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Unlabelled: The liver is essential for inducing immunological tolerance toward harmless antigens to maintain immune system homeostasis. However, the precise cellular mechanisms of tolerance induction against particle-bound antigens, the role of the local hepatic microenvironment, and implications for therapeutic targets in immune-mediated diseases are currently unclear. In order to elucidate cellular mechanisms of tolerance induction in healthy and injured liver, we developed a novel in vivo system combining the systemic delivery of low-dose peptide antigens coupled to inert particles, immunological readouts, and sophisticated intravital multiphoton microscopy-based imaging of liver in mice. We show that liver resident macrophages, Kupffer cells (KCs), but not hepatic monocyte-derived macrophages or dendritic cells (DCs), are the central cellular scavenger for circulating particle-associated antigens in homeostasis. KC-associated antigen presentation induces CD4 T-cell arrest, expansion of naturally occurring Foxp3(+) CD25(+) interleukin-10-producing antigen-specific regulatory T cells (Tregs) and tolerogenic immunity. Particle-associated tolerance induction in the liver protected mice from kidney inflammation in T-cell-mediated glomerulonephritis, indicating therapeutic potential of targeting KC for immune-mediated extrahepatic disorders. Liver inflammation in two independent experimental models of chronic liver injury and fibrosis abrogated tolerance induction and led to an immunogenic reprogramming of antigen-specific CD4 T cells. In injured liver, infiltrating monocyte-derived macrophages largely augment the hepatic phagocyte compartment, resulting in antigen redistribution between myeloid cell populations and, simultaneously, KCs lose signature markers of their tolerogenic phenotype., Conclusions: Hepatic induction of tissue-protective immunological tolerance against particulate antigens is dependent on KCs as well as on a noninflamed liver microenvironment, thereby providing mechanistic explanations for the clinical observation of immune dysfunction and tolerance break in patients with advanced liver diseases., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: Immunity against cancer is impeded by local mechanisms promoting development of tumor-specific T cell tolerance, such as regulatory T cells, myeloid-derived suppressor cells, or immunosuppressive factors in the tumor microenvironment. The release of soluble antigens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, has been investigated for diagnostic purposes, but not for its immunological consequences. Here, we address the question of whether soluble CEA influences tumor-specific immunity. Mice were injected with soluble CEA protein, and CEA-specific CD8 T cells were analyzed for their phenotype and functionality by means of restimulation ex vivo or antitumor efficacy in vivo. We furthermore characterized the CD8 T cell population in peripheral blood mononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. In mice, circulating CEA was preferentially taken up in a mannose receptor-dependent manner and cross-presented by liver sinusoidal endothelial cells, but not dendritic cells, to CD8 T cells. Such systemically circulating CEA promoted tolerization of CEA-specific CD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1. These CD8 T cells were not deleted but were rendered nonresponsive to antigen-specific stimulation and failed to control growth of CEA-expressing tumor cells. These nonresponsive CD8 T cells were phenotypically similar to central memory T cells being CD44(high) CD62L(high) CD25(neg) . We found T cells with a similar phenotype in PBMCs of healthy donors and at increased frequency also in patients with colorectal carcinoma., Conclusion: Our results provide evidence for the existence of an unrecognized tumor immune escape involving cross-presentation of systemically circulating tumor antigens that may influence immunotherapy of cancer., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Unlabelled: The liver has a role in T cell tolerance induction, which is mainly achieved through the functions of tolerogenic hepatic antigen-presenting cells (APCs) and regulatory T cells. Hepatic stellate cells (HSCs) are known to have various immune functions, which range from immunogenic antigen presentation to the induction of T cell apoptosis. Here we report a novel role for stellate cells in vetoing the priming of naive CD8 T cells. Murine and human HSCs and stromal cells (but not hepatocytes) prevented the activation of naive T cells by dendritic cells, artificial APCs, and phorbol 12-myristate 13-acetate/ionomycin by a cell contact-dependent mechanism. The veto function for inhibiting T cell activation was directly correlated with the activation state of HSCs and was most pronounced in HSCs from fibrotic livers. Mechanistically, high expression levels of CD54 simultaneously restricted the expression of interleukin-2 (IL-2) receptor and IL-2 in T cells, and this was responsible for the inhibitory effect because exogenous IL-2 overcame the HSC veto function., Conclusion: Our results demonstrate a novel function of HSCs in the local skewing of immune responses in the liver through the prevention of local stimulation of naive T cells. These results not only indicate a beneficial role in hepatic fibrosis, for which increased CD54 expression on HSCs could attenuate further T cell activation, but also identify IL-2 as a key cytokine in mediating local T cell immunity to overcome hepatic tolerance., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: In vivo evaluation of CD8 T cell effector (cytotoxic T lymphocyte [CTL]) function in peripheral organs such as the liver is currently not possible but would greatly improve our understanding of local immune regulation, because simple determination of antigen-specific CTL numbers does not predict the outcome of immune responses. In particular, measurement of alanine aminotransferase serum levels is not sensitive enough to detect T cell immunity against low numbers of target hepatocytes. We developed a procedure that detects virus-specific effector function of CTLs in the liver after simultaneous adenoviral transfer of reporter and immune target genes into hepatocytes, followed by bioluminescence imaging of reporter genes. Bioluminescence imaging enabled detection of as few as 10,000 infected hepatocytes in vivo, and even more importantly, quantification of antiviral effector function of as few as 50,000 CTLs., Conclusion: Our results provide evidence that low numbers of antigen-specific CTLs are sufficient to control viral gene expression and eliminate viral infection from hepatocytes. The experimental system established here is a highly sensitive method to simultaneously detect viral infection of hepatocytes and to quantify antiviral CTL function in the liver in vivo and will help in characterizing principles of hepatic immune regulation.

Unlabelled: Cross-presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ-resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross-presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross-presenting circulating soluble antigen ex vivo as DCs at a per-cell basis. However, antigen uptake in vivo was 100-fold more pronounced in LSECs, indicating distinct mechanisms of cross-presentation. In contrast to mannose-receptor-mediated antigen uptake and routing into stable endosomes dedicated to cross-presentation in DCs, we observed distinct antigen-uptake and endosomal routing with high antigen turnover in LSECs that resulted in short-lived cross-presentation. Receptor-mediated endocytosis did not always lead to cross-presentation, because immune-complexed antigen taken up by the Fc-receptor was not cross-presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity., Conclusion: These results provide a mechanistic explanation how organ-resident LSECs accommodate continuous scavenger function with the capacity to cross-present circulating antigens using distinct kinetics and dynamics of antigen-uptake, routing and cross-presentation compared to DCs.

The reason the adaptive immune system fails in advanced liver tumors is largely unclear. To address this question, we have developed a novel murine model that combines c-myc-induced autochthonous tumorigenesis with expression of a cognate antigen, ovalbumin (OVA). When c-myc/OVA transgenic mice were crossed with liver-specific inducer mice, multifocal hepatocellular carcinomas co-expressing OVA developed in a tetracycline-dependent manner with a short latency and 100% penetrance. Transferred OVA-specific T cells, although infiltrating the tumor at high numbers, were hyporesponsive, as evidenced by a lack of in vivo cytotoxicity and interferon gamma production. This allowed the tumor to progress even in the presence of large numbers of antigen-specific T cells and even after vaccination (OVA+CpG-DNA). Interestingly, T cell receptor down-modulation was observed, which may explain antigen-specific hyporesponsiveness. This model is helpful in understanding liver cancer-specific mechanisms of T cell tolerance and dissection of antigen-specific and nonspecific mechanisms of immunotherapies in the preclinical phase.