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Dendritic cells (DCs) are the most effective professional antigen-presenting cell. They ferry antigen from the extremities to T cells and are essential for the initiation of an adaptive immune response. Despite interest in how DCs respond to chemical stimuli, there have been few attempts to model DC migration. In this paper, we simulate the motility of DCs by modeling the generation of forces by filopodia and a force balance on the cell. The direction of fliopodial extension is coupled to differential occupancy of cognate chemokine receptors across the cell. Our model simulates chemokinesis and chemotaxis in a variety of chemical and mechanical environments. Simulated DCs undergoing chemokinesis were measured to have a speed of 5.1 ± 0.07 μm·min-1 and a persistence time of 3.2 ± 0.46 min, consistent with experiment. Cells undergoing chemotaxis exhibited a stronger chemotactic response when exposed to lower average chemokine concentrations, also consistent with experiment. We predicted that when placed in two opposing gradients, cells will cluster in a line, which we call the “line of equistimulation;” this clustering has also been observed. We calculated the effect of varying gradient steepness on the line of equistimulation, with steeper gradients resulting in tighter clustering. Moreover, gradients are found to be most potent when cells are in a gradient of chemokine whose mean concentration is close to the binding of the Kd to the receptor, and least potent when the mean concentration is 0.1Kd. Comparing our simulations to experiment, we can give a quantitative measure of the strength of certain chemokines relative to others. Assigning the signal of CCL19 binding CCR7 a baseline strength of 1, we found CCL21 binding CCR7 had a strength of 0.28, and CXCL12 binding CXCR4 had a strength of 0.30. These differences emerge despite both chemokines having virtually the same Kd, suggesting a mechanism of signal amplification in DCs requiring further study. [ABSTRACT FROM AUTHOR]

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying (“replicon”) mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers – CD80, CD86 and MHC-II – and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction. [ABSTRACT FROM AUTHOR]

Oncolytic virus (OV) is a replication competent virus that selectively invades cancer cells; as these cells die under the viral burden, the released virus particles proceed to infect other cancer cells. Oncolytic viruses are designed to also be able to stimulate the anticancer immune response. Thus, one may represent an OV by two parameters: its replication potential and its immunogenicity. In this paper we consider a combination therapy with OV and a checkpoint inhibitor, anti-PD-1. We evaluate the efficacy of the combination therapy in terms of the tumor volume at some later time, for example, 6 months from initial treatment. Since T cells kill not only virus-free cancer cells but also virus-infected cancer cells, the following question arises: Does increasing the amount of the checkpoint inhibitor always improve the efficacy? We address this question, by a mathematical model consisting of a system of partial differential equations. We use the model to construct, by simulations, an efficacy map in terms of the doses of the checkpoint inhibitor and the OV injection. We show that there are regions in the map where an increase in the checkpoint inhibitor actually decreases the efficacy of the treatment. We also construct efficacy maps with checkpoint inhibitor vs. the replication potential of the virus that show the same antagonism, namely, an increase in the checkpoint inhibitor may actually decrease the efficacy. These results have implications for clinical trials. [ABSTRACT FROM AUTHOR]

Cross-presentation of apoptotic cell associated antigens by immature dendritic cells prevents the activation of self reactive CD8 T cells. Tolerized self reactive CD8 T cells down-regulate IL-7R expression on their surface. Whether over-expression of IL-7R can reverse their fate and function has not been examined. In this paper, we showed forced expression of IL-7R in OT-I T cells by a transgene enhanced CD8 T cell mediated diabetes in the RIP-mOVA model. Although IL-7R Tg (transgenic) did not completely reverse the deletion of OT-I T cells, it provided a significant survival advantage over w.t OT-I T cells. Furthermore, IL7R Tg OT-I T cells isolated from diabetic pancreata displayed increased production of IFN-γ, higher expression of T-bet, and increased externalization of CD107a. We also found that immature DCs containing apoptotic cells expressed high levels of PDL-1 on their surface. Although IL-7R Tg did not change PD1 expression on activated OT-I cells in vivo, the transgene enabled a significantly lower number of OT-I T cells to induce diabetes in the absence of PDL-1. Our results demonstrated that forced expression of IL-7R not only improved the functionality of tolerized CD8 T cells, it also acted in synergy with PDL-1 deficiency to further promote CD8 T cell cytotoxicity to self antigens. [ABSTRACT FROM AUTHOR]

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated. [ABSTRACT FROM AUTHOR]

In this paper we consider a combination therapy of cancer. One drug is a vaccine which activates dendritic cells so that they induce more T cells to infiltrate the tumor. The other drug is a checkpoint inhibitor, which enables the T cells to remain active against the cancer cells. The two drugs are positively correlated in the sense that an increase in the amount of each drug results in a reduction in the tumor volume. We consider the question whether a treatment with combination of the two drugs at certain levels is preferable to a treatment by one of the drugs alone at ‘roughly’ twice the dosage level; if that is the case, then we say that there is a positive ‘synergy’ for this combination of dosages. To address this question, we develop a mathematical model using a system of partial differential equations. The variables include dendritic and cancer cells, CD4+ and CD8+ T cells, IL-12 and IL-2, GM-CSF produced by the vaccine, and a T cell checkpoint inhibitor associated with PD-1. We use the model to explore the efficacy of the two drugs, separately and in combination, and compare the simulations with data from mouse experiments. We next introduce the concept of synergy between the drugs and develop a synergy map which suggests in what proportion to administer the drugs in order to achieve the maximum reduction of tumor volume under the constraint of maximum tolerated dose. [ABSTRACT FROM AUTHOR]

This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named “L-DC” since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties. [ABSTRACT FROM AUTHOR]

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens. [ABSTRACT FROM AUTHOR]

Antibody dependent enhancement (ADE) has most often been associated with dengue virus (DENV). Studies using leukemia cell lines suggest that DENV specific antibodies can enhance Zika virus (ZIKV) infectivity, and vice versa. To examine the mechanisms of ADE of ZIKV infection in primary human cells, we assessed 40 serum samples obtained from convalescent DENV-1 or DENV-3 infected subjects. All sera tested exhibited high binding potency, while modest or none neutralization activities against ZIKV. Primary CD14+ monocytes, rather than B and T cells in peripheral blood mononuclear cells (PBMCs), were found to be the mediators of the enhancement of ZIKV infectivity by DENV immune sera. Monocyte-derived immature dendritic cells (DCs), but not mature DCs were highly permissive to ZIKV infection, whereas neither immature nor mature DCs could mediate enhanced ZIKV infection in the presence of DENV immune sera. In addition, antibody blocking of either FcγRI (CD64), or FcγRII (CD32), or FcγRIII (CD16) resulted in diminished ADE of ZIKV infection. Our findings provide an improved understanding of the pathogenesis of ZIKV infection, and inform rational vaccine design. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is a bacterial agent that causes sexually transmitted infections worldwide. The regulatory functions of dendritic cells (DCs) play a major role in protective immunity against Chlamydia infections. Here, we investigated the role of ASC in DCs metabolism and the regulation of DCs activation and function during Chlamydia infection. Following Chlamydia stimulation, maturation and antigen presenting functions were impaired in ASC-/- DCs compared to wild type (WT) DCs, in addition, ASC deficiency induced a tolerant phenotype in Chlamydia stimulated DCs. Using real-time extracellular flux analysis, we showed that activation in Chlamydia stimulated WT DCs is associated with a metabolic change in which mitochondrial oxidative phosphorylation (OXPHOS) is inhibited and the cells become committed to utilizing glucose through aerobic glycolysis for differentiation and antigen presenting functions. However, in ASC-/- DCs Chlamydia-induced metabolic change was prevented and there was a significant effect on mitochondrial morphology. The mitochondria of Chlamydia stimulated ASC-/- DCs had disrupted cristae compared to the normal narrow pleomorphic cristae found in stimulated WT DCs. In conclusion, our results suggest that Chlamydia-mediated activation of DCs is associated with a metabolic transition in which OXPHOS is inhibited, thereby dedicating the DCs to aerobic glycolysis, while ASC deficiency disrupts DCs function by inhibiting the reprogramming of DCs metabolism within the mitochondria, from glycolysis to electron transport chain. [ABSTRACT FROM AUTHOR]

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic cutaneous inflammatory disease. The immunopathogenesis is a complex interplay between T cells, dendritic cells and the epidermis in which T cells and dendritic cells maintain skin inflammation. Anti-tumour necrosis factor (anti-TNF)-α agents have been approved for therapeutic use across a range of inflammatory disorders including psoriasis, but the anti-inflammatory mechanisms of anti-TNF-α in lesional psoriatic skin are not fully understood. We investigated early events in skin from psoriasis patients after treatment with anti-TNF-α antibodies by use of bioinformatics tools. We used the Human Gene 1.0 ST Array to analyse gene expression in punch biopsies taken from psoriatic patients before and also 4 and 14 days after initiation of treatment with the anti-TNF-α agent adalimumab. The gene expression was analysed by gene set enrichment analysis using the Functional Annotation Tool from DAVID Bioinformatics Resources. The most enriched pathway was visualised by the Pathview Package on Kyoto Encyclopedia of Genes and Genomes (KEGG) graphs. The analysis revealed new very early events in psoriasis after adalimumab treatment. Some of these events have been described after longer periods of anti-TNF-α treatment when clinical and histological changes appear, suggesting that effects of anti-TNF-α treatment on gene expression appear very early before clinical and histological changes. Combining microarray data on biopsies from psoriasis patients with pathway analysis allowed us to integrate in vitro findings into the identification of mechanisms that may be important in vivo. Furthermore, these results may reflect primary effect of anti-TNF-α treatment in contrast to studies of gene expression changes following clinical and histological changes, which may reflect secondary changes correlated to the healing of the skin. [ABSTRACT FROM AUTHOR]

Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity. [ABSTRACT FROM AUTHOR]

The human peripheral leukocyte subset composition depends on genotype variation and pre-natal and post-natal environmental influence diversity. We quantified this composition in adults and neonates, and compared the median values and dispersal ranges of various subsets in them. We confirmed higher frequencies of monocytes and regulatory T cells (Tregs), similar frequencies of neutrophils, and lower frequencies of CD8 T cells, NKT cells, B1 B cells and gamma-delta T cells in neonatal umbilical cord blood. Unlike previous reports, we found higher frequencies of eosinophils and B cells, higher CD4:CD8 ratios, lower frequencies of T cells and iNKT cells, and similar frequencies of CD4 T cells and NK cells in neonates. We characterized monocyte subsets and dendritic cell (DC) subsets in far greater detail than previously reported, using recently described surface markers and gating strategies and observed that neonates had lower frequencies of patrolling monocytes and lower myeloid dendritic cell (mDC):plasmacytoid DC (pDC) ratios. Our data contribute to South Asian reference values for these parameters. We found that dispersal ranges differ between different leukocyte subsets, suggesting differential determination of variation. Further, some subsets were more dispersed in adults than in neonates suggesting influences of postnatal sources of variation, while some show the opposite pattern suggesting influences of developmental process variation. Together, these data and analyses provide interesting biological possibilities for future exploration. [ABSTRACT FROM AUTHOR]

Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4β7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity. [ABSTRACT FROM AUTHOR]

The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3+ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3+ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3+ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3+ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3+ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3+ DC numbers are increased in CHB patients. BDCA3+ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3+ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3+ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3+ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy. [ABSTRACT FROM AUTHOR]

Introduction: Tolerogenic dendritic cells (DCs) have the potential to prolong graft survival after transplantation. Tolerogenic DCs are in general characterized by a low expression of co-stimulatory molecule and a high IL-10:IL-12 production ratio. Based on promising results with earlier used alternatively activated DCs, we aimed to generate in culture potentially tolerogenic DC by simultaneously blocking GSK3 by lithium chloride (LiCl) and stimulating TLR2 by PAM3CysSerLys4. Materials and Methods: Bone marrow-derived LiClPAM3 DCs were generated by the addition of LiCl 24 hours before harvesting, and one hour later PAM3CysSerLys4. The phenotype of the DCs was assessed by determining the expression of co-stimulatory molecules in flow cytometry and cytokine production in ELISA, whereas their functional properties were tested in a mixed lymphocyte reaction. A fully MHC mismatched heterotopic heart transplant preceded by infusion of donor-derived LiClPAM3 DC was performed to assess the tolerogenic potential of LiClPAM3 DCs in vivo. Results: LiClPAM3 DCs displayed a tolerogenic phenotype accompanied with a low expression of co-stimulatory molecules and a high IL-10:IL-12 production ratio. However, in mixed lymphocyte reaction, LiClPAM3 DCs appeared superior in T cell stimulation, and induced Th1 and Th17 differentiation. Moreover, mice pretreated with LiClPAM3 DC displayed a reduced graft survival. Analysis of LiClPAM3 DC culture supernatant revealed high levels of CXCL-1, which was also found in supernatants of co-cultures of LiClPAM3 DC and T cells. Nevertheless, we could not show a role for CXCL-1 in T cell proliferation or activation in vitro. Discussion: LiClPAM3 DCs display in vitro a tolerogenic phenotype with a high IL-10:IL-12 ratio, but appeared to be highly immunogenic, since allograft rejection was accelerated. As yet unidentified LiClPAM3 DC-derived factors, may explain the immunogenic character of LiClPAM3 DCs in vivo. [ABSTRACT FROM AUTHOR]

HIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia. [ABSTRACT FROM AUTHOR]

The incidence of obesity has risen to epidemic proportions in recent decades, most commonly attributed to an increasingly sedentary lifestyle, and a ‘western’ diet high in fat and low in fibre. Although non-allergic asthma is a well-established co-morbidity of obesity, the influence of obesity on allergic asthma is still under debate. Allergic asthma is thought to result from impaired tolerance to airborne antigens, so-called respiratory tolerance. We sought to investigate whether a diet high in fats affects the development of respiratory tolerance. Mice fed a high fat diet (HFD) for 8 weeks showed weight gain, metabolic disease, and alteration in gut microbiota, metabolites and glucose metabolism compared to age-matched mice fed normal chow diet (ND). Respiratory tolerance was induced by repeated intranasal (i.n.) administration of ovalbumin (OVA), prior to induction of allergic airway inflammation (AAI) by sensitization with OVA in alum i.p. and subsequent i.n. OVA challenge. Surprisingly, respiratory tolerance was induced equally well in HFD and ND mice, as evidenced by decreased lung eosinophilia and serum OVA-specific IgE production. However, in a pilot study, HFD mice showed a tendency for impaired activation of airway dendritic cells and regulatory T cells compared with ND mice after induction of respiratory tolerance. Moreover, the capacity of lymph node cells to produce IL-5 and IL-13 after AAI was drastically diminished in HFD mice compared to ND mice. These results indicate that HFD does not affect the inflammatory or B cell response to an allergen, but inhibits priming of Th2 cells and possibly dendritic cell and regulatory T cell activation. [ABSTRACT FROM AUTHOR]

Innate immune cells shape the host response to microbial pathogens. Here we elucidate critical differences in the molecular response of macrophages vs. dendritic cells (DCs) to Histoplasma capsulatum, an intracellular fungal pathogen of humans. It has long been known that macrophages are permissive for Histoplasma growth and succumb to infection, whereas DCs restrict fungal growth and survive infection. We used murine macrophages and DCs to identify host pathways that influence fungal proliferation and host-cell viability. Transcriptional profiling experiments revealed that DCs produced a strong Type I interferon (IFN-I) response to infection with Histoplasma yeasts. Toll-like receptors 7 and 9 (TLR7/9), which recognize nucleic acids, were required for IFN-I production and restriction of fungal growth in DCs, but mutation of TLR7/9 had no effect on the outcome of macrophage infection. Moreover, TLR7/9 were essential for the ability of infected DCs to elicit production of the critical cytokine IFNγ from primed CD4+ T cells in vitro, indicating the role of this pathway in T cell activation. In a mouse model of infection, TLR7/9 were required for optimal production of IFN-I and IFNγ, host survival, and restriction of cerebral fungal burden. These data demonstrate the critical role of this pathway in eliciting an appropriate adaptive immune response in the host. Finally, although other fungal pathogens have been shown to elicit IFN-I in mouse models, the specific host cell responsible for producing IFN-I has not been elucidated. We found that CD103+ conventional DCs were the major producer of IFN-I in the lungs of wild-type mice infected with Histoplasma. Mice deficient in this DC subtype displayed reduced IFN-I production in vivo. These data reveal a previously unknown role for CD103+ conventional DCs and uncover the pivotal function of these cells in modulating the host immune response to endemic fungi. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated (abscopal) tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated (contralateral) secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects. [ABSTRACT FROM AUTHOR]

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability. [ABSTRACT FROM AUTHOR]

Macrophage and dendritic cell (DC) populations residing in the intestinal lamina propria (LP) are highly heterogeneous and have disparate yet collaborative roles in the promotion of adaptive immune responses towards intestinal antigen. Under steady-state conditions, macrophages are efficient at acquiring antigen but are non-migratory. In comparison, intestinal DC are inefficient at antigen uptake but migrate to the mesenteric lymph nodes (mLN) where they present antigen to T cells. Whether such distinction in the roles of DC and macrophages in the uptake and transport of antigen is maintained under immunostimulatory conditions is less clear. Here we show that the scavenger and phosphatidylserine receptor T cell Immunoglobulin and Mucin (TIM)-4 is expressed by the majority of LP macrophages at steady-state, whereas DC are TIM-4 negative. Oral treatment with the mucosal adjuvant cholera toxin (CT) induces expression of TIM-4 on a proportion of CD103+ CD11b+ DC in the LP. TIM-4+ DC selectively express high levels of co-stimulatory molecules after CT treatment and are detected in the mLN a short time after appearing in the LP. Importantly, intestinal macrophages and DC expressing TIM-4 are more efficient than their TIM-4 negative counterparts at taking up apoptotic cells and soluble antigen ex vivo. Taken together, our results show that CT induces phenotypic changes to migratory intestinal DC that may impact their ability to take up local antigens and in turn promote the priming of mucosal immunity. [ABSTRACT FROM AUTHOR]

Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy. [ABSTRACT FROM AUTHOR]

Although CD4+ Foxp3+ T cells are largely described in the regulation of CD4+ T cell responses, their role in the suppression of CD8+ T cell priming is much less clear. Because the induction of CD8+ T cells during experimental infection with Trypanosoma cruzi is remarkably delayed and suboptimal, we raised the hypothesis that this protozoan parasite actively induces the regulation of CD8+ T cell priming. Using an in vivo assay that eliminated multiple variables associated with antigen processing and dendritic cell activation, we found that injection of bone marrow-derived dendritic cells exposed to T. cruzi induced regulatory CD4+ Foxp3+ T cells that suppressed the priming of transgenic CD8+ T cells by peptide-loaded BMDC. This newly described suppressive effect on CD8+ T cell priming was independent of IL-10, but partially dependent on CTLA-4 and TGF-β. Accordingly, depletion of Foxp3+ cells in mice infected with T. cruzi enhanced the response of epitope-specific CD8+ T cells. Altogether, our data uncover a mechanism by which T. cruzi suppresses CD8+ T cell responses, an event related to the establishment of chronic infections. [ABSTRACT FROM AUTHOR]

In order for a virus to persist, there must be a balance between viral replication and immune clearance. It is commonly believed that adaptive immunity drives clearance of viral infections and, thus, dysfunction or viral evasion of adaptive immunity is required for a virus to persist. Type I interferons (IFNs) play pleiotropic roles in the antiviral response, including through innate control of viral replication. Murine norovirus (MNoV) replicates in dendritic cells (DCs) and type I IFN signaling in DCs is important for early control of MNoV replication. We show here that the non-persistent MNoV strain CW3 persists systemically when CD11c positive DCs are unable to respond to type I IFN. Persistence in this setting is associated with increased early viral titers, maintenance of DC numbers, increased expression of DC activation markers and an increase in CD8 T cell and antibody responses. Furthermore, CD8 T cell function is maintained during the persistent phase of infection and adaptive immune cells from persistently infected mice are functional when transferred to Rag1-/- recipients. Finally, increased early replication and persistence are also observed in mixed bone marrow chimeras where only half of the CD11c positive DCs are unable to respond to type I IFN. These findings demonstrate that increased early viral replication due to a cell-intrinsic innate immune deficiency is sufficient for persistence and a functional adaptive immune response is not sufficient for viral clearance. [ABSTRACT FROM AUTHOR]

Conventional dendritic cells (cDCs) comprise a heterogeneous population of cells that are important regulators of immunity and homeostasis. CD1c+ cDCs are present in human blood and tissues, and found to efficiently activate naïve CD4+ T cells. While CD1c is thought to specifically identify this subset of human cDCs, we show here that also classical and intermediate monocytes express CD1c. Accordingly, the commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two distinct cell populations from blood: CD1c+CD14− cDCs and CD1c+CD14+ monocytes. CD1c+ cDCs and CD1c+ monocytes exhibited strikingly different properties, including their differential regulation of surface marker expression, their levels of cytokine production, and their ability to stimulate naïve CD4+ T cells. These results demonstrate that a commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two functionally different cell populations, which has important implications for the interpretation of previously generated data using this kit to characterize CD1c+ cDCs. [ABSTRACT FROM AUTHOR]

Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines. [ABSTRACT FROM AUTHOR]

Skeletal muscle injury causes a local sterile inflammatory response. In parallel, a state of immunosuppression develops distal to the site of tissue damage. Granulocytes and monocytes that are rapidly recruited to the site of injury contribute to tissue regeneration. In this study we used a mouse model of traumatic skeletal muscle injury to investigate the previously unknown role of dendritic cells (DCs) that accumulate in injured tissue. We injected the model antigen ovalbumin (OVA) into the skeletal muscle of injured or sham-treated mice to address the ability of these DCs in antigen uptake, migration, and specific T cell activation in the draining popliteal lymph node (pLN). Immature DC-like cells appeared in the skeletal muscle by 4 days after injury and subsequently acquired a mature phenotype, as indicated by increased expression of the costimulatory molecules CD40 and CD86. After the injection of OVA into the muscle, OVA-loaded DCs migrated into the pLN. The migration of DC-like cells from the injured muscle was enhanced in the presence of the microbial stimulus lipopolysaccharide at the site of antigen uptake and triggered an increased OVA-specific T helper cell type 1 (Th1) response in the pLN. Naïve OVA-loaded DCs were superior in Th1-like priming in the pLN when adoptively transferred into the skeletal muscle of injured mice, a finding indicating the relevance of the microenvironment in the regenerating skeletal muscle for increased Th1-like priming. These findings suggest that DC-like cells that accumulate in the regenerating muscle initiate a protective immune response upon microbial challenge and thereby overcome injury-induced immunosuppression. [ABSTRACT FROM AUTHOR]

Cell-permeable peptides (CPPs) have been widely studied as an attractive drug delivery system to deliver therapeutic macromolecules such as DNA, RNA, and protein into cells. However, its clinical application is still limited and controversial due to the lack of a complete understanding of delivery efficiency in target cells. Previously we identified and characterized the novel and superior CPP, named dNP2, and here we comparatively analyzed intracellular delivery efficiency of dNP2 and TAT in various immune cells of mouse spleen to demonstrate their cell type preference. dNP2- or TAT-conjugated fluorescent proteins were most efficiently taken up by phagocytic cells such as dendritic cells and macrophages while little protein uptake was seen by lymphocytes including T cells, B cells, and NK cells. Interestingly CD8+ lymphoid dendritic cells and CD62LloCD44hi memory like T cell subsets showed significantly better uptake efficiency in vitro and in vivo relative to other dendritic cells or T cells, respectively. In addition, activated macrophages, T cells, and B cells took up the proteins more efficiently relative to when in the resting state. Importantly, only dNP2, not TAT, shows significant intracellular protein delivery efficiency in vivo. Collectively, this study provides important information regarding heterogeneous intracellular delivery efficiency of CPPs such as dNP2 and TAT with cell type preference in the spleen needed for its application in phagocytic cells or activated immune cells. [ABSTRACT FROM AUTHOR]

HIV transmission across the genital mucosa is a major mode of new HIV infections in women. The probability of infection may be influenced by several factors including recruitment and activation of HIV target cells, such as dendritic cells (DCs) and cytokine production, associated with genital inflammation. We evaluated the role of inflammatory cytokines and TLR signaling in migration and activation of genital tract DCs in the human cervical explant model. Hysterectomy tissues from 10 HIV-negative and 7 HIV-positive donor women were separated into ecto- and endocervical explants, and incubated with inflammatory cytokines (TNF-α, IL-1β, IL-8, MIP-1β) or agonists for TLR4 (LPS), TLR2/1 (PAM3) and TLR7/8 (R848). Migration (frequency) and activation (HLA-DR expression) of myeloid and plasmacytoid DCs and Langerhans cells were measured by flow cytometry. We observed that cytokines, LPS and PAM3 induced activation of migrating myeloid and plasmacytoid DCs. LPS induced a 3.6 fold lower levels of migration of plasmacytoid DCs from HIV-infected women compared with HIV-uninfected women (median activation indices of 2.932 vs 0.833). There was however a 4.5 fold increase in migration of Langerhans cells in HIV-infected compared with HIV-uninfected women in response to cytokines (median activation indices of 3.539 vs 0.77). Only TLR agonists induced migration and activation of DCs from endocervical explants. Hormonal contraception use was associated with an increase in activation of DC subsets in the endo and ectocervical explants. We conclude that inflammatory signals in the female genital tract induced DC migration and activation, with possible important implications for HIV susceptibility of cervical tissues. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques. [ABSTRACT FROM AUTHOR]

CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential. [ABSTRACT FROM AUTHOR]

Infection by Listeria monocytogenes (Lm) causes serious sepsis and meningitis leading to mortality in neonates. This work explored the ability of CD11chigh lineage DCs to induce CD8+ T-cell immune protection against Lm in mice before 7 days of life, a period symbolized by the absence of murine IL-12p70-producing CD11chighCD8α+ dendritic cells (DCs). We characterized a dominant functional Batf3-dependent precursor of CD11chigh DCs that is Clec9A+CD205+CD24+ but CD8α- at 3 days of life. After Lm-OVA infection, these pre-DCs that cross-present Ag display the unique ability to produce high levels of IL-12p40 (not IL-12p70 nor IL-23), which enhances OVA-specific CD8+ T cell response, and regulatory IL-10 that limits OVA-specific CD8+ T cell response. Targeting these neonatal pre-DCs for the first time with a single treatment of anti-Clec9A-OVA antibody in combination with a DC activating agent such as poly(I:C) increased the protection against later exposure to the Lm-OVA strain. Poly(I:C) was shown to induce IL-12p40 production, but not IL-10 by neonatal pre-DCs. In conclusion, we identified a new biologically active precursor of Clec9A+ CD8α- DCs, endowed with regulatory properties in early life that represents a valuable target to augment memory responses to vaccines. [ABSTRACT FROM AUTHOR]

We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4+ and CD8+ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8+ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology. [ABSTRACT FROM AUTHOR]

Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that one of the PI3K subunits, p110γ, is in fact critically required for mediating the host’s antiviral response. PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus. We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions. The defect in lung dendritic cells leads to deficient CD8+ T cell priming, which is associated with higher viral titers and more severe disease course during the infection. We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context. [ABSTRACT FROM AUTHOR]

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs. [ABSTRACT FROM AUTHOR]

Systems biology offers promising approaches for identifying response-specific signatures to vaccination and assessing their predictive value. Here, we designed a modelling strategy aiming to predict the quality of late T-cell responses after vaccination from early transcriptome analysis of dendritic cells. Using standardized staining with tetramer, we first quantified antigen-specific T-cell expansion 5 to 10 days after vaccination with one of a set of 41 different vaccine vectors all expressing the same antigen. Hierarchical clustering of the responses defined sets of high and low T cell response inducers. We then compared these responses with the transcriptome of splenic dendritic cells obtained 6 hours after vaccination with the same vectors and produced a random forest model capable of predicting the quality of the later antigen-specific T-cell expansion. The model also successfully predicted vector classification as low or strong T-cell response inducers of a novel set of vaccine vectors, based on the early transcriptome results obtained from spleen dendritic cells, whole spleen and even peripheral blood mononuclear cells. Finally, our model developed with mouse datasets also accurately predicted vaccine efficacy from literature-mined human datasets. [ABSTRACT FROM AUTHOR]

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner. [ABSTRACT FROM AUTHOR]

Effective search strategies have evolved in many biological systems, including the immune system. T cells are key effectors of the immune response, required for clearance of pathogenic infection. T cell activation requires that T cells encounter antigen-bearing dendritic cells within lymph nodes, thus, T cell search patterns within lymph nodes may be a crucial determinant of how quickly a T cell immune response can be initiated. Previous work suggests that T cell motion in the lymph node is similar to a Brownian random walk, however, no detailed analysis has definitively shown whether T cell movement is consistent with Brownian motion. Here, we provide a precise description of T cell motility in lymph nodes and a computational model that demonstrates how motility impacts T cell search efficiency. We find that both Brownian and Lévy walks fail to capture the complexity of T cell motion. Instead, T cell movement is better described as a correlated random walk with a heavy-tailed distribution of step lengths. Using computer simulations, we identify three distinct factors that contribute to increasing T cell search efficiency: 1) a lognormal distribution of step lengths, 2) motion that is directionally persistent over short time scales, and 3) heterogeneity in movement patterns. Furthermore, we show that T cells move differently in specific frequently visited locations that we call “hotspots” within lymph nodes, suggesting that T cells change their movement in response to the lymph node environment. Our results show that like foraging animals, T cells adapt to environmental cues, suggesting that adaption is a fundamental feature of biological search. [ABSTRACT FROM AUTHOR]

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4+ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund’s Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated. [ABSTRACT FROM AUTHOR]

Background: Streptococcus suis is an important swine pathogen and zoonotic agent. Infection with this highly pathogenic strain can cause streptococcal toxic shock-like syndrome (STSLS), characterized by a Th-1 inflammatory cytokine storm, and a high mortality rate. Monocyte derived dendritic cells (MoDCs) are known to stimulate Th-1 cell differentiation, but the role of MoDCs in STSLS remains to be elucidated. Methodology and Findings: Porcine CD14-positive monocytes, purified from peripheral blood mononuclear cells (PBMCs), were used to generate MoDCs using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Highly pure MoDCs were generated, as proved by their morphology, phenotype analysis, phagocytic ability, and induction of T cells proliferation. The MoDCs were further stimulated by the virulent S. suis serotype 2 (SS2) SC19 strain which triggered a strong release of several pro-inflammatory cytokines, including IL-1β, IL-8, TNF-α, IFN-γ, and IL-12. Furthermore, the stimulated MoDCs induced CD4+ T cell differentiation towards Th-1 cells in vitro. Conclusions: The results of this study indicated that the porcine MoDCs stimulated by SS2 could release high levels of Th-1 inflammatory cytokines and induce CD4+ T cell differentiation towards Th-1 cells. Hence, it is likely that porcine MoDCs play an important role in the STSLS caused by SS2. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, has recently been demonstrated to regulate T cell differentiation. Whether AhR activation participates in allergic airway inflammation remains unknown. In the current study, using a non-eosinophilic asthma model, we demonstrate that 2, 3, 7, 8-tetrachlorodibenzo-P-dioxin (TCDD), a potent AhR ligand, reduced the airway infiltration of neutrophils, airway hyperresponsiveness and Th17 cytokine expression. Furthermore, stimulation with TCDD promoted Treg differentiation and inhibited Th17 differentiation. However, the maturation of dendritic cells may not be inhibited by AhR activation. This study thus indicates a critical role of TCDD-induced AhR activation in the regulation of non-eosinophilic airway inflammation. [ABSTRACT FROM AUTHOR]

In the immune system, neuropilins (NRPs), including NRP-1 and NRP-2, are expressed in thymocytes, dendritic cells, regulatory T cells and macrophages. Their functions on immune cells around the neoplastic cells vary into pro-angiogenesis, tumor progression and anti-angiogenesis according to their ligands. Even though NRPs expression on malignant tumors and immune system has studied, a PubMed-based literature query did not yield any articles describing NRPs expression on tissue-specific macrophages. The aims of this study were (i) to detect NRPs expression on tissue-specific macrophages in the brain, liver, spleen, lymph node and lung; (ii) to observe NRPs expression in classes of macrophages, including alveolar macrophages (AMs), bronchial macrophages (BMs), interstitial macrophages (IMs), intravascular macrophages (IVMs) and macrophage subsets (M1, M2 and Mox) in lung; and (iii) to detect the co-expression of NRPs and dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in AMs. Both NRPs were specifically detected in AMs among tissue-specific macrophages by immunohistochemistry (IHC). NRPs mRNA expression levels were characterized in normal lung by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ-polymerase chain reaction (in situ-PCR). The expression of both NRPs was detected in AMs, BMs and IVMs by IHC. The frequency of NRPs+ AMs in lung tissue adjacent to the cancer margin was significantly higher than the frequencies in inflamed and normal lung tissue. Double and triple IHC demonstrated that NRPs are expressed on all macrophage subsets in lung. Double IHC showed co-expression of DC-SIGN and NRPs in AMs. This study demonstrated for the first time the specific expression of both NRPs in AMs among tissue-specific macrophages and their expression on M1, M2 and Mox macrophages. Furthermore, the possible origin of AMs from blood monocytes could be suggested from a co-expression of NRPs and DC-SIGN. [ABSTRACT FROM AUTHOR]

Allergen-specific immunotherapy (AIT) induces tolerance and shifts the Th2 response towards a regulatory T-cell profile. The underlying mechanisms are not fully understood, but dendritic cells (DC) play a vital role as key regulators of T-cell responses. DCs interact with allergens via Fc receptors (FcRs) and via certain C-type lectin receptors (CLRs), including CD209/DC-SIGN, CD206/MR and Dectin-2/CLEC6A. In this study, the effect of AIT on the frequencies as well as the FcR and CLR expression profiles of human DC subsets was assessed. PBMC was isolated from peripheral blood from seven allergic donors before and after 8 weeks and 1 year of subcutaneous AIT, as well as from six non-allergic individuals. Cells were stained with antibodies against DC subset-specific markers and a panel of FcRs and CLRs and analyzed by flow cytometry. After 1 year of AIT, the frequency of CD123+ DCs was increased and a larger proportion expressed FcεRI. Furthermore, the expression of CD206 and Dectin-2 was reduced on CD141+ DCs after 1 year of treatment and CD206 as well as Dectin-1 was additionally down regulated in CD1c+ DCs. Interestingly, levels of DNGR1/CLEC9A on CD141+ DCs were increased by AIT, reaching levels similar to cells isolated from non-allergic controls. The modifications in phenotype and occurrence of specific DC subsets observed during AIT suggest an altered capacity of DC subsets to interact with allergens, which can be part of the mechanisms by which AIT induces allergen tolerance. [ABSTRACT FROM AUTHOR]

Introduction: Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods: We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results: Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion: Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer. [ABSTRACT FROM AUTHOR]

The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response. [ABSTRACT FROM AUTHOR]

Dermatophytes are the most common agents of superficial mycoses that are caused by mold fungi. Trichophyton rubrum is the most common pathogen causing dermatophytosis. The immunology of dermatophytosis is currently poorly understood. Recently, our group investigated the interaction of T. rubrum conidia with peritoneal mouse macrophages. We found that macrophages phagocytose T. rubrum conidia resulted in a down-modulation of class II major histocompatibility complex (MHC) antigens and in the expression of co-stimulatory molecules. Furthermore, it induced the production of IL-10, and T. rubrum conidia differentiated into hyphae that grew and killed the macrophages after 8 hrs of culture. This work demonstrated that dendritic cells (DCs) and macrophages, from patients or normal individuals, avidly interact with pathogenic fungus T. rubrum. The dermatophyte has two major receptors on human monocyte-derived DC: DC-SIGN and mannose receptor. In contrast macrophage has only mannose receptor that participates in the phagocytosis or bound process. Another striking aspect of this study is that unlike macrophages that permit rapid growth of T. rubrum, human DC inhibited the growth and induces Th activation. The ability of DC from patients to interact and kill T. rubrum and to present Ags to T cells suggests that DC may play an important role in the host response to T. rubrum infection by coordinating the development of cellular immune response. [ABSTRACT FROM AUTHOR]