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The pathogenesis of the murine model of autoimmune pancreatitis associated with IgG4-related disease (AIP/IgG4-RD) induced by administration of polyinosinic-polycytidylic acid (poly[I:C]) is incompletely understood. While it is known that murine and human AIP/IgG4-RD is driven by plasmacytoid dendritic cells (pDCs) producing IFN-α, the origin of these cells and their relation to effector T cells is not known. Here, we show that murine AIP was initiated by TLR3-bearing conventional DCs in the uninflamed pancreas whose activation by the TLR3 ligand poly(I:C) caused IFN-α, CXCL9, and CXCL10 secretion. This, in turn, induced pancreatic recruitment of CXCR3+ T cells and these T cells, via their secretion of CCL25, facilitated migration of pDCs bearing CCR9 into the pancreas. This established a feedback loop anchored by the now dominant pDC production of IFN-α and the continued CXCR3+ T cell facilitation of pDC migration. Remarkably, the interaction between CXCR3+ T cells and pDCs also existed at the functional level since this interaction enhanced the production of CCL25 and IFN-α by CXCR3+ T cells and pDCs, respectively. Evidence presented here that a similar disease mechanism was present in human AIP/IgG4-RD creates new avenues of disease treatment.

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3α genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized 1 and 6 month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory T H 1 cell response. Antigen targeting to cDC2s induced T FH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the T H 1 cell response and induced T H 1-like T FH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8 + T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8 + T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8 + T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy., (Copyright © 2020 by The American Association of Immunologists, Inc.)

CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs., (Copyright © 2020 Álvarez, Nieto-Pelegrín, Martínez de la Riva, Toki, Poderoso, Revilla, Uenishi, Ezquerra and Domínguez.)

Viral infections are associated with increased incidence of severe sepsis. Particularly during the early stages, type I interferons (IFNs) are known mediators of detrimental effects. However, the functional role of early interferon β (IFNβ) and its cellular source during sepsis in the context of preexisting viral infections has not been defined. Using the colon ascendens stent peritonitis (CASP) model, we demonstrate that IFNβ -/- and type I IFN receptor (IFNAR1) -/- mice were less susceptible to sepsis after pre-stimulation with the viral mimetic poly(I:C). Wild type (WT) mice treated with poly(I:C) exhibited altered expression patterns of TNF and IL-12p40 during CASP which were dependent on IFNβ or IFNAR1, suggesting a mechanism for the increased sepsis susceptibility of WT mice. Using a double cytokine reporter mouse model, we present novel data on the simultaneous expression of IFNβ and IL-12p40 on a single cell level during polymicrobial sepsis in vivo. Conventional dendritic cells (cDCs) were identified as primary source of IFNβ and the protective cytokine IL-12p40 after CASP surgery irrespective of poly(I:C) pre-stimulation. These data demonstrated that if polymicrobial sepsis is preceded by a viral infection, IFNβ and IL-12p40 are expressed by polyfunctional cDCs suggesting that these cells can play both detrimental and beneficial roles during sepsis development.

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8 + memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cellular immune responses are implicated in resistance to HIV and have been considered for the development of an effective vaccine. Despite their safety profile, subunit vaccines need to be delivered combined with an adjuvant. In the last years, in vivo antigen targeting to dendritic cells (DCs) using chimeric monoclonal antibodies (mAb) against the DC endocytic receptor DEC205/CD205 was shown to support long-term T cell immunity. Here, we evaluated the ability of different adjuvants to modulate specific cellular immune response when eight CD4 + HIV-derived epitopes (HIVBr8) were targeted to DEC205 + DCs in vivo . Immunization with two doses of αDECHIVBr8 mAb along with poly(I:C) induced Th1 cytokine production and higher frequency of HIV-specific polyfunctional and long-lived T cells than MPL or CpG ODN-assisted immunization. Although each adjuvant elicited responses against the 8 epitopes present in the vaccine, the magnitude of the T cell response was higher in the presence of poly(I:C). Moreover, poly(I:C) up regulated the expression of costimulatory molecules in both cDC1 and cDC2 DCs subsets. In summary, the use of poly(I:C) in a vaccine formulation that targets multiple epitopes to the DEC205 receptor improved the potency and the quality of HIV-specific responses when compared to other vaccine-adjuvant formulations. This study highlights the importance of the rational selection of antigen/adjuvant combination to potentiate the desired immune responses.

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI -/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cross-talk between NK cells and dendritic cells (DCs) is important in Th1 immune responses, including antitumor immunity and responses to infections. DCs also play a crucial role in polarizing Th2 immunity, but the impact of NK cell-DC interactions in this context remains unknown. In this study, we stimulated human monocyte-derived DCs in vitro with different pathogen-associated molecules: LPS or polyinosinic-polycytidylic acid, which polarize a Th1 response, or soluble egg Ag from the helminth worm Schistosoma mansoni , a potent Th2-inducing Ag. Th2-polarizing DCs were functionally distinguishable from Th1-polarizing DCs, and both showed distinct morphology and dynamics from immature DCs. We then assessed the outcome of autologous NK cells interacting with these differently stimulated DCs. Confocal microscopy showed polarization of the NK cell microtubule organizing center and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarizing DCs but not Th1-polarizing DCs, indicative of the assembly of an activating immune synapse. Autologous NK cells lysed immature DCs but not DCs treated with LPS or polyinosinic-polycytidylic acid as reported previously. In this study, we demonstrated that NK cells also degranulated in the presence of Th2-polarizing DCs. Moreover, time-lapse live-cell microscopy showed that DCs that had internalized fluorescently labeled soluble egg Ag were efficiently lysed. Ab blockade of NK cell-activating receptors NKp30 or DNAM-1 abrogated NK cell lysis of Th2-polarizing DCs. Thus, these data indicate a previously unrecognized role of NK cell cytotoxicity and NK cell-activating receptors NKp30 and DNAM-1 in restricting the pool of DCs involved in Th2 immune responses., (Copyright © 2018 The Authors.)

The bone marrow hosts NK cells whose distribution, motility and response to systemic immune challenge are poorly understood. At steady state, two-photon microscopy of the bone marrow in Ncr1 gfp/+ mice captured motile NK cells interacting with dendritic cells. NK cells expressed markers and effector molecules of mature cells. Following poly (I:C) injection, RNA-Seq of NK cells revealed three phases of transcription featuring immune response genes followed by posttranscriptional processes and proliferation. Functionally, poly (I:C) promoted upregulation of granzyme B, enhanced cytotoxicity in vitro and in vivo, and, in the same individual cells, triggered proliferation. Two-photon imaging revealed that the proportion of sinusoidal NK cells decreased, while at the same time parenchymal NK cells accelerated, swelled and divided within the bone marrow. MVA viremia induced similar responses. Our findings demonstrate that the bone marrow is patrolled by mature NK cells that rapidly proliferate in response to systemic viral challenge while maintaining their effector functions., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the steady state, tumors harbor several populations of dendritic cells (DCs) and myeloid cells that are key regulators of the intratumoral immune environment. Among these cells, migratory CD103 + cross-presenting DCs are thought to be critical for tumor-specific CTL responses and tumor resistance. However, it is unclear whether this prominent role also extends to immunotherapy. We used a murine orthotopic mammary tumor model, as well as Clec9A-diphtheria toxin receptor mice that can be depleted of the specialized cross-presenting CD8α + and CD103 + DC1 subsets, to investigate the role of these DCs in immunotherapy. Treatment with monosodium urate crystals and mycobacteria at the tumor site delayed tumor growth and required DC1s for efficacy. In contrast, treatment with poly I:C was equally effective regardless of DC1 depletion. Neither treatment affected myeloid-derived suppressor cell numbers in the spleen or tumor. Similar experiments using subcutaneous B16 melanoma tumors in BATF3-knockout mice confirmed that CD103 + DCs were not necessary for successful poly I:C immunotherapy. Nevertheless, adaptive immune responses were essential for the response to poly I:C, because mice depleted of CD8 + T cells or all DC subsets were unable to delay tumor growth. In vivo experiments showed that DC1 and DC2 subsets were able to take up tumor Ags, with DC2s making up the larger proportion of lymph node DCs carrying tumor material. Both DC subsets were able to cross-present OVA to OT-I T cells in vitro. Thus, immunotherapy with poly I:C enables multiple DC subsets to cross-present tumor Ag for effective antitumor immune responses., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Intranasal inoculation with influenza hemagglutinin subunit with polyinosine-polycytidylic (polyI:C), a synthetic analog for double-stranded RNA, enhances production of vaccine-specific immunoglobulin (Ig) A, which is superior to IgG in prophylactic immunity. The mechanism whereby polyI:C skews to IgA production in the nasal-associated lymph tissue (NALT) was investigated in mouse models. Nasally instilled polyI:C was endocytosed into CD103 + dendritic cells (DCs) and induced T-cell activation, including interferon (IFN)-γ production. According to knockout mouse studies, polyI:C activated the Toll-like receptor 3 signal via the adapter TICAM-1 (also called TRIF), that mainly caused T-cell-dependent IgA production. Nasal CD103 + DCs activated transforming growth factor-β signaling and activation-induced cytidine deaminase upon polyI:C stimulation. IgA rather than IgG production was impaired in Batf3 -/- mice, where CD103 + DCs are defective. Genomic recombination occurred in IgA-producing cells in association with polyI:C-stimulated DCs and nasal microenvironment. PolyI:C induced B-cell-activating factor expression and weakly triggered T-cell-independent IgA production. PolyI:C simultaneously activated mitochondrial antiviral signaling and then type I IFN receptor pathways, which only minimally participated in IgA production. Taken together, CD103 + DCs in NALT are indispensable for the adjuvant activity of polyI:C in enhancing vaccine-specific IgA induction and protective immunity against influenza viruses.

Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac 5 3F ax Neu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac 5 3F ax Neu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac 5 3F ax Neu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac 5 3F ax Neu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.

Dendritic cell (DC) maturation involves widespread changes in cellular function and gene expression. The regulatory role of IFNAR in the program of DC maturation remains incompletely defined. Thus, the time evolution impact of IFNAR on this process was evaluated. Changes in DC phenotype, function, and gene expression induced by poly I:C were measured in wild-type and IFNAR(-/-) DC at 9 time points over 24 h. Temporal gene expression profiles were filtered on consistency and response magnitude across replicates. The number of genes whose expression was altered by poly I:C treatment was greatly reduced in IFNAR(-/-) DC, including the majority of the downregulated gene expression program previously observed in wild-type (WT) DC. Furthermore, the number of genes upregulated was almost equal between WT and IFNAR(-/-) DC, yet the identities of those genes were distinct. Integrating these data with protein-protein interaction data revealed several novel subnetworks active during maturation, including nucleotide synthesis, metabolism, and repair. A subnetwork associated with redox activity was uniquely identified in IFNAR(-/-) DC. Overall, temporal gene expression and network analyses identified many genes regulated by the type I interferon response and revealed previously unidentified aspects of the DC maturation process.

Background: Generation of antibodies against red blood cell (RBC) antigens can be a clinically significant problem. The underlying mechanisms that regulate the production of RBC antibodies are only partially understood; however, factors such as inflammation significantly increase the rates of RBC antibody generation. Humoral alloimmunization begins with consumption of transfused RBCs by antigen-presenting cells (APCs). Recently, it has become appreciated that there are multiple different types of APCs. The relative contribution of APC subsets to RBC antibodies has not been described in either the quiescent or the inflamed states., Study Design and Methods: To evaluate the types of APCs that consume RBCs, and how inflammation affects this process, C56Bl/6 mice were treated with polyinosinic-polycytidylic acid (poly(I:C)) to induce an inflammatory response and/or were transfused with 3,3'-dihexadecyloxacarbocyanine perchlorate-labeled syngeneic RBCs. Erythrophagocytosis (both at baseline and during inflammation) was analyzed for different subsets of macrophages (MΦ), dendritic cells (DCs), B cells, and monocytes, by a combined approach using flow cytometry and fluorescent microscopy technology., Results: In four independent experiments, erythrophagocytosis at baseline was predominately performed by red pulp MΦ; however, during inflammation both plasmacytoid DCs (pDCs) and monocytes increased RBC consumption. Furthermore, pDCs up regulated MHC-II and activation markers CD80 and CD86. In addition to changing patterns of erythrophagocytosis, inflammation also led to a significant decrease in CD11c+ conventional DC populations and an increase in granulocytes., Conclusions: The nature of APCs that consume transfused RBCs is changed by inflammation. Given that APCs initiate humoral immune responses, these findings provide potential mechanistic insight into how inflammation regulates RBC alloimmunization., (© 2016 AABB.)

Besides T helper (Th) cells, natural killer (NK) cells have also been described to participate in the shaping of dendritic cell (DC)-mediated adaptive immune responses. At present, it remains unclear to what extent the induction of these NK helper cell immune mechanisms is coupled with Th responses and whether both helper immune responses are induced by the same DC upon specific pathogen recognition receptor (PRR) stimulation. In this study, we demonstrate that maturation of DCs with a cocktail containing FMKp (membrane fragments of Klebsiella pneumoniae) mounts both Th cell and NK cell helper responses in a PRR-triggered dose-dependent manner as determined by the capacity of the helper cells to produce IFN-γ. Furthermore, by triggering an additional PRR pathway [FMKp in combination with poly(I:C) lyovec], we reveal that both approaches modulate the amount of DC-derived IL-12p70 and that this cytokine is the key determinant of the DC-induced Th1 and NK cell helper responses. Moreover, all PRR triggers able to induce IL-12-producing mature DCs are sufficient to induce these helper responses. We propose the existence of a single program used by DCs to induce potent cellular immune responses by stimulating both T helper and NK cell helper processes. This knowledge can help to select the proper PRR triggers in preventive and therapeutic vaccine design.

Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although cancer vaccine-based immunotherapy holds great potential for cancer treatment, tumor-induced dendritic cell (DC) dysfunction remains to be the major obstacle for developing effective vaccines. Compared with normal DCs, tumor-associated DCs (TADCs) are less matured with poor responsiveness to Toll-like receptor (TLR) stimulation, which has been related with STAT3 hyperactivity. In the present study, Poly I:C (PIC, a TLR3 agonist), STAT3 siRNA and OVA antigen were co-encapsulated by poly (ethylene glycol)-b-poly (L-lysine)-b-poly (L-leucine) (PEG-PLL-PLLeu) polypeptide micelles to generate PMP/OVA/siRNA nanovaccine, which was aimed to effectively overcome DC dysfunction in vivo by deleting STAT3 gene in situ. The results showed that PMP/OVA/siRNA simultaneously facilitated the cellular uptake of OVA antigen and siRNA about 3-200 folds, and decreased STAT3 expression in TADCs over 50% both in vitro and in vivo. PMP/OVA/siRNA also elevated CD86 and CD40 expression as well as IL-12 production by TADCs more effectively than PMP/OVA did, indicating its strong potency of inducing TADC maturation and activation. Moreover, the immunization of PMP/OVA/siRNA rather than PMP/OVA effectively abrogated immunosuppression in the tumor microenvironment by increasing mature DCs and decreasing immunosuppressive cells in tumor-draining lymph nodes, which thereby led to potent anti-tumor immune responses and dramatic tumor regression with prolonged survival. Hence, in vivo co-delivery of immunopotentiator (PIC) and immunosuppressive gene silencer (STAT3 siRNA) by nanovaccines are expected to be a promising strategy to improve the therapeutic efficacy of cancer vaccines by modulating TADCs and overcoming tumor immunosupression., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFNγ was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFNγ production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism., (©2014 American Association for Cancer Research.)

Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1(+) DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1(+) human DC. Assessment of the immunoactivation potential of XCR1(+) human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1(+) and XCR1(-) human DC in CD34(+) progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1(-) CD34-DC are similar to canonical MoDC, whereas XCR1(+) CD34-DC resemble XCR1(+) blood DC (bDC). XCR1(+) DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1(+) DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1(+) CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1(+) bDC. Hence, it is feasible to generate high numbers of bona fide XCR1(+) human DC in vitro as a model to decipher the functions of XCR1(+) bDC and as a potential source of XCR1(+) DC for clinical use., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: Dendritic cells (DCs) are professional antigen-presenting cells that are capable of inducing immune responses. DC-based vaccines are normally generated using a standard 5- to 7-day protocol. To shorten the DC-based vaccine production for use in cancer immunotherapy, we have developed a fast DC protocol by comparing standard DCs (Day 5 DCs) and fast DCs (Day 3 DCs)., Methods: We tested the generation of Day 5 versus Day 3 DCs using CellGro media and subsequent activation by two activation stimuli: Poly (I:C) and LPS. We evaluated DC morphology, viability, phagocyte activity, cytokine production and ability to stimulate antigen-specific T cells., Results: Day 5 and Day 3 DCs exhibited similar phagocytic capacity. Poly (I:C)-activated Day 5 DCs expressed higher levels of the costimulatory and surface molecules CD80, CD86 and HLA-DR compared to Poly (I:C)-activated Day 3 DCs. Nevertheless, LPS-activated Day 5 and Day 3 DCs were phenotypically similar. Cytokine production was generally stronger when LPS was used as the maturation stimulus, and there were no significant differences between Day 5 and Day 3 DCs. Importantly, Day 5 and Day 3 DCs were able to generate comparable numbers of antigen-specific CD8(+) T cells. The number of Tregs induced by Day 5 and Day 3 DCs was also comparable., Conclusion: We identified monocyte-derived DCs generated in CellGro for 3 days and activated using Poly (I:C) similarly potent in most functional aspects as DCs produced by the standard 5 day protocol. These results provide the rationale for the evaluation of faster protocols for DC generation in clinical trials., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) is a major cause of liver disease. The innate immune system is essential for controlling HCV replication, and HCV is recognized by RIG-I and TLR3, which evoke innate immune responses through IPS-1 and TICAM-1 adaptor molecules, respectively. IL-28B is a type III IFN, and genetic polymorphisms upstream of its gene are strongly associated with the efficacy of polyethylene glycol-IFN and ribavirin therapy. As seen with type I IFNs, type III IFNs induce antiviral responses to HCV. Recent studies established the essential role of TLR3-TICAM-1 pathway in type III IFN production in response to HCV infection. Contrary to previous studies, we revealed an essential role of IPS-1 in type III IFN production in response to HCV. First, using IPS-1 knockout mice, we revealed that IPS-1 was essential for type III IFN production by mouse hepatocytes and CD8(+) dendritic cells (DCs) in response to cytoplasmic HCV RNA. Second, we demonstrated that type III IFN induced RIG-I but not TLR3 expression in CD8(+) DCs and augmented type III IFN production in response to cytoplasmic HCV RNA. Moreover, we showed that type III IFN induced cytoplasmic antiviral protein expression in DCs and hepatocytes but failed to promote DC-mediated NK cell activation or cross-priming. Our study indicated that IPS-1-dependent pathway plays a crucial role in type III IFN production by CD8(+) DCs and hepatocytes in response to HCV, leading to cytoplasmic antiviral protein expressions.

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-β autocrine loop, dsRNA-elicited IFN-β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-β produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3(-/-) mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1(-/-) mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-β production and contribute to the antitumoral response., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Receptor internalization is a common mechanism underlying surface receptor down-regulation (and thus receptor signaling) upon its engagement with the cognate ligand. Tight regulation of surface CD40 expression is critical in regulating different functional properties of dendritic cell (DC). Engagement of CD40 on mature DC and the cognate CD40 ligand on T cell activates c-Jun N-terminal MAPK, p38 and ERK1/2 MAPK pathways in mature DC. JNK-associated leucine zipper protein (JLP) is a scaffolding protein that interacted with p38 and JNK. The molecular mechanism underlying CD40 internalization and its physiological impact on DC functions remained unclear. Here we reported that the engagement of CD40 on the LPS-activated DC down-regulated the surface expression of CD40. We examined the role of the JLP protein in DC differentiation, and in the regulation of DC function(s) in vitro. In contrast to the abundant JLP expression observed in immortal cell lines, primary immature DC expressed low levels of the JLP proteins. The induction of the JLP protein expression was observed in the LPS-mature DC that were activated by CD40 ligation, and also in the poly I:C stimulated DC. JLP-silenced DC was impaired in regulating CD40 surface expression upon LPS stimulation and CD40 induced receptor internalization. Such aberrant change in the regulation of surface CD40 expression was associated with an augmented capacity of the JLP-silenced DC in IL-12 production. Collectively, our data identified a novel role of a scaffolding protein JLP in the regulation of surface CD40 expression and fine-tuning of DC function., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

RIG-I-like receptors (RLRs) sense virus-derived RNA or polyinosinic-polycytidylic acid (poly IC) to exert antiviral immune responses. Here, we examine the mechanisms underlying the adjuvant effects of poly IC. Poly IC was taken up by dendritic cells (DCs), and it induced lysosomal destabilization, which, in turn, activated an RLR-dependent signaling pathway. Upon poly IC stimulation, cathepsin D was released into the cytoplasm from the lysosome to interact with IPS-1, an adaptor molecule for RLRs. This interaction facilitated cathepsin D cleavage of caspase 8 and the activation of the transcription factor NF-κB, resulting in enhanced cytokine production. Further recruitment of the kinase RIP-1 to this complex initiated the necroptosis of a small number of DCs. HMGB1 released by dying cells enhanced IFN-β production in concert with poly IC. Collectively, these findings suggest that cathepsin D-triggered, IPS-1-dependent necroptosis is a mechanism that propagates the adjuvant efficacy of poly IC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF(V600) mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF(V600) mutant melanoma., Methods: We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib., Results: Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF(V600E) mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations., Conclusions: BRAF(V600E) mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1β, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection.

Dendritic cell-based vaccines offer promise for therapy of ovarian cancer. Previous studies have demonstrated that oxidation of several antigens, including ovarian cancer cells, using hypochlorous acid strongly enhances their immunogenicity and their uptake and presentation by dendritic cells. The response of T cells and dendritic cells to autologous tumour from patients with active disease has not previously been investigated. Monocyte-derived dendritic cells were generated from patients with active disease and activated by co-culture with oxidised tumour cells and the TLR agonist poly I:C. The dendritic cells showed an activated phenotype, but secreted high levels of TGFβ. Co-culture of the antigen-loaded dendritic cells with autologous T cells generated a population of effector T cells that showed a low level of specific lytic activity against autologous tumour, as compared to autologous mesothelium. The addition of neutralising antibody to TGFβ in DC/T cell co-cultures increased the levels of subsequent tumour killing in three samples tested. Co-culture of monocytes from healthy volunteers with the ovarian cell line SKOV-3 prior to differentiation into dendritic cells reduced the ability of dendritic cells to stimulate cytotoxic effector cells. The study suggests that co-culture of dendritic cells with oxidised tumour cells can generate effector cells able to kill autologous tumour, but that the high tumour burden in patients with active disease may compromise dendritic cell and/or T cell function.

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4(+) T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4(+) T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Complement activation modulates DC-mediated T-cell activation, but whether complement affects DC-mediated priming of NK cells is unknown. Here, we demonstrated that conventional DCs (cDCs) from C3(-/-) and C5aR(-/-) mice are hyperresponsive to polyI:C, a TLR3 ligand, leading to enhanced NK-cell activation. We found that cDCs lack C5a receptor (C5aR) and do not respond to C5a directly. Depletion of Gr-1(+) myeloid cells augments polyI:C-induced cDC activation in WT but not in C3(-/-) or C5aR(-/-) mice, indicating that the effect of complement activation on cDCs is indirectly mediated through C5aR-expressing Gr-1(+) myeloid cells. We further demonstrated that the mechanism by which Gr-1(+) myeloid cells regulate the activity of cDCs involves C5a-dependent TGF-β1 production in Gr-1(+) myeloid cells. C5a enhances and blocking C5aR decreases TGF-β1 production in cultured bone marrow Gr-1(+) CD11b(+) cells. C5aR deficiency is associated with reduced circulating TGF-β1 levels, while depleting Gr-1(+) myeloid cells abrogates this difference between WT and C5aR(-/-) mice. Lastly, we showed that enhanced cDC-NK-cell activity in C3(-/-) mice led to delayed melanoma tumor growth. Thus, complement activation indirectly regulates cDC-NK-cell activation in response to inflammatory stimuli such as TLR3 by promoting TGF-β1 production in Gr-1(+) myeloid cells at steady state., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC+ dendritic cells (DCs) in a mouse breast cancer model., Methods: We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4+/CD8+ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated., Results: We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4+ T cell immunity, CD8+ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4+ and CD8+ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice., Conclusions: Immunization of mice with HER2 protein vaccine targeting DEC+ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4+ and CD8+ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 μg of HER2 protein incorporated in the vaccine. Vaccine-induced CD4+ and CD8+ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.

Dendritic cells (DCs) have the ability to generate peptide epitopes for MHC class I molecules derived from apoptotic tumour cells for direct recognition by cytotoxic T cells. This function has lead to DCs being used in vaccine strategies. In this study, we investigate the effect of inducing apoptosis in tumour cell lines using IFN-γ and poly(I:C), the subsequent maturation of the endocytosing DC and its ability to direct the resulting T cell response. We show that uptake of poly(I:C)-induced apoptotic tumour cells leads to DC maturation and activation with a Th1 cell polarising capacity. In contrast, these effects are not seen by DCs loaded with γ-irradiated apoptotic tumour cells. We propose that the manner in which tumour cells are induced to die can have a profound effect on the endocytosing DC and the resulting T cell response.

Multiple physiological and pathological conditions interfere with the function of the endoplasmic reticulum (ER). However, much remains unknown regarding the impact of ER stress on inflammatory responses in dendritic cells (DCs) upon the recognition of pathogen molecules. We show that ER stress greatly potentiates the expression of inflammatory cytokines and IFN-β in murine DCs stimulated by polyIC, a synthetic mimic of virus dsRNA. Both toll-like receptor 3 and melanoma differentiation-associated gene-5 are involved in the enhanced IFN-β production, which is associated with increased activation of NF-κB and IRF3 signaling as well as the splicing of X-box-binding protein-1 (XBP-1), an important regulator involved in ER stress response. Surprisingly, silencing of XBP-1 reduces polyIC-stimulated IFN-β expression in the presence or absence of ER stress, indicating that XBP-1 may be essential for polyIC signaling and ER stress-amplified IFN-β production. Overexpression of a spliced form of XBP-1 (XBP-1s) synergistically augments polyIC-induced inflammatory response. For the first time, we show that XBP-1s overexpression-enhanced IFN-β production in DCs markedly suppresses vesicular stomatitis virus infection, revealing a previously unrecognized role for XBP-1 in an antiviral response. Our findings suggest that evolutionarily conserved ER stress response and XBP-1 may function collaboratively with innate immunity to maintain cellular homeostasis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host's response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects., Methods: Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2., Results: MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells., Conclusion: These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.

Background: Effective vaccination against human papillomavirus (HPV) represents an opportunity to control cervical cancer. Peptide-based vaccines targeting HPV E6 and/or E7 antigens while safe, will most likely require additional strategies to enhance the vaccine potency., Methods: We tested the HPV-16 E7 peptide-based vaccine in combination with a strategy to enhance CD4+ T help using a Pan HLA-DR epitope (PADRE) peptide and a strategy to enhance dendritic cell activation using the toll-like receptor 3 ligand, poly(I:C)., Results: We observed that mice vaccinated with E7 peptide-based vaccine in combination with PADRE peptide and poly(I:C) generated better E7-specific CD8+ T cell immune responses as well as significantly improved therapeutic anti-tumor effects against TC-1 tumors compared to E7 peptide-based vaccine with either PADRE peptide or poly(I:C) alone. Furthermore, we found that intratumoral vaccination with the E7 peptide in conjunction with PADRE peptide and poly(I:C) generates a significantly higher frequency of E7-specific CD8+ T cells as well as better survival compared to subcutaneous vaccination with the same regimen in treated mice., Conclusions: The combination of PADRE peptide and poly(I:C) with antigenic peptide is capable of generating potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice. Our study has significant clinical implications for peptide-based vaccination.

HIV-Tat based vaccines have been proposed as an attractive option to prevent or treat AIDS. A vaccine to induce optimal anti-Tat neutralizing antibody responses was designed by inserting this protein, or its dominant B-cell epitope, into the CyaA vector, which targets dendritic cells (DC). Tat was inserted into various sites of CyaA, including regions that do not translocate into the cytosol of the targeted DC. The presentation of the Tat CD4(+) T-cell epitope delivered by the CyaA-Tat proteins was observed with a recombinant CyaA in which the entire AC domain was replaced by the entire Tat protein (Tat-Δ373 CyaA) but was abolished with large deletions of the N-terminal region. Moreover, CyaA carrying multiple copies of the dominant Tat: 1-21 B-cell epitope were shown to induce high titers of anti-Tat antibodies, even after a single immunization, that persisted up to 10 weeks post-immunization., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Dendritic cells (DC) play a central role in primary immune responses and become potent stimulators of the adaptive immune response after undergoing the critical process of maturation. Understanding the dynamics of DC maturation would provide key insights into this important process. Time course microarray experiments can provide unique insights into DC maturation dynamics. Replicate experiments are necessary to address the issues of experimental and biological variability. Statistical methods and averaging are often used to identify significant signals. Here a novel strategy for filtering of replicate time course microarray data, which identifies consistent signals between the replicates, is presented and applied to a DC time course microarray experiment., Results: The temporal dynamics of DC maturation were studied by stimulating DC with poly(I:C) and following gene expression at 5 time points from 1 to 24 hours. The novel filtering strategy uses standard statistical and fold change techniques, along with the consistency of replicate temporal profiles, to identify those differentially expressed genes that were consistent in two biological replicate experiments. To address the issue of cluster reproducibility a consensus clustering method, which identifies clusters of genes whose expression varies consistently between replicates, was also developed and applied. Analysis of the resulting clusters revealed many known and novel characteristics of DC maturation, such as the up-regulation of specific immune response pathways. Intriguingly, more genes were down-regulated than up-regulated. Results identify a more comprehensive program of down-regulation, including many genes involved in protein synthesis, metabolism, and housekeeping needed for maintenance of cellular integrity and metabolism., Conclusions: The new filtering strategy emphasizes the importance of consistent and reproducible results when analyzing microarray data and utilizes consistency between replicate experiments as a criterion in both feature selection and clustering, without averaging or otherwise combining replicate data. Observation of a significant down-regulation program during DC maturation indicates that DC are preparing for cell death and provides a path to better understand the process. This new filtering strategy can be adapted for use in analyzing other large-scale time course data sets with replicates.

Polyelectrolyte microcapsules (MCs) are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells (DCs). We investigated the immune stimulatory capacity of monocyte-derived DC (Mo-DC) loaded with these MCs, containing p24 antigen from human immunodeficiency virus type 1 (HIV-1) alone [p24-containing MC (MCp24)] or with the Toll-like receptor ligand 3 (TLR3) ligand poly I:C (MCp24pIC) as a maturation factor. MO-DC, loaded with MCp24pIC, upregulated CCR7, CD80, CD83, and CD86 and produced high amounts of interleukin-12 (IL-12) cytokine, to a similar extent as MCp24 in the presence of an optimized cytokine cocktail. MO-DC from HIV-infected patients under highly active antiretroviral therapy (HAART) exposed to MCp24 together with cytokine cocktail or to MCp24pIC expanded autologous p24-specific CD4(+) and CD8(+) T-cell responses as measured by interferon-gamma (IFN-gamma) and IL-2 cytokine production and secretion. In vivo relevance was shown by immunizing C57BL/6 mice with MCp24pIC, which induced both humoral and cellular p24-specific immune responses. Together these data provide a proof of principle that both antigen and DC maturation signal can be delivered as a complex with polyelectrolyte capsules to stimulate virus-specific T cells both in vitro and in vivo. Polyelectrolyte MCs could be useful for in vivo immunization in HIV-1 and other infections.

Profound type I interferon (IFN-I)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic at 2 to 4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cells ex vivo for activated caspases but with only low levels of ex vivo staining with annexin V, probably due to the rapid clearance of apoptotic cells in vivo. Instead, a high frequency of annexin V-reactive CD8alpha(+) dendritic cells (DCs), which are known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After short in vitro incubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 h) displayed substantial DNA fragmentation, as detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the proapoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlate with reduced immunodomination by cross-reactive memory cells.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-D-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34(+) hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE(D)) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-alpha), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rgamma(-/-) mouse model engrafted with human CD34(+) hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-alpha effectively suppressed poly(I:C)-induced TNF-alpha production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a broadly applicable, comprehensive, and unbiased approach to reveal the wiring and functions of a regulatory network controlling a major transcriptional response in primary mammalian cells.

Infection with RNA viruses presents a typical pattern of virus products, double-stranded RNA (dsRNA), and induces the maturation of antigen-presenting dendritic cell (mDC). There are several dsRNA sensors that are differentially distributed on the cell membrane and in the cytoplasm and are variably expressed depending on the cell type. Among these sensors, TLR3 links to the adaptor TICAM-1 (TRIF), which is characterized by its unique multipronged signaling cascades for cytokine/chemokine production, apoptosis and autophagy in both immune and tumor cells. In the context of mDC maturation, various cellular events are further induced in response to dsRNA; these include cross-priming followed by CD8+ CTL induction, NK activation and proliferation of CD4+ T cells including Th1, Th2, Treg and Th17 cells. In this review, we focus on the potential role of dsRNA in modulating the inflammatory milieu around mDCs and tumor-associated antigens to drive specific cellular effectors against the tumor.

Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4(+) T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205(+) DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4(+) immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.

Phenotypic and functional property changes of bone marrow-derived immature dendritic cells (BM-imDCs) after porcine reproductive and respiratory syndrome virus (PRRSV) infection have been detailed in a previous report. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 were observed in BM-imDCs after the exposure to PRRSV. In this study, we further investigate the expression of surface phenotypes of BM-imDCs in relation to their infection status. Exposure of PRRSV to BM-imDCs resulted in a down-regulated expression of MHC I and an up-regulated expression of CD80/86 in infected cells, as demonstrated by significant alterations in both percentage of expressing cells and mean fluorescence intensity (MFI) in PRRSV-positive cells. A significant suppression in MFI of MHC I and an increase in percentage of cells expressing CD80/86 were observed in noninfected bystander cells. We also demonstrated that exposure of BM-imDCs to PRRSV resulted in a significantly increased secretion of IL-1, IL-6, IL-8, IL-10 and IFN-gamma but not IL-12 or TNF-alpha. In addition, the PRRSV infection modulates cytokine expressions of BM-imDCs through their response to microbial pathogen-associated molecular patterns. These results will prove helpful in clarification of the factors that mediate host defense against PRRSV, as well as the possible interaction mechanisms between PRRSV and other microbes in the pathogenesis of PRRSV infection in pigs.