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

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 Ca2+–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.


Article
https://doi.org/10.1038/s43018-023-00624-w We engineered the CRISPR vector to also express Cre recombinase (Extended Data Fig. 1a).The transposon and Cre-encoding CRISPR vectors were delivered into Rosa26 Confetti/+ mice, animals in which cells stochastically express one of four fluorescent proteins after Cre-mediated recombination 24 .We found that most tumor nodules expressed a single fluorescent marker, suggesting that these malignancies were monoclonal (Extended Data Fig. 1b,c).

Immunosurveillance is elicited in our HCC model
The immune microenvironment shapes the clonal evolution of tumor cells 7,25 .We investigated whether immune responses were evoked during tumorigenesis in our model.We found that hepatic CD4 + T cells and CD8 + T cells expanded as HCC development progressed, whereas the percentage of natural killer T (NKT) cells was reduced (Fig. 1e,f and Extended Data Fig. 1d).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).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).Therefore, immune responses, particularly those mediated by T cells, are activated in our HCC model.
To determine the overall role of the immune system in our model, we induced HCC in severely immunodeficient NOD scid gamma (NSG) mice.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).Thus, immune cells participate in protection against liver cancer development in this setting.

ChAT-expressing T cells are induced during HCC development
Our group has been studying the function of cholinergic T cells in various contexts [26][27][28] .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).In contrast to the extensive cholinergic neural fibers and plexuses in the small intestine, we found no ChAT-expressing neural fibers in either the parenchyma of normal liver or in HCC (Extended Data Fig. 2a), findings in line with earlier reports [15][16][17] .However, we observed accumulation of lymphocyte-like ChAT-expressing cells in HCC (Extended Data Fig. 2a).These ChAT-GFP + CD4 + T cells were comprised mainly of CD44 + activated T cells that significantly increased during HCC progression (Fig. 2a,b).The percentages of ChAT-GFP + CD8 + T cells and NKT cells were also significantly elevated in HCC but to a lesser extent (Fig. 2b).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).The overall level of Chat mRNA in bulk intrahepatic mononuclear cells (MNCs) from HCC-bearing livers was enhanced, reflecting the induction of ChAT-expressing T cells (Extended Data Fig. 2c).
The immune system plays a dual role in liver cancer and can sense and eliminate preneoplastic and malignant hepatocytes 5,6 ; it can also promote the selection of tumor cells and favor cancer progression in situations of chronic inflammation or immunosuppression [7][8][9] .This duality renders current immunotherapies that target immune checkpoints suboptimal for HCC 8 .Further exploration of the molecular determinants of immune responses in liver cancer is necessary to understand HCC biology and guide the design of effective therapeutic strategies.
The nervous system is involved in the development of cancer in multiple tissues [10][11][12] .For example, cholinergic fibers infiltrate prostate tumors, promoting their invasion and metastasis 11 .Engagement of nicotinic acetylcholine (ACh) receptors (nAChRs) mediates lung cancer growth 13 .Vagal innervation promotes gastric tumorigenesis through the M3 muscarinic ACh receptor (mAChR) 10 .Extensive efforts have been directed at delineating the hepatic nervous system.Despite some discrepancies among studies, sympathetic and parasympathetic neural markers have been detected in regions of the hepatic artery, portal vein and bile ducts in the majority of species investigated 14 .However, the liver parenchyma of rodents and humans appears to be devoid of vagal or cholinergic innervation, as determined by immunohistochemistry, retrograde tracing and advanced three-dimensional imaging [15][16][17] .Thus, whether and how cholinergic signaling plays a role in HCC regulation remains an open question.
In this study, we establish that 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.Importantly, we show that genetic ablation of Chat in T cells impairs HCC immunosurveillance.Examination of data from human HCC samples revealed parallels to our mouse findings.Our results demonstrate an unexpected aspect of the regulation of cancer immunosurveillance: mediation by an immune cell-derived neurotransmitter.

Induction of HCC using CRISPR and transposon technology
We sought to model HCC in mice by combining genetic alterations recurrently observed in human disease.These changes included mutation of the TP53 and PTEN tumor suppressor genes and overexpression of the MYC oncogene [18][19][20][21] .CRISPR-mediated somatic knockout of tumor suppressor genes and transposon-based expression of oncogenes induce HCC in mice 6,22,23 .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).These vectors were delivered in combination to mice via hydrodynamic injection, allowing for specific plasmid delivery to hepatocytes.Synergism between Myc expression and Trp53/Pten ablation induced rapid HCC development.Neoplasms were visible on the liver surface by 15 d after injection, with substantial tumor nodules present by day 25 (Fig. 1b).Immunostaining confirmed that the majority of these tumor clones were negative for p53 and PTEN and positive for MYC (Fig. 1c,d).

Transcriptional landscape of cholinergic CD4 + T cells in HCC
To delineate the heterogeneity of cholinergic CD4 + T cells in HCC, we conducted single-cell RNA sequencing (scRNA-seq) on sorted ChAT-GFP + and ChAT-GFP − CD4 + T cells from four control and four HCC-bearing livers.Cells from individual mice were labeled with distinct antibody barcodes, pooled and processed for CITE-seq coupled with TCR-seq.In total, 11 clusters of CD4 + T cells were identified: clusters C1 and C8 were two naive T cell clusters; C2 was composed of T helper 17 (T H 17)  markers; C7 cells expressed Cxcr6 but were negative for Pdcd1; C9 cells were canonical regulatory T (T reg ) cells; C10 cells expressed Eomes, Prf1, Gzmk, Fasl, Gzmb and other cytotoxic genes; and C11 was a minor cluster expressing interferon (IFN)-stimulated genes (Fig. 2c-e).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.
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).Cells in the C3, C4, C9 and C10 clusters were overrepresented among HCC ChAT-GFP + T cells (Fig. 2d).We identified two subsets of Foxp3-expressing T cells, one in C4 and the other in C9.Comparing their transcriptomes, we discerned that the Foxp3 + cells in C4 were T FR cells that overexpressed Bcl6, Tcf7, Gpm6b and other T FR -associated
Cxcr6, a marker for resident T cells in the liver 31 , was enriched primarily in the C3 and C7 clusters.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).These C3 cells showed high expression of inhibitory immunoreceptors and exhaustion markers, such as Pdcd1, Havcr2, Ptpn11, Lag3, Tox and Tigit.These Pdcd1 + T cells also expressed Cd247, the gene encoding PD-L1, potentially providing an autologous ligand for PD-1 binding in addition to the PD-L1 expressed on APCs and HCC cells (Fig. 2f and Extended Data Fig. 2g).By contrast, C7 cells strongly expressed differentiation, cytotoxicity-related and other functional genes, such as Il2ra, Il4, Il2, Fasl, Gzmb and Csf2.These results suggest that Chat expression is associated with the appearance of dysfunctional Pdcd1 + T cells.
Examination of a published scRNA-seq dataset derived from immune cells isolated from individuals with HCC 32 showed that ChAT-expressing T cells, including CD4 + FOXP3 + T reg cells, CD8 + MKI67 + proliferating T cells, CD8 + GZMK + T cells and CD8 + PDCD1 + T cells, were present in liver tumor tissues but not in adjacent normal liver tissues (Extended Data Fig. 3a,b).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.To complement our scRNA-seq results, we profiled ChAT-expressing T cells in mouse HCC by flow cytometry.T reg cell accumulation is an immune hallmark of liver cancer 33,34 .In our model, we observed a marked increase in Foxp3 + ChAT-GFP + T cells alongside expansion of T reg cells during HCC development (Fig. 3a-c).
Histologically, ChAT-GFP + Foxp3 + T reg cells and ChAT-GFP + T conv 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).

Tumor antigens drive cholinergic CD4 + T cell expansion
The proliferation characteristic of ChAT-expressing T cells in HCC livers led us to investigate the driving force behind their expansion.Single-cell TCR-seq revealed dominant TCR types across all animals (as defined by shared amino acid sequences for both the TCRα and TCRβ chains; Fig. 4a,b).Among the top 30 most prevalent TCR types, only 5 were present in control mice, while the remaining 25 were observed in their HCC-bearing littermates.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).These results demonstrate a TCR-specific expansion of ChAT-GFP + T cells in liver cancer.
The most dominant TCR type in HCC was denoted TCR 1, which was encoded by 25 clonotypes (as defined by shared mRNA sequences for both the TCRα and TCRβ chains).These clonotypes consisted of synonymous mRNA variants using the same combination of V, D and J genes.The variations arose from distinct junctions of the V, D and J segments occurring in different HCC-bearing mice, but the resulting amino acid sequences were identical due to codon redundancy (Fig. 4c).The majority of TCR 1 clonotypes were predominantly observed in ChAT-GFP + cells (Fig. 4d).This TCR convergence across HCC-bearing mice suggests that TCRs specific for HCC antigens elicit the expansion of CD4 + T cells, particularly within the ChAT-GFP + CD4 + T cell compartment.
We next characterized the cells bearing particular TCR types.T cells carrying TCR 1 were mainly from the C3 cluster that harbored ChAT-expressing Pdcd1 + T conv cells (Fig. 4d and Extended Data Fig. 4a).T cells carrying TCR 22 were chiefly ChAT-expressing Foxp3 + T reg cells (Extended Data Fig. 4b).These results reveal an accumulation of ChAT-expressing, clonally expanded PD-1 + T conv cells and Foxp3 + T reg cells in HCC.Pertinently, clonal expansion of T reg cells has also been revealed by TCR usage analysis at the single-cell level in human HCC 34 .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).Among the 25 clonotypes of TCR 1, clones 14 and 25 were predominantly C3 cells but ChAT-GFP -, although they carried the same TCR and shared a similar composition of cell clusters with many other ChAT-GFP + clonotypes of TCR 1 (Fig. 4c,d).We postulate that this clonal divergence in ChAT expression likely reflects the complexity of the tumor microenvironment.
To determine the role of tumor antigens in the induction of ChAT-expressing T cells, we crossed Chat-GFP mice with chicken ovalbumin (OVA)-specific TCR transgenic OT-II mice.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).
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.We introduced this vector alongside the CRISPR Trp53/Pten deletion vector into Chat-GFP; OT-II mice (Fig. 4h).When HCC tumors were palpable, doxycycline (Dox) was administered to these mice through their drinking water to induce OVA expression (Fig. 4i).When we compared ChAT expression in Dox-treated and untreated mice, we found that about 40% of OVA-specific (TCR Vβ5 + ) CD4 + T cells expressed ChAT after OVA induction in HCC (Fig. 4j,k).In untreated mice, only about 2% of Vβ5 + CD4 + T cells expressed ChAT, comparable to the percentage in Chat-GFP; OT-II mice bearing OVA - HCC (Fig. 4f,g,j,k).Interestingly, we also observed an elevated percentage of ChAT-GFP + cells among CD4 + T cells carrying natural TCRs (TCR Vβ5 -) (Fig. 4j,k), suggesting that ChAT expression in T cells can also be induced through 'antigen spreading' 35 .
We further characterized the OVA-specific ChAT + T cells in our HCC model with inducible OVA expression and found that Dox-mediated activation of OVA expression markedly induced T reg cells, especially among OVA-specific T cells (Vβ5 + ; Extended Data Fig. 5a).There was also an induction of ChAT-GFP + T cells among both Foxp3 + T reg cells and Foxp3 -T conv cells harboring OVA-specific TCRs (Extended Data Fig. 5a).PD-1 was highly expressed by these OVA-specific T conv cells, and ~40% of these cells were ChAT-GFP + (Extended Data Fig. 5a).Taken together, these data confirm that tumor antigens can induce ChAT-expressing T reg cells and PD-1 + T conv cells and demonstrate that the expansion of ChAT-expressing T cells in liver cancer depends on TCR activation by tumor antigens.

Ablation of Chat in T cells dampens HCC immunosurveillance
To determine the role of ChAT-expressing T cells in the onset of liver cancer, we deleted Chat specifically in T cells by crossing mice carrying    the conditional Chat fl allele with mice expressing the Cd4-cre transgene, thereby obtaining Chat fl/fl ; Cd4-cre progeny.When we subjected these animals (and Chat fl/fl controls) to HCC induction, we found that Chat fl/fl ; Cd4-cre mice developed liver cancer much faster than their Chat fl/fl littermates (Fig. 5a).The numbers of tumor nodules and liver weights were also significantly increased in Chat fl/fl ; Cd4-cre mice (Fig. 5b-e).
To further substantiate the role of ChAT-expressing T cells in liver tumorigenesis, we used an alternative disease model in which Chat fl/fl ; Cd4-cre and Chat fl/fl mice were fed long term on a Western diet (high fat, high cholesterol and high sugar) to induce non-alcoholic steatohepatitis (NASH).NASH sets the stage for liver cirrhosis, which eventually progresses to spontaneous HCC 8,36 .After 15 months on the Western diet, we found that the incidence of NASH-derived HCC was significantly higher in mice bearing T cells lacking Chat (Fig. 5f,g).These consistent results from two models of HCC development establish that a deficiency of Chat in T cells renders mice susceptible to liver tumorigenesis.
Before overt tumor nodules appeared in our HCC model, preneoplastic cells could be identified by their high MYC expression (Fig. 5h), reflecting successful transfection and oncogene expression.The proportion of preneoplastic hepatocytes showing high MYC expression was significantly elevated in livers of Chat fl/fl ; Cd4-cre mice, and this increase was not due to a difference in vector delivery (Fig. 5h,i and Extended Data Fig. 6a-c).Immune cells are critical for clearing preneoplastic cells from the liver 5 .In Chat fl/fl mice, we observed immune cell clusters around MYC-expressing preneoplastic cells (Fig. 5h).These clusters were reduced in frequency and size in Chat fl/fl ; Cd4-cre mice (Fig. 5h,j,k), suggesting a defect in immunosurveillance of preneoplastic cells.Accordingly, T cell infiltration into HCC-bearing livers was significantly decreased in the absence of Chat (Fig. 5l and Extended Data Fig. 6d,e).
IFNγ, a hallmark of the type 1 helper T cell immune response, has a pivotal function in antitumor immunity 37 .We found that IFNγ production by HCC-associated T cells was decreased in Chat fl/fl ; Cd4-cre mice (Fig. 5m-p and Extended Data Fig. 6f).In addition to adaptive immune responses, innate cytotoxic NK cells play a crucial role in antitumor immune response in HCC 38,39 .We observed that mRNA levels of genes encoding IFNγ, granzymes and perforin, which are cytotoxic effectors shared by cytotoxic T cells and NK cells, were significantly decreased in Chat fl/fl ; Cd4-cre mice (Fig. 5p, left).These deficits correlated with reduced expression of NK cell marker genes (Fig. 5p, right).Therefore, both adaptive and innate antitumor immune responses are hampered by the ablation of Chat in T cells.

T reg cells blunt HCC immunosurveillance in Chat fl/fl ; Cd4-cre mice
To delve more deeply into the mechanism of antitumor immunity mediated by ChAT-expressing T cells, we devised a vector mediating the expression of OVA alongside MYC.We introduced this vector plus our Trp53/Pten deletion vector into OVA-immunized and non-immunized mice (Extended Data Fig. 7a).In non-immunized mice, tumor progression to endpoint was significantly accelerated by Chat ablation in T cells (Extended Data Fig. 7b,c), consistent with our previous observations (Fig. 5a).However, OVA-immunized Chat fl/fl and Chat fl/fl ; Cd4-cre mice were equally protected against HCC development (Extended Data Fig. 7b,c).These results showed that the antitumor immune response elicited by a potently immunogenic tumor antigen was not compromised in the absence of ChAT in T cells.In addition, depletion of cytotoxic T lymphocytes (CTLs) by treatment with anti-CD8 had little effect on liver tumor burden (Extended Data Fig. 7d,e).Thus, CTLs do not play a non-redundant role in the immunosurveillance of liver cancer in this setting.
To determine if loss of adaptive immune cells in general would compromise antitumor immunity in our model, we compared the onset of HCC in lymphocyte-deficient Rag1 -/-mice to that in Chat fl/fl ; Cd4-cre mice.In contrast to the significantly increased tumor burden in Chat fl/fl ; Cd4-cre mice, HCC progression was not exacerbated in Rag1 -/-mice but instead was alleviated (Extended Data Fig. 7f,g).We reasoned that this unexpected observation could be attributed to the absence of T reg cells and dysfunctional effector T cells in Rag1 -/-mice.This loss of control by adaptive immune cells (especially T reg cells) causes Rag1 -/-mice to exhibit an excessive innate immune response by NK cells 40 .The fact that HCC progression was aggravated in NSG mice (which lack both adaptive immune cells and NK cells; Fig. 1h) bolsters our contention that the antitumor activity of NK cells in our model is curbed by adaptive immune cells, particularly T reg cells.
To investigate our hypothesis that the accelerated tumor onset linked to T cell-specific Chat ablation could be due to suppression of antitumor responses by elevated T reg cell activity and/or T conv cell dysfunction, we examined how T reg cells modulate the antitumor activity of ChAT-expressing T cells.We observed no difference in the abundance of Foxp3 + T reg cells in liver tumors of Chat fl/fl and Chat fl/fl ; Cd4-cre mice (Extended Data Fig. 8a).However, CD25 expression by Foxp3 + T reg cells in Chat fl/fl ; Cd4-cre mice was substantially increased compared to in control mice (Fig. 6a-c), whereas CTLA-4 expression levels were similar (Extended Data Fig. 8b-d).We then purified CD25 -CD4 + T cells from spleens of Chat fl/fl and Chat fl/fl ; Cd4-cre mice and analyzed CD25 induction following TCR stimulation by anti-CD3/CD28 beads.CD25 expression in Foxp3 + T reg cells from Chat fl/fl ; Cd4-cre mice was significantly higher than in T reg cells from Chat fl/fl mice (Extended Data Fig. 8e).This induction of CD25 by TCR activation was much stronger on T reg cells than on T conv cells (Extended Data Fig. 8e).Therefore, TCR-induced expression of CD25 in T reg cells is modulated by ChAT in T cells.When we used anti-CD25 to deplete CD25-expressing T reg cells (Fig. 6d and Extended Data Fig. 8f), we observed that the enhanced tumor burden in Chat fl/fl ; Cd4-cre mice was partially decreased (Fig. 6e,f).Together, these results point toward the involvement of T reg cells in the suppression of antitumor immune responses in Chat fl/fl ; Cd4-cre mice.
CD25-expressing T reg cells inhibit the antitumor activities of cytotoxic T cells and NK cells 41 .Chat fl/fl ; Cd4-cre mice showed significantly increased expression of CD25 on T reg cells (Fig. 6a-c) and reduced numbers of IFNγ + CD4 + T cells and NK cells (Fig. 5m-p).When we removed CD4 + T cells during HCC induction using anti-CD4 (Fig. 6g and Extended Data Fig. 8g), we observed that CD4 + T cell depletion did not affect HCC progression in Chat fl/fl mice but tended to reduce it in Chat fl/fl ; Cd4-cre mice (Fig. 6h,i).This result suggested that the collective functions of T reg cells and T conv cells in HCC are neutral in Chat fl/fl mice, whereas Chat deletion in T cells tilts the balance toward HCC promotion.Because CD4 + T cell depletion abolished the difference in HCC development between Chat fl/fl mice and Chat fl/fl ; Cd4-cre mice, the independent contributions to HCC of other ChAT-expressing T lineage cells, including CD8 + T cells and NKT cells, appear to be minor.When we used anti-NK1.1 to deplete NK cells from our model (Fig. 6g and Extended Data Fig. 8h), HCC development was promoted in Chat fl/fl mice but not in Chat fl/fl ; Cd4-cre mice, essentially eliminating the differences in tumor progression (Fig. 6h,i).Thus, NK cells are indispensable for HCC immunosurveillance in our model, and the antitumor functions of NK cells are impeded in the absence of cholinergic T cells.

ChAT loss in T cells exacerbates T cell dysfunction in HCC
To investigate the role of ChAT specifically in T reg cells, we crossed Chat fl/fl mice with Foxp3 Cre mice and induced HCC in Foxp3 Cre and Fox-p3 Cre ; Chat fl/fl littermates.We found that deleting Chat only in T reg cells had a milder effect on HCC progression than did deleting Chat in all T cells (Extended Data Fig. 8i,j), suggesting that ChAT-expressing T conv cells are indispensable for a full-fledged anti-HCC immune response.ChAT-expressing T conv 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).PD-1 expression was significantly higher in Foxp3 -CD4 + T conv cells from Chat fl/fl ; Cd4-cre mice than in those from Chat fl/fl mice (Fig. 7a,b).Notably, PD-1 levels strongly correlated with HCC grade in Chat fl/fl ; Cd4-cre mice but not in Chat fl/fl mice (Fig. 7c).We previously reported that loss of ChAT in T cells promotes the expression of PD-1, Tim-3 and Lag-3 during chronic viral infection 27 .
In the present study, both CD4 + T cells and CD8 + T cells in HCC-bearing livers showed a broad trend of upregulation of these inhibitory receptors in the absence of cholinergic T cells (Extended Data Fig. 9a-f).Thus, in the absence of Chat in T cells, an unleashing of PD-1 inhibitory
To test this hypothesis, we administered PD-1 blockade antibodies to Chat fl/fl and Chat fl/fl ; Cd4-cre mice during HCC development (Fig. 7d).As reported for NASH-induced HCC 8 , we did not observe a therapeutic effect of PD-1 blockade in control Chat fl/fl mice, perhaps due to ChAT expression by PD-1 + T conv cells and the negative effects of cholinergic activity on PD-1 expression.However, PD-1 blockade significantly reduced HCC development in Chat fl/fl ; Cd4-cre mice, substantially eliminating the differences in tumor progression (Fig. 7e,f).We did observe two tumor-free animals among anti-PD-1-treated Chat fl/fl ; Cd4-cre mice (Fig. 7f), a status rarely seen for this genotype.These data suggest that ChAT invigorates dysfunctional T cells in HCC.

Cholinergic modulation of TCR-induced Ca 2+ -NFAT signaling
CD25 expression is controlled by the transcription factor NFAT, which is regulated by Ca 2+ signaling 42 .TCR-induced Ca 2+ -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).NFAT-induced transcription factors, including NR4A and TOX, drive T cell exhaustion/dysfunction [44][45][46][47] .We therefore sought to determine if cholinergic activity affects Ca 2+ signaling in T cells.Ca 2+ influx elicited by TCR engagement was significantly stronger in Chat fl/fl ; Cd4-cre T cells than in their Chat fl/fl counterparts (Fig. 8a-c).Furthermore, TCR activation-induced nuclear translocation of NFAT was higher in Chat fl/fl ; Cd4-cre T cells than in Chat fl/fl T cells (Fig. 8d,e).Interestingly, when Chat fl/fl ; Cd4-cre T cells were prestimulated with ACh, NFAT nuclear translocation was no longer inducible by TCR activation (Fig. 8d,e).Thus, cholinergic activity in T cells constrains TCR-induced Ca 2+ -NFAT signaling.
ACh regulates intracellular Ca 2+ levels by binding to nAChRs and mAChRs 48 .We found that T reg cells and T conv cells in livers of HCC-bearing mice expressed similar arrays of AChRs (Fig. 8f).We then applied either a nicotinic agonist or a muscarinic agonist to Chat fl/fl ; Cd4-cre T cells in vitro.Nicotine, which activates nAChRs, had no detectable influence on TCR-induced NFAT translocation.By contrast, oxotremorine methiodide (Oxo-M), an mAChR agonist, by itself induced an increase in NFAT nuclear translocation and abolished subsequent TCR-induced NFAT translocation (Fig. 8d,e).
The G q/11 -coupled M1, M3 and M5 mAChRs elicit Ca 2+ release from the endoplasmic reticulum (ER) by activating the downstream phospholipase C/inositol 1,4,5-trisphosphate/inositol 1,4,5-trisphosphate receptor (PLC/IP 3 /IP 3 R) cascade 49 , the same pathway triggered by TCR signaling.Mobilization of Ca 2+ in T cells is a biphasic event divided into the initial releasing of intracellular ER Ca 2+ stores and the subsequent extracellular Ca 2+ influx from 'Ca 2+ release-activated Ca 2+ ' (CRAC) channels 50 .The CRAC channels can also be triggered by mAChR activation in T cells 51,52 .However, the activation of mAChR depletes the IP 3 -sensitive

Parallels to human HCC
We examined an scRNA-seq dataset 34 and noted that T cells from individuals with HCC expressed CHAT and an array of mAChRs and nAChRs (Extended Data Fig. 10a).To further study this association between cholinergic signaling and human HCC, we examined HCC cases profiled by The Cancer Genome Atlas (TCGA).High expression of CHRM3 or CHRM5 in samples from individuals with HCC was positively correlated with a favorable prognosis (Extended Data Fig. 10b-d).These observations prompted us to use our mouse model to determine if Chrm3 and Chrm5 expressed by HCC cells were acting to directly suppress HCC development.To this end, we designed CRISPR vectors to target M3 and M5 mAChRs and induced Chrm3/Chrm5-knockout HCC.We found that the knockout of M3 and M5 AChRs in mouse HCC cells did not significantly affect HCC development (Extended Data Fig. 10e,f).Therefore, our data do not support the direct suppression of HCC by CHRM3 and CHRM5 in HCC cells.We do acknowledge that there could be compensation by other receptors or interspecies differences.Collectively, our study supports a model (Fig. 8g) whereby tumor antigens induce the expansion of ChAT-expressing T reg cells and PD-1 + T conv cells.ACh produced by these T cells modulates TCR-induced Ca 2+ signaling to prevent its hyperactivity.In the absence of such cholinergic modulation, the Ca 2+ -NFAT pathway becomes hyperactivated to increase immunosuppression by T reg cells and to impose dysfunction on T conv cells, resulting in compromised antitumor immunity.

Discussion
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.Liver T reg cells have a crucial function in the maintenance of this peripheral tolerance state 53 .In the livers of individuals with HCC, T reg cell infiltration is prominently elevated, alongside the exhaustion and impaired function of CD8 + T cells 34,54 .The clonal expansion of T reg cells and exhausted T cells in individuals with HCC has been revealed by scRNA-seq 34 .Using scRNA-seq and flow cytometric analysis, we have demonstrated the induction of Foxp3 + T reg cells and PD-1 + T conv cells in our mouse HCC model and have shown that ChAT-expressing T cells predominantly belong to these two populations.
The clonal expansion of ChAT-expressing T reg cells and PD-1 + T conv cells in our model appears to be driven by tumor antigens.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.Such proteins are postulated to be weakly immunogenic, particularly within the tolerogenic immune milieu in the liver.Indeed, the T cell immune response to HCC antigens is reportedly dysfunctional and unreactive to HCC 55 .Our data suggest that ChAT deficiency in T cells exacerbates the dysfunction of T conv cells and reinforces the immunosuppressive function of T reg cells, significantly compromising immunosurveillance against liver cancer.We used OVA to mimic a 'foreign' tumor antigen by inducing OVA HCC in immunized mice.We expected that HCC onset in this situation would induce a potent antitumor immune response rather than the accumulation of T reg cells and dysfunctional T conv cells.Indeed, OVA HCC development was prevented to the same degree in Chat fl/fl and Chat fl/fl ; Cd4-cre mice.Thus, the cholinergic activity of T cells is most relevant when triggered by 'self' tumor antigens.
Many actions of ACh in the nervous system are mediated through Ca 2+ signaling via either G-protein-coupled mAChRs or ionotropic nAChRs 48 .nAChRs are ACh-gated ionic channels with a variable range of permeability to Ca 2+ .The M1, M3 and M5 mAChRs are coupled with G q/11 and elicit the PLC/IP 3 /IP 3 R cascade to trigger ER Ca 2+ release, whereas M2 and M4 mAChRs are generally linked via G i/o to cAMP production 48 .We demonstrated that these three classes of AChRs are expressed by both T conv cells and T reg cells in HCC-bearing livers.In T lymphocytes, M1 mAChR activation and TCR engagement rely on the same molecular pathway to trigger Ca 2+ influx.Due to this overlap, Ca 2+ signaling events triggered by these pathways are mutually exclusive 52 .In the Premack study, the maximal cholinergic stimulation mediated by overexpressed M1 receptor completely emptied the ER Ca 2+ store, the Ca 2+ pool essential for TCR-induced Ca 2+ signaling.Moreover, the ER Ca 2+ release mediated by cholinergic signaling occurred in a quantal manner: a submaximal concentration of cholinergic agonist rapidly released a fraction of the ER Ca 2+ store, followed by a slower or terminated Ca 2+ release 56,57 .Certain mechanisms, including the inactivation of IP 3 receptors, can be triggered to attenuate ER Ca 2+ release 57,58 .During persistent submaximal cholinergic stimulation, the ER Ca 2+ release elicited by other IP 3 -dependent agonists is dampened 56 .Due to the modest expression levels of AChRs by T cells and the ubiquitous presence of acetylcholinesterase 59 , we speculate that autocrine/paracrine ACh signaling by T cells might operate in such a submaximal way.Accordingly, although we did not observe significant differences in basal levels of Ca 2+ influx and NFAT nuclear translocation between Chat fl/fl and Chat fl/fl ; Cd4-cre T cells, both TCR-induced Ca 2+ influx and NFAT nuclear translocation were elevated in the absence of cholinergic activity in T cells.Moreover, ACh pretreatment inhibited TCR-induced NFAT nuclear translocation.Collectively, these data allow us to propose a model in which autocrine/paracrine ACh produced by ChAT-expressing T cells affects their Ca 2+ homeostasis.This altered Ca 2+ status restrains the expression of CD25 by T reg cells and the expression of PD-1 and other exhaustion markers by T conv cells.In the absence of ChAT in T cells, hyperimmunosuppressive T reg cells and dysfunctional T conv cells interfere with adaptive and innate antitumor responses and permit HCC progression.In conclusion, our study shows that lymphocytes are the dominant cholinergic cells in HCC-bearing livers and that ChAT-expressing T cells orchestrate immune responses against HCC.Our results may prompt investigation of how lymphocyte-mediated cholinergic regulation of liver carcinogenesis can be exploited to enhance antitumor immune responses in individuals with liver cancer.

CRISPR and transposon vectors
Single guide RNAs (sgRNAs) targeting Trp53 and Pten were as previously described 21 .The guide oligonucleotides were designed to express mouse Pten sgRNA and Trp53 sgRNA (Supplementary Table 1).The annealed double-stranded guide oligonucleotides were cloned into the BbsI cut sites of the pX330 vector 60 .The Trp53 sgRNA cassette in pX330-p53 was amplified by PCR with primers (Supplementary Table 1).This additional sgRNA cassette was then cut with NheI and XbaI and subcloned into the NheI site of pX330-Pten to obtain the duplex CRISPR vector pX330-p53-Pten.
The transposon system using SB100X and pT2/BH was as described previously 61 .The mouse Myc coding sequence was cloned into pT2/ BH with EcoR1 and NotI restriction enzymes to obtain the pT2-Myc plasmid.
A modified puromycin-T2A-NLS-Cre version of the pX330 vector was generated.In brief, this vector contained an additional expression cassette under the control of the mouse PGK promoter (driving expression of the puromycin-resistance gene), a T2A self-cleaving peptide flanked by flexible GSG linkers and a P1 bacteriophage Cre recombinase engineered with an N-terminal nuclear localization site and an HSVpA signal.This vector was further modified to express both mouse Pten and Trp53 U6 promoter-driven gRNAs following the process used to build pX330-p53-Pten (see above).
For the pT2-OVA-P2A-Myc plasmid, a 660-base pair (bp) fragment of the cytosolic region (amino acids 173-386, containing both major histocompatibility class I-and II-restricted epitopes) of OVA cDNA was amplified by PCR and engineered with a Kozak consensus methionine and EcoRI and NheI cloning sites using the following primers: OVA_ERI_ U1 and OVA_Nhe1_L1 (Supplementary Table 1).A P2A self-cleaving 2A peptide cassette, flanked by a flexible GSG linker region, was then added in-frame to the 3′ end of the OVA region using NheI and XhoI restriction sites.Finally, the mouse Myc cDNA was subcloned in-frame 3′ of the P2A cassette using PCR primers Cmyc_XhoI_LE_U1 and Cmyc_BstB1_L1 (Supplementary Table 1).This primer set removed the mouse c-Myc start methionine and engineered an additional BstBI site at the 3′ end of mouse Myc that allowed subcloning of the entire Kozak OVA-P2A-Myc cassette into the pT2/BH-CAG-GS-Myc plasmid using EcoRI and BstBI restriction enzyme cloning sites.
To induce Chrm3/Chrm5-knockout HCC, the guide oligonucleotides were designed to express mouse Chrm3 sgRNA and Chrm5 sgRNA (Supplementary Table 1).The annealed double-stranded guide oligonucleotides were cloned into the BbsI cut sites of a modified version of the pX330 vector from which the Cas9 cassette had been removed.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.

Hydrodynamic injections to induce HCC
For delivery of transposon and CRISPR vectors, mice (8-20 weeks old) were injected with a volume of 100 ml per kg (body weight) containing 25 μg of pX330-p53-Pten (or its modified form) plus 0.66 μg of SB100X and 5 μg of pT2-Myc (or its modified forms).The molar ratio of SB100X to pT2-Myc was 1 to 5. Hydrodynamic injection into the lateral tail vein took 5-7 s.Blinding was achieved during injection by putting littermates of different genotypes into new cages lacking mouse information.
To evaluate and quantify HCC development, mice were monitored daily for the appearance of palpable tumors, which were defined as a discernable enlargement of the abdomen.The humane endpoint was reached when one of the following criteria was met: a 10% increase in body weight from baseline, an estimated liver weight of over 5 g determined by the degree of abdominal distension, moribund condition or persistent facial displays of pain and distress.For the survival curve, the exact dates when mice reached the humane endpoint were determined after killing mice bearing significant HCC.Livers collected at this stage were 3-7 g in weight, and the exact endpoints (liver weight over 5 g) were adjusted by 1 d per g (live weight).Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.The number of liver tumors was quantified by counting tumor nodules on the surface of the liver or by normalizing the number of tumor clones to the area of liver sections.Blinding was performed in quantification of liver sections but not surface tumor nodules.
For OVA immunization, mice were injected into the base of their tails with 50 μl of OVA/complete Freund's adjuvant emulsion (Hooke Laboratories, EK-0301).
To induce OVA expression in HCC, HCC was induced in mice by injection of pT2-EF1a-rtTA-P2A-Myc+TRE-OVA.When mice showed a discernable enlargement of the abdomen, they received Dox-containing drinking water (600 mg liter -1 ; Sigma) for 15 d, followed by euthanasia and fluorescence-activated cell sorting analysis of liver cells isolated as described below.

Hepatic MNC isolation
Mice were killed by CO 2 asphyxiation and immediately subjected to whole-body perfusion with ice-cold PBS containing 10 mM EDTA.Liver tissues were collected, disrupted and passed through 70-μm sieves to obtain single-cell suspensions.MNCs were enriched by centrifugation through a 40%/80% Percoll gradient for 20 min at 2,000 r.p.m.
For intracellular cytokine staining, MNCs were stimulated for 4 h with 1 mg ml -1 ionomycin plus 25 μg ml -1 phorbol myristate acetate in the presence of BD GolgiPlug.Cytokine staining was performed with Cytofix/Cytoperm kits (BD) following the manufacturer's instructions.For staining of transcription factors, the eBioscience Foxp3/Transcription Factor Staining Buffer Set was used following the manufacturer's instructions.Anti-GFP Alexa Fluor 488 (Thermo Fisher, A21311) were used to label GFP in intracellular staining analyses.
Flow cytometric analyses were performed using BD LSRFortessa cell analyzers at the Princess Margaret Flow Facility.

scRNA-seq and data analysis
HCC was induced in Chat-GFP mice using pX330-p53-Pten plus SB100X and pT2-Myc.HCC livers (from two male and two female mice) and control livers (from sex-matched littermates) were collected for hepatic MNC isolation on day 26 of HCC induction.Hepatic MNCs were stained with antibodies to CD4, CD8, CD19, TCRβ, NK1.1, CD45 and CD1d tetramer as well as with barcode antibodies (TotalSeq-C0304, C0305, C0306 or C0307) to hashtag cells from individual mice of the HCC or control group.CD45 + DAPI -NK1.1 -CD1dTetramer -CD19 -TCRβ + CD4 + CD8 -GFP + (ChAT-GFP + CD4 + T cells) and CD45 + DAPI -NK1.1 -CD1dTetramer - CD19 − TCRβ + CD4 + CD8 -GFP -(ChAT-GFP -CD4 + T cells) populations were sorted on a FACSAria Fusion cell sorter (BD) at the Princess Margaret Flow Facility, with the two CD4 + T cell compartments individually sorted from each mouse.The same CD4 + T cell compartments from mice receiving the same treatment were pooled into four samples: control ChAT-GFP + , control ChAT-GFP -, HCC ChAT-GFP + and HCC ChAT-GFP -.After sorting and pooling, the samples were immediately submitted to the Princess Margaret Genomics Centre for downstream processing.The four samples were loaded onto a 10x Chromium Controller, and libraries were prepared using a Chromium Next GEM Single Cell 5′ HT Reagent kit v2 (Dual Index, 10x Genomics).The libraries were sequenced on an Illumina NovaSeq 6000 instrument.The sequencing depths were GEX ~50,000 reads per cell, TCR ~5,000 reads per cell and cell hashing TotalSeq C ~2,000 reads per cell.
For scRNA-seq data analysis, sequencing data were processed using Cell Ranger (version 7.0.0)and aligned to the annotated mouse genome (mm10).The Cell Ranger VDJ pipeline was used to call TCR sequences.The clonotype analysis was performed on the merged contig annotations of four samples.The junctions of the V, D and J segments were determined with the IMGT database.The filtered feature barcode matrices in the hierarchical data format (.h5 files) and .csvfiles of filtered contig annotations from the four samples were analyzed with Partek Flow software (version 10.0.23.0214, license from the Centre for PanorOmic Sciences Bioinformatics Core) and analyzed together.With the 'split by feature type' tool, the single-cell counts were split into two data nodes: gene expression and antibody capture.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.Antibody capture was normalized with the recommended method (add 1.0, divide by geometric mean, add 1.0 and log 2.0), and the multiplets and cells with ambiguous hashing were filtered out.These two sets of data were then merged with the 'merge matrices' tool to obtain the filtered, uniquely hashtagged and normalized counts (15,703 cells in total).This counts data node was resplit to generate new gene expression and antibody capture nodes.Dimensionality reduction and visualization were performed on the new gene expression data node using 'PCA' (number of principal components: 100; features contribute: by variance; split by sample: no), 'graph-based clusters' (with default parameters except the resolution was set to 1.0) and 'UMAP' (with default parameters) tools.'Compute biomarkers' was performed on graph-based clustering results to identify marker genes of each cell cluster.Differential analyses were performed using 'GSA' and visualized using heat maps and volcano plots.

Immunohistological analyses
Sections cut from formalin-fixed, paraffin-embedded blocks of mouse livers were used for immunohistochemistry (IHC).After dewaxing and rehydration, endogenous peroxidase was deactivated in 3% hydrogen peroxide (20 ml of 30% hydrogen peroxide + 180 ml of PBS) for 15 min at room temperature.Antigen retrieval with 10 mM sodium citrate buffer (pH 6.0) was performed before immunostaining.

Article
https://doi.org/10.1038/s43018-023-00624-wImmunostained histological sections were scanned using a Nano-Zoomer 2.0-HT slide scanner from Hamamatsu.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.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 .
For the immunofluorescence staining shown in Extended Data Fig. 1f, Alexa Fluor 568-conjugated goat anti-rabbit IgG (Thermo Fisher, A-11036) was used.
For detection of fluorescent proteins in livers of Rosa26 Confetti mice, frozen liver sections were fixed with 2% paraformaldehyde for 8 min at room temperature and directly observed using an Olympus FluoView FV1000 confocal laser-scanning microscope.

Analysis of NFAT nuclear translocation
CD4 + T cells were purified from spleens of Chat fl/fl and Chat fl/fl ; Cd4-cre mice using a CD4 + T Cell Isolation kit (Miltenyi Biotec) and autoMACS following the manufacturer's instructions.Purified CD4 + T cells were resuspended in serum-free RPMI medium at 5 × 10 5 cells per ml.These CD4 + T cell suspensions were incubated with ACh (Sigma), nicotine (Tocris Bioscience) or Oxo-M (Tocris Bioscience) at 37 °C for 15 min.Anti-CD3/CD28 microbeads (Thermo Fisher) were then added to the treated T cell suspensions at a 1:1 bead:cell ratio, and the cells were cultured for another 15 min.After removing the anti-CD3/CD28 microbeads with a magnetic block, CD4 + T cells were fixed and prepared for cytospins with a cytocentrifuge (Thermo Fisher).The cytospins were permeabilized with 1% Triton X-100 for 30 min, blocked with 3% fetal bovine serum, 1% bovine serum albumin and 0.3% Triton X-100 in PBS for 30 min and stained with Alexa Fluor 488-conjugated anti-NFAT1 diluted 1:50 (clone D43B1, Cell Signaling) overnight at 4 °C.After washing and counterstaining with DAPI, the cytospins were mounted with cover slides and examined with a Nikon A1R confocal microscope.Fluorescence micrograms were acquired using a ×40 objective lens.Consecutive images across the diameter of each cytospin (about 16 shots) were collected and quantified.CellProfiler (v4.2.1) was used for the quantification of NFAT nuclear translocation.The minimum cross-entropy thresholding method was used to identify the objects.The MeasureObjectIntensityDistribution module was used for the analysis of NFAT distribution.Briefly, the radial distribution of NFAT staining was measured with three concentric rings (bins) starting from the center of DAPI staining.The ratio of the fraction of total NFAT intensity in the first bin (innermost ring) versus the third bin (outermost ring) was designated as the parameter for NFAT nuclear translocation.The CellProfiler project file will be shared upon inquiry.

Analysis of human HCC datasets
Expression levels of CHAT in various clusters of T cells isolated from tumor tissue and adjacent liver tissue of individuals with HCC were determined by examining the scRNA-seq dataset of immune cells of human HCC previously published by Zhang et al. 32 .Violin plots were generated using their interactive web-based tool (http://cancer-pku.cn:3838/HCC/).Expression levels of CHAT, mAChRs and nAChRs (α-subunits) in T cells isolated from samples from individuals with HCC were determined by extracting data from a published scRNA-seq dataset (GSE98638) on T cells from individuals with HCC 34 .Survival curves of individuals with HCC with high expression of CHRM3 (cutoff was set to log 2 (fragments per kilobase of transcript per million mapped reads upper quartile (FPKM-UQ) + 1) = 11) and CHRM5 (cutoff was set to log 2 (FPKM-UQ + 1) = 9.6) were generated using data obtained from the TCGA Research Network (https://www.cancer.gov/tcga)and analyzed with University of California, Santa Cruz, Xena (http://xena.ucsc.edu/).

Statistics and reproducibility
Pilot experiments were used to estimate the sample size necessary to generate statistically significant results using the appropriate statistical tests.Genetically modified mice and their littermate controls were used for all experiments where possible.For comparing liver weights and nodule numbers, five to ten mice per group were sufficient to achieve statistical significance.For survival curves, a cohort of 10-20 mice per group was used.To account for potential technical failures, including missed hydrodynamic injection and early mortality associated with injection, we usually included an extra 10% of mice per group.Early mortalities (<5 d) were considered to be due to injection-associated death and were removed from the analysis.The numbers of replicates and independent experiments have been stated in the figure legends.The attempts at replication were successful.
To generate statistically appropriate numbers, it was usually necessary to use more than three litters of mice for each experiment.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.This grouping was performed ahead of each experiment.For vector delivery by hydrodynamic injection, blinding was achieved during injection by placing littermates of different genotypes into new cages lacking mouse information.Blinding was also performed for quantitative analyses of liver sections.

Statistical analyses
The Kolmogorov-Smirnov test was used to evaluate normality.Pair-wise comparisons were assessed using two-tailed unpaired Student's t-tests unless otherwise denoted in the figure legends.Data are presented as mean ± s.e.m. unless otherwise indicated.GraphPad Prism 8 and Microsoft Excel (version 2019) were used for the statistical analyses.P values of <0.05 were considered statistically significant.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE231322.The scRNA-seq datasets of human HCC analyzed in this study include those published by Zheng et al. 34 and Zhang et al. 32 2c.e Volcano plot comparing transcripts between Foxp3 mRNA-expressing cells from cluster 4 and those from cluster 9. Gene-set analysis (GSA) was performed to identify sets of differentially expressed genes.f Percentages of Foxp3 mRNA + cells among Chat mRNA + CD4 + T cells and Chat mRNA -CD4 + T cells from HCC-bearing livers as determined from scRNAseq data.Cells from each mouse were identified using antibody barcodes.Each dot represents an individual mouse (n=4 mice per group).Data are the mean ± SEM.P values were determined by unpaired, twotailed t-test.g Representative flow cytometry plots showing the expression of PD-L1 by CD4 + T cells, CD45 + CD3 -immune cells and CD45 -cells from HCC-bearing liver.Related to Fig. 2.

Fig. 1 |
Fig. 1 | Immunosurveillance is present in CRISPR-and transposon-induced HCC in mice.a, Schematic diagrams of the plasmids used to induce CRISPR-Cas9-mediated deletion of Trp53 and Pten (top) and transposon-mediated overexpression of Myc in mouse livers (bottom).b, Representative macroscopic views of livers from mice injected with the combination of plasmids shown in a at the indicated days after injection.Control mice received transposase vector only.Arrowheads indicate tumor nodules.c, Representative histological sections of tumor-burdened livers immunostained to detect MYC, p53 and PTEN.Images are representative of two independent experiments.d, Distribution of immunostained liver tumor nodules from the livers in c.Numbers of tumor nodules with the indicated immunostaining patterns are labeled in the pie plot, which is a summary of two independent experiments.Sections were resected from three mice per group on day 25 of HCC induction.e,f, Representative flow cytometry plots (e) and quantification (f) showing changes in the percentages of the indicated immune cell populations during the development of liver cancer in mice.In f, each dot represents an individual mouse (n = 6 mice per condition).Data are shown as mean ± s.e.m.Significance was assessed by unpaired, twotailed t-test, and data are representative of three independent experiments.Control mice received transposase vector only.g, Representative histological sections from HCC-bearing livers that were immunostained to detect MYC or CD3 in areas of either preneoplastic cells (left) or HCC cells (right).Images represent immunostaining of liver sections from ten mice in one experiment.h, Survival of immunodeficient (NSG) and control wild-type (WT) mice (n = 10 per group) following injection of Trp53/Pten CRISPR and Myc overexpression plasmids to induce HCC development.P = 0.001 by log-rank test.

Fig. 2 |
Fig. 2 | ChAT-expressing T cells are induced during HCC development.a,b, Representative flow cytometry plots (a) and quantification (b) of GFP expression in the indicated T cell subsets during HCC progression in Chat-GFP reporter mice.In b, each dot represents an individual mouse (n = 6 mice per condition).Data are shown as mean ± s.e.m.P values were determined by unpaired, two-tailed t-test, and data are representative of three independent experiments.c,d, Transcriptional landscape of ChAT-GFP + and ChAT-GFP − CD4 + T cells in livers from control mice and mice with HCC.Uniform manifold approximation and projection (UMAP) representation of total hepatic

Fig. 3 |
Fig. 3 | T reg cells and PD-1 + CD4 + T cells are overrepresented among HCCinduced ChAT-expressing T cells.a-d, Representative flow cytometric plots (a) and quantification (b-d) of the percentages of the indicated CD4 + T cell subsets expressing Foxp3 and/or GFP in livers that were isolated on the indicated days from control or HCC-bearing Chat-GFP reporter mice.In b-d, each dot represents an individual mouse (n = 6 per condition).Data are shown as mean ± s.e.m.P values were determined by unpaired, two-tailed t-test, and data are representative of three independent experiments.e,f, Representative flow cytometric plots (e) and quantification (f) of the percentages of the indicated CD4 + T cell subsets expressing PD-1 and/or GFP in livers from control (n = 5) or HCC-bearing (n = 7) Chat-GFP reporter mice.In f, each dot represents an individual mouse (n = 5 mice in control and n = 7 mice in HCC).Data are shown as mean ± s.e.m.P values were determined by unpaired, two-tailed t-test.Data

Fig. 4 |
Fig. 4 | Tumor antigens drive clonal expansion of ChAT-expressing CD4 + T cells in HCC-bearing livers.a,b, Circos plots showing the distribution of TCR types among GFP + and GFP -T cells from control (a) and HCC-bearing (b) mice.T cells of the same TCR type share TCRα and TCRβ chains of the same amino acid sequences.The top 30 TCRs are numbered and highlighted with different colors.c, CDR3 sequences of clonotypes encoding TCR 1. V, D and J segments and N nucleotides and P nucleotides at V(D)J junctions are denoted with different colors.Nucleotides that are mismatched between clonotypes are shaded.d, Composition of ChAT-GFP + and ChAT-GFP -cells among T cells bearing the indicated TCR 1 clonotypes color-coded by cell clusters.Each bar represents an individual clonotype and is labeled with the clone ID as shown in c.Horizontal axis labels indicate cell numbers.e, Percentage of ChAT-GFP + cells among CD4 + T cells from normal or HCC-bearing livers of Chat-GFP or Chat-GFP; OT-II mice (n = 6 in the control, HCC and OT-II control groups; n = 5 in the OT-II HCC group).Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, and data are representative of three independent experiments.f,g, Representative flow cytometry plots (f) and quantification (g) of percentages of ChAT-GFP + cells among CD4 + T cells expressing TCR Vβ5 + (transgenic TCR) or TCR Vβ5 -(natural TCRs) in livers of Chat-GFP; OT-II mice (n = 7 in control; n = 6 in HCC).h, Plasmids for simultaneous CRISPR-Cas9-mediated deletion of Trp53/Pten plus overexpression of Myc and tetracycline-on (Tet-On) inducible OVA.i, Experimental protocol for inducing OVA expression.Dox was added to drinking water following palpable HCC onset; FACS, fluorescence-activated cell sorting.j,k, Representative flow cytometry plots (j) and quantification (k) of percentages of ChAT-GFP + cells among CD4 + T cells expressing TCR Vβ5 + or TCR Vβ5 -in livers of mice that were left untreated or treated with Dox-containing drinking water following HCC onset.Data are the summary of two independent experiments (n = 3 mice per group).In e, g and k, each dot represents an individual mouse.Data are shown as mean ± s.e.m.In g and k, P values were determined by two-way ANOVA with Sidak's multiple comparisons test. Articlehttps://doi.org/10.1038/s43018-023-00624-w

Fig. 5 |
Fig. 5 | Ablation of Chat in T cells inhibits the immunosurveillance of liver cancer in mice.a, Curves showing latency to palpable HCC development (left, black lines) and survival to humane endpoint (right, red lines) of Chat fl/fl (n = 14) and Chat fl/fl ; Cd4-cre (n = 20) mice.P values were determined by log-rank (Mantel-Cox) test, and data are representative of two independent experiments.b-e, Representative images (b), numbers of tumor nodules (c), liver weights (d) and ratios of liver weight (LW) to body weight (BW; e) of mice on day 35 of HCC induction.For c-e, n = 11 mice in the Chat fl/fl group and n = 12 mice in Chat fl/fl ; Cd4-cre group; data are representative of five independent experiments.f,g, Representative images (f) and quantification (g) of tumor incidence in mice fed for 15 months on a Western diet (WD).P values were determined by Fisher's exact test.h,i, Representative histological sections (h) and quantification (i) of immunostaining to detect MYC + preneoplastic cells in non-tumor areas of liver sections from the mice in b-e.In i, n = 11 Chat fl/fl mice and n = 9 Chat fl/fl ; Cd4-cre mice.The inset box in h shows a higher-magnification view of the smaller boxed area.Arrowheads indicate immune cell clusters.j,k, Quantification of the number (j) and size (k) of immune cell clusters in hematoxylin and eosin (H&E)stained sections of livers from the mice in b-e (n = 11 Chat fl/fl mice and n = 9 Chat fl/fl ; Cd4-cre mice).l, Percentage of CD3 + T cells among MNCs isolated from HCCbearing livers as assessed by flow cytometry (n = 11 Chat fl/fl mice and n = 12 Chat fl/fl ; Cd4-cre mice); data are representative of three independent experiments.m-o, Representative flow cytometry plots (m) and quantification of IFNγ + CD4 + (n) and IL-17A + CD4 + T cells (o) in HCC-bearing livers (n = 11 mice per group); data are representative of two independent experiments.p, Quantitative PCR (qPCR) determination of mRNA levels (relative to Actb) of the indicated cytotoxicity genes and NK cell marker genes in HCC-bearing livers (n = 5 mice per group); data are representative of two independent experiments.For c-e, i-l and n-p, each dot represents an individual mouse.Data are shown as mean ± s.e.m., and P values were determined by unpaired, two-tailed t-tests.

Fig. 6 |
Fig. 6 | T cell-specific loss of Chat causes alterations to T reg cells that are linked to compromised antitumor immunity.a-c, Representative flow cytometry plots (a), quantification of the percentages of Foxp3 + CD4 + T cells expressing CD25 (b) and the CD25 mean fluorescent intensity (MFI; c) in HCC-bearing livers from Chat fl/fl and Chat fl/fl ; Cd4-cre mice on day 35 of HCC induction (n = 9 mice per group).P values were determined by unpaired, two-tailed t-test; data are representative of two independent experiments.d, Experimental protocol used to deplete CD25-expressing T reg cells during HCC induction.e,f, Representative low-magnification histological images of H&E-stained sections (e) and quantification of numbers of tumor nodules per mm 2 in these sections (f) of livers from Chat fl/fl and Chat fl/fl ; Cd4-cre mice treated as in d.In f, n = 7 mice in the Chat fl/fl + IgG, Chat fl/fl + anti-CD25 and Chat fl/fl ; Cd4-cre + anti-CD25 groups, and n = 9 mice in the Chat fl/fl + anti-CD25 group.P values were determined by two-way ANOVA with Tukey's multiple comparisons tests.The 'X' symbols indicate animals that reached the humane endpoint before day 20.g, Experimental protocol used to deplete CD4 + T cells and NK cells during HCC induction.Each mouse received 100 μg of depleting antibody per injection.Mice in the control group received either 100 μg of rat IgG or PBS.h,i, Quantification of numbers of tumor nodules per mm 2 in H&E-stained sections (h) and ratios of liver weight to body weight (i) of Chat fl/fl and Chat fl/fl ; Cd4-cre mice treated as in g.The 'X' symbols indicate animals that reached the humane endpoint before day 20.In h, n = 8 mice in the Chat fl/fl + IgG/PBS and anti-CD4 groups; n = 7 mice in the Chat fl/fl ; Cd4-cre + IgG/ PBS group; n = 9 mice in the Chat fl/fl + anti-NK1.1 group; and n = 5 mice in the Chat fl/fl ; Cd4-cre + anti-NK1.1 group.In i, n = 8 mice in the IgG/PBS and anti-CD4 groups; n = 9 mice in the Chat fl/fl + anti-NK1.1 group; and n = 5 mice in the Chat fl/fl ; Cd4-cre + anti-NK1.1 group.In b, c, f, h and i, each dot represents an individual mouse.Data are shown as means ± s.e.m.P values in h were determined by paired, two-tailed t-tests.P values in i were determined by two-tailed Mann-Whitney test because the Chat fl/fl group did not pass the normality test; NS, not significant.

Fig. 7 |
Fig. 7 | PD-1 inhibitory activity is unleashed in the absence of Chat in T cells in HCC.a,b, Representative flow cytometry histogram overlay plot (a) and quantification of the percentages of Foxp3 -CD4 + T conv cells expressing PD-1 (b) in HCC-bearing livers from Chat fl/fl and Chat fl/fl ; Cd4-cre mice.Each dot represents an individual mouse (n = 8 Chat fl/fl mice and n = 9 Chat fl/fl ; Cd4-cre mice).Data are shown as mean ± s.e.m.The P value was determined by unpaired, two-tailed t-test.c, Correlation of PD-1 expression in T conv cells with HCC grade in Chat fl/fl and Chat fl/fl ; Cd4-cre mice.HCC grade is represented by the ratio of liver weight to body weight.P values were determined by two-tailed Pearson correlation.d,

Fig. 8 |
Fig. 8 | TCR-induced Ca 2+ -NFAT signaling is restrained by cholinergic activity in T cells.a-c, Representative overlaid kinetics plots of flow cytometry curves (a), quantification of Ca 2+ influx peaks (b) and 'area under curve' (AUC; c) for the Ca 2+ flux occurring in Chat fl/fl and Chat fl/fl ; Cd4-cre CD4 + T cells.In b and c, each dot represents T cells from an individual mouse.Chat fl/fl and Chat fl/fl ; Cd4-cre littermates of both sexes across various ages were paired for analysis, with at least two measurements taken per mouse.P values were determined by paired, two-tailed t-test; data are a summary of two independent experiments.d,e, Representative fluorescence micrographs (d) and quantification (e) of NFAT immunofluorescent staining of splenic CD4 + T cells purified from Chat fl/fl and Chat fl/fl ; Cd4-cre mice.Ratios of nuclear NFAT to cytoplasmic NFAT are displayed in each image in d and are statistically compared in e.Each dot in e represents one cell (n = 160 control Chat fl/fl T cells; n = 180 anti-CD3-treated Chat fl/fl T cells; n = 101 control Chat fl/fl ; Cd4-cre T cells; and n = 269, 128, 119, 246, 114, 157 and 212 Chat fl/fl ; Cd4-cre T cells treated with anti-CD3, ACh, ACh + anti-CD3, nicotine (Nic), nicotine + anti-CD3, Oxo-M or Oxo-M + anti-CD3).Data are shown as mean ± s.e.m.P values were determined by one-way ANOVA with Tukey's multiple comparisons test and are representative of two independent experiments.f, qPCR determination of mRNA levels (relative to Actb) of the indicated nAChR (Chrna1-Chrna9) and mAChR (Chrm1-Chrm5) genes in T conv and T reg CD4 + T cells sorted from livers of HCC-bearing Foxp3-YFP mice (n = 4); data are representative of three independent experiments.g, Diagram summarizing our proposed model of ChAT function in T cells during HCC.In wild-type mice, HCC antigens induce the expression of ChAT in T cells.Autocrine/paracrine cholinergic signaling by ChAT-expressing T cells influences T cell Ca 2+ homeostasis and regulates TCR-induced Ca2+  signaling.Without such cholinergic modulation (as occurs in Chat fl/fl ; Cd4-cre mice), TCR-induced Ca 2+ signaling is hyperactivated, leading to T cell exhaustion, overexpression of PD-1 in T conv cells and increased CD25 in T reg cells.The inhibitory activity of PD-1 is unleashed, and T reg -mediated suppression is enhanced, compromising antitumor responses mounted by NK cells and T conv cells.In the absence of ChAT, HCC progression proceeds unabated; Ag, antigen; MHC-II, major histocompatibility complex class II.
Article https://doi.org/10.1038/s43018-023-00624-wChat fl/fl ; Cd4-cre Article https://doi.org/10.1038/s43018-023-00624-w . The accession code of the Zheng et al. data is GSE98638.The accession codes of the Zhang et al. data are GSE140228 and EGAS00001003449.The human liver HCC (TCGA-LIHC) data analyzed in this study were derived from the TCGA Research Network (http://cancergenome.nih.gov/).All other data supporting the findings of this study are available from the corresponding author upon reasonable request.Source data are provided with this paper.Extended Data Fig. 1 | The immunosurveillance of murine liver cancer.a Schematic diagrams of the plasmids used to simultaneously induce CRISPR/Cas9-mediated deletion of Trp53 and Pten, Cre expression, and Myc overexpression in Rosa26 Confetti/+ reporter mice.b Representative fluorescence microscopy images depicting (left) a monoclonal and (right) a polyclonal tumor from the livers of the mice in (a).Clonality was determined based on the expression of one or multiple fluorescent markers encoded by the Rosa26 Confetti reporter, representative of one experiment.c Quantification of monoclonal and polyclonal tumors expressing the indicated fluorescent markers in livers from the mice in (a).Numbers of tumor nodules expressing the indicated fluorescent marker(s) are labeled in the pie plot.Data are from examination of 113 tumor nodules in liver sections from 10 Rosa26 Confetti reporter mice.d Gating strategy used for flow cytometric analysis of murine hepatic immune cells.e Percentage of OX40 + CD4 + T cells in livers on the indicated days of liver cancer development.Each dot represents an individual mouse (n=6 mice per group).Data are the mean ± SEM.P values were determined by unpaired, two-tailed t-test.f Representative immunofluorescence images showing CD3 and CD11b expression in adjacent sections of an HCC-bearing liver, representative of one experiment.g Representative macroscopic images (upper) and microscopic images (lower) of tumor-bearing livers resected at the humane endpoint from NSG and control mice subjected to HCC induction, representative of one experiment.Related to Fig. 1.Extended Data Fig. 2 | Identification of cholinergic cells in liver cancer.a Representative histological sections showing GFP immunoreactivity in (left) small intestine, (middle) normal liver, and (right) HCC-bearing liver resected from Chat-GFP mice.Arrowheads, cells with GFP immunoreactivity.Inset boxes show higher magnification views of the GFP + cells in the small boxes.Scale bars, 100 μm.T, tumor tissue.NT, non-tumor tissue.The micrographs displayed are representative of sections obtained from three independent experiments.b Percentage of GFP + cells among the indicated cell subsets in livers of Chat-GFP mice at the indicated time points following HCC induction.Each dot represents an individual mouse (n=6 mice per group).Data are the mean ± SEM. n.s., not significant by unpaired, two-tailed t-test was performed.Results are representative of three independent experiments.c qPCR determinations of Chat mRNA levels (relative to Actb) in hepatic mononuclear cells of Chat-GFP mice at the indicated time points following standard HCC induction.Each dot represents an individual mouse.P values were determined by unpaired, twotailed t-test.d Expression of Foxp3 in CD4 + T cells from control and HCC-bearing livers as determined in the scRNAseq UMAP plot shown in Fig.

Fig. 3 |
Identification of CHAT-expressing T cells in human HCC.a,b Violin plots showing the expression of CHAT in the indicated clusters of T cells that were isolated from tumor tissue (a) and adjacent liver tissue (b) of HCC patients.Data are from a published single-cell RNAseq dataset on immune cells of HCC patients (GSE140228).Related to Fig. 2. Extended Data Fig. 4 | Expression of Chat by clonally expanded PD-1 + Tconvs and Foxp3 + Tregs in HCC. a UMAP representation of Chat-GFP + and Chat-GFP - CD4 + T cells bearing TCR #1 induced in HCC, color-coded by cell clusters and shape-coded by mouse ID. b UMAP representation of Chat-GFP + and Chat-GFP - CD4 + T cells bearing TCR #22 induced in HCC, color-coded by expression level of Foxp3 and shape-coded by mouse ID. c UMAP representation of Chat-GFP + and Chat-GFP -CD4 + T cells bearing TCR #3 induced in HCC, color-coded by cell clusters (upper) and expression levels of Cxcr6, Pdcd1, and Foxp3 (lower), and shape-coded by mouse ID.Related to Fig. 4. Extended Data Fig. 5 | Induction of OVA specific Chat-GFP + Tregs and PD-1 + Tconvs in HCC with inducible OVA expression.a Representative flow cytometry plots showing the expression of Foxp3 and PD-1 in OVA-specific Chat-GFP + CD4 + T cells in livers of mice that were left untreated or treated with Dox-containing drinking water to induce OVA expression following HCC onset, representing two independent experiments.Related to Fig. 4. Extended Data Fig. 9 | Elucidating the effects of Chat on inhibitory immunoreceptor expression by T cells in HCC.a-f Expression levels of the immune inhibitory receptors PD-1, Tim-3 and Lag-3 in CD4 + T cells (a-c), and in CD8 + T cells (d-f), from HCC-bearing livers of Chat fl/fl and Chat fl/fl ;CD4-Cre mice on day 35 of HCC induction.Each dot represents an individual mouse (n=11 mice in Chat fl/fl and n=12 mice in Chat fl/fl ;CD4-Cre).Data are the mean ± SEM.P values were determined by unpaired, two-tailed t-test.Related to Fig. 7. Extended Data Fig. 10 | Cholinergic signaling is associated with human HCC. a Violin plots showing the expression of CHAT and the indicated muscarinic acetylcholine receptors and nicotinic acetylcholine receptors (alpha subunits) in T cells isolated from patient HCC samples (GSE98638).b-d Survival of HCC patients with high expression of CHRM3 (b), CHRM5 (c), or both CHRM3 and CHRM5 (d).Data are from the TCGA database.e,f Ratios of liver weight to body weight (e) and numbers of tumor nodules (f) of Chat fl/fl and Chat fl/fl ;CD4-Cre mice on day 20 of HCC induction with standard vectors or plus vectors carrying gRNAs for Chrm3 and Chrm5.Each dot represents an individual mouse (n=7 mice per group).Data are the mean ± SEM. n.s., not significant, determined by unpaired, two-tailed t-test.β γ μ