Abstract Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity, successful anticancer vaccination and therapy. We have discovered a novel molecular mechanism operating in antigen donor cells, which regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We show that cellular peptidases, dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1) present in non-immunogenic necrotic cells eliminate proteasomal degradation products and block antigen cross-presentation. While sterile necrotic tumor cells fail to induce CD8+ T cell responses, their non-immunogenicity can be reversed in vitro and in vivo by inactivation of peptidases, DPP-3 and TOP-1. Thus control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on the proteasome dependent oligopeptide generation and functional status of peptidases in antigen donor cells. Citation Format: Jaba Gamrekelashvili, Tamar Kapanadze, Josef Wissing, Chi Ma, Lothar Jaensch, Firouzeh Korangy, Tim F. Greten. Cross-priming of CD8+ T cells is controlled by dipeptidyl peptidase 3 and thimet oligopeptidase 1 present in necrotic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B30.
Abstract The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent in vivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated in vitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti–CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function in vivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.
Meeting abstracts Myeloid-derived suppressor cells (MDSC) represent a heterogeneous population of immature myeloid cells that accumulate in blood, liver, spleen and tumors upon chronic inflammation and tumor development in patients and mice. Acute hepatitis is characterized by a fast infiltration
Hepatocellular carcinoma (HCC) represents the third most common cause of cancer-related deaths worldwide and efficient treatment options are urgently needed. Based on its pathogenesis as well as a number of correlative studies, immunotherapy represents a potential therapeutic option for patients with HCC. However, tumors have also evolved numerous immune escape mechanisms, such as the generation of cells with immune suppressor functions, including regulatory T cells and myeloid-derived suppressor cells. It has been shown that these suppressor cells mask tumor-specific immune responses in patients with HCC. We propose that targeting suppressor cells either alone or in combination with conventional immunotherapy should be further evaluated in HCC patients.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Conventional cytotoxic chemotherapy has failed to show a substantial benefit for patients with HCC. Recently, a number of new drugs targeting molecular mechanisms involved in liver cell transformation have entered into clinical trials and led to encouraging results. In this review we summarise this data and point to a number of new compounds, which are currently being tested and can potentially broaden our therapeutic arsenal even further.
Innate lymphoid cells (ILC) are a heterogeneous and plastic population of cells of the innate immune system. Their role in cancer and specifically in hepatocellular carcinoma is unraveling. The presence of ILCs in peripheral blood of HCC patients has not been explored yet. Their role and function in response to checkpoint inhibitor therapy have also not been explored. Here, we characterized ILCs in PBMC of HCC patients at baseline and after treatment with immune checkpoint inhibitors (ICI) by flow cytometry and single-cell sequencing. Characterization of ILC subsets in PBMCs of HCC patients showed a significant increase in ILC1 and a decrease in ILC3 frequencies. Single-cell RNA-sequencing identified a subgroup of NK-like ILCs which expressed cytotoxicity markers as well as NKp80/ KLRF1 . This KLRF1 high NK-like population showed low abundance in patients with HCC and was enhanced after combined anti-CTLA-4 and anti-PD-1immunotherapy. Trajectory analysis placed this population in between ILC1 and ILC3 cells. The transcriptomic signature of KLRF1 high NK-like ILCs was associated with better progression-free survival in large HCC cohorts. This study shows a previously unknown effect of ICI on the composition and plasticity of ILCS in peripheral blood. Thus, ILCs from PBMC can be used to study changes in the innate immune system under immunotherapy.
Liver cancer remains the third leading cause of worldwide cancer-related deaths.1 Despite clinical advances, established immunotherapies largely fail patients due to poor immune responses.1 2 Peripheral nerves influence tumors, but brain-liver interactions remain largely unstudied.3 A recent study reported a decreased risk of liver cancers in patients that underwent truncal vagotomy (vagus snip) compared to simple suture procedure.4 Here, we identify a vagal-immune arc regulating hepatic cancer.
Methods
C57BL/6 or BALB/c mice (9–12 weeks old) underwent a surgical hepatic vagotomy (HV) or sham procedure (SV). Following established protocols,5 HV and SV mice received orthotopic tumors via intrahepatic injection (2.5x105 RIL-175 or B16-F10 cells) or tail vein/flank injection (1.0x106 A20, RIL-175 cells) to model primary and metastatic liver cancer. Growth of luciferase-labeled cells was measured via in vivo imaging and immune profiling was conducted via flow cytometry and scRNASEQ. Open field test, Y maze, and phenotyper cages assessed mouse behavior. Highly-multiplexed immunofluorescence (CODEX platform) revealed peripheral nerves in clinical resection samples.
Results
Precise liver denervation reduced tumor growth in three models of primary (RIL-175) and metastatic (B16-F10, A20) tumors (figure 1A). Outcomes remained organ specific as HV mice exhibited reduced tumor burden of intrahepatic, but not subcutaneous, models. HV livers exhibited decreased levels of vagal neurotransmitter acetylcholine (ACh). As immunofluorescent analyses revealed colocalization of peripheral nerves and lymphocytes in clinical liver cancer, we profiled HV immunity. HV livers exhibited broad anti-tumor immunity, notably increased CD8+ T cells and higher expression of intracellular cytokines (IFNγ, TNFα) (figure 1B). We then examined whether immune alterations were a cause or consequence of HV tumor burden. ACh exposure reduced intracellular cytokine levels in ex vivo CD8+ T cells following anti-CD3/CD8 activation. Treatment with bethanechol (ACh receptor agonist, 400 μg/mL drinking water) increased tumor burden and reduced CD8+TNFα+ subsets. Bethanechol failed to promote tumor growth in Rag1KO mice lacking mature B and T cells, and targeted depletion of CD8+ T cells abrogated the effects of vagotomy (figure 1C). Finally, as the vagus nerve is largely comprised of afferent fibers, we assessed murine behavior and ambulation. Tumor-bearing HV mice displayed decreased anxiety-life features and fatigue compared to sham controls.
Conclusions
Our findings highlight a vagal-CD8+ T cell axis modulating hepatic tumor burden and behavior. This work furthers the emerging field of cancer neuroscience and identifies ACh signaling targets to alter hepatic immunosuppression and cancer outcomes.
References
Kim E, Viatour P. Hepatocellular carcinoma: old friends and new tricks. Exp Mol Med. 2020;52:1898–1907. Greten T, et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of hepatocellular carcinoma. 2021. JITC. 2021;9:e002794. Monje M, et al. Roadmap for the Emerging Field of Cancer Neuroscience. Cell. 2020;181:219–222. Wu S, et al. Decreased risk of liver and intrahepatic cancer in non-H. pylori-infected perforated peptic ulcer patients with truncal vagotomy: a nationwide study. Sci Rep. 2021;11:e15594. Brown Z, Heinrich B, Greten T. Establishment of orthotopic liver tumors by surgical intrahepatic tumor injection in mice with underlying non-alcoholic fatty liver disease. Methods protoc, 2018;2:e21.
Ethics Approval
This research was approved by the NCI Division of Intramural Research Animal Care and Use Committee, proposal numbers: MOB-028 and TGOB-015. Patients provided informed consent for clinical tissue acquisition: Institutional Review Board protocol #2017–0365.
Abstract Liver cancer ranks amongst the deadliest cancers. Nerves have emerged as an understudied regulator of tumor progression. The parasympathetic vagus nerve influences systemic immunity via acetylcholine (ACh). Whether cholinergic neuroimmune interactions influence hepatocellular carcinoma (HCC) remains uncertain. Liver denervation via hepatic vagotomy (HV) significantly reduced liver tumor burden, while pharmacological enhancement of parasympathetic tone promoted tumor growth. Cholinergic disruption in Rag1KO mice revealed that cholinergic regulation requires adaptive immunity. Further scRNA-seq and in vitro studies indicated that vagal ACh dampens CD8+ T cell activity via muscarinic ACh receptor (AChR) CHRM3. Depletion of CD8+ T cells abrogated HV outcomes and selective deletion of Chrm3 on CD8 + T cells inhibited liver tumor growth. Beyond tumor-specific outcomes, vagotomy improved cancer-associated fatigue and anxiety-like behavior. As microbiota transplantation from HCC donors was sufficient to impair behavior, we investigated putative microbiota-neuroimmune crosstalk. Tumor, rather than vagotomy, robustly altered fecal bacterial composition, increasing Desulfovibrionales and Clostridial taxa. Strikingly, in tumor-free mice, vagotomy permitted HCC-associated microbiota to activate hepatic CD8+ T cells. These findings reveal that gut bacteria influence behavior and liver anti-tumor immunity via a dynamic and pharmaceutically targetable, vagus-liver axis.
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