Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

IL-17 regulates DC migration to the peribronchial LNs and allergen presentation in experimental allergic asthma.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

A comparison of curated gene sets versus transcriptomics-derived gene signatures for detecting pathway activation in immune cells.

BACKGROUND: Despite the significant contribution of transcriptomics to the fields of biological and biomedical research, interpreting long lists of significantly differentially expressed genes remains a challenging step in the analysis process. Gene set enrichment analysis is a standard approach for summarizing differentially expressed genes into pathways or other gene groupings. Here, we explore an alternative approach to utilizing gene sets from curated databases. We examine the method of deriving custom gene sets which may be relevant to a given experiment using reference data sets from previous transcriptomics studies. We call these data-derived gene sets, "gene signatures" for the biological process tested in the previous study. We focus on the feasibility of this approach in analyzing immune-related processes, which are complicated in their nature but play an important role in the medical research. RESULTS: We evaluate several statistical approaches to detecting the activity of a gene signature in a target data set. We compare the performance of the data-derived gene signature approach with comparable GO term gene sets across all of the statistical tests. A total of 61 differential expression comparisons generated from 26 transcriptome experiments were included in the analysis. These experiments covered eight immunological processes in eight types of leukocytes. The data-derived signatures were used to detect the presence of immunological processes in the test data with modest accuracy (AUC = 0.67). The performance for GO and literature based gene sets was worse (AUC = 0.59). Both approaches were plagued by poor specificity. CONCLUSIONS: When investigators seek to test specific hypotheses, the data-derived signature approach can perform as well, if not better than standard gene-set based approaches for immunological signatures. Furthermore, the data-derived signatures can be generated in the cases that well-defined gene sets are lacking from pathway databases and also offer the opportunity for defining signatures in a cell-type specific manner. However, neither the data-derived signatures nor standard gene-sets can be demonstrated to reliably provide negative predictions for negative cases. We conclude that the data-derived signature approach is a useful and sometimes necessary tool, but analysts should be weary of false positives.

B cells control maternofetal priming of allergy and tolerance in a murine model of allergic airway inflammation.

BACKGROUND: Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. OBJECTIVE: We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming. METHODS: Wild-type (WT) and B cell-deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation. RESULTS: Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell-deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and antigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell-deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3+ regulatory T cells after in vitro coincubation with OVA/IgG-containing AF. CONCLUSION: Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future.

B cell subsets are modulated during allergic airway inflammation but are not required for the development of respiratory tolerance in a murine model.

Allergic asthma is a widespread chronic inflammatory disease of the airways. The role of different B cell subsets in developing asthma and respiratory tolerance is not well known. Especially regulatory B (Breg) cells are proposed to be important in asthma regulation. Using wild-type (WT) and B cell-deficient (µMT) mice we investigated how B cells are affected by induction of allergic airway inflammation and respiratory tolerance and whether they are necessary to develop these conditions. WT mice with an asthma-like phenotype, characterized by increased airway hyper reactivity, eosinophilic airway inflammation, mucus hypersecretion and elevated Th2 cytokines, exhibited increased MHCII and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen, which contributed to allergen-specific T cell proliferation in vitro. Germinal center B cell numbers were elevated and associated with increased production of allergen-specific immunoglobulins especially in bLN. In contrast, respiratory tolerance clearly attenuated these B cell alterations and directly enhanced marginal zone precursor B cells, which induced regulatory T cells in vitro. However, µMT mice developed asthma-like and tolerized phenotypes like WT mice. Our data indicate that although B cell subsets are affected by asthma-like and respiratory tolerant phenotypes, B cells are not required for tolerance induction.

IL-27 Is Essential for Suppression of Experimental Allergic Asthma by the TLR7/8 Agonist R848 (Resiquimod).

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell-driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death-ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.

Improved protocol for simultaneous analysis of leukocyte subsets and epithelial cells from murine and human lung.

PURPOSE: To study and isolate lung cells by flow cytometry, enzymatic digestion and generation of single cell suspensions is required. This significantly influences expression of cellular epitopes and protocols need to be adapted for the best isolation and subsequent analysis of specific cellular subsets. MATERIALS AND METHODS: We optimized protocols for the simultaneous isolation and characterization of specific human and murine lung cell types. For alveolar epithelial cells (AEC), a primarily dispase based digestion method and for leukocytes, a primarily collagenase based technique was adapted. Protocols were applied in parallel in either single experimental mice or human lung specimens. RESULTS: Optimized dispase/DNase digestion yielded a high percentage of Epcam+CD45-CD31- AEC as assessed by flow cytometry. Epcam+CD45-CD3-CD11b-CD11c-CD16/32-CD19-CD31-F4/80- AEC were readily sortable with high purity and typical morphology and function upon in vitro stimulation with lipopolysaccharide or respiratory-syncytial-virus (RSV) infection. To analyze lung leukocytes, specimens were digested with an adapted collagenase/DNase protocol yielding high percentages of viable leukocytes with typical morphology, function, and preserved subset specific leukocyte markers. Both protocols could be applied simultaneously in a single experimental mouse post mortem. Application of both digestion methods in primary human lung specimens yielded similar results with high proportions of Epcam+CD45- human AEC after dispase/DNase digestion and preservation of human T cell epitopes after collagenase/DNase digestion. CONCLUSION: The here described protocols were optimized for the simple and efficient isolation of murine and human lung cells. In contrast to previously described techniques, they permit simultaneous in-depth characterization of pulmonary epithelial cells and leukocyte subsets such as T helper, cytotoxic T, and B cells from one sample. As such, they may help to comprehensively and sustainably characterize murine and human lung specimens and facilitate studies on the role of lung immune cells in different respiratory pathologies.

Gain-of-function STAT1 mutations are associated with intracranial aneurysms.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor ß1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-ß type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Priming of CD8+ T cells against cytomegalovirus-encoded antigens is dominated by cross-presentation.

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8(+) T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB498 epitope from HSV-1 were unable to stimulate in vitro naive gB498-specific CD8(+) T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB498-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α(+) DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8(+) T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I-mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8(+) T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.

Polysialic acid on neuropilin-2 is exclusively synthesized by the polysialyltransferase ST8SiaIV and attached to mucin-type o-glycans located between the b2 and c domain.

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.

Competitive survival of clonal serial Pseudomonas aeruginosa isolates from cystic fibrosis airways in human neutrophils.

Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.

Single cell RNA sequencing reveals distinct clusters of Irf8-expressing pulmonary conventional dendritic cells.

A single population of interferon-regulatory factor 8 (Irf8)-dependent conventional dendritic cell (cDC type1) is considered to be responsible for both immunogenic and tolerogenic responses depending on the surrounding cytokine milieu. Here, we challenge this concept of an omnipotent single Irf8-dependent cDC1 cluster through analysis of pulmonary cDCs at single cell resolution. We report existence of a pulmonary cDC1 cluster lacking Xcr1 with an immunogenic signature that clearly differs from the Xcr1 positive cDC1 cluster. The Irf8+Batf3+Xcr1- cluster expresses high levels of pro-inflammatory genes associated with antigen presentation, migration and co-stimulation such as Ccr7, Cd74, MHC-II, Ccl5, Il12b and Relb while, the Xcr1+ cDC1 cluster expresses genes corresponding to immune tolerance mechanisms like Clec9a, Pbx1, Cadm1, Btla and Clec12a. In concordance with their pro-inflammatory gene expression profile, the ratio of Xcr1- cDC1s but not Xcr1+cDC1 is increased in the lungs of allergen-treated mice compared to the control group, in which both cDC1 clusters are present in comparable ratios. The existence of two distinct Xcr1+ and Xcr1- cDC1 clusters is furthermore supported by velocity analysis showing markedly different temporal patterns of Xcr1- and Xcr1+cDC1s. In summary, we present evidence for the existence of two different cDC1 clusters with distinct immunogenic profiles in vivo. Our findings have important implications for DC-targeting immunomodulatory therapies.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Interleukin-17A and interleukin-22 production by conventional and non-conventional lymphocytes in three different end-stage lung diseases.

Objectives: The contribution of adaptive vs. innate lymphocytes to IL-17A and IL-22 secretion at the end stage of chronic lung diseases remains largely unexplored. In order to uncover tissue- and disease-specific secretion patterns, we compared production patterns of IL-17A and IL-22 in three different human end-stage lung disease entities. Methods: Production of IL-17A, IL-22 and associated cytokines was assessed in supernatants of re-stimulated lymphocytes by multiplex assays and multicolour flow cytometry of conventional T cells, iNKT cells, γδ T cells and innate lymphoid cells in bronchial lymph node and lung tissue from patients with emphysema (n = 19), idiopathic pulmonary fibrosis (n = 14) and cystic fibrosis (n = 23), as well as lung donors (n = 17). Results: We detected secretion of IL-17A and IL-22 by CD4+ T cells, CD8+ T cells, innate lymphoid cells, γδ T cells and iNKT cells in all end-stage lung disease entities. Our analyses revealed disease-specific contributions of individual lymphocyte subpopulations to cytokine secretion patterns. We furthermore found the high levels of microbial detection in CF samples to associate with a more pronounced IL-17A signature upon antigen-specific and unspecific re-stimulation compared to other disease entities and lung donors. Conclusion: Our results show that both adaptive and innate lymphocyte populations contribute to IL-17A-dependent pathologies in different end-stage lung disease entities, where they establish an IL-17A-rich microenvironment. Microbial colonisation patterns and cytokine secretion upon microbial re-stimulation suggest that pathogens drive IL-17A secretion patterns in end-stage lung disease.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

In vitro neutrophil migration is associated with inhaled corticosteroid treatment and serum cytokines in pediatric asthma.

Background: Different asthma phenotypes are driven by molecular endotypes. A Th1-high phenotype is linked to severe, therapy-refractory asthma, subclinical infections and neutrophil inflammation. Previously, we found neutrophil granulocytes (NGs) from asthmatics exhibit decreased chemotaxis towards leukotriene B4 (LTB4), a chemoattractant involved in inflammation response. We hypothesized that this pattern is driven by asthma in general and aggravated in a Th1-high phenotype. Methods: NGs from asthmatic nd healthy children were stimulated with 10 nM LTB4/100 nM N-formylmethionine-leucyl-phenylalanine and neutrophil migration was documented following our prior SiMA (simplified migration assay) workflow, capturing morphologic and dynamic parameters from single-cell tracking in the images. Demographic, clinical and serum cytokine data were determined in the ALLIANCE cohort. Results: A reduced chemotactic response towards LTB4 was confirmed in asthmatic donors regardless of inhaled corticosteroid (ICS) treatment. By contrast, only NGs from ICS-treated asthmatic children migrate similarly to controls with the exception of Th1-high donors, whose NGs presented a reduced and less directed migration towards the chemokines. ICS-treated and Th1-high asthmatic donors present an altered surface receptor profile, which partly correlates with migration. Conclusions: Neutrophil migration in vitro may be affected by ICS-therapy or a Th1-high phenotype. This may be explained by alteration of receptor expression and could be used as a tool to monitor asthma treatment.

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

Contribution of direct and cross-presentation to CTL immunity against herpes simplex virus 1.

Dendritic cells (DC), which can be subdivided into different phenotypic and functional subsets, play a pivotal role in the generation of cytotoxic T cell immunity against viral infections. Understanding the modes of Ag acquisition, processing and presentation by DC is essential for the design of effective antiviral vaccines. We aimed to assess the contribution of direct vs cross-presentation for the induction of HSV1-specific CD8(+) T lymphocyte responses in mice. Using HSV1 strains expressing fluorescence proteins, we provide evidence for the ability of HSV1 to induce viral transcription. Using HSV1-wild-type as well as gB- or gH-deficient mutants to either directly inoculate DC or to infect target cells, which then were given to DC, we show that DC acquired viral Ag via phagocytosis of target cells and via direct inoculation of virus being released from target cells. Our study corroborates the function of the CD8(+) DC specialized in Ag cross-presentation and confirms this specific feature for Ags that these DC acquire directly from HSV1. However, although infection of cross-presenting DC amplified T cell responses, it was not a requirement for presentation of viral Ags, both in vitro and in vivo. Finally, we provide evidence that direct presentation did not contribute to the Ag presentation capacity of CD8(+) DC after phagocytosis of infected target cells. We conclude that cross-presentation is of major importance for the induction of CTL immunity in mice.

Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages.

Tissue macrophages are important components of tissue homeostasis and inflammatory pathologies. In the peritoneal cavity, resident macrophages interact with a variety of immune cells and can exhibit broad range of phenotypes and functions. Forkhead-box-P3 (FOXP3)+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance, yet whether, and how the pathological condition that results from the lack of functional Tregs affects peritoneal macrophages (PM) is largely unknown. We used FOXP3-deficient scurfy (Sf) mice to investigate PM behavior in terms of the missing crosstalk with Tregs. Here, we report that Treg deficiency induced a marked increase in PM numbers, which was reversed after adoptive transfer of CD4+ T cells or neutralization of macrophage colony-stimulating factor. Ex vivo assays demonstrated a pro-inflammatory state of PM from Sf mice and signs of excessive activation and exhaustion. In-depth immunophenotyping of Sf PM using single-cell chipcytometry and transcriptome analysis revealed upregulation of molecules involved in the initiation of innate and adaptive immune responses. Moreover, upon transfer to non-inflammatory environment or after injection of CD4+ T cells, PM from Sf mice reprogramed their functional phenotype, indicating remarkable plasticity. Interestingly, frequencies, and immune polarization of large and small PM subsets were dramatically changed in the FOXP3-deficient mice, suggesting distinct origin and specialized function of these subsets in inflammatory conditions. Our findings demonstrate the significant impact of Tregs in shaping PM identity and dynamics. A better understanding of PM function in the Sf mouse model may have clinical implication for the treatment of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and other forms of immune-mediated enteropathies.