1019 CSF1R⁺PD-L1⁺ tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin

Abstract

<h3>Background</h3> 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 influences 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 are assigned crucial roles in regulating immunity and inflammation in the context of infection, albeit their role in HCC remains elusive. <h3>Methods</h3> Study design is displayed in figure 1. High-dimensional flow cytometry (n=37) and scRNA sequencing (n=8) was used to analyze MAIT cell phenotypic changes in patient tissue samples. Highly-multiplexed immunofluorescence microscopy was used to quantify immune cell infiltration in paired human HCC samples. We developed and validated a 37-plex antibody panel and applied CODEX technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in n=15 HCC patient samples using whole slide scanning. We established an image analysis pipeline using a machine learning (ML) algorithm (S³-CIMA) to quantify the MAIT cell interaction network at the HCC invasive front. Murine models of orthotopic HCC using transgenic mouse strains were used for <i>in vivo</i> validation, a co-culture system using sorted MAITs from primary human liver cancer tissue was established to gain further mechanistic insight. <h3>Results</h3> Hepatic MAIT cells in n=37 patient samples are characterized by impaired infiltration (p&lt;0.001) into tumor lesions, increasing dysfunction (e.g. upregulation of PD-1 (p&lt;0.05) &amp; reduced IFN-γ (p&lt;0.01) within the HCC TiME and show previously underappreciated heterogeneity as seen by scRNA-seq. CODEX imaging revealed the distinct cellular composition of the MAIT neighborhood in human HCC tissue. This allowed for in-depth characterization of cellular interaction networks underlying the MAIT cell dysfunction in HCC. S³-CIMA, a novel ML method, to analyze our spatially resolved immune cell atlas of human liver cancer identified interactions of CSF1R⁺PD-L1⁺ tumor-associated macrophages (TAMs) and MAIT cells localized in the adjacent (non-tumor) liver as key regulatory elements of MAIT cell dysfunction. Finally, perturbation of this detrimental cell-cell interaction using PD-L1 and CSF1R blocking strategies or depletion of TAMs using <i>Lysm^Cre x Csf1r^LsL-DTR</i> transgenic mice increased MAIT cell infiltration (p&lt;0.05) into murine HCC lesions and reinvigorated the cytotoxic MAIT cell phenotype (p&lt;0.01). <h3>Conclusions</h3> This work provides evidence that MAIT antitumor immunity and response to ICB therapies relies on organized, spatially nuanced interactions between MAITs and PD-L1+CSF1R+ TAMs within the tumor immune microenvironment. <h3>Acknowledgements</h3> B.R. was supported by the International Liver Cancer Association (ILCA) Fellowship Award 2021, M.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany9s Excellence Strategy – EXC number 2064/1. S.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany9s Excellence Strategy – EXC number 2180. N.K was supported by the Intramural Research Program of the NIH, NCI (ZIC BC 011434). S.W. was funded by the Deutsche Forschungsgemeinschaft (WA-4610/1-1), A.K. acknowledges funding support from the National Institute of Allergy and Infectious Diseases (R01AI132389; R21AI130800). X.W.W. was supported by the Intramural Research program of the NIH, NCI (ZIA BC 010313). T.F.G. was supported by the Intramural Research Program of the NIH, NCI (ZIA BC 011345). <h3>Ethics Approval</h3> All patients gave informed consent for collection of clinical information, tissue acquisition under the Institutional Review Board (IRB)-approved protocol IRB #2017-0365 and the material transfer agreement (M.T.A. #43655-18) between the GUH and the U.S. National Cancer Institute (NCI).