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1.  The transcription factor Zeb2 regulates development of conventional and plasmacytoid DCs by repressing Id2 
Lambrecht et al. show that the transcription factor Zeb2 regulates commitment toward both the pDC and cDC2 lineages by repressing Id2.
Plasmacytoid dendritic cells (DCs [pDCs]) develop from pre-pDCs, whereas two lineages of conventional DCs (cDCs; cDC1s and cDC2s) develop from lineage-committed pre-cDCs. Several transcription factors (TFs) have been implicated in regulating the development of pDCs (E2-2 and Id2) and cDC1s (Irf8, Id2, and Batf3); however, those required for the early commitment of pre-cDCs toward the cDC2 lineage are unknown. Here, we identify the TF zinc finger E box–binding homeobox 2 (Zeb2) to play a crucial role in regulating DC development. Zeb2 was expressed from the pre-pDC and pre-cDC stage onward and highly expressed in mature pDCs and cDC2s. Mice conditionally lacking Zeb2 in CD11c+ cells had a cell-intrinsic reduction in pDCs and cDC2s, coupled with an increase in cDC1s. Conversely, mice in which CD11c+ cells overexpressed Zeb2 displayed a reduction in cDC1s. This was accompanied by altered expression of Id2, which was up-regulated in cDC2s and pDCs from conditional knockout mice. Zeb2 chromatin immunoprecipitation analysis revealed Id2 to be a direct target of Zeb2. Thus, we conclude that Zeb2 regulates commitment to both the cDC2 and pDC lineages through repression of Id2.
doi:10.1084/jem.20151715
PMCID: PMC4886362  PMID: 27185854
2.  A Benchmark for Evaluation of Algorithms for Identification of Cellular Correlates of Clinical Outcomes 
The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets.
doi:10.1002/cyto.a.22732
PMCID: PMC4874734  PMID: 26447924
flow cytometry; bioinformatics; clustering; classification; data analysis; HIV; clinical outcome; supervised analysis
3.  The potential of radiotherapy to enhance the efficacy of renal cell carcinoma therapy 
Oncoimmunology  2015;4(10):e1042198.
Renal cell carcinoma (RCC) is an immunogenic tumor, but uses several immune-suppressive mechanisms to shift the balance from tumor immune response toward tumor growth. Although RCC has traditionally been considered to be radiation resistant, recent evidence suggests that hypofractionated radiotherapy contributes to systemic antitumor immunity. Because the efficacy of antitumor immune responses depends on the complex balance between diverse immune cells and progressing tumor cells, radiotherapy alone is unlikely to induce persistent antitumor immunity. Therefore, the combination of radiotherapy with drugs having synergistic immunomodulatory properties holds great promise with the optimal timing and sequence of modalities depending on the agent used. We highlight the immunomodulatory properties of targeted therapies, such as tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors and vascular endothelial growth factor (VEGF) neutralizing antibodies, and will suggest a combination schedule with radiotherapy based on the available literature. We also address the combination of radiotherapy with innovative treatments in the field of immunotherapy.
doi:10.1080/2162402X.2015.1042198
PMCID: PMC4590014  PMID: 26464810
antitumor immunity; immunotherapy; radiotherapy; renal cell carcinoma; targeted therapy; treatment combination
4.  β-Glucan exacerbates allergic airway responses to house dust mite allergen 
Respiratory Research  2016;17:35.
β-(1,3)-Glucan is present in mould cell walls and frequently detected in house dust mite (HDM) faeces. β-Glucan exposure is thought to be associated with pulmonary allergic inflammation in mouse and man, although the published data are inconsistent. Here, we show that highly purified β-glucan exacerbates HDM-induced eosinophilic, T helper 2 type airway responses by acting as an adjuvant, promoting activation, proliferation and polarisation of HDM-specific T cells (1-Derβ T cells). We therefore provide definitive evidence that β-glucan can influence allergic pulmonary inflammation.
doi:10.1186/s12931-016-0352-5
PMCID: PMC4818888  PMID: 27039089
β-glucans; Allergy; Eosinophil; T helper 2; House dust mite
5.  Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species 
Immunity  2016;45(3):669-684.
Summary
Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.
Graphical Abstract
Image 1
Highlights
•A conserved gating strategy aligns dendritic cells (DCs) in mouse and human tissues•Unsupervised computational analysis of flow cytometry data outperforms manual analysis•Mass cytometry reveals heterogeneity of DC subsets across mouse and human tissues•DC activation upon inflammation tracked by automated analysis of mass cytometry
Using unsupervised analysis of flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues, Guilliams et al. provide a universal toolbox for the automated identification of dendritic cells. This framework represents the way forward to high-throughput and standardized analysis of dendritic cells from mutant mice and patients.
doi:10.1016/j.immuni.2016.08.015
PMCID: PMC5040826  PMID: 27637149
6.  Early IL-1 Signaling Promotes iBALT Induction after Influenza Virus Infection 
Inducible bronchus-associated lymphoid tissue (iBALT) is a long lasting tertiary lymphoid tissue that can be induced following influenza A virus (IAV) infection. Previous studies have shown that iBALT structures containing germinal center (GC) B cells protect against repeated infection by contributing locally to the cellular and humoral immune response. If we are to exploit this in vaccination strategies, we need a better understanding on how iBALT structures are induced. One hypothesis is that the strength of the initial innate response dictates induction of iBALT. In the present study, we investigated the role of interleukin (IL)-1 and IL-1R signaling on iBALT formation. Mice lacking the IL-1R had a delayed viral clearance and, thus, a prolonged exposure to viral replication, leading to increased disease severity, compared to wild-type mice. Contradictorily, iBALT formation following clearance of the virus was heavily compromised in Il1r1−/− mice. Quantification of gene induction after IAV infection demonstrated induction of IL-1α and to a much lesser extent of IL-1β. Administration of recombinant IL-1α to the lungs of wild-type mice, early but not late, after IAV infection led to more pronounced iBALT formation and an increased amount of GC B cells in the lungs. Bone marrow chimeric mice identified the stromal compartment as the crucial IL-1 responsive cell for iBALT induction. Mechanistically, Q-PCR analysis of lung homogenates revealed a strongly diminished production of CXCL13, a B cell-attracting chemokine, in Il1r−/− mice during the early innate phase of IAV infection. These experiments demonstrate that appropriate innate IL-1α–IL-1R signaling is necessary for IAV clearance and at the same time instructs the formation of organized tertiary lymphoid tissues through induction of CXCL13 early after infection. These findings are discussed in the light of recent insights on the pathogenesis of tertiary lymphoid organ formation in the lung in various diseases where the IL-1 axis is hyperactive, such as rheumatoid arthritis and COPD.
doi:10.3389/fimmu.2016.00312
PMCID: PMC4985557  PMID: 27579026
influenza; innate immunity; TLO; IL-1; iBALT; CXCL13
7.  Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy 
Scientific Reports  2016;6:31621.
Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.
doi:10.1038/srep31621
PMCID: PMC4992859  PMID: 27546726
8.  Emerging functions of the unfolded protein response in immunity 
Nature immunology  2014;15(10):910-919.
The unfolded protein response (UPR) has traditionally been viewed as an adaptive response triggered upon accumulation of unfolded proteins in the endoplasmic reticulum (ER), aimed at restoring ER function. The UPR can also be an anticipatory response that is activated well before the disruption of protein homeostasis. UPR signaling intersects at many levels with the innate and adaptive immune response. In some immune cell types like dendritic cells and B cells, particular UPR sensors appear constitutively active in the absence of traditional UPR gene program induction, necessary for antigen presentation and immunoglobulin synthesis. The UPR also influences Toll-like receptor signaling and NF-κB activation, and some pathogens subvert the UPR. This review summarizes these emerging non-canonical functions of the UPR in immunity.
doi:10.1038/ni.2991
PMCID: PMC4388558  PMID: 25232821
9.  Cholesterol‐sensing liver X receptors stimulate Th2‐driven allergic eosinophilic asthma in mice 
Abstract
Introduction
Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known.
Methods
To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin‐ and a house dust mite‐driven eosinophilic asthma mouse model.
Results
In both models, airway inflammation, airway hyper‐reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXRα and LXRβ isoforms (LXRα−/−β−/−) as compared to wild‐type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXRα−/−β−/− mice showed strongly reduced protein levels of IL‐5 and IL‐13 in the lungs as well as reduced expression of these cytokines by CD4+ lung cells and lung‐draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung‐draining lymph node cells.
Conclusions
In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production.
doi:10.1002/iid3.118
PMCID: PMC5004289  PMID: 27621817
asthma; cholesterol; eosinophilic airway inflammation; liver X receptor
10.  Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting 
Bio-protocol  2015;5(5):e1415.
The spleen is a vastly vasculated organ and consists of a complex organized network of innate and adaptive immune cells. This permits the specialized functions of the spleen such as antibacterial and antifungal immunity and iron metabolism among others (Mebius and Kraal, 2005). Different dendritic cell (DC) subsets reside in the spleen and can be defined by the expression of unique surface markers. These DC subsets are recognized to perform non-redundant functions in the immune system (Merad et al., 2013). In our recent study, we found that Inositol Requiring Enzyme (IRE)-1 is specifically activated in splenic CD8a+ DCs. Furthermore, loss of X-box binding protein (XBP)-1 – the transcription factor regulated by IRE-1 – resulted in defective cross-presentation of dead cell associated antigens by splenic CD8a+ DCs (Osorio et al., 2014). This protocol allows the isolation of specific DC subsets for experimental use ex-vivo.
PMCID: PMC4928688  PMID: 27376108
11.  Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection 
The Journal of Experimental Medicine  2015;212(12):2015-2025.
The transcription factor Zeb2 cooperates with T-bet to control NK cell maturation, viability, and exit from the bone marrow and is essential for rejection of melanoma lung metastasis.
Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2−/− NK cells closely resembled that of Tbx21−/− NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21−/− NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells.
doi:10.1084/jem.20150809
PMCID: PMC4647267  PMID: 26503444
12.  Immunity induced by a broad class of inorganic crystalline materials is directly controlled by their chemistry 
The Journal of Experimental Medicine  2014;211(6):1019-1025.
Immune responses can be predicted by the chemical properties of systematically variable inorganic crystalline materials.
There is currently no paradigm in immunology that enables an accurate prediction of how the immune system will respond to any given agent. Here we show that the immunological responses induced by members of a broad class of inorganic crystalline materials are controlled purely by their physicochemical properties in a highly predictable manner. We show that structurally and chemically homogeneous layered double hydroxides (LDHs) can elicit diverse human dendritic cell responses in vitro. Using a systems vaccinology approach, we find that every measured response can be modeled using a subset of just three physical and chemical properties for all compounds tested. This correlation can be reduced to a simple linear equation that enables the immunological responses stimulated by newly synthesized LDHs to be predicted in advance from these three parameters alone. We also show that mouse antigen–specific antibody responses in vivo and human macrophage responses in vitro are controlled by the same properties, suggesting they may control diverse responses at both individual component and global levels of immunity. This study demonstrates that immunity can be determined purely by chemistry and opens the possibility of rational manipulation of immunity for therapeutic purposes.
doi:10.1084/jem.20131768
PMCID: PMC4042647  PMID: 24799501
13.  A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection 
PLoS Pathogens  2016;12(1):e1005410.
A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.
Author Summary
Influenza viruses are a major public health threat. Each year, the typical seasonal flu epidemic affects millions of people with sometimes fatal outcomes, especially in high risk groups such as young children and elderly. The sporadic pandemic outbreaks can have even more disastrous consequences. The protein A20 is an important negative regulator of antiviral immune responses. We show that the specific deletion of A20 in bronchial epithelial cells improves the protection against influenza virus infections. This increased protection correlates with a dampened pulmonary cytotoxic T cell response and a strongly suppressed expression of the chemokine CCL2 during later stages of infection.
doi:10.1371/journal.ppat.1005410
PMCID: PMC4731390  PMID: 26815999
14.  Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells 
Nature Communications  2016;7:10321.
Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them.
Tissue-resident macrophages are mostly derived from embryonic progenitors. Scott et al. develop a mouse model to specifically deplete Kupffer cells (KC) in vivo and show that monocyte-derived cells can repopulate KC niche and behave similar to their embryonically-derived counterparts.
doi:10.1038/ncomms10321
PMCID: PMC4737801  PMID: 26813785
15.  Impaired survival of regulatory T cells in pulmonary sarcoidosis 
Respiratory Research  2015;16(1):108.
Background
Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.
Methods
Patients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).
Results
In sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO+ Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs - but not Th cells - showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs.
Conclusion
In untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0265-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12931-015-0265-8
PMCID: PMC4574219  PMID: 26376720
17.  Microbial Ligand Costimulation Drives Neutrophilic Steroid-Refractory Asthma 
PLoS ONE  2015;10(8):e0134219.
Asthma is a heterogeneous disease whose etiology is poorly understood but is likely to involve innate responses to inhaled microbial components that are found in allergens. The influence of these components on pulmonary inflammation has been largely studied in the context of individual agonists, despite knowledge that they can have synergistic effects when used in combination. Here we have explored the effects of LPS and β-glucan, two commonly-encountered microbial agonists, on the pathogenesis of allergic and non-allergic respiratory responses to house dust mite allergen. Notably, sensitization with these microbial components in combination acted synergistically to promote robust neutrophilic inflammation, which involved both Dectin-1 and TLR-4. This pulmonary neutrophilic inflammation was corticosteroid-refractory, resembling that found in patients with severe asthma. Thus our results provide key new insights into how microbial components influence the development of respiratory pathology.
doi:10.1371/journal.pone.0134219
PMCID: PMC4532492  PMID: 26261989
18.  Imaging regulatory T cell dynamics and suppression of T cell priming mediated by CTLA4 
Nature communications  2015;6:6219.
Foxp3+ regulatory T cells (Tregs) maintain immune homeostasis through mechanisms that remain incompletely defined. Here, by two-photon imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node. In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes, and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters, and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T cell priming.
doi:10.1038/ncomms7219
PMCID: PMC4347855  PMID: 25653051
Immunoimaging; two-photon microscopy; immunoregulation; Foxp3; CTLA4; regulatory T cell
19.  Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen 
The Journal of Experimental Medicine  2010;207(10):2097-2111.
It is unclear how Th2 immunity is induced in response to allergens like house dust mite (HDM). Here, we show that HDM inhalation leads to the TLR4/MyD88-dependent recruitment of IL-4 competent basophils and eosinophils, and of inflammatory DCs to the draining mediastinal nodes. Depletion of basophils only partially reduced Th2 immunity, and depletion of eosinophils had no effect on the Th2 response. Basophils did not take up inhaled antigen, present it to T cells, or express antigen presentation machinery, whereas a population of FceRI+ DCs readily did. Inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required. We favor a model whereby DCs initiate and basophils amplify Th2 immunity to HDM allergen.
doi:10.1084/jem.20101563
PMCID: PMC2947072  PMID: 20819925
20.  The lung vascular filter as a site of immune induction for T cell responses to large embolic antigen 
The Journal of Experimental Medicine  2009;206(12):2823-2835.
The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 µm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11chi cells or Ly6C/G+ cells but restored upon adoptive transfer of Ly6Chi monocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.
doi:10.1084/jem.20082401
PMCID: PMC2806611  PMID: 19858325
21.  Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice 
The Journal of Experimental Medicine  2009;206(11):2339-2349.
Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11chi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.
doi:10.1084/jem.20090410
PMCID: PMC2768850  PMID: 19808255
22.  Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells 
The Journal of Experimental Medicine  2008;205(7):1621-1634.
Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.
doi:10.1084/jem.20071365
PMCID: PMC2442640  PMID: 18591406
23.  Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells 
Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)–resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c+ monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting “nature's adjuvant,” the inflammatory DC through induction of the endogenous danger signal uric acid.
doi:10.1084/jem.20071087
PMCID: PMC2292225  PMID: 18362170
24.  CCR2+CD103− intestinal dendritic cells develop from DC committed precursors and induce interleukin 17 production by T cells 
Mucosal immunology  2014;8(2):327-339.
The identification of intestinal macrophages (mϕs) and dendritic cells (DCs) is a matter of intense debate. While CD103+ mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103− MPs remain controversial. We show here that intestinal CD103−CD11b+ MPs can be separated clearly into DCs and mϕs based on phenotype, gene profile and kinetics. CD64-CD103−CD11b+ MPs are classical DCs, being derived from Flt3L-dependent, DC-committed precursors, not Ly6Chi monocytes. Surprisingly, a significant proportion of these CD103−CD11b+ DCs expresses CCR2 and there is a selective decrease in CD103−CD11b+ DCs in mice lacking this chemokine receptor. CCR2+CD103− DCs are present in both the murine and human intestine, drive IL-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2+ DC, which is involved in priming mucosal Th17 responses.
doi:10.1038/mi.2014.70
PMCID: PMC4270738  PMID: 25138666
25.  Activation of the D prostanoid 1 receptor suppresses asthma by modulation of lung dendritic cell function and induction of regulatory T cells 
Prostaglandins (PGs) can enhance or suppress inflammation by acting on different receptors expressed by hematopoietic and nonhematopoietic cells. Prostaglandin D2 binds to the D prostanoid (DP)1 and DP2 receptor and is seen as a critical mediator of asthma causing vasodilation, bronchoconstriction, and inflammatory cell influx. Here we show that inhalation of a selective DP1 agonist suppresses the cardinal features of asthma by targeting the function of lung dendritic cells (DCs). In mice treated with DP1 agonist or receiving DP1 agonist-treated DCs, there was an increase in Foxp3+ CD4+ regulatory T cells that suppressed inflammation in an interleukin 10–dependent way. These effects of DP1 agonist on DCs were mediated by cyclic AMP–dependent protein kinase A. We furthermore show that activation of DP1 by an endogenous ligand inhibits airway inflammation as chimeric mice with selective hematopoietic loss of DP1 had strongly enhanced airway inflammation and antigen-pulsed DCs lacking DP1 were better at inducing airway T helper 2 responses in the lung. Triggering DP1 on DCs is an important mechanism to induce regulatory T cells and to control the extent of airway inflammation. This pathway could be exploited to design novel treatments for asthma.
doi:10.1084/jem.20061196
PMCID: PMC2118726  PMID: 17283205

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