A Structurally Dynamic Pathogen-Mimicking Biomaterial Is an Efficient Activator of Dendritic Cells.

Nanoparticles and the immune system: challenges and opportunities.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DC) are the only antigen-presenting cells that can activate naïve T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c+ and CD11c- DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin- = CD3-, CD19-, CD14-, CD16-) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86+ and CD40+ lin- cells. Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c+ and CD11c- DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86+ and CD40+ DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44+ antigen was mainly associated with CD11c+ DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86+ and CD40+ DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation.

Dendritic cells (DCs) are specialized antigen presentation cells that play critical roles in the initiation and regulation of immune responses. The molecular determinants of DC differentiation and maturation are target of extensive investigation. VentX is a human homeobox transcriptional factor that regulates proliferation and differentiation of hematopoietic cells. In the current study, we report that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs. Conversely, overexpression of VentX in monocytic THP1 cells accelerated their differentiation toward DCs. We showed that VentX regulates DC differentiation, in part, through modulating IL6 expression. Clinically, we found that VentX expression was elevated in intestinal lamina propria DCs (LPDCs) of inflamed mucosa from inflammatory bowel disease patients. Knockdown experiments suggested that VentX is essential for the maturation of LPDCs. In addition, corticosteroid treatment markedly decreased VentX expression in LPDCs and enforced expression of VentX counteracted the effects of corticosteroid on DCs maturation. Our data suggest that VentX is a critical transcriptional regulator of DC differentiation and maturation, and a potential target of immune regulation and therapy.

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

Objective: The effect of melanoma cell coculture on generating human dendritic cells(DC) from CD14+ monocytes has not been extensively studies yet. We investigated how melanoma cells and membrane-bound granulocyte/macrophage colony stimulating factor (mbGM-CSF) melanoma cell lines affect the differentiation of DC obtained by in vitro coculture of plastic-adherent blood monocytes with the presence or absence of GM-CSF and interleukin(IL)-4. Method: The malignant melanoma cell lines(Conley and Jorp) were used in this study. Conley B-F8 and Jorp C-E6 are mbGM-CSF-positive cells derived from Conley and Jorp respectively. Each cell-free supernatants were collected and used to assess the level of GM-CSF using ELISA. Peripheral blood was collected from healthy donors. Adherent monocytes were cocultured for 6-7 days with irradiated mbGM-CSF and wild type melanoma cells(50 Gy) at each 1:1 and 0.1:1 ratio in 6-well culture plates in X-vivo culture medium containing IL-4. In control experiments, GM-CSF was added at the time of culture initiation. Immunophenotyping was performed by triple color immunofluorescence staining with flow cytometry analysis. Results: In this study, GM-CSF was detected at low levels in the culture supernatants of Conley B-F8 (0.48 ng/106 cells/24h), whereas there was no detectable GM-CSF in that of Conley melanoma cell line. ELISA demonstrated that Jorp C-E6 cells produced 18 ng/106 cells/24h, whereas Jorp wild type produced 3.6 pg/106 cells/24h. Monocytes cultured with GM-CSF/IL-4 generated the expression of high levels of HLA DR, CD1a and CD86, while the expression of CD14 and CD83 were in low amounts. After culture in medium containing IL-4 alone, monocytes cocultured with Conley B-F8 showed low levels of CD1a and CD86 and relatively low value of HLA DR. However, the addition of GM-CSF to these cultures resulted in an increased expression of these markers and decreased that of CD14. Monocytes cocultured with Jorp C-E6 illustrated similar expression pattern of CD1a, HLA DR and CD14 in the presence or absence of GM-CSF as Conley B-F8 melanoma cell line. Monocytes cocultured with Conley B-F8 melanoma cells at 1:1 and 0.1:1 ratio showed no significant difference in expression of CD1a, CD14 and CD83 between two ratio. Conclusion: Our results illustrate the feasibility to generate monocyte-derived DC from coculture with melanoma tumor cells in the presence of GM-CSF and IL-4. However, mbGM-CSF tumor cells did not significantly enhance the DC differentiation through juxtacrine stimulation unless soluble GM-CSF was added in the culture media.

Mucosal mast cells (MMCs) are distinguished from connective tissue mast cells (MCs) by the specific cell‐surface expression of integrin CD103 (also known as integrin αE/β7; αE is encoded by Itgae) and mast cell protease 1 and 2 (Mcpt1 and Mcpt2, respectively). Although the expression of the Mcpt1 and Mcpt2 genes is cooperatively regulated by the transcription factor GATA‐binding protein 2 (GATA2) and transforming growth factor beta (TGF‐β) signaling in MMCs, the transcriptional mechanism of the cell‐surface expression of CD103 remains unknown. We herein found that surface CD103 and Itgae mRNA levels were significantly increased by the knockdown (KD) of Gata2 in mouse bone marrow‐derived MCs (BMMCs), which was accelerated by TGF‐β stimulation. Since the mRNA levels of Spi1 (encoding transcription factor PU.1) were increased in Gata2 KD BMMCs, we examined the effects of PU.1 on the cell‐surface expression of CD103. As expected, CD103 levels on BMMCs were significantly decreased by Spi1 KD and increased by Spi1 overexpression. Spi1 KD suppressed Itgae expression even in the presence of TGF‐β in BMMCs and peritoneal MCs, whereas Gata2 KD amplified the TGF‐β‐induced increase in Itgae expression. The amount of PU.1 binding to the cis‐element in the Itgae gene was significantly and moderately increased by Gata2 KD and TGF‐β stimulation, respectively. Since PU.1 is an essential transcription factor for dendritic cells (DCs), we examined the role of PU.1 in CD103 cell‐surface expression on DCs. The KD experiment using bone marrow‐derived DCs (BMDCs) showed a significant decrease in CD103 levels in Spi1‐siRNA‐transfected BMDCs. We concluded that PU.1 affected CD103 expression on MMCs and DCs by transactivating the Itgae gene, and also that GATA2, which positively regulated the MMC‐specific expression of Mcpt1 and Mcpt2, inhibited the cell‐surface expression of CD103 by repressing PU.1.

Mesenchymal stem cells (MSCs) possess a wide range of immunomodulatory functions mostly in immune cells including dendritic cells (DCs). DCs are the key cells in the immune response and play an important role in initiating cell-mediated immunity. To evaluate the immunomodulatory effects of MSCs supernatant on maturation and function of DCs. Bone marrow derived mice MSCs were isolated and cultured. Twenty-four, forty-eight and seventy-two hours after passage 6, supernatants were collected and MSCs were assessed by flow cytometric analysis for the expression of CD34, CD44, CD45 and SCA-1. Splenic DCs were isolated using MACS and then co-cultured with MSCs supernatant. Expression of CD86, CD40 and MHC-II on DCs were also evaluated by flow cytometry. H3-thymidine incorporation by proliferating T cells was determined in two separate MLR assay settings. In one setting, DCs were co-cultured with T cells in the presence of MSCs supernatant, and in the other setting DCs were treated with MSCs supernatant and then were co-cultured with T cells. Production of IL-12, IL-6 and IL-10 cytokines was measured in the supernatant of DCs treated with MSCs supernatant. We also measured IFN-γ and IL-4 levels in MLR supernatant. The results showed that 72h MSCs supernatant could decrease the expression of MHC-II and CD86. The T cell proliferation was inhibited in the presence of MSCs supernatant and MSCs supernatant treated DCs as demonstrated by MLR assay. A significant increase in IL-4 level and a non significant decrease in IFN-γ level in MLR supernatant was observed. However, IL-6, IL-10 and IL-12 production did not change significantly. MSCs supernatant has a time dependent effect on the maturation of DCs. Also, it could alter cytokine production from responding T cells toward Th2. Generally, the findings of this study supported the immunomodulatory effect of MSCs supernatant on DCs maturation and function.

Targeted immunotherapies make substantial strides in clinical cancer care due to their ability to counteract the tumor's capacity to suppress immune responses. Advances in biomimetic technology with minimally immunogenic and highly targeted, are addressing issues of targeted drug delivery and disrupting the tumor's immunosuppressive environment to trigger immune activation. Specifically, the use of dendritic cell (DC) membranes to coat nanoparticles ensures targeted delivery due to DC's unique ability to activate naive T cells, spotlighting their role in immunotherapy aimed at disrupting the tumor microenvironment. The potential of DC's biomimetic membrane to mediate immune activation and target tumors is gaining momentum, enhancing the effectiveness of cancer treatments in conjunction with other immune responses. This review delves into the methodologies behind crafting DC membranes and the fusion of dendritic and tumor cell membranes for encapsulating therapeutic nanoparticles. It explores their applications and recent advancements in combating cancer, offering an all-encompassing perspective on DC biomimetic nanosystems, immunotherapy driven by antigen presentation, and the collaborative efforts of drug delivery in chemotherapy and photodynamic therapies. Current evidence shows promise in augmenting combined therapeutic approaches for cancer treatment and holds translational potential for various cancer treatments in a clinical setting.Graphical

Recent insights into immune system function have fostered a better appreciation of the role of specialized antigen-presenting cells (i.e., dendritic cells). Initially described by Steinman and Cohn (1) in 1973 as an adherent cell type among mouse splenocytes with a distinctive stellate morphology, dendritic cells soon became known for their role as the sentinels of the immune system (2). In their immature state, dendritic cells reside in peripheral tissues, where they survey for incoming pathogens. An encounter with pathogens leads to dendritic cell activation and migration to secondary lymphoid organs, where they trigger a specific T-cell response. Dendritic cells are cells that not only can stimulate quiescent naive CD4+ and CD8+ T and B cells and initiate primary immune responses but also can induce a strong secondary immune response at relatively low numbers and with low amounts of antigen. Furthermore, dendritic cells are involved in polarization of T-cell response via secreted cytokines and in induction of tolerance through deletion of self-reactive thymocytes and anergy of mature T cells (3). Given their central role in controlling immunity, dendritic cells are logical delivering vehicles for many clinical disease states that involve T cells, such as transplantation, allergy, autoimmune disease, resistance to infection and to tumors, immunodeficiency, and vaccination.In vivo, dendritic cells are continuously produced from hematopoietic stem cells in the bone marrow and are widely distributed as immature cells, such as epidermal Langerhan's cells, splenic marginal zone dendritic cells, and interstitial dendritic cells, in both lymphoid and nonlymphoid tissues (2). However, circulating dendritic cells are rare (they account for <1% of human peripheral blood mononuclear cells) and are difficult to maintain in culture. Most experimental and clinical studies currently rely on the in vitro development of dendritic cell–like cells from CD34+ progenitor cells or nonproliferating blood monocytes (4, 5). Commonly, monocytes are cultured for 5 to 7 days with granulocyte macrophage colony-stimulating factor and interleukin-4 to generate immature, monocyte-derived dendritic cells that have to be activated for another 1 to 2 days with microbial, proinflammatory, or T-cell–derived stimuli to obtain mature dendritic cells, which are fully capable of activating T cells and inducing an immune response (4, 5). With advances in cell culture, it is now possible as well as also feasible to obtain substantial numbers of these cells for preclinical and clinical studies.Due to their ready availability, monocyte-derived dendritic cells have been widely used as stimulators for in vitro or ex vivo generation of tumor-specific CTLs. To activate CD8+ T cells, presentation of tumor antigens on MHC class I molecules is needed. Dendritic cells are proving to be quite specialized in their capacity to form MHC class I-peptide complexes, via a mechanism of “cross-presentation” of proteins derived from immune complexes or inactivated microbes, or antigens originally synthesized in other cells (6). Several dendritic cell receptors lead to MHC class I-peptide complex formation via the exogenous pathway, including FcγR, the integrin αvβ5 and the phosphatidylserine receptor, and various receptors for heat shock proteins (6). The ability of dendritic cells to cross-present antigens on MHC class I molecules to CD8+ T cells has prompted many preclinical studies pulsing dendritic cells with soluble tumor antigens or lysates or loading dendritic cells with necrotic or apoptotic tumor bodies to generate tumor-specific CTLs in various tumor settings, including solid tumors (7) and hematologic malignancies (8, 9).In this issue of Clinical Cancer Research, Dr. Tokunaga and coworkers provide another good example of using the ability of dendritic cells to cross-present soluble tumor antigen to CD8+ T cells. In their study, monocyte-derived dendritic cells from HLA-A2-positive or HLA-A24-positive healthy individuals were used and pulsed with purified, full-length, wild-type p53 protein. The antigen-pulsed monocyte-derived dendritic cells were then used as stimulator cells in culture with autologous T cells to generate p53-specific CTLs. The resulting CTLs were able to kill p53-overexpressing tumor cells, and the cytotoxicity was MHC class I restricted. Thus, it is evident that the exogenously pulsed p53 protein was successfully presented, in the form of antigenic peptides in the context of MHC class I molecules, to CD8+ T cells. It should be noted that, although many investigators exploit dendritic cells for their ability to cross-present tumor antigens to CD8+ T cells, dendritic cells simultaneously present exogenous proteins and cellular antigens on both MHC class I and II molecules (2). By doing so, dendritic cells are also able to activate specific CD4+ helper T cells, which not only are important for antibody production and amplification and sustaining of CD8+ CTL response but also may by themselves exert antitumor activity, especially the type 1 helper T cells (2).Monocyte-derived dendritic cells are also the favorite choice of cells for dendritic cell–based immunotherapy in human patients. Among different clinical studies in cancer patients reported to date, most have used monocyte-derived dendritic cells as the vaccines (10, 11). Based on preliminary data, dendritic cell vaccines may prime and boost antigen-specific T-cell responses in patients, and dendritic cell–based immunotherapy has been proven to be feasible, nontoxic, and effective in some patients, especially if the dendritic cells have been appropriately matured and activated (10, 11). Nevertheless, as the full potential of these cells has not yet been entirely exploited, many strategies could improve the immunogenicity of these vaccines.

Maturation profiles of peripheral blood dendritic cells in patients with endogenous uveitis

Tu1651 Increased Activation of Dendritic Cells Contributes to T-Cell Mediated Colitis but Not Gastritis in DC-TGFBR2 Ko Mice

The immune stimulating ability of mannosylated liposomes containing FITC-ovalbumin as a model antigen and displaying either a branched tri-mannose or a mono-mannose ligand on the liposome surface was investigated in human monocyte-derived dendritic cells (MoDCs) and murine bone-marrow-derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8(+) T cells from OT-I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri-mannose ligand was enhanced in human MoDCs compared with both non-mannosylated liposomes and liposomes displaying mono-mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri-mannose- nor mono-mannose-containing liposomes were taken up by murine BMDCs to a greater extent than non-mannose-containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated liposomes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8(+) T cells to any greater extent than BMDCs incubated with non-mannosylated liposomes. These findings suggest that while mannose-containing ligands can enhance the uptake of antigen-containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate-binding receptors for mannose-containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.