Smad7 Deficiency in Myeloid Cells Does Not Affect Liver Injury, Inflammation or Fibrosis after Chronic CCl4 Exposure in Mice.

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-ß) is pivotal for scarring and fibrosis induction and progression. TGF-ß signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-ß-signalling pathway, rendering cells that express high levels of it resistant to TGF-ß-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-ß-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

Pioglitazone-Mediated Peroxisome Proliferator-Activated Receptor γ Activation Aggravates Murine Immune-Mediated Hepatitis.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPARγ activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPARγ activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPARγ activation by the agonist Pioglitazone on the function of hepatic immune cells and its effect in a murine model of immune-mediated hepatitis. Cytokine production by both liver sinusoidal endothelial cells (IL-6) and in T cells ex vivo (IFNγ) was decreased in cells from Pioglitazone-treated mice. However, PPARγ activation did not decrease pro-inflammatory tumor necrosis factor alpha TNFα production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPARγ activation was shown to ameliorate chronic inflammatory diseases, it did not improve hepatic injury in a model of immune-mediated hepatitis. In contrast, Pioglitazone-induced PPARγ activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNFα by Kupffer cells and increased sensitivity of hepatocytes towards TNFα after in vivo Pioglitazone administration. These results unravel liver-specific effects of Pioglitazone that fail to attenuate liver inflammation but rather exacerbate liver injury in an experimental hepatitis model.

Carcinoembryonic antigen-related cell adhesion molecule 1 controls IL-2-dependent regulatory T-cell induction in immune-mediated hepatitis in mice.

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4+ T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1-/- mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic forkhead box protein P3+ (Foxp3+ )CD4+ Treg numbers. CEACAM1-/- CD4+ T cells showed impaired IL-2-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation, which correlated with a failure of naïve CEACAM1-/- CD4+ T cells to convert into Tregs in vitro. Furthermore, CEACAM1-/- Tregs expressed reduced levels of Foxp3, CD25, and B-cell lymphoma 2. Adoptive transfer experiments demonstrated that hepatic Treg expansion and suppressive activity required CEACAM1 expression on both CD4+ T cells and Tregs. We identified predominant CEACAM1S expression on hepatic CD4+ T cells and Tregs from mice with acute liver injury and expression of both isoforms in liver-derived CD4+ T-cell clones from patients with liver injury. CONCLUSION: Our data suggest that CEACAM1S expression in CD4+ T cells augments IL-2 production and STAT5 phosphorylation leading to enhanced Treg induction and stability, which, ultimately, confers protection from T-cell-mediated liver injury. (Hepatology 2018;68:200-214).

Acute Liver Injury after CCl4 Administration is Independent of Smad7 Expression in Myeloid Cells.

Myeloid cells are essential for the initiation and termination of innate and adaptive immunity that create homeostasis in the liver. Smad7 is an inhibitor of the transforming growth factor ß (TGF-ß) signaling pathway, which regulates inflammatory cellular processes. Knockdown of Smad7 in hepatocytes has been shown to promote liver fibrosis, but little is known about the effects of Smad7 in myeloid cells during inflammatory responses in the liver. Using mice with a myeloid-specific knockdown of Smad7 (LysM-Cre Smad7fl/fl), we investigated the impact of Smad7 deficiency in myeloid cells on liver inflammation and regeneration using the well-established model of CCl4-mediated liver injury. Early (24/48 h) and late (7 d) time points were analyzed. We found that CCl4 induces severe liver injury, with elevated serum ALT levels, centrilobular and periportal necrosis, infiltrating myeloid cells and an increase of inflammatory cytokines in the liver. Furthermore, as expected, inflammation peaked at 24 h and subsided after 7 d. However, the knockdown of Smad7 in myeloid cells did not affect any of the investigated parameters in the CCl4-treated animals. In summary, our results suggest that the inhibition of TGF-ß signaling via Smad7 expression in myeloid cells is dispensable for the induction and control of acute CCl4-induced liver injury.

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.

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.

CD44 expression on murine hepatic stellate cells promotes the induction of monocytic and polymorphonuclear myeloid-derived suppressor cells.

In chronic inflammation, regulatory immune cells, such as regulatory T cells and myeloid-derived suppressor cells, can develop. Local signals in the inflamed tissue, such as cytokines and eicosanoids, but also contact-dependent signals, can promote myeloid-derived suppressor cell development. In the liver, hepatic stellate cells may provide such signals via the expression of CD44. Myeloid-derived suppressor cells generated in the presence of hepatic stellate cells and anti-CD44 antibodies were functionally and phenotypically analyzed. We found that both monocytic and polymorphonuclear myeloid-derived suppressor cells generated in the presence of αCD44 antibodies were less suppressive toward T cells as measured by T-cell proliferation and cytokine production. Moreover, both monocytic and polymorphonuclear myeloid-derived suppressor cells were phenotypically altered. Monocytic myeloid-derived suppressor cells mainly changed their expression of CD80 and CD39, and polymorphonuclear myeloid-derived suppressor cells showed altered expression of CD80/86, PD-L1, and CCR2. Moreover, both polymorphonuclear and monocytic myeloid-derived suppressor cells lost expression of Nos2 messenger RNA, whereas monocytic myeloid-derived suppressor cells showed reduced expression of TGFb messenger RNA and polymorphonuclear myeloid-derived suppressor cells reduced expression of Il10 messenger RNA. In summary, the presence of CD44 in hepatic stellate cells promotes the induction of both monocytic and polymorphonuclear myeloid-derived suppressor cells, although the mechanisms by which these myeloid-derived suppressor cells may increase suppressive function due to interaction with CD44 are only partially overlapping.

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

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.

Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

Murine hepatic stellate cells veto CD8 T cell activation by a CD54-dependent mechanism.

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.

Four-and-a-half LIM domain protein 2 is a novel regulator of sphingosine 1-phosphate receptor 1 in CCL19-induced dendritic cell migration.

We identified the four-and-a-half LIM domain protein 2 (FHL2) as a novel regulator of CCL19-induced dendritic cell (DC) migration. Initiation of migration is a hallmark of DC function and plays a central role in the induction and regulation of immune responses. In vivo, DCs continuously acquire Ag in the periphery and migrate to draining lymph nodes, under the influence of local environmental chemotactic factors like CCL19/21 or sphingosine 1-phosphate (S1P). We investigated the role of S1P- and RhoA-regulated FHL2 in this process. We found reduced nuclear localization of FHL2 in mature bone marrow-derived DCs (BMDCs), compared with immature BMDCs, following stimulation with CCL19. Furthermore, in vitro-generated murine FHL2(-/-) BMDCs displayed a significantly increased migratory speed, directionality, and migratory persistence toward the chemokine CCL19 compared with wild-type BMDCs. Moreover, in vivo, FHL2(-/-) BMDCs showed increased migration toward lymphoid organs. FHL2(-/-) BMDCs increased the expression of S1PR1, which was associated with greater Rac activation. An S1PR1 antagonist and knock-down of S1PR1 abrogated the increased migratory speed of FHL2(-/-) BMDCs. Our results identify FHL2 as an important novel regulator of DC migration via regulation of their sensitivity toward environmental migratory cues like S1P and CCL19.

Dynamic regulation of CD8 T cell tolerance induction by liver sinusoidal endothelial cells.

Cross-presentation of soluble Ag on MHC class I molecules to naive CD8 T cells by liver sinusoidal endothelial cells (LSECs) leads to induction of T cell tolerance that requires interaction between coinhibitory B7-H1 on LSECs and programmed cell death-1 on CD8 T cells. In this study, we investigate whether cross-presentation of high as well as low Ag concentrations allowed for LSEC-induced tolerance. Ag concentration directly correlated with the cross-presentation capacity of murine LSECs and thus strength of TCR stimulation. Although LSEC cross-presentation at low-Ag concentrations resulted in tolerance, they induced differentiation into effector T cells (CTL) at high-Ag concentrations. CTL differentiation under these conditions was not caused by increased expression of costimulatory CD80/86 on cross-presenting LSECs but was determined by early IL-2 release from naive CD8 T cells. B7-H1 signals from LSECs and TCR avidity reciprocally controlled early T cell release of IL-2 and CTL differentiation. B7-H1 expression directly correlated with cross-presentation at low- but not high-Ag concentrations, indicating an imbalance between TCR and coinhibitory signals regulating T cell release of IL-2. Exogenous IL-2 overrode coinhibitory B7-H1-mediated signals by LSECs and induced full CTL differentiation. Our results imply that LSEC-mediated T cell tolerance can be broken in situations where T cells bearing high-avidity TCR encounter LSECs cross-presenting high numbers of cognate MHC class I peptide molecules, such as during viral infection of the liver. Furthermore, we attribute a novel costimulatory function to IL-2 acting in a T cell autonomous fashion to promote local induction of immunity in the liver even in the absence of CD80/86 costimulation.

The ADP-Ribosylation Factor 4d Restricts Regulatory T-Cell Induction via Control of IL-2 Availability.

Interleukin-2 is central to the induction and maintenance of both natural (nTreg) and induced Foxp3-expressing regulatory T cells (iTreg). Thus, signals that modulate IL-2 availability may, in turn, also influence Treg homeostasis. Using global knockout and cell-specific knockout mouse models, we evaluated the role of the small GTPase ADP-ribosylation factor 4d (Arl4d) in regulatory T-cell biology. We show that the expression of Arl4d in T cells restricts both IL-2 production and responsiveness to IL-2, as measured by the phosphorylation of STAT5. Arl4d-deficient CD4 T cells converted more efficiently into Foxp3+ iTreg in vitro in the presence of αCD3ε and TGFß, which was associated with their enhanced IL-2 secretion. As such, Arl4d-/- CD4 T cells induced significantly less colonic inflammation and lymphocytic infiltration in a model of transfer colitis. Thus, our data reveal a negative regulatory role for Arl4d in CD4 T-cell biology, limiting iTreg conversion via the restriction of IL-2 production, leading to reduced induction of Treg from conventional CD4 T cells.

Antigen Cross-Presentation by Murine Proximal Tubular Epithelial Cells Induces Cytotoxic and Inflammatory CD8+ T Cells.

Immune-mediated glomerular diseases are characterized by infiltration of T cells, which accumulate in the periglomerular space and tubulointerstitium in close contact to proximal and distal tubuli. Recent studies described proximal tubular epithelial cells (PTECs) as renal non-professional antigen-presenting cells that stimulate CD4+ T-cell activation. Whether PTECs have the potential to induce activation of CD8+ T cells is less clear. In this study, we aimed to investigate the capacity of PTECs for antigen cross-presentation thereby modulating CD8+ T-cell responses. We showed that PTECs expressed proteins associated with cross-presentation, internalized soluble antigen via mannose receptor-mediated endocytosis, and generated antigenic peptides by proteasomal degradation. PTECs induced an antigen-dependent CD8+ T-cell activation in the presence of soluble antigen in vitro. PTEC-activated CD8+ T cells expressed granzyme B, and exerted a cytotoxic function by killing target cells. In murine lupus nephritis, CD8+ T cells localized in close contact to proximal tubuli. We determined enhanced apoptosis in tubular cells and particularly PTECs up-regulated expression of cleaved caspase-3. Interestingly, induction of apoptosis in the inflamed kidney was reduced in the absence of CD8+ T cells. Thus, PTECs have the capacity for antigen cross-presentation thereby inducing cytotoxic CD8+ T cells in vitro, which may contribute to the pathology of immune-mediated glomerulonephritis.

Virally infected mouse liver endothelial cells trigger CD8+ T-cell immunity.

BACKGROUND & AIMS: Dendritic cell activation through ligation of pattern recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increased delivery of costimulatory signals instead of tolerance. Here we investigate whether organ-resident antigen-presenting cells, such as liver sinusoidal endothelial cells (LSECs), also switch from tolerogenic to immunogenic CD8(+) T-cell activation upon such stimulation. METHODS: Murine LSECs were isolated by immunomagnetic separation and analyzed for functional maturation upon triggering pattern recognition receptors or viral infection employing gene expression analysis and T cell coculture assays. In vivo relevance of the findings was confirmed with bone-marrow chimeric animals. RESULTS: LSECs expressed numerous pattern recognition receptors that allowed for sentinel function, but ligand-induced activation of these receptors was not sufficient to overcome tolerance induction of CD8(+) T cells. Importantly, viral infection with murine cytomegalovirus caused functional maturation of antigen-presenting LSECs and was sufficient to promote antigen-specific differentiation into effector CD8(+) T cells in the absence of dendritic cells and independent of CD80/86. CONCLUSIONS: These results shed new light on the generation of organ-specific immunity and may contribute to overcoming tolerance in relevant situations, such as cancer.

Antigen presentation, autoantibody production, and therapeutic targets in autoimmune liver disease.

The liver is an important immunological organ that controls systemic tolerance. The liver harbors professional and unconventional antigen-presenting cells that are crucial for tolerance induction and maintenance. Orchestrating the immune response in homeostasis depends on a healthy and well-toned immunological liver microenvironment, which is maintained by the crosstalk of liver-resident antigen-presenting cells and intrahepatic and liver-infiltrating leukocytes. In response to pathogens or autoantigens, tolerance is disrupted by unknown mechanisms. Intrahepatic parenchymal and nonparenchymal cells exhibit unique antigen-presenting properties. The presentation of microbial and endogenous lipid-, metabolite- and peptide-derived antigens from the gut via conventional and nonconventional mechanisms can educate intrahepatic immune cells and elicit effector responses or tolerance. Perturbation of this balance results in autoimmune liver diseases, such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis. Although the exact etiologies of these autoimmune liver diseases are unknown, it is thought that the disruption of tolerance towards self-antigens and microbial metabolites and lipids, as well as alterations in bile acid composition, may result in changes in effector cell activation and polarization and may reduce or impair protective anti-inflammatory regulatory T and B cell responses. Additionally, the canonical and noncanonical transmission of antigens and antigen:MHC complexes via trogocytosis or extracellular vesicles between different (non) immune cells in the liver may play a role in the induction of hepatic inflammation and tolerance. Here, we summarize emerging aspects of antigen presentation, autoantibody production, and the application of novel therapeutic approaches in the characterization and treatment of autoimmune liver diseases.

Comparison of Resistance Spectra after First and Second Line Osimertinib Treatment Detected by Liquid Biopsy.

Since 2009, several first, second, and third generation EGFR tyrosine kinase inhibitors (TKI) have been approved for targeted treatment of EGFR mutated metastatic non-small lung cancer (NSCLC). A vast majority of patients is improving quickly on treatment; however, resistance is inevitable and typically occurs after one year for TKI of the first and second generation. Osimertinib, a third generation TKI, has recently been approved for first line treatment in the palliative setting and is expected to become approved for the adjuvant setting as well. Progression-free survival (PFS) under osimertinib is superior to its predecessors but its spectrum of resistance alterations appears significantly more diverse compared to first and second generation EGFR TKI. As resistance mechanisms to osimertinib are therapeutically targetable in some cases, it is important to comprehensively test for molecular alterations in the relapse scenario. Liquid biopsy may be advantageous over tissue analysis as it has the potential to represent tumor heterogeneity and clonal diversification. We have previously shown high concordance of hybrid capture (HC) based next generation sequencing (NGS) in liquid biopsy versus solid tumor biopsies. In this study, we now present real-word data from 56 patients with metastatic NSCLC that were tested by liquid biopsy at the time of disease progression on mostly second line treated osimertinib treatment. We present examples of single and multiple TKI resistance mechanisms, including mutations in multiple pathways, copy number changes and rare fusions of RET, ALK, FGFR3 and BRAF. In addition, we present the added value of HC based NGS to reveal polyclonal resistance development at the DNA level encoding multiple EGFR C797S and PIK3CA mutations.

Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model.

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

Systemic antigen cross-presented by liver sinusoidal endothelial cells induces liver-specific CD8 T-cell retention and tolerization.

UNLABELLED: Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. CONCLUSION: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

TP53 co-mutations in EGFR mutated patients in NSCLC stage IV: A strong predictive factor of ORR, PFS and OS in EGFR mt+ NSCLC.

INTRODUCTION: The impact of TP53 co-mutations in EGFR mutated patients on PFS and OS is controversial. Different classifications of TP53 mutations with respect to functional and potential clinical impact have been published. Therefore, we retrospectively analyzed the impact of TP53 co-mutations on ORR, PFS and OS in a cohort of EGFR mutated NSCLC IV patients (UICC 7) using different classifications of TP53 mutations. METHODS: 75 EGFR mutated NSCLC IV patients homogeneously treated with 1st line EGFR TKI were analyzed for TP53 co-mutations. TP53 mutations were classified according to three different types of classifications. The endpoints ORR, PFS and OS were investigated. RESULTS: TP53 co-mutations were found in 29/59 patients (49.2%). TP53 co-mutations were a statistically significant independent negative predictive factor for ORR, PFS and OS. TP53 co-mutations were associated with inferior mPFS and mOS: mPFS/mOS 12 vs. 18/24 vs. 42 months for non-disruptive/disruptive mutations vs. WT (p < 0.004)/(p < 0.009), 11 vs. 17/23 vs. 42 months for pathogenic vs. non-pathogenic/WT (p < 0.001)/(p < 0.001), and 7 vs. 12 vs. 18/12 vs. 28 vs. 42 months for exon 8 vs. non-exon 8 vs. WT (p < 0.001)/(p < 0.002). CONCLUSIONS: TP53 co-mutations are frequent in EGFR mt+ NSCLC and have a strong negative impact on all clinical endpoints of TKI therapy.