Bastian Höchst

Functional classification of memory CD8(+) T cells by CX3CR1 expression.

Localization of memory CD8+ T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX3CR1 distinguishes memory CD8+ T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8+ T cells with effector function. We find CD62LhiCX3CR1+ memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1+ memory CD8+ T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8+ T-cell memory.

Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion

BACKGROUND & AIMS Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice.

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 CD44highCD62LhighCD25neg. 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. (HEPATOLOGY 2012;56:1924–1933)

Differential Induction of Ly6G and Ly6C Positive Myeloid Derived Suppressor Cells in Chronic Kidney and Liver Inflammation and Fibrosis

CD11b+Gr1+ myeloid derived suppressor cells (MDSC) are known to be very potent suppressors of T cell immunity and can be further stratified into granulocytic MDSC and monocytic MDSC in mice based on expression of Ly6G or Ly6C, respectively. Here, using these markers and functional assays, we aimed to identify whether MDSC are induced during chronic inflammation leading to fibrosis in both kidney and liver and whether additional markers could more specifically identify these MDSC subsets. In an adenine-induced model of kidney inflammation/fibrosis suppressive Ly6Gpos MDSC were induced. The suppressive function within the Ly6G+ MDSC population was exclusively present in IFNγRβ expressing cells. In contrast, in chronic inflammation in the liver induced by bile duct ligation, suppressive capacity was exclusively present in the Ly6Cpos MDSC subset. Gene expression analyses confirmed the differential origins and regulation of those MDSC subsets. Additionally, depletion of MDSC in either kidney or liver fibrosis enhanced fibrosis markers, indicating a protective role for MDSC in organ fibrosis. Thus, our data demonstrate that during liver inflammation and kidney fibrosis MDSC with similar function arise bearing a distinct marker profile and arising from different cell populations.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

The induction of human myeloid derived suppressor cells through hepatic stellate cells is dose-dependently inhibited by the tyrosine kinase inhibitors nilotinib, dasatinib and sorafenib, but not sunitinib

Abstract Increased numbers of immunosuppressive myeloid derived suppressor cells (MDSCs) correlate with a poor prognosis in cancer patients. Tyrosine kinase inhibitors (TKIs) are used as standard therapy for the treatment of several neoplastic diseases. However, TKIs not only exert effects on the malignant cell clone itself but also affect immune cells. Here, we investigate the effect of TKIs on the induction of MDSCs that differentiate from mature human monocytes using a new in vitro model of MDSC induction through activated hepatic stellate cells (HSCs). We show that frequencies of monocytic CD14+HLA-DR−/low MDSCs derived from mature monocytes were significantly and dose-dependently reduced in the presence of dasatinib, nilotinib and sorafenib, whereas sunitinib had no effect. These regulatory effects were only observed when TKIs were present during the early induction phase of MDSCs through activated HSCs, whereas already differentiated MDSCs were not further influenced by TKIs. Neither the MAPK nor the NFκB pathway was modulated in MDSCs when any of the TKIs was applied. When functional analyses were performed, we found that myeloid cells treated with sorafenib, nilotinib or dasatinib, but not sunitinib, displayed decreased suppressive capacity with regard to CD8+ T cell proliferation. Our results indicate that sorafenib, nilotinib and dasatinib, but not sunitinib, decrease the HSC-mediated differentiation of monocytes into functional MDSCs. Therefore, treatment of cancer patients with these TKIs may in addition to having a direct effect on cancer cells also prevent the differentiation of monocytes into MDSCs and thereby differentially modulate the success of immunotherapeutic or other anti-cancer approaches.

Targeting myeloid derived suppressor cells with all-trans retinoic acid is highly time-dependent in therapeutic tumor vaccination

ABSTRACT Tumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action. Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival. When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after ex vivo treatment with TLR-ligation. Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.

Checkpoint Inhibition in Head and Neck Cancer: Immune Therapeutic Options, Limitations, and Beyond

The immune system functions to defend the organism against infectious microorganisms but also against transformed cells. This key role of the immune system, in particular cancer-specific T cells, in eliminating cancer cells is compromised by various immune escape strategies employed by cancer cells and the cancer microenvironment. Here, we review the current knowledge about the immune escape mechanisms of cancer and the attempts to reconstitute cancer-specific immunity by using checkpoint inhibitors in head and neck squamous cell carcinoma. We discuss the different options of immune therapy based on a mechanistic understanding of the relevance of co-inhibitory signaling, regulatory T cells, and myeloid-derived suppressor cells. A thorough mechanistic understanding of cancer immune escape mechanisms and their presence in the individual patient is required in order to design effective multicomponent immune therapies in the future.

Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions

Background Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH), and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1) on liver regeneration in a subseptic setting. Methods Wild-type (WT) and LFA-1-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS) application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined. Results Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3+ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3+NK1.1+ NKT cells in the liver parenchyma of LFA-1-/- mice after PH and LPS application compared to WT controls, while CD3-NK1.1+ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice. Conclusion A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration.