Spleen Dendritic Cells Research Articles

Dendritic cell activation by iron oxide nanoparticles depends on the extracellular environment.

Nanoparticles can exert immune modulating effects in a host depending on composition, mode of administration, and type of disease. Although the specific mechanisms of nanoparticle-induced immune responses remain unclear, their uptake by macrophages and other phagocytic innate immune cells is considered to be a key event. Our objective here was to ascertain if nanoparticle-mediated activation of dendritic cells (DCs) occurs in vitro or in vivo when exposed to hydroxyethyl starch-coated iron oxide nanoparticles. For the present studies, our choice of nanoparticles, animal model, and experimental design is motivated by our previously published observations that systemic exposure can induce antitumor adaptive immune responses in mouse models of metastatic breast cancer. Here, we began by assessing the potential toxicity of systemic exposure to commercially available starch-coated Bionized Nanoferrite® nanoparticles (BP) by measuring body weight, complete blood count, and enzyme parameters in healthy FVB/NJ mice after repeated BP dosing. We observed no evidence of toxicity at doses up to 25 mg Fe per mouse, five-fold higher than those used in subsequent in vivo experiments. We then measured the expression of surface maturation markers (CD86, MHC II) in DCs incubated with BP in vitro. Although DCs cultured with BP revealed high levels of nanoparticle uptake, neither JAWSII dendritic cells nor bone marrow derived dendritic cells (BMDCs) showed significant changes in marker expression to indicate stimulation of maturation and effector function. To assess whether BP interactions in vivo produced different effects, we analyzed CD80, CD86, and MHC II expression of DCs recovered from the livers, spleens, bone marrows, and lymph nodes of mice injected once with BP (5 mg Fe). Interestingly, only DCs in spleens and bone marrow cells responded to BP exposure. DCs recovered from other organs showed no evidence of increased activation. These findings highlight complex interactions between living systems and nanoparticles, and their potential to mediate context-specific and selective activation of innate immune cells. Our study also emphasizes that results obtained from in vitro experiments must be interpreted with caution, as they may not faithfully represent responses in living systems.

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.

NLRP12-expressing dendritic cells mediate both dissemination of infection and adaptive immune responses in visceral leishmaniasis

The NLR protein NLRP12 contributes to innate immunity, but the mechanism remains elusive. Infection of Nlrp12 -/- or wild-type mice with Leishmania infantum led to aberrant parasite tropism. Parasites replicated to higher levels in livers of Nlrp12 -/- mice than in the livers of WT mice and failed to disseminate to spleens. Most retained liver parasites resided in dendritic cells (DCs), with correspondingly fewer infected DCs in spleens. Furthermore, Nlrp12 -/- DCs expressed lower CCR7 than WT DCs, failed to migrate toward CCL19 or CCL21 in chemotaxis assays, and migrated poorly to draining lymph nodes after sterile inflammation. Leishmania-infected Nlpr12 -/- DCs were significantly less effective at transporting parasites to lymph nodes than WT DCs. Consistently, adaptive immune responses were also impaired in infected Nlrp12 -/- mice. We hypothesize that Nlrp12-expressing DCs are required for efficient dissemination and immune clearance of L.infantum from the site of initial infection. This is at least partly due to the defective expression of CCR7.

Mitochondrial DNA mediates immunoparalysis of dendritic cells in sepsis via STING signalling.

Mitochondrial DNA (mtDNA) is a potent activator for pro-inflammatory response. Dendritic cells (DCs) are immunosuppressed in sepsis, whether mtDNA mediates immunoparalysis in sepsis remains unknown. The mRNAs were assessed by qPCR. Flow cytometry was used to measure the expression of costimulatory molecules and the proliferation of CD4+ T cells. Western blot and immunofluorescence staining were used to analyse the expression of proteins. Cytokine secretion was detected by ELISA. Histology of lung tissue was used to assess the inflammatory injury. Lipopolysaccharide-induced endotoxemia increased plasma mtDNA levels and immunoparalysis of spleen DCs, while hydrolysing mtDNA reversed immunoparalysis of spleen DCs in vivo. Moreover, cytoplasmic mtDNA of DCs was accumulated in endotoxemia and sepsis. mtDNA transfection into bone marrow-derived DCs (BMDCs) inhibited the expression of costimulatory molecules (e.g., CD40, CD80 and CD86) and the release of IL-12p70, while increasing the secretion of IL-10. Cytoplasmic mtDNA also inhibited the ability of BMDCs to promote the proliferation of CD4+ T cells. Mechanistic analysis revealed that STING signalling was required for mtDNA-mediated immunoparalysis of DCs in vivo and in vitro. Further studies showed deletion of STING reversed mtDNA-mediated immunoparalysis of DCs and improved the prognosis of endotoxemia and sepsis. Our results demonstrated that mtDNA promotes immunoparalysis of DCs, and contributes to sepsis-associated immunosuppression by activating STING signalling. Our study may provide new insights to elucidate the molecular pathogenesis of immunosuppressive DCs in sepsis.

Multi-Modal Imaging Monitored M2 Macrophage Targeting Sono-Responsive Nanoparticles to Combat MRSA Deep Infections.

BackgroundMRSA with high morbidity and mortality is prone to cause serious infection, SDT has become a new antibiotic-free modality for bacterial infection treatment. Switching from proinflammatory M1 macrophages to anti-inflammatory M2 macrophages dominant could activate the immune system to generate an anti-infection immune response.MethodsHerein, we developed M2 macrophages derived cell membranes coated PLGA nanoparticles with IR780 encapsulation (M2/IR780@PLGA) for antibacterial SDT and subsequent M2 macrophage polarization to enhance the therapeutic efficacy of MRSA myositis. For in situ visualization of antibacterial SDT, both diagnostic high-frequency US and magnetic resonance imaging (MRI) were introduced to monitor the sono-therapeutic progression of M2/IR780@PLGA nanoparticles in mice with bacterial myositis.ResultsOur developed M2/IR780@PLGA nanoparticles exhibited excellent antibacterial effects due to the IR780 under low-frequency US irradiation in vitro. In an MRSA-infected mice model, a great deal of M2/IR780@PLGA nanoparticles accumulated at the site of inflammation due to M2 macrophage coating. The infected legs in the M2/IR780@PLGA nanoparticles-based SDT group were significantly smaller, fewer blood flow signals, a slight muscular edema without obvious intermuscular abscesses under high-frequency US and MR images guidance. Histopathology proved the infected legs in the M2/IR780@PLGA nanoparticles-mediated SDT group had less clumped bacteria infiltration, more M2 macrophage expression and less M1 macrophage expression. The percentage of mature dendritic cells in spleens was much higher in the group of mice with M2/IR780@PLGA nanoparticles-based SDT.ConclusionThis study provides a promising nanoparticles-based SDT anti-bacterial strategy, which could effectively enhance the antibacterial SDT and subsequent promote M2 macrophage polarization to boost the therapeutic efficacy of MRSA myositis.

Immunological Effects of Cold Atmospheric Plasma-Treated Cells in Comparison with Those of Cells Treated with Lactaptin-Based Anticancer Drugs

The ability of dying cancer cells to induce an anticancer immune response can increase the effectiveness of anticancer therapies, and such type of death is termed immunogenic cell death (ICD). Cells can die along the ICD pathway when exposed not only to chemo- and immunotherapeutics, but also to various types of radiation, such as ionizing radiation and cold atmospheric plasma jets (CAP). We have previously shown that CAP, lactaptin, and a recombinant vaccinia virus encoding lactaptin induce in vitro molecular changes typical of ICD in cancer cells. In the current work, we treated MX-7 rhabdomyosarcoma cells with CAP and lactaptin-based anticancer drugs and evaluated the immunological effects of the treated cells. We showed that dendritic cells (DCs) captured cells treated with various ICD inducers with different efficiency. CAP-treated cells were weakly potent in inducing the maturation of DCs according to MHC II externalization. Moreover, CAP-treated cells were worse in the stimulation of IFN-α release in vitro and were poorly captured by spleen DCs in vivo. Under the irradiation conditions used, CAP was not capable of activating a significant immunological anti-tumor effect in vivo. It is possible that modifications of the CAP irradiation regimen will enhance the activation of the immune system.

Widely Targeted Lipidomics and Transcriptomics Analysis Revealed Changes of Lipid Metabolism in Spleen Dendritic Cells in Shrimp Allergy.

Shrimp allergy (SA) is pathological type 2 inflammatory immune responses against harmless shrimp protein allergen, which is caused by complex interactions between dendritic cells (DCs) and other immune cells. Lipid metabolism in different DCs states are significantly changed. However, the lipid metabolism of spleen DCs in SA remain ambiguous. In this study, we established a BALB/c mouse shrimp protein extract-induced allergy model to determine the lipid profile of spleen DCs in SA, and the molecular mechanism between lipid metabolism and immune inflammation was preliminarily studied. Spleen DCs were sorted by fluorescence-activated cell sorting, and then widely targeted lipidomics and transcriptomics analysis were performed. Principal component analysis presented the lipidome alterations in SA. The transcriptomic data showed that Prkcg was involved in lipid metabolism, immune system, and inflammatory signaling pathway. In the correlation analysis, the results suggested that Prkcg was positively correlated with triacylglycerol (Pearson correlation coefficient = 0.917, p = 0.01). The lipidomics and transcriptomics integrated pathway analysis indicated the activated metabolic conversion from triacylglycerol to 1,2-diacyl-sn-glycerol and the transmission of lipid metabolism to immune inflammation (from triacylglycerol and ceramide to Prkcg) in SA spleen DCs, and cellular experiments in vitro showed that glyceryl trioleate and C16 ceramide treatment induced immune function alteration in DCs.

Photocurable Hydrogel Substrate-Better Potential Substitute on Bone-Marrow-Derived Dendritic Cells Culturing.

Dendritic cells (DCs) are recognized as the most effective antigen-presenting cells at present. DCs have corresponding therapeutic effects in tumor immunity, transplantation immunity, infection inflammation and cardiovascular diseases, and the activation of T cells is dependent on DCs. However, normal bone-marrow-derived Dendritic cells (BMDCs) cultured on conventional culture plates are easy to be activated during culturing, and it is difficult to imitate the internal immune function. Here, we reported a novel BMDCs culturing with hydrogel substrate (CCHS), where we synthesized low substituted Gelatin Methacrylate-30 (GelMA-30) hydrogels and used them as a substitute for conventional culture plates in the culture and induction of BMDCs in vitro. The results showed that 5% GelMA-30 substrate was the best culture condition for BMDCs culturing. The low level of costimulatory molecules and the level of development-related transcription factors of BMDCs by CCHS were closer to that of spleen DCs and were capable of better promoting T cell activation and exerting an immune effect. CCHS was helpful to study the transformation of DCs from initial state to activated state, which contributes to the development of DC-T cell immunotherapy.

Chemo- and mechanosensing by dendritic cells facilitate antigen surveillance in the spleen.

Spleen dendritic cells (DC) are critical for initiation of adaptive immune responses against blood-borne invaders. Key to DC function is their positioning at sites of pathogen entry, and their abilities to selectively capture foreign antigens and promptly engage T cells. Focusing on conventional DC2 (cDC2), we discuss the contribution of chemoattractant receptors (EBI2 or GPR183, S1PR1, and CCR7) and integrins to cDC2 positioning and function. We give particular attention to a newly identified role in cDC2 for adhesion G-protein coupled receptor E5 (Adgre5 or CD97) and its ligand CD55, detailing how this mechanosensing system contributes to splenic cDC2 positioning and homeostasis. Additional roles of CD97 in the immune system are reviewed. The ability of cDC2 to be activated by circulating missing self-CD47 cells and to integrate multiple red blood cell (RBC)-derived inputs is discussed. Finally, we describe the process of activated cDC2 migration to engage and prime helper T cells. Throughout the review, we consider the insights into cDC function in the spleen that have emerged from imaging studies.

CD97 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells.

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα13 and adhesion G protein-coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

Dendritic cell-mediated chronic low-grade inflammation is regulated by the RAGE-TLR4-PKC\u03b21 signaling pathway in diabetic atherosclerosis

BackgroundThe unique mechanism of diabetic atherosclerosis has been a central research focus. Previous literature has reported that the inflammatory response mediated by dendritic cells (DCs) plays a vital role in the progression of atherosclerosis. The objective of the study was to explore the role of DCs in diabetes mellitus complicated by atherosclerosis.MethodsApoE−/− mice and bone marrow-derived DCs were used for in vivo and in vitro experiments, respectively. Masson’s staining and Oil-red-O staining were performed for atherosclerotic lesion assessment. The content of macrophages and DCs in plaque was visualized by immunohistochemistry. The expression of CD83 and CD86 were detected by flow cytometry. The fluctuations in the RNA levels of cytokines, chemokines, chemokine receptors and adhesions were analyzed by quantitative RT-PCR. The concentrations of IFN-γ and TNF-α were calculated using ELISA kits and the proteins were detected using western blot. Coimmunoprecipitation was used to detect protein–protein interactions.ResultsCompared with the ApoE−/− group, the volume of atherosclerotic plaques in the aortic root of diabetic ApoE−/− mice was significantly increased, numbers of macrophages and DCs were increased, and the collagen content in plaques decreased. The expression of CD83 and CD86 were significantly upregulated in splenic CD11c+ DCs derived from mice with hyperglycemia. Increased secretion of cytokines, chemokines, chemokine receptors, intercellular cell adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) also were observed. The stimulation of advanced glycation end products plus oxidized low-density lipoprotein, in cultured BMDCs, further activated toll-like receptor 4, protein kinase C and receptor of AGEs, and induced immune maturation of DCs through the RAGE-TLR4-PKCβ1 signaling pathway that was bound together by intrinsic structures on the cell membrane. Administering LY333531 significantly increased the body weight of diabetic ApoE−/− mice, inhibited the immune maturation of spleen DCs, and reduced atherosclerotic plaques in diabetic ApoE−/− mice. Furthermore, the number of DCs and macrophages in atherosclerotic plaques was significantly reduced in the LY333531 group, and the collagen content was increased.ConclusionsDiabetes mellitus aggravates chronic inflammation, and promotes atherosclerotic plaques in conjunction with hyperlipidemia, which at least in part through inducing the immune maturation of DCs, and its possible mechanism of action is through the RAGE-TLR4-pPKCβ1 signaling pathway.

FMS-like tyrosine kinase 3 ligand (FLT3L) administration alters rat classical and plasmacytoid dendritic cell phenotype in a tissue-specific manner

Abstract Background: Characterization of tissue-resident dendritic cell (DC) subsets, including classical DCs (cDCs) and plasmacytoid DCs (pDCs), is complicated experimentally by their rare nature. Many investigators have artificially increased DC yields through administration of the cytokine FMS-like tyrosine kinase 3 ligand (FLT3L). Despite its previous use experimentally, no study has yet described the effects of in vivo FLT3L administration on rat tissue-resident DCs. This study provides an advanced characterization of rat tissue-resident cDCs and pDCs following FLT3L injection. Methods: Lewis rats were injected once daily for 10 days with PBS vehicle or with 10, 50, or 100μg of FLT3L. Animals were sacrificed on the 11th day and dissected to obtain liver, spleen, and mesenteric lymph nodes. Cells were stained for 13-color flow cytometry and data was acquired on the Cytek Aurora spectral cytometer. Results: cDC and pDC enumeration increased in a dose-dependent manner following FLT3L administration. Expression levels of MHC I, MHC II, CD40, CD80, CD86, and PD-L1 were evaluated on liver, spleen, and mesenteric lymph node-resident cells. MHC II, CD80, and CD86 levels were significantly decreased on liver-resident cDCs but remained unchanged or increased on spleen and lymph node cDCs. Changes in MHC I/II and costimulatory molecule expression levels on pDCs varied by tissue and marker. Conclusions: FLT3L administration in vivo increased cDC and pDC enumeration, generated immature cDCs in the liver, and increased cDC maturation in the spleen and mesenteric lymph nodes. This comprehensive characterization of rat liver, spleen, and lymph node-resident DCs is key to understanding the effects of FLT3L on cDC and pDC phenotype.

Polysaccharide from Astragalus membranaceus promotes the activation of human peripheral blood and mouse spleen dendritic cells.

Astragalus membranaceus (A. membranaceus) is a widely used traditional herb in China and Korea. A. membranaceus polysaccharides (AMP), which make up a major part of the root extract, have been shown to modulate immune modulations, especially activation of bone marrow-derived dendritic cells (BMDCs) and T cells. However, the immune stimulatory effect of AMP in the mouse in vivo and human peripheral blood DCs (PBDCs) has not been well investigated. In this study, we found that intravenous (i.v.) injection of AMP in C57BL/6 mice induced remarkable elevations in co-stimulatory and MHC class I and II molecule levels in the splenic DCs and its subsets. The stimulatory effect of DCs by AMP was elevated 6 h after treatment, which rapidly decreased 18 h after injection. Furthermore, AMP promoted intracellular production of pro-inflammatory cytokines in spleen DC subsets, which contributed elevation of serum cytokine levels. Finally, the AMP promoted PBDC activation. Thus, these results demonstrate that AMP can be used as an immune stimulatory molecules in human and mouse.

CXCR3 chemokine receptor contributes to specific CD8+T cell activation by pDC during infection with intracellular pathogens.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.

MiRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy.

Dendritic cell (DC) based immunotherapy is a promising approach to clinical cancer treatment. miRNAs are a class of small non-coding RNA molecules that bind to RNAs to mediate multiple events which are important in diverse biological processes. miRNA mimics and antagomirs may be potent agents to enhance DC-based immunotherapy against cancers. miRNA array analysis was used to identify a representative miR-5119 potentially regulating PD-L1 in DCs. We evaluated levels of ligands of immune cell inhibitory receptors (IRs) and miR-5119 in DCs from immunocompetent mouse breast tumor-bearing mice, and examined the molecular targets of miR-5119. We report that miRNA-5119 was downregulated in spleen DCs from mouse breast cancer-bearing mice. In silico analysis and qPCR data showed that miRNA-5119 targeted mRNAs encoding multiple negative immune regulatory molecules, including ligands of IRs such as PD-L1 and IDO2. DCs engineered to express a miR-5119 mimic downregulated PD-L1 and prevented T cell exhaustion in mice with breast cancer homografts. Moreover, miR-5119 mimic-engineered DCs effectively restored function to exhausted CD8+ T cells in vitro and in vivo, resulting in robust anti-tumor cell immune response, upregulated cytokine production, reduced T cell apoptosis, and exhaustion. Treatment of 4T1 breast tumor-bearing mice with miR-5119 mimic-engineered DC vaccine reduced T cell exhaustion and suppressed mouse breast tumor homograft growth. This study provides evidence supporting a novel therapeutic approach using miRNA-5119 mimic-engineered DC vaccines to regulate inhibitory receptors and enhance anti-tumor immune response in a mouse model of breast cancer. miRNA/DC-based immunotherapy has potential for advancement to the clinic as a new strategy for DC-based anti-breast cancer immunotherapy.

CD8α- conventional dendritic cells control Vβ T-cell immunity in response to Staphylococcus aureus infection in mice.

Dendritic cells (DCs) are potent immune cells that control innate and adaptive immune responses. Previous studies have shown that the DCs are required for protection against Staphylococcus aureus infection. However, the role of conventional DC (cDC) subsets during S.aureus infection in vivo has not been well investigated. In this study, we examined the function of spleen DC subsets in the activation of immunity against S.aureus infection. C57BL/6 mice were infected intravenously with S.aureus and DC and T-cell activation were analyzed in vivo. We found that the spleen CD8α- cDCs phagocytosed S.aureus more efficiently than type-1 conventional DCs (cDC1s) did. Moreover, the CD8α- cDCs contributed to the production of pro-inflammatory cytokines in response to S.aureus infection, whereas the cDC1s did not. In addition, infection with S.aureus promoted an increase in the number of Vβ T cells. The CD4+ and CD8+ Vβ T cells up-regulated the production of interferon-γ (IFN-γ) and interleukin-17 (IL-17) in response to S.aureus infection. Importantly, the induction of IFN-γ and IL-17 production in CD4+ and CD8+ Vβ T cells was mediated by S.aureus-stimulated CD8α- cDCs, whereas cDC1s failed to promote IFN-γ and IL-17 production in the cells. Therefore, these data suggested that the spleen CD8α- cDCs are the main DC subsets for induction of S.aureus superantigen-specific immunity.

Cancer immunotherapy using a polysaccharide from Codium fragile in a murine model

ABSTRACT Natural polysaccharides have shown immune modulatory effects with low toxicity in both animal and human models. A previous study has shown that the polysaccharide from Codium fragile (CFP) promotes natural killer (NK) cell activation in mice. Since NK cell activation is mediated by dendritic cells (DCs), we examined the effect of CFP on DC activation and evaluated the subsequent induction of anti-cancer immunity in a murine model. Treatment with CFP induced activation of bone marrow-derived dendritic cells (BMDCs). Moreover, subcutaneous injection of CFP promoted the activation of spleen and lymph node DCs in vivo. CFP also induced activation of DCs in tumor-bearing mice, and combination treatment with CFP and ovalbumin (OVA) promoted OVA-specific T cell activation, which consequently promoted infiltration of IFN-γ-and TNF-α-producing OT-1 and OT-II cells into the tumors. Moreover, combination treatment using CFP and cancer self-antigen efficiently inhibited B16 tumor growth in the mouse model. Treatment with CFP also enhanced anti-PD-L1 antibody mediated anti-cancer immunity in the CT-26 carcinoma-bearing BALB/c mice. Taken together these data suggest that CFP may function as an adjuvant in the treatment of cancer by enhancing immune activation. Abbreviations CFP: Codium fragile polysaccharide; NK: natural killer; IFN: interferon; TNF: tumor necrosis factor; IL: interleukin; tdLN: tumor draining lymph node; BMDC: bone marrow-derived dendritic cell; OVA: ovalbumin; Ab: antibody; Ag: antigen; DC: dendritic cell; CTL: cytotoxic T lymphocyte; APC: antigen-presenting cell; pDC: plasmacytoid dendritic cell; mDC: myeloid dendritic cell; MHC: major histocompatibility complex; CR3: complement receptor type 3; TLR: Toll-like receptor; LPS: lipopolysaccharide; SP: sulfated polysaccharide; TRP2: tyrosinase-related protein 2; SR-A: scavenger receptor-A

IDO inhibitor synergized with radiotherapy to delay tumor growth by reversing T cell exhaustion

Previous studies suggest that radiotherapy (RT) can induce immune activation, which not only reduces the progression of tumors, but also causes the release of tumor antigens. The combination of RT and immune checkpoint blockade, such as the inhibition of programmed cell death 1 (PD‑1) and programmed cell death ligand 1 (PD‑L1), has been demonstrated to yield impressive response rates. However, a substantial proportion of patients develop resistance such therapies. Previous studies have shown that indoleamine 2,3‑dioxygenase (IDO) causes T cell exhaustion and increased formation of regulatory T cells (Tregs), upregulating the expression of inhibitory receptors and ligands. Therefore, the application of IDO inhibitors combined with RT may have a synergistic effect by relieving immunosuppression. Lewis lung cancer cell‑bearing mice were treated with the IDO inhibitor 1‑methyl‑tryptophan (1MT) and/or 10Gy RT. Tumor size was measured every day, flow cytometry was performed to measure the expression of dendritic cell (DC) maturation markers, inhibitory receptors, ligands, cytotoxic T cells and Treg phenotypic markers. Reverse transcription‑quantitative PCR was used to evaluate the mRNA expression levels of IDO, PD‑L1, PD‑1, T cell immunoglobulin domain and mucin domain 3 (TIM‑3), B‑ and T‑lymphocyte attenuator (BTLA) and galectin‑9. Compared with the control group, treatment with 1MT or RT reduced tumor growth, however, the combination therapy was more effective than either treatment alone. Flow cytometry showed the upregulation of CD80, CD86 and the major histocompatibility complex II in spleen DCs and the concurrent downregulation of CD4, CD25 and forkhead box protein P3 in lymphocytes in the treatment groups. Compared with the control group, inhibitory receptors and ligands that affect DCs and T cells were observed, expression levels of PD‑L1, PD‑1, TIM‑3, BTLA and galectin‑9 are decreased in treatment group compared with control. IDO inhibition synergized with RT to downregulate Tregs, inhibitory receptors and ligands to prevent T cell exhaustion. By activating DCs and T cells, this combination therapy may strongly enhance antitumor immunity and inhibit tumor progression.

P6306Hyperlipidemia inhibits the protective effect of lisinopril after myocardial infarction via activation of dendritic cells

Abstract Background In clinical situation, patients with myocardial infarction (MI) usually suffered from hyperlipidemia, hypertension, renal dysfunction or diabetes as well and the interaction of those risk factors may affect the effectiveness of ACEI. Dendritic cell (DC) is a potent central immunoregulator that orchestrates various types of inflammatory cells in innate and adoptive immunity. The role of DCs in the inflammation of cardiovascular disease is a hotspot of international research. Purpose To investigate the improvement of cardiac remodeling and inflammatory immune response after MI via ACEI regulating DCs and explore that whether the protective effect of ACEI is under the circumstance of hyperlipidemia. Methods In vivo, MI mice models were created via coronary artery ligation. We detected the survival and left ventricular function on day 7 after MI. Tissue samples of the myocardium, spleen, bone marrow and peripheral blood were obtained and assessed for Ang2 concentration, inflammatory cytokines and DCs expression via immunohistochemistry, ELISA, flow cytometry and western blot. In vitro, we treated DCs with ox-LDL+AngIIfor 48 hours simulating the internal environment of MI in ApoE−/− mice to explore the mechanism involved in the DCs maturation and inflammation. Results Under the circumstance of hyperlipidemia, the cardioprotective effect of ACEI is attenuated through regulating DCs maturation and inflammation after MI, including survival rate (Figure 1) and left ventricular function. The number of DCs in cardiac tissue were increased in MI mice with hyperlipidemia (Figure 2), when compared with the WT group. The expression of the maturity phenotype related molecules, such as CD83, on the DCs in spleen were also increased in the ApoE−/− mice. Then, the expressions of inflammatory cytokines, such as IL-6 and TNF-α. In vitro, it was found that the immune maturation and inflammation of DCs could be further induced by ox-LDL on the basis of AngIItreatment, as indicated by the upregulation of CD83 and CD86, the expressions of cytokines and chemokines. Furthermore, ox-LDL could activate the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88) signaling pathway, promoting the phosphorylation of interleukin-1 receptor associated kinase (IRAK)-4 and the nuclear transferring of nuclear factor-kappaB (NF-κB). Conclusion We proved ACEI reduces the recruitment of DCs to the infarct, leading to a higher survival rate and improved function and remodeling through decreased inflammation resulting from immune response after MI. However, this effect was inhibited under the circumstance of hyperlipidemia. TLR4-MyD88 signaling pathway might be the possible molecular mechanism involved in the immune maturation and inflammation of DCs induced by ox-LDL on the basis of AngII. Acknowledgement/Funding the Youth fund of the National Natural Science Foundation of China, Grant 81600280

Deubiquitinating Enzyme UCH-L1 Promotes Dendritic Cell Antigen Cross-Presentation by Favoring Recycling of MHC Class I Molecules.

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow-derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1-deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.