, 488053)
  • Cancer Research Institute
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Psychiatry and Mental health › Cancer, Stress, Anesthesia, and Immune Response
Keywords
hepatocellular carcinoma, CD4+ T cells, choline acetyltransferase, antitumor immunity, tumor surveillance, regulatory T cells, PD-1+ T cells, T cell receptor signaling
MeSH terms
Animals, Carcinoma, Hepatocellular, Liver Neoplasms, Monitoring, Immunologic, T-Lymphocytes, Regulatory, Mice, Programmed Cell Death 1 Receptor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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