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Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease with unknown etiology. Dextran sulfate sodium (DSS) induced colitis is a widely used mouse model in IBD research. DSS colitis involves activation of the submucosal immune system and can be used to study IBD-like disease characteristics in acute, chronic, remission and transition phases. Insight into colon inflammatory parameters is needed to understand potentially irreversible adaptations to the chronification of colitis, determining the baseline and impact of further inflammatory episodes. We performed analyses of non-invasive and invasive colitis parameters in acute, chronic and remission phases of the DSS colitis in C57BL/6 mice. Non-invasive colitis parameters poorly reflected inflammatory aspects of colitis in chronic remission phase. We found invasive inflammatory parameters, positively linked to repeated DSS-episodes, such as specific colon weight, inflamed colon area, spleen weight, absolute cell numbers of CD4+ and CD8+ T cells as well as B cells, blood IFN-γ level, colonic chemokines BLC and MDC as well as the prevalence of Turicibacter species in feces. Moreover, microbial Lactobacillus species decreased with chronification of disease. Our data point out indicative parameters of recurrent gut inflammation in context of DSS colitis., Competing Interests: The authors declare no conflict of interest.

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6 ) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11 ) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11 D61Y in T cells. We observed reduced numbers of CD8 + and increased numbers of CD4 + T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8 + T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4 + and CD8 + T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cammann, Israel, Frentzel, Jeron, Topfstedt, Schüler, Simeoni, Zenker, Fehling, Schraven, Bruder and Seifert.)

Mitochondrial functionality is crucial for the execution of physiologic functions of metabolically active cells in the respiratory tract including airway epithelial cells (AECs). Cigarette smoke is known to impair mitochondrial function in AECs. However, the potential contribution of mitochondrial dysfunction in AECs to airway infection and airway epithelial barrier dysfunction is unknown. In this study, we used an in vitro model based on AECs exposed to cigarette smoke extract (CSE) followed by an infection with Streptococcus pneumoniae ( Sp ). The levels of oxidative stress as an indicator of mitochondrial stress were quantified upon CSE and Sp treatment. In addition, expression of proteins associated with mitophagy, mitochondrial content, and biogenesis as well as mitochondrial fission and fusion was quantified. Transcriptional AEC profiling was performed to identify the potential changes in innate immune pathways and correlate them with indices of mitochondrial function. We observed that CSE exposure substantially altered mitochondrial function in AECs by suppressing mitochondrial complex protein levels, reducing mitochondrial membrane potential and increasing mitochondrial stress and mitophagy. Moreover, CSE-induced mitochondrial dysfunction correlated with reduced enrichment of genes involved in apical junctions and innate immune responses to Sp , particularly type I interferon responses. Together, our results demonstrated that CSE-induced mitochondrial dysfunction may contribute to impaired innate immune responses to Sp .

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies., (© 2022. The Author(s).)

Chronic exposure to environmental pollutants is a major contributor to the development and progression of obstructive airway diseases, including asthma and COPD. Understanding the mechanisms underlying the development of obstructive lung diseases upon exposure to inhaled pollutants will lead to novel insights into the pathogenesis, prevention and treatment of these diseases. The respiratory epithelial lining forms a robust physicochemical barrier protecting the body from inhaled toxic particles and pathogens. Inhalation of airborne particles and gases may impair airway epithelial barrier function and subsequently lead to exaggerated inflammatory responses and airway remodelling, which are key features of asthma and COPD. In addition, air pollutant-induced airway epithelial barrier dysfunction may increase susceptibility to respiratory infections, thereby increasing the risk of exacerbations and thus triggering further inflammation. In this review, we discuss the molecular and immunological mechanisms involved in physical barrier disruption induced by major airborne pollutants and outline their implications in the pathogenesis of asthma and COPD. We further discuss the link between these pollutants and changes in the lung microbiome as a potential factor for aggravating airway diseases. Understanding these mechanisms may lead to identification of novel targets for therapeutic intervention to restore airway epithelial integrity in asthma and COPD., Competing Interests: Conflict of interest: M. Aghapour reports personal fees from Kommission zur Förderung des wissenschaftlichen Nachwuchses, Medical Faculty, Otto-von-Guericke University, Magdeburg, non-financial support from German Research Foundation, funded by grant 361210922/RTG 2408, outside the submitted work. Conflict of interest: N.D. Ubags has nothing to disclose. Conflict of interest: D. Bruder reports grants from German Research Foundation, grant number 361210922/RTG 2408, outside the submitted work. Conflict of interest: P.S. Hiemstra reports grants from Boehringer Ingelheim, outside the submitted work. Conflict of interest: V. Sidhaye has nothing to disclose. Conflict of interest: F. Rezaee has nothing to disclose. Conflict of interest: I.H. Heijink has nothing to disclose., (Copyright ©The authors 2022.)

Purpose: Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion. Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT)., Methods: Pigr gene expression in lung, trachea, and nasal-associated lymphoid tissue (NALT) of germ-free mice, specific pathogen-free mice, mice with an undefined microbiome, as well as LPS- and IFN-γ-treated mice was determined by quantitative real-time PCR. IgA levels in bronchoalveolar lavage (BAL), nasal lavage (NAL), and serum were determined by ELISA. LPS- and IFN-γ-treated mice were colonized with Streptococcus pneumoniae and bacterial CFUs were determined in URT and LRT., Results: Respiratory Pigr expression and IgA levels were dependent on the degree of exposure to environmental microbial stimuli. While immunostimulation with LPS and IFN-γ differentially impacts respiratory Pigr expression and IgA in URT vs. LRT, only prophylactic IFN-γ treatment reduces nasal colonization with S. pneumoniae., Conclusion: Airway-associated secretory immunity can be partly modulated by exposure to microbial ligands and proinflammatory stimuli. Prophylactic IFN-γ-treatment modestly improves antibacterial immunity in the URT, but this does not appear to be mediated by SIgA or pIgR., (© 2021. The Author(s).)

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a β-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8 + T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMV HA ) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMV S ). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMV HA -vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens., (© 2021. The Author(s).)

Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marquardt, Jakob, Scheibel, Hofmann, Klawonn, Neumann-Schaal, Gerhard, Bruder and Jänsch.)

Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes ( Lm ) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm -infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Böning, Parzmair, Jeron, Düsedau, Kershaw, Xu, Relja, Schlüter, Dunay, Reinhold, Schraven and Bruder.)

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N- glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β -1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the β -1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N -glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N -glycans were converted to complex-type galactosylated N -glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N -glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N -glycosylation on immunity and to improve the efficacy of protein subunit vaccines., Competing Interests: ER and UR hold shares in glyXera GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruhnau, Grote, Juarez-Osorio, Bruder, Mahour, Rapp, Rexer and Reichl.)

COPD and asthma are two distinct but sometimes overlapping diseases exhibiting varying degrees and types of inflammation on different stages of the disease. Although several biomarkers are defined to estimate the inflammatory endotype and stages in these diseases, there is still a need for new markers and potential therapeutic targets. We investigated the levels of a phytohormone, abscisic acid (ABA) and its receptor, LANCL2, in COPD patients and asthmatics. In addition, PPAR-γ that is activated by ABA in a ligand-binding domain-independent manner was also included in the study. In this study, we correlated ABA with COPD-propagating factors to define the possible role of ABA, in terms of immune regulation, inflammation, and disease stages. We collected blood from 101 COPD patients, 52 asthmatics, and 57 controls. Bronchoscopy was performed on five COPD patients and 29 controls. We employed (i) liquid chromatography-tandem mass spectrometry and HPLC to determine the ABA and indoleamine 2,3-dioxygenase levels, respectively; (ii) real-time PCR to quantify the gene expression of LANCL2 and PPAR-γ; (iii) Flow cytometry to quantify adipocytokines; and (iv) immunoturbidimetry and ELISA to measure CRP and cytokines, respectively. Finally, a multinomial regression model was used to predict the probability of using ABA as a biomarker. Blood ABA levels were significantly reduced in COPD patients and asthmatics compared to age- and gender-matched normal controls. However, PPAR-γ was elevated in COPD patients. Intriguingly, ABA was positively correlated with immune-regulatory factors and was negatively correlated with inflammatory markers, in COPD. Of note, ABA was increased in advanced COPD stages. We thereby conclude that ABA might be involved in regulation of COPD pathogenesis and might be regarded as a potential biomarker for COPD stages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hoang, Nguyen, Oberacher, Fuchs, Hernandez-Vargas, Borucki, Waldburg, Wippermann, Schreiber, Bruder and Veluswamy.)

Foxp3 + Treg cells, which are crucial for maintenance of self-tolerance, mainly develop within the thymus, where they arise from CD25 + Foxp3 - or CD25 - Foxp3 + Treg cell precursors. Although it is known that infections can cause transient thymic involution, the impact of infection-induced thymus atrophy on thymic Treg (tTreg) cell development is unknown. Here, we infected mice with influenza A virus (IAV) and studied thymocyte population dynamics post infection. IAV infection caused a massive, but transient thymic involution, dominated by a loss of CD4 + CD8 + double-positive (DP) thymocytes, which was accompanied by a significant increase in the frequency of CD25 + Foxp3 + tTreg cells. Differential apoptosis susceptibility could be experimentally excluded as a reason for the relative tTreg cell increase, and mathematical modeling suggested that enhanced tTreg cell generation cannot explain the increased frequency of tTreg cells. Yet, an increased death of DP thymocytes and augmented exit of single-positive (SP) thymocytes was suggested to be causative. Interestingly, IAV-induced thymus atrophy resulted in a significantly reduced T-cell receptor (TCR) repertoire diversity of newly produced tTreg cells. Taken together, IAV-induced thymus atrophy is substantially altering the dynamics of major thymocyte populations, finally resulting in a relative increase of tTreg cells with an altered TCR repertoire., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205 + DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Allergic airway inflammation (AAI) involves T helper cell type 2 (Th2) and pro-inflammatory responses to aeroallergens and many predisposing factors remain elusive. Influenza A virus (IAV) is a major human pathogen that causes acute respiratory infections and induces specific immune responses essential for viral clearance and resolution of the infection. Beyond acute infection, IAV has been shown to persistently affect lung homeostasis and respiratory immunity. Here we asked how resolved IAV infection affects subsequently induced AAI. Mice infected with a sublethal dose of IAV were sensitized and challenged in an ovalbumin mediated mouse model for AAI after resolution of the acute viral infection. Histological changes, respiratory leukocytes, cytokines and airway hyperreactivity were analyzed in resolved IAV infection alone and in AAI with and without previous IAV infection. More than five weeks after infection, we detected persistent pneumonia with increased activated CD4 + and CD8 + lymphocytes as well as dendritic cells and MHCII expressing macrophages in the lung. Resolved IAV infection significantly affected subsequently induced AAI on different levels including morphological changes, respiratory leukocytes and lymphocytes as well as the pro-inflammatory cytokine responses, which was clearly diminished. We conclude that IAV has exceptional persisting effects on respiratory immunity with substantial consequences for subsequently induced AAI.

Co-infections by multiple pathogens have important implications in many aspects of health, epidemiology and evolution. However, how to disentangle the non-linear dynamics of the immune response when two infections take place at the same time is largely unexplored. Using data sets of the immune response during influenza-pneumococcal co-infection in mice, we employ here topological data analysis to simplify and visualise high dimensional data sets. We identified persistent shapes of the simplicial complexes of the data in the three infection scenarios: single viral infection, single bacterial infection, and co-infection. The immune response was found to be distinct for each of the infection scenarios and we uncovered that the immune response during the co-infection has three phases and two transition points. During the first phase, its dynamics is inherited from its response to the primary (viral) infection. The immune response has an early shift (few hours post co-infection) and then modulates its response to react against the secondary (bacterial) infection. Between 18 and 26 h post co-infection the nature of the immune response changes again and does no longer resembles either of the single infection scenarios., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; and in the decision to publish the results., (© 2020 Elsevier B.V. All rights reserved.)

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date only limited and moreover conflicting data exist regarding the role of ADAP in NK cells. To extend existing knowledge we investigated ADAP-dependency of NK cells in the context of in vivo infection with the intracellular pathogen Listeria monocytogenes ( Lm ). Ex vivo analysis of infection-primed NK cells revealed impaired cytotoxic capacity in NK cells lacking ADAP as indicated by reduced CD107a surface expression and inefficient perforin production. However, ADAP-deficiency had no global effect on NK cell morphology or intracellular distribution of CD107a-containing vesicles. Proteomic definition of ADAPko and wild type NK cells did not uncover obvious differences in protein composition during the steady state and moreover, similar early response patterns were induced in NK cells upon infection independent of the genotype. In line with protein network analyses that suggested an altered migration phenotype in naïve ADAPko NK cells, in vitro migration assays uncovered significantly reduced migration of both naïve as well as infection-primed ADAPko NK cells compared to wild type NK cells. Notably, this migration defect was associated with a significantly reduced expression of the integrin CD11a on the surface of splenic ADAP-deficient NK cells 1 day post- Lm infection. We propose that ADAP-dependent alterations in integrin expression might account at least in part for the fact that during in vivo infection significantly lower numbers of ADAPko NK cells accumulate in the spleen i.e., the site of infection. In conclusion, we show here that during systemic Lm infection in mice ADAP is essential for efficient cytotoxic capacity and migration of NK cells., (Copyright © 2020 Böning, Trittel, Riese, van Ham, Heyner, Voss, Parzmair, Klawonn, Jeron, Guzman, Jänsch, Schraven, Reinhold and Bruder.)

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity, all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1 int CD11b hi CD11c int ) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4 + T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4 + T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

Hemodialysis (HD) patients exhibit chronic inflammation and leukocyte activation. We investigated the surface-marker profile of monocytes by flow cytometry to assess the chronic effect of uremia and the acute effect of dialysis on their phenotypical and functional features in 16 healthy controls (CON) and 15 HD patients before and after a polysulfone-based dialysis session. Median fluorescence intensities were analyzed indicating expression of CD14, CD16, integrins (CD11b, CD18), chemokine receptors (CCR2, CX3CR1), scavenger receptors (CD36, CD163) and Toll-like receptor-2 (TLR2). Before and after dialysis, HD patients harbour 0.9-fold less CD14 ++ CD16 - (Mo1), 1.8-fold more CD14 ++ CD16 + (Mo2) and CD14 + CD16 ++ (Mo3) monocytes than CON. HD patients' Mo1 showed elevated expression of CD11b (1.7-fold), CD18 (1.2-fold) and CD36 (2.1-fold), whereas CD163 expression was reduced in Mo1 and Mo2 (0.6-fold) compared to CON. These markers remained unaffected by dialysis. CX3CR1 expression on Mo2 and Mo3 was lower in HD patients before (0.8-fold) and further diminished after dialysis (0.6-fold). Stimulation of monocytes resulted in diminished responses in HD patients compared to CON. In conclusion, a systematic analysis of the expression of particular surface markers on distinct monocyte subsets may help to distinguish between uremia and/or dialysis induced effects and to evaluate the functionality of monocytes and biocompatibility of HD.

The local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach to dampen inflammation during pulmonary diseases. For the local therapeutic treatment of pulmonary inflammation, we produced multi-shell nanoparticles consisting of a calcium phosphate core, coated with siRNAs directed against pro-inflammatory mediators, encapsulated into poly(lactic-co-glycolic acid), and coated with a final outer layer of polyethylenimine. Nasal instillation of nanoparticles loaded with a mixture of siRNAs directed against different cytokines to mice suffering from T H 1 cell-mediated lung inflammation, or of siRNA directed against NS-1 in an influenza infection model led to a significant reduction of target gene expression which was accompanied by distinct amelioration of lung inflammation in both models. Thus, this study provides strong evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of inflammatory disorders of the lung., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκB NS are important regulators of Foxp3 induction in Treg precursors upon γ-chain cytokine stimulation. In c-REL/IκB NS double-deficient mice, Treg numbers were dramatically reduced, indicating that together, c-REL and IκB NS are pivotal for Treg development. However, despite the highly reduced Treg compartment, double-deficient mice did not develop autoimmunity even when aged to more than 1 y, suggesting that c-REL and IκB NS are required for T cell effector function as well. Analyzing Treg development in more detail, we identified a CD122 + subset within the CD25 - Foxp3 - precursor population, which gave rise to classical CD25 + Foxp3 - Treg precursors. Importantly, c-REL, but not IκB NS , controlled the generation of classical CD25 + Foxp3 - precursors via direct binding to the Cd25 locus. Thus, we propose that CD4 + GITR + CD122 + CD25 - Foxp3 - cells represent a Treg pre-precursor population, whose transition into Treg precursors is mediated via c-REL., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Background: COPD represents a multifactorial lung disorder with high morbidity and mortality. Despite intensive research concerning the underlying disease mechanisms, the involvement of the CD200/CD200R axis in supporting or preventing the onset of COPD has not yet been addressed. Since the CD200/CD200R axis is crucially implicated in the maintenance of pulmonary immune homeostasis, we hypothesized that it might be involved in controlling the onset of COPD., Methods: To address this, we analyzed the serum samples from COPD patients and normal controls for soluble (s) CD200 and correlated the data to COPD-relevant clinical parameters. In addition, basic studies were conducted in CD200-deficient and wild-type mice in which COPD-like inflammation was induced with elastase/LPS followed by lung and serum component analysis., Results: We observed a positive correlation between serum sCD200 and IL-6 levels as well as a trend toward a negative correlation of sCD200 with vitamin D3 in COPD patients. Further investigations in mice revealed that despite elevated serum concentration of MMP-9 in CD200KO mice, the early onset of COPD-like lung inflammation was similar in CD200-deficient and wild-type animals in terms of immune cell infiltration, emphysematous changes, and mucus overproduction., Conclusions: While our murine studies suggest that the co-inhibitory molecule CD200 does not appear to play a prominent role in the early onset of COPD-like features, correlation of sCD200 serum levels with COPD-related parameters in humans with established disease revealed that the CD200/CD200R axis may be mechanistically linked to the disease course in COPD patients.

The adhesion and degranulation promoting adaptor protein (ADAP) is a multifunctional scaffold involved in many different signaling pathways that are important for the function of T cells, including the inside-out and outside-in signaling of integrins, the activation of NF-κB, and the subsequent production of proinflammatory cytokines (e.g., IFN-γ and IL-2). Strikingly, despite its well-established role in T cells, previous studies did not distinguish between CD4 + and CD8 + T cells, and thus, it is unknown whether ADAP fulfills equally important functions in both T cell subsets. We show here that despite comparable ADAP expression levels in CD4 + and CD8 + T cells, their function is differentially dependent on ADAP. Whereas in vitro TCR-stimulation experiments revealed that activation, proliferation, and adhesion are severely compromised in CD4 + T cells lacking ADAP, their CD8 + counterparts are hardly affected by ADAP deficiency. Accordingly, antigen-specific in vivo stimulation of adoptively transferred CD8 + T cells during Listeria monocytogenes (Lm) and influenza A virus (IAV) infection revealed only moderate effects of ADAP deficiency in terms of CD8 + T cell activation, proliferation, and differentiation, which, however, did not impair pathogen-specific immunity. Thus, we show for the first time that ADAP fulfills different functions in CD4 + and CD8 + T cells, with CD8 + T cells being less dependent on ADAP. Our data identify ADAP as a potential molecular target for T cell subset-specific therapeutic interventions., (© Society for Leukocyte Biology.)

Purpose of Review: The formation of noncaseating granuloma is a hallmark of pulmonary sarcoidosis. This review summarizes recent progress made to explain the cellular dynamics within the granuloma structure that may considerably differ between the two clinically distinct variants, that is, acute and chronic sarcoidosis., Recent Findings: Compelling evidence exists that in acute but not chronic sarcoidosis CD4 T lymphocytes specifically recognizing the auto-antigen vimentin on human leukocyte antigen-DR3 molecules accumulate in sarcoid granuloma. These so-called TH17.1 cells produce high amounts of the TH17-related cytokines interleukin-17 (IL-17) and IL-22 in addition to interferon-γ. Moreover, regulatory T cells from patients with acute sarcoidosis are ICOS, providing a mechanistic link to the comparably high concentration of IL-10 exclusively found in the airways of these patients. Next to obvious differences in T effector cell and Treg subsets, alveolar macrophages harbor a functional mitochondrial system in acute sarcoidosis patients, while this system is impaired in patients with chronic disease., Summary: We provide a comprehensive update on the cellular components and their functional implications in sarcoid granuloma formation, with special emphasis on the specific characteristics of granuloma in acute versus chronic sarcoidosis. Moreover, the specific antigens thought to be involved in both forms of the disease are discussed.

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening studies requiring fast purification protocols., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Purpose: Chronic lung inflammation commonly induces a multitude of structural and functional adaptations within the lung tissue and airspaces. Yet the impact of a persistent inflammatory environment on alveolar macrophages is still incompletely understood. Here, we examined morphology and function of alveolar macrophages in a transgenic mouse model of chronic lung disease., Methods: Imaging flow cytometry, flow cytometry, and microscopic evaluation of alveolar macrophages isolated from healthy and inflamed lungs were performed. Gene expression of polarization markers was compared by quantitative real-time RT-PCR. The pro-inflammatory immune response of alveolar macrophages toward bacterial ligands was assessed in in vivo clodronate-liposome depletion studies., Results: Chronic lung inflammation is associated with a substantially altered, activated alveolar macrophage morphology, and blunted TNF-α response by these cells following stimulation with ligands derived from the respiratory pathogen Streptococcus pneumoniae., Conclusions: These results demonstrate pleiotropic effects of pulmonary inflammation on alveolar macrophage phenotype and function and suggest a functional impairment of these cells during infection with airborne pathogens.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although the contribution of CD8(+) T cells to the pathogenesis of noncommunicable lung diseases has become increasingly appreciated, our knowledge about the mechanisms controlling self-reactive CD8(+) T cells in the respiratory tract remains largely elusive. The outcome of the encounter between pulmonary self-antigen and naive CD8(+) T cells, in the presence or absence of inflammation, was traced after adoptive transfer of fluorescence-labeled CD8(+) T cells specific for the neo-self-antigen influenza A hemagglutinin into transgenic mice expressing hemagglutinin specifically in alveolar type II epithelial cells in order: to study the outcome of alveolar antigen encounter in the steady state and under inflammatory conditions; to define the phenotype and fate of CD8(+) T cells primed in the respiratory tract; and, finally, to correlate these findings with the onset of autoimmunity in the lung. We found that CD8(+) T cells remain ignorant in the steady state, whereas transient proliferation of self-reactive CD8(+) T cells is induced by forced maturation or licensing of dendritic cells, increases in the antigenic threshold, and targeted release of alveolar self-antigen by epithelial injury. However, these cells fail to acquire effector functions, lack the expression of the high-affinity IL-2 receptor CD25, and do not precipitate autoimmunity in the lung. We conclude that inadvertent activation of CD8(+) T cells in the lung is prevented in the absence of "danger signals," whereas tissue damage after infection or noninfectious inflammation creates an environment that allows the priming of previously ignorant T cells. Failure in effector cell differentiation after abortive priming, however, precludes the establishment of self-perpetuating autoimmunity in the lung.

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 Nras(G12V). 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., (©2011 Macmillan Publishers Limited. All rights reserved)

Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8alpha. Here we describe a subset of CD8alpha(+) DC in lymphoid organs of naïve mice characterized by expression of the CX(3)CR1 chemokine receptor. CX(3)CR1(+) CD8alpha(+) DC lack hallmarks of classical CD8alpha(+) DC, including IL-12 secretion, the capacity to cross-present antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX(3)CR1(+) CD8alpha(+) DC resemble CD8alpha(-) cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX(3)CR1(+) CD8alpha(+) DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D-J Ig gene rearrangements and that development of CX(3)CR1(+) CD8alpha(+) DC requires E2-2, the critical transcriptional regulator of PDC. Thus, CX(3)CR1(+) CD8alpha(+) DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8alpha(+) and CD8alpha(-) DC, and should assist the identification of human counterparts of murine DC subsets.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-beta as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-beta gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-beta following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-beta is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-beta under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

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.

The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4+CD25+ regulatory T (TR) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4+ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4+CD25+ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25- TR cells are able to interfere with an Ag-specific CD8+ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3-CD25- TR cells with regulatory capacity both in vitro and in vivo.

Regulatory T cells play a crucial role in the control of immune responses in the intestinal mucosa and their absence may predispose to inflammatory bowel disease (IBD). However, the induction of regulatory T cells at sites of mucosal inflammation is not yet fully understood and may involve antigen presentation by local immature dendritic cells and/or intestinal epithelial cells (IECs). VILLIN-HA mice, which express the hemagglutinin (HA) from influenza virus A exclusively in enterocytes of the intestinal epithelium, were matched with T cell receptor (TCR)-HA mice expressing an alphabeta-TCR which recognizes a major histocompatibility complex (MHC) class II-restricted epitope of HA in order to determine the impact of antigen presentation by IECs on CD4(+) T cell immunity. In VILLIN-HA x TCR-HA mice, peripheral HA-specific lymphocytes showed an activated phenotype and increased infiltration into the intestinal mucosa without destruction of the intestinal epithelium. Mucosal lymphocytes from VILLIN-HA x TCR-HA mice secreted lower amounts of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) and exhibited an increased expression of interleukin-10 (IL-10), Nrp-1, and Foxp3, molecules published as markers for regulatory T cells. IECs can take up and process antigen but the antigen presentation capacity of these cells is often inefficient. Functional and molecular characterization of IECs from VILLIN-HA and VILLIN-HA x TCR-HA transgenic mice revealed a direct role in the induction of CD4(+) T cells with a regulatory phenotype that maintain intestinal homeostasis.

CD8+ T cell recognition of viral antigens presented by lung epithelial cells is important in the clearance of respiratory viral infection but may cause considerable injury to the lung. We have shown that a critical event of this type of injury is the activation of target epithelial cells and expression of chemokines by these cells. In this study, epithelial gene expression and transcription factor activation triggered by specific CD8+ T cell antigen recognition was examined in vitro and in vivo. T cell recognition triggers expression profiles of tumor necrosis factor-alpha (TNF-alpha)-dependent and interferon-gamma (IFN-gamma)-dependent genes in epithelial target cells. Consistent with these profiles, transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were activated in lung epithelial cells of wild-type (WT) mice but not TNF receptor 1 (TNFR1)-deficient mice after CD8+ T cell recognition in vivo. In contrast, Stat1 activation and Stat1-dependent genes, such as IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-2 (GBP-2), were induced to a similar extent in epithelial cells of both WT and TNFR1-deficient mice, indicating that this pathway is insufficient to induce pulmonary immunopathology in the absence of NF-kappaB-dependent transcriptional activation. Antibody neutralization of TNF-alpha abrogated epithelial monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) production in vitro as well as pulmonary immunopathology in vivo, confirming the primary importance of this cytokine in CD8+ T cell-mediated immunopathology.

Immunodominance in CD8+ T cell responses against Listeria monocytogenes is a well-recognized but still not fully understood phenomenon. From listeriolysin, the major virulence factor of L. monocytogenes, only a single epitope, pLLO91-99, is presented by MHC class I molecules in BALB/c mice which dominates the cytotoxic T cell response against this bacterial pathogen. To obtain more insights into the molecular and cellular mechanisms underlying immunodominance of this particular epitope, we compared the various steps involved in the presentation and recognition of pLLO91-99 derived from a wild-type toxin with an equivalent epitope from a mutated toxin. This fully functional variant contains within the pLLO91-99 epitope a conservative isoleucine to alanine replacement at the C-terminal anchor residue which results in loss of antigenicity. The binding properties of the variant peptide to soluble Kd remained unaffected and cytotoxic T cells capable of recognizing the pLLO99A/Kd complex were detectable in BALB/c mice. However, such T cells required higher concentrations of antigen in order to be optimally activated in vitro. A comparison between the TAP translocation efficiency of wild-type and mutant peptide demonstrated that the mutation at the C-terminus leads to a reduced transportation rate. Furthermore, the amino acid substitution changes the in vitro proteasomal cleavage pattern, resulting in a reduced liberation of the correct peptide from a polypeptide precursor. Thus, in all assays employed the immunodominant epitope performs optimally while the variant was found to be inferior. The synergy of all these steps most likely is the decisive factor in the immunodominance of pLLO91-99.

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up-regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4+CD25- T cells after TCR stimulation. Furthermore, CD4+Nrp1(high) T cells express high levels of Foxp3 and suppress CD4+CD25- T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non-regulatory T cells.