Marcus Gereke

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Alveolar Type II Epithelial Cells Present Antigen to CD4+ T Cells and Induce Foxp3+ Regulatory T Cells

RATIONALE Although the contribution of alveolar type II epithelial cells (AECIIs) in respiratory immunity has become increasingly appreciated, their precise function in the induction and regulation of T-cell reactivity to self-antigen remains poorly understood. OBJECTIVES To investigate the role of AECII in the initiation of T-cell reactivity to alveolar self-antigen, and to clarify their function in the peripheral induction of Foxp3(+) regulatory CD4(+) T cells. METHODS To dissect the complex cellular and molecular functions of AECIIs in lung inflammation and immune regulation, we use a transgenic mouse model for CD4(+) T-cell-mediated pulmonary inflammation. MEASUREMENTS AND MAIN RESULTS Here we report that AECIIs present endogenously expressed antigen on major histocompatibility complex class II molecules to CD4(+) T cells. Epithelial antigen display was sufficient to induce primary T-cell activation and pulmonary inflammation. Upon inflammation, AECIIs induce the differentiation of Foxp3(+) regulatory T cells by a mechanism involving antiproliferative soluble factors, including transforming growth factor (TGF)-beta. Whereas, in acute inflammation, TGF-beta appears to be the dominant factor to induce regulatory T cells, other AECII-derived factors can substitute for and/or synergize with TGF-beta in chronic pulmonary inflammations. CONCLUSIONS AECIIs are capable of priming naive CD4(+) T cells, demonstrating an active participation of these cells in respiratory immunity. Moreover, AECIIs display as yet unrecognized functions in balancing inflammatory and regulatory T-cell responses in the lung by connecting innate and adaptive immune mechanisms to establish peripheral T-cell tolerance to respiratory self-antigen.

New insights into the molecular pathology of radiation-induced pneumopathy.

BACKGROUND AND PURPOSE Pneumonitis and fibrosis constitute dose-limiting side effects of thorax or total body irradiation. An improved understanding of the underlying mechanisms is a prerequisite for the development of effective radioprotective strategies. Here we characterized the behavior of resident and immune cells in a murine model of radiation-induced pneumopathy. MATERIALS AND METHODS Wild type (WT) or RAG-2 deficient C57BL/6 mice received 15 Gray of (hemi)-thorax irradiation in a single dose. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected at defined time points post-irradiation for the determination of apoptosis, microvascular injury, and histological and immunohistochemical analyses. RESULTS Higher albumin levels and increased apoptosis were detected in the BALF 21 days after irradiation, indicative for delayed damage to resident cells. Irradiation also induced time-dependent changes in the BALF cytokine profile, the recruitment of activated T-cells into the lung and the formation of lipid-loaded resident cells. Lung fibrosis occurred earlier in RAG-2(-/-) mice, which lack mature T and B cells, compared to WT mice. CONCLUSIONS Thorax irradiation triggers a delayed disturbance of tissue integrity and lipid metabolism in the lung. Activated T-lymphocytes infiltrating the lung tissue upon thorax irradiation participate in the protection of the lung from radiation-induced fibrosis.

Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.

The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti‐tumor, both in mice and human. In the absence of IFN‐β, pro‐tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF‐α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life‐span. Notably, interferon therapy in mice altered TAN polarization towards anti‐tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti‐tumor agent.

Increased Susceptibility for Superinfection with Streptococcus pneumoniae during Influenza Virus Infection Is Not Caused by TLR7-Mediated Lymphopenia

Influenza A virus (IAV) causes respiratory tract infections leading to recurring epidemics with high rates of morbidity and mortality. In the past century IAV induced several world-wide pandemics, the most aggressive occurring in 1918 with a death toll of 20–50 million cases. However, infection with IAV alone is rarely fatal. Instead, death associated with IAV is usually mediated by superinfection with bacteria, mainly Streptococcus pneumoniae. The reasons for this increased susceptibility to bacterial superinfection have not been fully elucidated. We previously demonstrated that triggering of TLR7 causes immune incompetence in mice by induction of lymphopenia. IAV is recognized by TLR7 and infections can lead to lymphopenia. Since lymphocytes are critical to protect from S. pneumoniae it has long been speculated that IAV-induced lymphopenia might mediate increased susceptibility to superinfection. Here we show that sub-lethal pre-infections of mice with IAV-PR8/A/34 strongly increased their mortality in non-lethal SP infections, surprisingly despite the absence of detectable lymphopenia. In contrast to SP-infection alone co-infected animals were unable to control the exponential growth of SP. However, lymphopenia forced by TLR7-triggering or antibody-mediated neutropenia did not increase SP-susceptibility or compromise the ability to control SP growth. Thus, the immune-incompetence caused by transient lympho- or leukopenia is not sufficient to inhibit potent antibacterial responses of the host and mechanisms distinct from leukodepletion must account for increased bacterial superinfection during viral defence.

Therapeutic Intervention in Cancer and Chronic Viral Infections: Antibody Mediated Manipulation of PD-1/PD-L1 Interaction

Programmed Death-1 (PD-1) is a negative regulator of T cell activation and proliferation that mediates suppressive action by binding to its ligands PD-L1 and PD-L2. The well-established immunosuppressive properties of PD-1/PD-L1 interaction resulting in the re-establishment of peripheral tolerance makes PD-1 an interesting target for therapeutic intervention in cancer patients. In addition to its relevance in tumor-specific immunity, recent studies demonstrate that PD-1 expression on T cells correlates with viral load in HIV and HCV infected patients and further identified PD-1 expression as a marker for exhausted virus-specific CD8+ T cells. In particular, PD-1+CD8+ T cells show impaired effector functions and PD-1 associated T cell exhaustion could be restored by blocking the PD-1/PD-L1 interaction. This results in recovery of virus-specific CD8+ T cell mediated immunity, suggesting that interrupting PD-1 signaling using an antagonistic antibody restores T-cell effector functions. Thus, immunotherapy based on the blockade of PD-1/PD-L1 interaction does not only result in breakdown of effector T-cell tolerance to tumor antigens, but in addition also represents a promising therapeutic strategy for reactivation of virus-specific effector T cells to exert pathogen eradication in chronic viral infections. In this review, we give a comprehensive overview about the immunological functions of PD-1 mediated signaling in T cells with special emphasis on its immune regulatory functions in the context of cancer and chronic viral infections. Moreover, we will summarize recent data obtained in animal models, in-vitro preclinical approaches in patients and their implementation in clinical trials for treating patients with cancer and chronic viral infections.

Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation

BackgroundAlthough the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4+ T cell recognition of alveolar antigen. This model was utilized to assess the profile of inflammatory mediators expressed by alveolar epithelial target cells triggered by antigen-specific recognition in CD4+ T cell-mediated lung inflammation.MethodsWe established a method that allows the flow cytometric negative selection and isolation of primary AEC II of high viability and purity. Genome wide transcriptional profiling was performed on mRNA isolated from AEC II isolated from healthy mice and from mice with acute and chronic CD4+ T cell-mediated pulmonary inflammation.ResultsT cell-mediated inflammation was associated with expression of a broad array of cytokine and chemokine genes by AEC II cell, indicating a potential contribution of epithelial-derived chemoattractants to the inflammatory cell parenchymal infiltration. Morphologically, there was an increase in the size of activated epithelial cells, and on the molecular level, comparative transcriptome analyses of AEC II from inflamed versus normal lungs provide a detailed characterization of the specific inflammatory genes expressed in AEC II induced in the context of CD4+ T cell-mediated pneumonitis.ConclusionAn important contribution of AEC II gene expression to the orchestration and regulation of interstitial pneumonitis is suggested by the panoply of inflammatory genes expressed by this cell population, and this may provide insight into the molecular pathogenesis of pulmonary inflammatory states. CD4+ T cell recognition of antigen presented by AEC II cells appears to be a potent trigger for activation of the alveolar cell inflammatory transcriptosome.

Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function

Immunological characterization of immune cells that reside in specific anatomic compartments often requires their isolation from the respective tissue on the basis of enzymatic tissue disintegration. Applying enzymatic digestion of primary splenocytes, we evaluated the impact of collagenase and dispase, two enzymes that are commonly used for the liberation of immune cells from tissues, on the detectability of 48 immunologically relevant surface molecules that are frequently used for flow cytometric identification, isolation, and characterization of immune cell subsets. Whereas collagenase treatment had only minor effects on surface expression of most molecules tested, dispase treatment considerably affected antibody-mediated detectability of the majority of surface markers in subsequent FACS analyses. This effect was long lasting and, in case of high-dose dispase treatment, evident for the majority of surface molecules even after 24 h of in vitro culture. Of note, high-dose dispase treatment not only affected surface expression of certain molecules but also impaired antigen-specific proliferation of CD4(+) and CD8(+) T cells. Together, our data indicate that enzymatic tissue disintegration can have profound effects on the expression of a variety of cell-surface molecules with direct consequences for phenotypic analysis, FACS- and MACS-based target cell isolation, and immune cell function in cell culture experiments.

CD4+CD25+Foxp3+ Regulatory T Cells Are Dispensable for Controlling CD8+ T Cell-Mediated Lung Inflammation

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8+ T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8+ T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8+ T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8+ T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8+ T cells in the lung. Notably, CD8+ T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8+ T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8+ T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103+CD62Llo Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8+ T cells.

Alveolar Type II Epithelial Cells Contribute to the Anti-Influenza A Virus Response in the Lung by Integrating Pathogen- and Microenvironment-Derived Signals

ABSTRACT Influenza A virus (IAV) periodically causes substantial morbidity and mortality in the human population. In the lower lung, the primary targets for IAV replication are type II alveolar epithelial cells (AECII), which are increasingly recognized for their immunological potential. So far, little is known about their reaction to IAV and their contribution to respiratory antiviral immunity in vivo. Therefore, we characterized the AECII response during early IAV infection by analyzing transcriptional regulation in cells sorted from the lungs of infected mice. We detected rapid and extensive regulation of gene expression in AECII following in vivo IAV infection. The comparison to transcriptional regulation in lung tissue revealed a strong contribution of AECII to the respiratory response. IAV infection triggered the expression of a plethora of antiviral factors and immune mediators in AECII with a high prevalence for interferon-stimulated genes. Functional pathway analyses revealed high activity in pathogen recognition, immune cell recruitment, and antigen presentation. Ultimately, our analyses of transcriptional regulation in AECII and lung tissue as well as interferon I/III levels and cell recruitment indicated AECII to integrate signals provided by direct pathogen recognition and surrounding cells. Ex vivo analysis of AECII proved a powerful tool to increase our understanding of their role in respiratory immune responses, and our results clearly show that AECII need to be considered a part of the surveillance and effector system of the lower respiratory tract. IMPORTANCE In order to confront the health hazard posed by IAV, we need to complete our understanding of its pathogenesis. AECII are primary targets for IAV replication in the lung, and while we are beginning to understand their importance for respiratory immunity, the in vivo AECII response during IAV infection has not been analyzed. In contrast to studies addressing the response of AECII infected with IAV ex vivo, we have performed detailed gene transcriptional profiling of AECII isolated from the lungs of infected mice. Thereby, we have identified an exceptionally rapid and versatile response to IAV infection that is shaped by pathogen-derived as well as microenvironment-derived signals and aims at the induction of antiviral measures and the recruitment and activation of immune cells. In conclusion, our study presents AECII as active players in antiviral defense in vivo that need to be considered part of the sentinel and effector immune system of the lung. In order to confront the health hazard posed by IAV, we need to complete our understanding of its pathogenesis. AECII are primary targets for IAV replication in the lung, and while we are beginning to understand their importance for respiratory immunity, the in vivo AECII response during IAV infection has not been analyzed. In contrast to studies addressing the response of AECII infected with IAV ex vivo, we have performed detailed gene transcriptional profiling of AECII isolated from the lungs of infected mice. Thereby, we have identified an exceptionally rapid and versatile response to IAV infection that is shaped by pathogen-derived as well as microenvironment-derived signals and aims at the induction of antiviral measures and the recruitment and activation of immune cells. In conclusion, our study presents AECII as active players in antiviral defense in vivo that need to be considered part of the sentinel and effector immune system of the lung.