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The gut microbiome is an important modulator of the host immune system. Here, we found that altering the gut microbiome by oral vancomycin increases liver NKT cell function. Enhanced NKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both antigen-specific and antigen-independent in vivo NKT stimulations, with a more prominent effect in liver as compared to the spleen. Fecal transplantation studies demonstrated that the NKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of NKT cell population size. Interestingly, when stimulated in vitro, NKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. NKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing antibody or using genetically deficient mice attenuated the enhanced NKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this NKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTRmice identified CSF-1R+ macrophages as a critical modulator of NKT function. Together, our results demonstrate that the gut microbiome controls liver NKT function via regulating CSF-1R+ macrophages to produce IL-18. Citation Format: Chi Ma, Justin McCallen, John C. McVey, Qianfei Zhang, Benjamin Ruf, Kylynda Bauer, Sophie Wang, Chunwei Walter Lai, Giorgio Trinchieri, Jay A. Berzofsky, Firouzeh Korangy, Tim F. Greten. CSF-1R+ macrophages control the gut microbiome-enhanced liver NKT function through IL-18 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 642.

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L

Introduction: Hepatocellular Carcinoma (HCC) is considered a prototype of inflammation-derived cancer arising from chronic liver injury. The cellular composition of the HCC tumor immune microenvironment (TiME) has a major impact on cancer biology as the TiME can influence tumor initiation, progress, and response to therapy. Mucosal-associated invariant T (MAIT) cells can represent the most abundant T cell subtype in the human liver and have been found to be impaired in both number and function in liver cancer. These innate-like T cells are assigned crucial roles in regulating immunity and inflammation in the context of infection, albeit their role in HCC remains elusive. Methods: High-dimensional flow cytometry was used to analyze MAIT cell phenotypic changes in murine and human liver cancer. Highly multiplexed immunofluorescence microscopy was used to quantify immune cell infiltration in primary human HCC samples. We developed and validated a comprehensive 37-plex antibody panel for immunofluorescence imaging of human fresh frozen HCC samples. We applied co-detection by indexing (CODEX) technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in 15 HCC patient samples using whole slide scanning. Initial image analysis was performed using HALO quantitative image analysis software. Finally, we established an image analysis pipeline to quantify the MAIT cell interaction network at the HCC invasive front. Results: Profiling of human and murine HCC using flow cytometry and highly multiplexed CODEX imaging revealed substantial dysregulation/aberrant activation of MAITs in liver cancer. In situ phenotyping of 4,500,000 single cells (including 1,500,000 CD45+ immune cells) allowed for the quantification of 20 distinct immune cell phenotype clusters, differential analysis of activation markers and spatial features of each individual cell. CODEX imaging revealed detailed composition of the MAIT cell niche in human liver cancer tissue allowing for further distinct spatial analysis including infiltration and nearest-neighbor analysis. Importantly, flow cytometry data of paired samples correlated well with image-based immune phenotyping. Beyond that, whole slide imaging revealed spatial relationships and interactions within the MAIT cell hub localized in distinct tissue regions. Conclusion: Here, we demonstrate that spatially resolved, single-cell analysis of human liver cancer tissue allows for in-depth characterization of interacting immune cellular programs underlying MAIT cell dysfunction in HCC. Citation Format: Benjamin Ruf, Noemi Kedei, Matthias Bruhns, Sepideh Babaei, Bernd Heinrich, Varun Subramanyam, Chi Ma, Simon Wabitsch, Benjamin Green, Kylynda C. Bauer, Yuta Myojin, Jonathan Qi, Amran Nur, Justin McCallen, Layla Greten, William G. Telford, Merrill K. Stovroff, Kesha Oza, Jiman Kang, Alexander Kroemer, Manfred Claassen, Firouzeh Korangy, Tim F. Greten. Spatially resolved immune cell atlas of human liver cancer identifies the cellular interaction network underlying mucosal-associated invariant T (MAIT) cell dysfunction in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2106.

Non-alcoholic fatty liver disease (NAFLD) has become an important etiology leading to liver cancer. NAFLD alters adaptive T cell immunity and has a profound influence on liver cancer development. However, it is unclear how NAFLD affects tumor antigen specific T cell responses. In this study, we generated a doxycycline inducible MHC-I and II antigen expressing HCC cell line which allowed us investigate tumor antigen specific T cell response in two NAFLD mouse models. The system proved to be an effective and efficient way to study tumor antigen specific T cells. Using this model, it was found that NAFLD impairs antigen specific CD8+ T cell immunity against liver cancer. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen specific CD8+ T cells but caused by accumulated macrophages in liver environment. The findings suggest that targeting macrophages in NAFLD driven HCC may improve therapeutic outcomes.