Stimulation Of Dendritic Cells Research Articles

Abstract 388: Antigen-loaded Fe3O4 nanoparticles for the stimulation and tracking of dendritic cell based vaccine

Abstract Background and purpose:Dendritic cells (DCs), as the antigen-presenting cells, play important roles in immunotherapy. The antigen uptake, cross-presentation, maturation and lymph node migration of DCs are crucial steps for immune system activation. Herein, we aim to construct a novel Fe3O4@SIINFEKL-TAT nanoparticle, which mainly consists of Fe3O4 nanosphere core and transmembrane peptide TAT on the shell, and also with the model antigen SIINFEKL peptide, a peptide of the ovalbumin (OVA) antigen. To explore the feasibility of Fe3O4@OVA-TAT to stimulate the maturation of DCs and the in vivo tracking of DCs using MRI-MPI dual-modal imaging. Methods: On the one hand, TAT peptide facilitates the uptake of the Fe3O4@OVA-TAT nanoparticles into DCs, and subsequently the OVA antigens are cross-presented to stimulate DCs maturation. On the other hand, Fe3O4 nanosphere core can function for both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) dual-modality imaging, which can realize the in vivo dynamic tracking of DCs migration to lymph nodes. Results: The laser confocal microscope experiments confirmed that Fe3O4@OVA-TAT can be taken up through the early endosome-late endosome pathway, but the fluorescent signal of the imaging probe is located outside the lysosome after 4 h, which suggested that Fe3O4@OVA-TAT could escape lysosomal uptake and cross-present. The CD80 and CD86 flow cytometry results showed that Fe3O4@OVA-TAT, LPS, and OVA can stimulate 90%, 60%, and 40% of BMDCs to mature, respectively (p < 0.05). After the Fe3O4@SIINFEKL-TAT labeled DCs was injected into the mouse footpad, the MRI T2WI signal decreased and the MPI signal increased in the popliteal and inguinal lymph nodes 24 h post-injection, suggesting that our DC vaccine can efficiently migrate to the lymph node. Conclusions: Our study showed that Fe3O4@SIINFEKL-TAT nanoparticles can stimulate and trace DCs in in vivo, which pave the way for further development and precision application of DC vaccine in both preclinical and clinical settings. Citation Format: Yafei Qi, Yang Du, Jie Tian, Zhengyu Jin, Yuan Li. Antigen-loaded Fe3O4 nanoparticles for the stimulation and tracking of dendritic cell based vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 388.

Dendritic cells' characteristics in patients with treated systemic lupus erythematosus.

The systemic lupus erythematosus (SLE) is a chronic autoimmune disease related to a loss of immune tolerance against autoantigens that leads to tissue inflammation and organ dysfunction. Constant stimulation of dendritic cells (DC) with autoantigens is hypothesized to increase the B cells' activity which are involved in production of autoantibodies that play an essential role in the SLE development. We focused our study on detecting alterations in DCs at the cellular and molecular levels in patients with treated SLE, depending on the disease activity and treatment. In order to phenotype subpopulations of DCs, multicolor flow cytometry was used. Transcriptional changes were identified with quantitative PCR, while soluble cytokine receptors were assessed with the Luminex technology. We show that SLE patients display a higher percentage of activated myeloid DCs (mDCs) when compared to healthy people. Both, the mDCs and plasmacytoid DCs (pDCs) of SLE patients were characterized by changes in expression of genes associated with their maturation, functioning and signalling, which was especially reflected by low expression of regulatory factor ID2 and increased expression of IRF5. pDCs of SLE patients also showed increased expression of IRF1. There were also significant changes in the expression of APRIL, MBD2, and E2-2 in mDCs that significantly correlated with some serum components, i.e. anti-dsDNA antibodies or complement components. However, we did not find any significant differences depending on the disease activity. While the majority of available studies focuses mainly on the role of pDCs in the disease development, our results show significant disturbances in the functioning of mDCs in SLE patients, thus confirming mDCs' importance in SLE pathogenesis.

Role of syndecan-1 in the interaction between dendritic cells and T cells.

Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that can bind cytokines and chemokines via its heparan sulfate side chains, and has immunomodulatory properties in experimental models. Sdc-1 expression has been reported on dendritic cells (DC) and T cells. The potential role of Sdc-1 in DC-T cell interaction has not been investigated yet. We postulate that Sdc-1 is involved in DC-T cell interaction and may influence graft survival in an allogeneic transplant model. Sdc-1 expression on bone marrow-derived DC and T cells was analyzed by flow cytometry. Unstimulated and LPS stimulated Sdc-1 deficient DC were evaluated in vitro for phenotype and stimulatory capacity in mixed lymphocyte reaction. Sdc-1 deficient T cells were evaluated for proliferative capacity and differentiation in a mixed lymphocyte reaction and a proliferation assay. Allograft survival was evaluated in a fully MHC mismatched heterotopic heart transplant model, with either Sdc-1 deficient donors or recipients. Sdc-1 was expressed on the cell surface of unstimulated and LPS matured DC. Sdc-1 deficiency had no effect on expression of co-stimulatory molecules, cytokine production or T cell stimulatory capacity as compared to WT DC. Sdc-1 expression was not detectable on WT T cells, although intracellular Sdc-1 expression could be demonstrated after ConA activation. Sdc-1 deficient T cells showed reduced proliferation upon DC or ConA stimulation and reduced IL-17 production upon ConA stimulation, compared to WT T cells. Sdc-1 deficiency of either allograft or recipient did not prolong allograft survival. In conclusion, Sdc-1 is expressed on the cell surface of DC, where its absence does not affect DC phenotype or T cell stimulatory capacity. Sdc-1 is intracellularly expressed in ConA activated T cells. Sdc-1 deficiency in T cells results in a reduced proliferative response in vitro, as induced by DC and ConA. Sdc-1 deficiency in donor or recipient does not affect allograft survival.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Poor stimulation of bovine dendritic cells by Mycobacterium bovis culture supernatant and surface extract is associated with decreased activation of ERK and NF-κB and higher expression of SOCS1 and 3.

The cell envelope of pathogenic mycobacteria interfaces with the host. As such, the interaction of bacterial products localized at or released from the cell surface with the host’s immune system can determine the fate of the bacterium in its host. In this study, the effects of three different types of Mycobacterium bovis cell envelope fractions—purified protein derivative, total cell wall lipids and culture supernatant and surface extract—on bovine dendritic cells were assessed. We found that the culture supernatant and surface extract fraction induced little to no production of the pro-inflammatory cytokines TNF-α and IL-12 in bovine dendritic cells. Moreover, this muted response was associated with poor activation of ERK and NF-κB, both of which are critical for the pro-inflammatory response. Furthermore, culture supernatant and surface extract treatment increased the expression of suppressor of cytokine signaling 1 and 3, both of which are negative regulators of pro-inflammatory signaling, in bovine dendritic cells. These observations taken together suggest the M. bovis culture supernatant and surface extract fraction contain immunomodulatory molecules that may aid in M. bovis pathogenesis.

STING-dependent type-1 interferon restrains schistosome immunopathology via down-regulation of the CD209a lectin receptor

Abstract Infection with the helminth parasite Schistosoma mansoni causes morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens. We now demonstrate that stimulation of dendritic cells (DCs) with schistosome eggs induces robust IFNβ production in a manner dependent on the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon genes (STING) cytosolic DNA sensing pathway, resulting in the suppression of proinflammatory IL-1β and IL-23 production and in Th17 cell activation. Consistent with these results, low-pathology BL/6 mice lacking STING exhibited markedly enhanced hepatic granulomatous inflammation associated with significantly increased Th17 and diminished Th2 cytokine responses. Mechanistically, IFNβ acts by suppressing DC expression and function of CD209a, a C-type lectin receptor associated with severe schistosome immunopathology. Importantly, there was an increased baseline CD209a expression in unstimulated DCs from STING−/− mice, suggesting a role for constitutive IFN signaling. Our findings demonstrate that innate, cGAS/STING-dependent sensing of parasite DNA represents a novel pathway inducing type I IFN production, which protects the host from excessive inflammation and immunopathology in schistosomiasis. This work is supported by NIAID grant R01 AI018919 to MJS.

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus

Hepatitis C virus (HCV) can cause chronic infection and evade the immune response. The generation and maintenance of an effective T-cell response is important for immune-mediated control of HCV infection. The purpose of this study was to obtain recombinant mosaic proteins containing the cytotoxic T lymphocyte (CTL) epitopes of HCV fused with different adjuvants and analyse their immunogenicity. A recombinant polyepitope protein comprising HLA-A2-restricted CTL epitopes of the NS3, NS4ab and NS5a proteins of HCV was designed. Adjuvant compounds, the T-helper (Th) epitope PADRE, lipopeptide from Neisseria meningiditis and interleukin 2 (IL-2) were included in the fusion proteins. Three proteins differing in their adjuvant content were expressed in Escherichia coli and purified. The purified proteins formed nanosized particles. The proteins were characterized by their ability to cause proliferation of spleen cells, induce expression of cytokine genes and production of interferon gamma by T lymphocytes of immunized mice. The obtained recombinant vaccine proteins effectively stimulate dendritic cells, which in turn specifically activate Th1 and Th2 lymphocytes. Adjuvant components act additively to enhance the stimulation of dendritic cells and polarize them in the direction of Th1 lymphocyte activation. Analysis of spleen cell proliferation, expression of Th1 and Th2 cytokines and production of interferon gamma by lymphocytes of immunized mice after specific stimulation in vitro revealed that recombinant protein comprising CTL epitopes of HCV, Th epitope PADRE, lipoprotein and IL-2 induced the highest response of T-lymphocytes.

Immunogenicity of Exosomes from Dendritic Cells Stimulated with Toxoplasma gondii Lysates in Ocularly Immunized Mice.

Immunogenicity of dendritic cell-derived exosomes stimulated with Toxoplasma gondii lysates (TLA exo), mixed with cholera toxin as an adjuvant, was investigated in mice immunized via 2 mucosal routes (ocular vs intranasal). BALB/c mice were injected 3 times with TLA exo vaccine at 2 week interval, and the levels of IgG in serum and IgA in tear, saliva, feces, and vaginal wash were measured. To observe the expression of T. gondii-specific B1 gene, mice infected with ME49 T. gondii cysts were immunized with TLA exo or PBS exo (not stimulated with TLA), and their brain tissues were examined. The mice vaccinated via intranasal route elicited significantly higher humoral and mucosal immune responses compared with mice treated with PBS alone. Also, mice immunized via ocular route (by eyedrop) induced significantly higher T. gondii-specific IgG in serum and IgA in tear and feces in comparison with PBS controls. B1 gene expression was significantly lower in TLA exo vaccinated mice than in PBS or PBS exo vaccinated mice. These results demonstrated that ocular immunization of mice with TLA exo vaccine has the potential to stimulate systemic or local antibody responses. This study also highlighted an advantage of an eyedrop vaccine as an alternative for T. gondii intranasal vaccines.

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants.

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL−1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL−1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL−1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response.

Indirectly stimulation of DCs by Ganoderma atrum polysaccharide in intestinal-like Caco-2/DCs co-culture model based on RNA-seq

Abstract Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) activated the immune response of dendritic cells (DCs) by direct contacting with surface receptors. However, as a kind of macromolecule compound, PSG-1 cannot directly stimulate DCs via passing through the intestine epithelial cells. To investigate the role of PSG-1 in the interaction between the intestinal epithelium and its internal immune cells, the intestinal-like Caco-2/DCs co-culture model was established, then all transcripts of DCs with or without treatment of PSG-1 were globally screened by RNA-seq. According to the RNA-seq analysis, cytokine receptor interaction, NF-κB and MAPK signaling pathway were three important signal pathways annotate to the environmental information processing. Moreover, cytokine and related proteins expression were in DCs indirectly stimulated by PSG-1 were raised. These above findings indicated that PSG-1 indirectly affected the immune function of DCs in intestinal-like Caco-2/DCs co-culture model, in which NF-κB and MAPK pathway played essential roles.

The deubiquitinase OTUB1 augments NF-\u03baB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasmagondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T.encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

Selective transmission of some HIV-1 subtype C variants might depend on Envelope stimulating dendritic cells to secrete IL-10.

Envelope (Env) phenotype(s) that provide transmitted founders (TF) with a selective advantage during HIV-1 transmission would be the ideal target for preventative therapy. We generated Env clones from four individuals infected with a single virus and one participant infected with multiple variants at transmission and compared phenotype with matched Envs from chronic infection (CI). When we determined whether pseudovirus (PSV) of the five TF and thirteen matched CI Env clones differed in their ability to 1) enter TZM-bl cells, 2) bind DC-SIGN, and 3) trans-infect CD4+ cells there was no association between time post-infection and variation in Env phenotype. However, when we compared the ability of PSV to induce monocyte-derived dendritic cells (MDDCs) to secrete Interleukin-10 (IL-10), we found that only TF Envs from single variant transmission cases induced MDDCs to secrete either higher or similar levels of IL-10 as the CI clones. Furthermore, interaction between MDDC DC-SIGN and Env was required for secretion of IL-10. When variants were grouped according to time post-infection, TF PSV induced the release of higher levels of IL-10 than their CI counterparts although this relationship varied across MDDC donors. The selection of variants during transmission is therefore likely a complex event dependent on both virus and host genetics. Our findings suggest that, potentially due to overall variation in N-glycosylation across variants, nuanced differences in binding of TF Env to DC-SIGN might trigger alternative DC immune responses (IRs) in the female genital tract (FGT) that favour HIV-1 survival and facilitate transmission.

Immune Response of Plasmacytoid Dendritic Cells Stimulated by Human Papillomavirus (HPV) E6 in an In Vitro System.

BackgroundThis study aimed to analyze the changes in plasmacytoid dendritic cell (pDC) immunophenotypes when co-cultured with Caski cells and stimulated by human papillomavirus (HPV) E6 in vitro, and thus to discuss the immunoregulatory roles of pDCs in the tumorigenesis of cervical cancer.Material/MethodsThe immunophenotypic expression of pDCs was analyzed under stimulation of HPV E6 and co-culturing with Caski cells in vitro.ResultsHPV E6 infection caused significantly increased expression of CD40 in HPV16 M and HPV16 H groups MyD88 in HPV16 M,HPV16 H, and HPV18L groups; and TRAF6 in HPV16 M, HPV16 H, and HPV18L groups. pDCs co-cultured with Caski cells showed significantly lower expression of MyD88 and TRAF6 compared with the control.ConclusionsThe expression of MyD88 and TRAF6 might vary in different stages of HPV infection. pDCs might regulate CD40 to participate in the tumorigenesis and progression of cervical cancer, but related mechanisms still need further investigation.

Comparative immunophenotyping of Saccharomyces cerevisiae and Candida spp. strains from Crohn’s disease patients and their interactions with the gut microbiome

Investigation of the fungal communities in animal models of Inflammatory Bowel Diseases (IBD) showed a controversial role of Saccharomyces cerevisiae and Candida spp. In health and disease. These conflicting observations could be ascribed to immunogenic differences among co-specific strains. To assess the relevance of intra-strains differences on yeast immunogenicity and impact on the microbiota, we screened S. cerevisiae and Candida spp. Strains isolated from fecal samples of IBD patients. We compared the cytokine profiles, obtained upon stimulation of Peripheral Blood Mononuclear Cells (PBMCs) and Dendritic Cells with different yeast strains, and evaluated the relationship between strain’s cell wall sugar amount and immune response. Moreover, the gut microbiota composition was explored in relation to fungal isolation from fecal samples by metabarcoding analysis. The comparison of cytokine profiles showed strain dependent rather than species-dependent differences in immune responses. Differences in immunogenicity correlated with the cell wall composition of S. cerevisiae intestinal strains. Stimulation of human healthy PBMCs with different strains showed a pro-inflammatory IL-6 response counterbalanced by IL-10 production. Interestingly, Crohn’s (CD) patients responded differently to “self” and “non-self” strains, eliciting pure Th1 or Th17 cytokine patterns. The differences observed in vitro were recapitulated in vivo, where different strains contributed in dramatically different ways to local epithelial activity and to the inflammation of wild type and Interleukin-deficient mice. Furthermore, we observed that the gut microbiota profiles significantly differentiated according to the presence of Saccharomyces or Candida spp. or the absence of fungal isolates in fecal samples. Our results show the importance to deepen metagenomics and immunophenotyping analyses to the strain level, to elucidate the role of fungal and bacterial communities in health and disease.

PARP inhibition and immune modulation: scientific rationale and perspectives for the treatment of gynecologic cancers.

Poly[adenosine diphosphate (ADP) ribose]polymerase (PARP) has multifaceted roles in the maintenance of genomic integrity, deoxyribonucleic acid (DNA) repair and replication, and the maintenance of immune-system homeostasis. PARP inhibitors are an attractive oncologic therapy, causing direct cancer cell cytotoxicity by propagating DNA damage and indirectly, by various mechanisms of immunostimulation, including activation of the cGAS/STING pathway, paracrine stimulation of dendritic cells, increased T-cell infiltration, and upregulation of death-ligand receptors to increase susceptibility to natural-killer-cell killing. However, these immunostimulatory effects are counterbalanced by PARPi-mediated upregulation of programmed cell-death-ligand 1 (PD-L1), which leads to immunosuppression. Combining PARP inhibition with immune-checkpoint blockade seeks to exploit the immune stimulatory effects of PARP inhibition while negating the immunosuppressive effects of PD-L1 upregulation.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Mannan Enhances IL-12 Production by Increasing Bacterial Uptake and Endosomal Degradation in L. acidophilus and S. aureus Stimulated Dendritic Cells.

The mannose receptor (MR) is a C-type lectin involved in endocytosis and with a poorly defined ability to modulate cellular activation. We investigated the effect of mannan treatment prior to stimulation of murine bone marrow-derived dendritic cells with the Gram-positive bacteria Lactobacillus acidophilus NCFM (L. acidophilus) on the induction of Interleukin (IL)-12. Mannan enhanced the IL-12 production induced by L. acidophilus in a dose dependent manner (up to 230% enhancement). Additionally, mannan-enhanced IL-12 induction was also demonstrated with another Gram-positive bacteria, Staphylococcus aureus (S. aureus), while an IL-12 reducing effect was seen on Escherichia coli stimulated cells. Furthermore, the expression of Interferon β (Ifnb) was increased in cells treated with mannan prior to stimulation with L. acidophilus. The addition of mannan but not of bacteria led to endocytosis of MR, while addition of mannan prior to L. acidophilus or S. aureus resulted in increased endocytosis of bacteria, a faster killing of endocytosed bacteria, and increased reactive oxygen species production. Expression of signaling lymphocytic activation molecule (SLAMF)1 shown previously to be involved in the facilitation of endosomal degradation was upregulated by mannan but not by L. acidophilus and S. aureus. The IL-12 enhancement by mannan but not the IL-12 induced by the bacteria was abrogated by addition of inhibitors of clathrin coated pits (chlorpromazine and monodansylcadaverine). Furthermore, the addition of acid sphingomyelinase, a facilitator of ceramide raft formation, prior to addition of L. acidophilus enhanced the IL-12 production and the endocytosis of bacteria. In summary, our results show that mannan increases the IL-12 production induced by some Gram-positive bacteria through MR-endocytosis, which increases bacterial endocytosis and endosomal killing. The differential effect of MR activation on the IL-12 production induced by Gram-positive and Gram-negative bacteria may influence the immune response toward allergens and other glycoproteins.

IL-25 Orchestrates Activation of Th Cells via Conventional Dendritic Cells in Tissue to Exacerbate Chronic House Dust Mite-Induced Asthma Pathology.

House dust mite (HDM) extract is a common trigger of asthma in humans. Chronic exposure to HDM also induces asthma-like pathology in mice. Allergic responses to HDM and other allergens are linked to release of IL-25, IL-33, and TSLP by epithelial cells; these cytokines, especially IL-33, target innate lymphoid cells type 2 to produce type 2 cytokines. To what extent and by what mechanisms IL-25 contributes to chronic HDM-induced pathology is not well understood. In humans, elevated levels of IL-25 appear to be associated with cases of uncontrolled asthma and exacerbated attacks. In this article, we demonstrate that blockade of IL-25 signaling in either lung conventional dendritic cells or in T cells resulted in similar decreases in production of IL-13 and IL-9 by T cells, reduced mast cell accumulation and tissue remodeling, and improved lung function but had only modest effects on eosinophilia. Stimulation of conventional dendritic cells by IL-25 promoted proximal accumulation of Th cells, and stimulation of Th cells by IL-25 locally promoted IL-13 and IL-9 production. IL-25 made notable contributions to chronic HDM-induced allergic asthma pathology by facilitating clustering and cross-stimulation of different cell types in tissue. Therapeutic targeting of IL-25 in combination with other treatments may be beneficial.

Diverse Immunomodulatory Effects of Individual IFNα Subtypes on Virus-Specific CD8+ T Cell Responses.

Clinical administration of Interferon α (IFNα) resulted in limited therapeutic success against some viral infections. Immune modulation of CD8+ T cell responses during IFNα therapy is believed to play a pivotal role in promoting viral clearance. However, these clinical studies primarily focused on IFNα subtype 2. To date, the immunomodulatory roles of the remaining 10–13 IFNα subtypes remains poorly understood, thereby precluding assessments of their potential for more effective treatments. Here, we report that virus-specific CD8+ T cell responses were influenced to various extents by individual IFNα subtypes. IFNα4, 6, and 9 had the strongest effects on CD8+ T cells, including antiproliferative effects, improved cytokine production and cytotoxicity. Interestingly, augmented cytokine responses were dependent on IFNα subtype stimulation of dendritic cells (DCs), while antiproliferative effects and cytotoxicity were mediated by IFNAR signaling in either CD8+ T cells or DCs. Thus, precise modulation of virus-specific CD8+ T cell responses may be feasible for specific antiviral immunotherapies through careful selection and administration of individual IFNα subtypes.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from na\xefve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.