TLR1/2 Ligand−Stimulated Mouse Liver Endothelial Cells Secrete IL-12 and Trigger CD8+ T Cell Immunity In Vitro This information is current as Jia Liu, Min Jiang, Zhiyong Ma, Kirsten K. Dietze, of October 2, 2021. Gennadiy Zelinskyy, Dongliang Yang, Ulf Dittmer, Joerg F. Schlaak, Michael Roggendorf and Mengji Lu J Immunol published online 13 November 2013 http://www.jimmunol.org/content/early/2013/11/12/jimmun ol.1301262 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/11/13/jimmunol.130126 Material 2.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published November 13, 2013, doi:10.4049/jimmunol.1301262 The Journal of Immunology TLR1/2 Ligand–Stimulated Mouse Liver Endothelial Cells Secrete IL-12 and Trigger CD8+ T Cell Immunity In Vitro Jia Liu,* Min Jiang,† Zhiyong Ma,* Kirsten K. Dietze,* Gennadiy Zelinskyy,* Dongliang Yang,‡ Ulf Dittmer,* Joerg F. Schlaak,† Michael Roggendorf,* and Mengji Lu* Liver sinusoidal endothelial cells (LSECs) are unique organ-resident APCs capable of Ag cross-presentation and subsequent tol- erization of naive CD8+ T cells. Under certain conditions, LSECs can switch from a tolerogenic to an immunogenic state and promote the development of T cell immunity. However, little is known about the mechanisms of LSECs to induce T cell immunity. In this study, we investigated whether functional maturation of LSECs can be achieved by TLR ligand stimulation and elucidated the mechanisms involved in LSEC-induced T cell immunity. We demonstrate that pretreatment of LSECs with palmitoyl-3- cysteine-serine-lysine-4 (P3C; TLR1/2 ligand) but not poly(I:C) (TLR3 ligand) or LPS (TLR4 ligand) reverted their suppressive Downloaded from properties to induce T cell immunity. Importantly, P3C stimulation caused functional maturation of Ag-presenting LSECs and enabled them to activate virus-specific CD8+ T cells. The LSEC-mediated CD8+ T cell immunity was initiated by soluble mediators, one of which was IL-12 secreted at a low but sustained level after P3C stimulation. P3C stimulation did not induce programmed death ligand 1 expression on LSECs, thereby favoring T cell proliferation and activation instead of suppression. Our data suggest that LSECs undergo maturation exclusively in response to TLR1/2 ligand stimulation and that the immunological status of LSECs was dependent upon the balance between programmed death ligand 1 and IL-12 expression. These results have implications for our http://www.jimmunol.org/ understanding of liver-specific tolerance and autoimmunity and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections or liver cancer. The Journal of Immunology, 2013, 191: 000–000. oll-like receptors, as evolutionarily conserved, germline- The liver is continuously exposed to food and microbial Ags that encoded pattern recognition receptors (PRRs), play a cru- are transported from the gut via the portal circulation, and it T cial role in early host defense by recognizing so-called exhibits barrier functions toward environmental Ags. Therefore, pathogen-associated molecular patterns (PAMP). Thus, they serve the risk of immune activation in the liver appears quite high in the as an important link between innate and adaptive immunity (1). body. It seems that the liver has in turn acquired special mecha- by guest on October 2, 2021 Ligation of TLRs or cytoplasmic PRRs causes functional matura- nisms of immune tolerance to avoid an overactivation of innate and tion of professional APCs, in particular dendritic cells (DCs) (2). adaptive immune responses (8, 9). The liver is known for its ability This leads to increased expression of costimulatory molecules such to induce Ag-specific immune tolerance rather than immunity as CD80/CD86 and CD40 as well as increased release of IL-12, (10). Liver nonparenchymal cells, including liver sinusoidal en- which are required to promote cytotoxic CD8+ T cell differentia- dothelial cells (LSECs) and Kupffer cells, play a crucial role in tion and activation (3–5). maintaining the homeostasis of the hepatic microenvironment Previously, TLR2 was able to mediate antiviral actions against through induction of Ag-specific T cell tolerance (11–13). Among hepatitis B virus (HBV) and was showntoplayanimportantrolein those different cell populations involved in the induction of he- HBV infection (6). Our previous experiments indicated that TLR2 patic tolerance, LSECs are particularly important because they are may activate various APCs, including liver nonparenchymal cells (7). strategically located in the liver sinusoids to interact with pas- senger leukocytes (14). In addition, LSECs have extraordinary *Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, scavenger function. For example, LSECs take up more hepato- 45122 Essen, Germany; †Department of Gastroenterology and Hepatology, Univer- tropic viruses (such as HBV and hepatitis C virus) from the blood sity Hospital of Essen, University of Duisburg-Essen, 45122 Essen, Germany; and ‡ than other hepatic cells during viral infection (15, 16), and they Department of Infectious Diseases, Union Hospital, Tongji Medical College, + + Huazhong University of Science and Technology, 430022 Wuhan, China are highly efficient in presenting Ags to CD4 and CD8 T cells. + Received for publication May 13, 2013. Accepted for publication October 12, 2013. However, cross-presentation to CD8 T cells by LSECs can in- duce Ag-specific immune tolerance in contrast to professional This work was supported by Deutsche Forschungsgemeinschaft Grants Transregio + TRR60 and GRK1045/2. APCs such as DCs (11). Naive CD8 T cells that are primed by Address correspondence and reprint requests to Prof. Mengji Lu, Institute of Virol- LSECs are initially activated to proliferate and express activation ogy, University Hospital of Essen, Hufelandstrasse 55, 45122 Essen, Germany. markers, like CD69 and CD25, but finally exhibit low IL-2 and E-mail address: [email protected] IFN-g production and low cytotoxicity (11). The induction of The online version of this article contains supplemental material. tolerance correlates with the expression of the negative costimu- Abbreviations used in this article: bmDC, bone marrow–derived dendritic cell; DC, latory molecule ligand programmed death ligand 1 (PD-L1) by dendritic cell; FDR, false discovery rate; FV, Friend virus; HBV, hepatitis B virus; LSEC, liver sinusoidal endothelial cell; P3C, palmitoyl-3-cysteine-serine-lysine-4; LSECs, as LSECs from PD-L1–deficient mice failed to induce + PAMP, pathogen-associated molecular pattern; PD-1, programmed death 1; PD-L1, CD8 T cell tolerance (13). programmed death ligand 1; PRR, pattern recognition receptor; tg, transgenic; However, it is still unclear whether ligation of TLRs or PRRs can WHcAg, woodchuck hepatitis virus core Ag. cause functional maturation of LSECs and revert their suppressive Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 properties to induce T cell immunity. Although it has been reported www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301262 2 ACTIVATION OF LSECs BY TLR1/2 LIGANDS that murine CMV-infected LSECs were able to trigger CD8+ T cell analyzed for activation/proliferation by flow cytometry. For determination of the maturation of LSEC function, corresponding peptide-loaded LSECs or immunity, the authors failed to clarify the mechanisms of the + + LSEC maturation (14). Physiologic exposure of the liver to bac- DCs were cocultured with FV TCR tg CD8 T cells or CD8 Tcellsfrom woodchuck hepatitis virus core Ag (WHcAg)-immunized mice for either 3 terial degradation products is normally not associated with local or 7 d. Blocking Abs were added when indicated. After 3 d, cytokine con- inflammatory responses (17). This suggests that organ-resident APCs centrations in supernatants were assessed by ELISA. After 7 d, intracellular such as LSECs may have evolved the ability to differentiate vari- IFN-g staining was performed to determine the percentage of IFN-g–pro- + + eties of TLR stimulations, and thus perform different sentinel ducing CD8 T cells. The ratio of CD8 T cells to LSECs/DCs was 2:1. functions such as inducing immune tolerance or immunity. There- DNA vaccination fore, we determined whether organ-resident LSECs show plasticity Recombinant eukaryotic expression plasmid pCGWHc, which expresses and switch from a tolerogenic to an immunogenic APC upon PAMP WHcAg, was constructed by our laboratory (A. Kosinska, unpublished stimulation. In this study, we show that LSECs undergo immuno- data). Mice were pretreated with cardiotoxin (Latoxan, Valence, France) by logical maturation