Semi-mature Phenotype Research Articles

TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells

Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M.scrofulaceum and we analyzed the effect on the activation of DCs. We found that M.scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M.scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M.scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M.scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M.scrofulaceum.

Immunomodulatory Effects of 1,25-Dihydroxyvitamin D3 on Dendritic Cells Promote Induction of T Cell Hyporesponsiveness to Myelin-Derived Antigens.

While emerging evidence indicates that dendritic cells (DC) play a central role in the pathogenesis of multiple sclerosis (MS), their modulation with immunoregulatory agents provides prospect as disease-modifying therapy. Our observations reveal that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment of monocyte-derived DC results in a semimature phenotype and anti-inflammatory cytokine profile as compared to conventional DC, in both healthy controls and MS patients. Importantly, 1,25(OH)2D3-treated DC induce T cell hyporesponsiveness, as demonstrated in an allogeneic mixed leukocyte reaction. Next, following a freeze-thaw cycle, 1,25(OH)2D3-treated immature DC could be recovered with a 78% yield and 75% viability. Cryopreservation did not affect the expression of membrane markers by 1,25(OH)2D3-treated DC nor their capacity to induce T cell hyporesponsiveness. In addition, the T cell hyporesponsiveness induced by 1,25(OH)2D3-treated DC is antigen-specific and robust since T cells retain their capacity to respond to an unrelated antigen and do not reactivate upon rechallenge with fully mature conventional DC, respectively. These observations underline the clinical potential of tolerogenic DC (tolDC) to correct the immunological imbalance in MS. Furthermore, the feasibility to cryopreserve highly potent tolDC will, ultimately, contribute to the large-scale production and the widely applicable use of tolDC.

Tolerogenic Dendritic Cells Generated with Tofacitinib Ameliorate Experimental Autoimmune Encephalomyelitis through Modulation of Th17/Treg Balance.

It is well known that dendritic cells (DCs) play a pivotal role in triggering self-specific responses. Conversely, tolerogenic DCs (tolDCs), a specialized subset, induce tolerance and negatively regulate autoreactive responses. Tofacitinib, a Janus kinase inhibitor developed by Pfizer for treatment of rheumatoid arthritis, is probable to be a promising candidate for inducing tolDCs. The aims of this study were to evaluate the effectiveness of tolDCs induced by tofacitinib in a myelin oligodendrocyte glycoprotein- (MOG-) specific experimental autoimmune encephalomyelitis (EAE) model and to investigate their effects on Th17/Treg balance in the animal model of multiple sclerosis (MS). Our results revealed that tofacitinib-treated DCs maintained a steady semimature phenotype with a low level of proinflammatory cytokines and costimulatory molecules. DCs treated by tofacitinib also induced antigen-specific T cells hyporesponsiveness in a concentration-dependent manner. Upon intravenous injection into EAE mice, MOG pulsed tolDCs significantly dampened disease activity, and adoptive cell therapy (ACT) disturbed Th17/Treg balance with a remarkable decrease of Th1/Th17 cells and an increase in regulatory T cells (Tregs). Overall, DCs modified by tofacitinib exhibited a typical tolerogenic phenotype, and the antigen-specific tolDCs may represent a new avenue of research for the development of future clinical treatments for MS.

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

Gene Expression Profiling Implicates Attenuation of NFkB Signalling By Regulatory T Cells in Modulating Dendritic Cell Function

Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays.Immature, mature and Treg-treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC, respectively) and moDC populations were generated using a well-established 6 day culture with GM-CSF and IL-4, followed by 24 hour LPS maturation. Treg were added on day 3 of culture at a 3:1 ratio. All cell populations were harvested on day 7 and sorted via FSC/SSC/CD3neg gating to remove Treg present in the co-culture and control for any changes in gene expression caused by shear stress. Gene expression profiling was carried out using the Illumina HumanHT12 microarray platform. Data was processed using R/Bioconductor workflows and the functional significance of differentially expressed genes was evaluated using Ingenuity Pathway Analysis software.Mat-DC and Treg-DC expression profiles were compared relative to the im-DC for data analysis. Upon LPS treatment, the levels of 1834 unique genes were differentially regulated in mat-DC by at least twofold (862 genes upregulated/972 downregulated) compared to the im-DC counterparts. In the Treg-DC, 1326 unique genes were differentially modulated (633 genes upregulated/693 genes downregulated). Microarray analysis of the CD markers identified a higher expression of the previously identified surface markers CD80, CD83 and CD86 in the mat-DC compared to the Treg-treated counterpart (validated by flow cytometry), confirming the semi-mature phenotype.Novel findings from the dataset include the reduction of the endocytotic-related genes, CD206 and CD209, in the Treg-DCs compared to the im-DC and this reduction manifested functionally in an impaired antigen uptake, as assessed by FITC-Dextran. Additionally, the surface marker, CD38, was downregulated in the Treg-DC compared to the mat-DC, confirmed by flow cytometry. CD38 has been shown to be NFκB-dependent and a marker of maturation in monocyte-derived DCs, further supporting the semi-mature phenotype. Furthermore, CD38 is functionally involved in CD83 expression and IL-12 induction. We assessed IL-12 cytokine secretion by Treg-treated DCs and showed a significantly reduced level of induction compared to mat-DC (p=0.0079).Pathway analysis revealed NFκB-related genes to be downregulated in the Treg-DC compared to the mat-DC. These differentially expressed genes included the TLR-adaptor protein, MYD88, the NFκB subunit, RELB and an inhibitor of NFκB, NFκB1A. This finding, coupled to the importance of NFκB signalling pathway in DC function, prompted us to investigate it at the functional level by measuring levels of phosphorylation of serine 536 of the RelA subunit as a marker of activity in response to LPS stimulation. DC cultured in the absence of Tregs (mat-DC) showed significantly higher levels of Ser536 phosphorylation when compared to those unstimulated cells (im-DC) (p= 0.0018). Concordant with the gene expression data, Treg-treated DCs (Treg-DC), showed a significantly attenuated NFκB activation when compared to their LPS-stimulated DCs counterparts (p = 0.0191), however, signalling was not completely abolished compared to those unstimulated DCs (p= 0.0003).In conclusion, gene expression profiles of Treg-treated DCs are significantly different to their mat-DC and im-DC counterparts. Here, we present the novel finding that Tregs modulate DC function, in part, by attenuation of the NFkB signalling pathway, arresting the DCs at a semi-mature phenotype, as evidenced by expression arrays and functional assays. DisclosuresNo relevant conflicts of interest to declare.

ID: 173: IκBζ mediates suppression of secretion of pro-inflammatory cytokines in dendritic cells

Maintaining intestinal homeostasis is essential to maintain a balanced immune response in order to fight intestinal intruders but not to overreact towards commensals and nutritional antigens. Dendritic cells (DC) play an important role in balancing the intestinal immune responses as they are among the first antigen presenting cells encountering bacteria at mucosal surfaces. Upon antigen uptake immature DC usually become mature, secrete high amounts of pro-inflammatory cytokines and promote T cell activation. If the intestinal microbiota contains high amounts of commensal Bacteroides vulgatus, DCs become semi-mature and prevent from TH1/TH17-triggered inflammatory responses in animal models, therefore contributing to homeostasis. Compared to mature DCs, smDCs only express very low amounts of pro-inflammatory cytokines like IL-6, IL-12p70, TNFa and IL-1β, that do not affect the activation status of T cells, with the exception of IL-6. Importantly, smDCs encountering a bacterial stimulus that usually induces complete maturation in DCs, do not develop into to mature DCs. This non-responsiveness of smDCs in terms of cytokine secretion is mainly mediated by the host nuclear protein IκBζ. IκBζ expression is caused by TLR induced NFκB transactivation and forms a NFκB feedback inhibition. We suppose that IκBζ expression is also induced by IL-6 binding to the IL-6 receptor via an auto- and paracrine loops. This IL-6 induced IκBζ expression in smDCs, not providing enhanced NFκB transactivation, leads to an inhibition of NFκB activated pro-inflammatory cytokines and therefore promotes the induction of the described non-responsive semi-mature DC phenotypes that helps maintaining the intestinal homeostasis.

ID: 178: Outer membrane vesicles derived from B. vulgatus mpk as a acellular therapeutical agent for treatment of inflammatory disorders

Outer membrane vesicles (OMVs) are produced by many pathogenic and non-pathogenic bacteria through ubiquitous blebbing of the outer membrane. These vesicles consist of lipids, proteins, lipopolysaccharides and other luminal molecules. Supplementary to the roles in pathogenesis, cell-to-cell communication and stress response, recent work has suggested that OMVs play important roles in immunomodulation and the establishment of a balanced gut microbiota for maintaining the intestinal homeostasis. We focus on the influences of OMVs derived from the symbiotic gram-negative B. vulgatus mpk on the innate immune system. These vesicles show anti-inflammatory functions resulting in inhibition of T cell proliferation. Responsible for this effect is the induction of semi-mature dendritic cells in the host, characterised by an intermediate MHC-class-II surface expression, low secretion of the proinflammatory cytokines TNFα, IL-1s and IL-12 with simultaneous remarkable production of IL-6. Stimulation of wt, TLR2−/−, TLR4−/− and TLR2−/−x TLR4−/− DCs indicate the crucial role of different Pathogen-associated molecular patterns (PAMPs) of the bacterial membrane for the development of this non-responsive semi-mature phenotype. In addition to bacterial LPS, binding to host TLR4, a yet unknown compound to host TLR2 is important for the production of this phenotypical cytokine profile. We propose that outer membrane vesicles derived from commensal B. vulgatus mpk are promising drugs for therapeutical treatment of systemic diseases like e.g. colitis, type-I-diabetes or multiple sclerosis. By the induction of semi-mature dendritic cells, OMVs have the ability to modulate the cytokine secretion in the gastrointestinal tract in order to maintaining permanent anti-inflammatory conditions.

Jagged-1 is required for the expansion of CD4+ CD25+ FoxP3+ regulatory T cells and tolerogenic dendritic cells by murine mesenchymal stromal cells.

IntroductionMesenchymal stromal cells (MSC) have well defined immunomodulatory properties including the suppression of lymphocyte proliferation and inhibition of dendritic cell (DC) maturation involving both cell contact and soluble factors. These properties have made MSC attractive candidates for cellular therapy. However, the mechanism underlying these characteristics remains unclear. This study sought to investigate the mechanisms by which MSC induce a regulatory environment.MethodAllogeneic bone marrow mesenchymal stromal cells were cultured with T cells or dendritic cells in the presence or absence of gamma secretase inhibitor to block Notch receptor signalling. T cells and dendritic cells were examined by flow cytometry for changes in phenotype marker expression. Stable knock down MSC were generated to examine the influence of Jagged 1 signalling by MSC. Both wildtype and knockdown MSC were subsequently used in vivo in an animal model of allergic airway inflammation.ResultsThe Notch ligand Jagged-1 was demonstrated to be involved in MSC expansion of regulatory T cells (Treg). Additionally, MSC-induced a functional semi-mature DC phenotype, which further required Notch signalling for the expansion of Treg. MSC, but not Jagged-1 knock down MSC, reduced pathology in a mouse model of allergic airway inflammation. Protection mediated by MSC was associated with enhanced Treg in the lung and significantly increased production of interleukin (IL)-10 in splenocytes re-stimulated with allergen. Significantly less Treg and IL-10 was observed in mice treated with Jagged-1 knock down MSC.ConclusionsThe current study suggests that MSC-mediated immune modulation involves the education and expansion of regulatory immune cells in a Jagged-1 dependent manner and provides the first report of the importance of Jagged-1 signalling in MSC protection against inflammation in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0021-5) contains supplementary material, which is available to authorized users.

Rapamycin-conditioned dendritic cells activated with monophosphoryl lipid-A promote allograft acceptance in vivo.

To date, there is no human dendritic cell (DC) based therapy to prevent allograft rejection in transplanted patients. Here, we evaluate a potential protocol using a murine in vivo transplant model. We generated murine bone marrow-derived DCs (BM-DCs), modulated with rapamycin (Rapa) and activated with monophosphoryl lipid A (Rapamycin-treated and monophosphoryl lipid A-matured DCs [Rapa-mDCs]). DCs phenotype was evaluated by flow cytometry, cytokine production by ELISA and their T-cell stimulatory ability was tested in co-cultures with CD4(+) T cells. Using an in vivo skin graft model, we evaluated DCs tolerogenicity. In vitro, Rapa-mDCs exhibit a semi-mature phenotype given by intermediate levels of co-stimulatory molecules and cytokines, and inhibit CD4(+) T-cell proliferation. In vivo, skin-grafted mice treated with Rapa-mDCs show high allograft survival, accumulation of Foxp3(+) Tregs and cytokine pattern modification. Rapa-mDCs re-educate the inflammatory microenvironment, promoting skin-allograft survival.

Inhibins regulate peripheral Treg induction through modulation of dendritic cell function.

Event Abstract Back to Event Inhibins regulate peripheral Treg induction through modulation of dendritic cell function. SANDRA ORTEGA1*, Marisol De La Fuente Granada1, Roxana Olguín Alor1, Jesús Ramsés Chávez-Ríos1, Laura Bonifaz Alfonso2, Eduardo A. García-Zepeda1 and Gloria Soldevila Melgarejo1 1 UNAM, Depto de Inmunología, Instituto de Investigaciones Biomédicas, Mexico 2 Centro Médico Nacional Siglo XXI, IMSS, Mexico Inhibins and Activins, members of the Transforming Growth Factorb superfamily, participate in the immune system as key regulators of several cellular functions. Despite some reports indicate that Activins may regulate DC maturation and function, the role of Inhibins in these processes remains elusive. Here, we investigated the role of Inhibins in DC maturation and function and its impact in Treg induction and Th effector differentiation. Analysis of mature Inhα-/- BMDCs obtained after lipopolysaccharide (LPS) stimulation (mBMDC) showed a significant reduction in the surface expression of MHC II and CD86 (a semi-mature phenotype), and a concomitant increase in PD-L1, compared to WT mBMDC. In addition, the Inhα-/- mBMDC showed reduced migration towards CCL19 and CCL21 both in vitro chemotaxis assays as well as and in vivo, in in vivo adoptive competitive transfer assays, in comparison to WT. To evaluate if these findings correlated with a “tolerogenic” phenotype, functional responses were assayed showing that mBMDC from Inhα-/- displayed reduced capacity to induce proliferation of allogeneic CD4+ naïve T cells. This correlated with an increased induction of iTregs (CD25+Foxp3+) from CD4+ naïve T cells in the presence of low concentrations of anti-CD3 and TGFb as well as with increased absolute numbers of iTregs and nTreg (CD4+CD25+Foxp3+Helios- and Helios+, respectively) in peripheral lymph nodes of 4 week-old Inhα-/- mice compared to WT. Interestingly, Inhα-/- T cells showed a reduced induction of Tregs in the absence of APCs, suggesting an intrinsic role of Inhibins in the differentiation of T cells. Analysis of Th effector differentiation is currently being performed using an OVA-specific transgenic mouse model. Our data demonstrate for the first time a direct role of Inhibins in the regulation of DC mediated functions and point out the importance of Inhibin signaling to maintain the balance between inflammation and tolerance. Acknowledgements Work supported by PAPIIT, DGAPA, UNAM (Grant IN209615). Keywords: Inhibins, T cells diferentiation, Dendritic cells function, T regs induction, Dendritic cells maturation Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Adaptive Immunity Citation: ORTEGA S, De La Fuente Granada M, Olguín Alor R, Chávez-Ríos J, Bonifaz Alfonso L, García-Zepeda E and Soldevila Melgarejo G (2015). Inhibins regulate peripheral Treg induction through modulation of dendritic cell function.. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00374 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 May 2015; Published Online: 16 Sep 2015. * Correspondence: Ms. SANDRA ORTEGA, UNAM, Depto de Inmunología, Instituto de Investigaciones Biomédicas, México, D.F., Mexico, amyrisortega@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers SANDRA ORTEGA Marisol De La Fuente Granada Roxana Olguín Alor Jesús Ramsés Chávez-Ríos Laura Bonifaz Alfonso Eduardo A. García-Zepeda Gloria Soldevila Melgarejo Google SANDRA ORTEGA Marisol De La Fuente Granada Roxana Olguín Alor Jesús Ramsés Chávez-Ríos Laura Bonifaz Alfonso Eduardo A. García-Zepeda Gloria Soldevila Melgarejo Google Scholar SANDRA ORTEGA Marisol De La Fuente Granada Roxana Olguín Alor Jesús Ramsés Chávez-Ríos Laura Bonifaz Alfonso Eduardo A. García-Zepeda Gloria Soldevila Melgarejo PubMed SANDRA ORTEGA Marisol De La Fuente Granada Roxana Olguín Alor Jesús Ramsés Chávez-Ríos Laura Bonifaz Alfonso Eduardo A. García-Zepeda Gloria Soldevila Melgarejo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1.

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

Tumor-infiltrating dendritic cells exhibit defective cross-presentation of tumor antigens, but is reversed by chemotherapy.

Cross-presentation defines the unique capacity of an APC to present exogenous Ag via MHC class I molecules to CD8(+) T cells. DCs are specialized cross-presenting cells and as such have a critical role in antitumor immunity. DCs are routinely found within the tumor microenvironment, but their capacity for endogenous or therapeutically enhanced cross-presentation is not well characterized. In this study, we examined the tumor and lymph node DC cross-presentation of a nominal marker tumor Ag, HA, expressed by the murine mesothelioma tumor AB1-HA. We found that tumors were infiltrated by predominantly CD11b(+) DCs with a semimature phenotype that could not cross-present tumor Ag, and therefore, were unable to induce tumor-specific T-cell activation or proliferation. Although tumor-infiltrating DCs were able to take up, process, and cross-present exogenous cell-bound and soluble Ags, this was significantly impaired relative to lymph node DCs. Importantly, however, systemic chemotherapy using gemcitabine reversed the defect in Ag cross-presentation of tumor DCs. These data demonstrate that DC cross-presentation within the tumor microenvironment is defective, but can be reversed by chemotherapy. These results have important implications for anticancer therapy, particularly regarding the use of immunotherapy in conjunction with cytotoxic chemotherapy.

Treg Induced Impairment of DC Function and Its Impact on GvH Reactions

Promising results from murine models and early stage clinical trials have shown that adoptive transfer of regulatory T cells (Treg) prevents graft-versus-host disease (GvHD). However, the primary target of Treg mediated protection against GvHD is yet to be fully defined. We have previously shown that the presence of Treg during effector T cell priming is able to ameliorate cutaneous GvH reactions in vitro by blocking effector cell migration. This has led to the hypothesis that Treg modulation of dendritic cells (DC) could be a key mechanism by which Treg exert their protective role in GvHD. DC are fundamental for the initiation of allo-reactive immune responses and are critical in GvHD pathogenesis. We investigated the effect of Treg on the phenotypic profile and allo-reactive functions of DCs. Furthermore, the impact of Treg treatment on the ability of DCs to induce GvH target tissue damage was examined for the first time using an in vitro human GvHD skin explant model.Immature, mature and Treg treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC respectively). The three moDC populations were generated using the well-established 6 day culture with GM-CSF and IL-4 followed by 24 hour LPS maturation. Treg were added on day 3 of moDC culture. Im-DC, mat-DC and Treg-DC were harvested on day 7 and tested in parallel as stated below. Prior to functional assays Treg-moDC were isolated by FACS sorting via FSC/SSC/CD3neg gating to remove Treg present in the co-culture.Our results revealed that Treg-DC displayed a semi-mature phenotype with CD83, CD80 and CD86 expression significantly lower than mat-moDC (p<0.005) but significantly higher than im-moDC (P<0.05) whilst HLA-DR levels were comparable to mat-moDC but significantly higher than imDC (p<0.005). Treg-DC also expressed CCR7 comparable to that of im-DC but markedly higher than mat-DC (p=0.052). Distinct morphology of Treg-DC, defined by Giemsa staining, corroborated their semi-mature status. Data from FITC-dextran uptake showed a significant reduction in antigen-capture capacity of Treg treated im-DC compared to untreated im-DC (p=0.047). The Treg mediated functional impairment of DCs was also associated with significantly higher expression of LAP-TGFβ1 on Treg-DC when compared to that of mat-DC (P<0.05). Treg-DC were also markedly defective in stimulating activation and proliferation of allo-reactive CD8 T cells, detected by CD25 expression and CFSE dilution respectively (p=0.009, p=0.046). Interestingly the presence of Treg throughout the entire allo-response induction resulted in a more potent reduction in activation and proliferation than when only the DC were treated with Treg (p=0.009, p=0.0085). Furthermore, allo-reactive CD8 T cells primed with Treg treated moDC were less able to mediate cutaneous GvH tissue damage (Figure 1).In conclusion, attenuation of DC signature and function is key for Treg mediated protection against GvHD. However, isolated DC modulation by Treg was less effective in suppressing CD8 T cell allo-responses compared to the continued presence of Treg during the CD8 T cell priming, activation and proliferation, suggesting that Treg exert most effective function via multidimensional modulation on the DC, T cells and DC-T cell interactions simultaneously. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alternatively activated dendritic cells derived from systemic lupus erythematosus patients have tolerogenic phenotype and function

Tolerogenic dendritic cells (DCs) are potential cell-based therapy in autoimmune diseases. In this study, we generated alternatively activated DCs (aaDCs) by treating monocyte-derived DCs from patients with systemic lupus erythematosus (SLE) and healthy subjects with combination of 1,25 dihydroxyvitamin D(3) (vitD3) and dexamethasone followed by lipopolysaccharide-induced maturation. Lupus aaDCs were found to acquire semi-mature phenotype that remained maturation-resistant to immunostimulants. They produced low level of IL-12 but high level of IL-10. They had attenuated allostimulatory effects on T cell activation and proliferation comparable to normal aaDCs and demonstrated differential immunomodulatory effects on naïve and memory T cells. These aaDCs were capable of inducing IL-10 producing regulatory T effectors from naïve T cells whereas they modulated cytokine profile with suppressed production of IFN-γ and IL-17 by co-cultured memory T cells with attenuated proliferation. These aaDCs were shown to be superior to those generated using vitD3 alone in lupus patients.

Interferon alpha induces generation of semi-mature dendritic cells with high pro-inflammatory and cytotoxic potential

Dendritic cell-based vaccines are considered as a new and promising immunotherapeutic approach for cancer treatment. However, the choice of optimal protocol of dendritic cell generation in vitro represents the major challenge. Here, we compared phenotype and functional characteristics of human monocyte-derived dendritic cells (DCs) generated in the presence of IL-4/GM-CSF (IL4-DCs) and IFNα/GM-CSF (IFN-DCs). We showed that IFN-DCs displayed semi-mature phenotype and expressed higher level of CD123, TNF-related apoptosis-inducing ligand (TRAIL) and B7-H1 molecules in comparison with IL4-DCs. LPS-stimulated IFN-DCs were characterized by greater production of Th1/pro-inflammatory (IFN-γ, IL-2, IL-1β, TNF-α, IL-17), Тh2/anti-inflammatory cytokines (IL-10, IL-5), hematopoietic growth factors (G-CSF) and chemokines (MCP-1). These data indicated more pronounced ability of IFN-DCs to induce cellular immune response as well as humoral immune response compared to IL4-DCs. LPS-stimulated IFN-DCs possessed higher direct cytotoxic activity against TRAIL-sensitive tumor cell line Jurkat and similar cytotoxicity against TRAIL-resistant tumor HEp-2 cells. Besides, IFN-DCs and IL4-DCs equally induced apoptosis of activated CD4+ and CD8+ T cells. These results suggest that IFN-DCs can be used as potent cell-based curative therapies for individuals with cancer.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.

Adrenomedullin protects from experimental autoimmune encephalomyelitis at multiple levels.

Adrenomedullin is a neuropeptide known for its cardiovascular activities and anti-inflammatory effects. Here, we investigated the effect of adrenomedullin in a model of experimental autoimmune encephalomyelitis (EAE) that mirrors chronic progressive multiple sclerosis. A short-term systemic treatment with adrenomedullin reduced clinical severity and incidence of EAE, the appearance of inflammatory infiltrates in spinal cord and the subsequent demyelination and axonal damage. This effect was exerted at multiple levels affecting both early and late events of the disease. Adrenomedullin decreased the presence/activation of encephalitogenic Th1 and Th17 cells and down-regulated several inflammatory mediators in peripheral lymphoid organs and central nervous system. Noteworthy, adrenomedullin inhibited the production by encephalitogenic cells of osteopontin and of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), two critical cytokines in the development of EAE. At the same time, adrenomedullin increased the number of IL-10-producing regulatory T cells with suppressive effects on the progression of EAE. Furthermore, adrenomedullin generated dendritic cells with a semi-mature phenotype that impaired encephalitogenic responses in vitro and in vivo. Finally, adrenomedullin regulated glial activity and favored an active program of neuroprotection/regeneration. Therefore, the use of adrenomedullin emerges as a novel multimodal therapeutic approach to treat chronic progressive multiple sclerosis.

Cladribine exerts an immunomodulatory effect on human and murine dendritic cells

Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.

The effect of cyclic mechanical strain on activation of dendritic cells cultured on adhesive substrates

Dendritic cells (DCs), key regulators of tolerance and immunity, have been found to reside in mechanically active tissues such as the interior layers of the arterial wall, which experience cyclic radial wall strain due to pulsatile blood flow. Although experimentally difficult to determine in vivo, it is reasonable to postulate DCs experience the mechanical forces in such mechanically active tissues. However, it is currently unknown how DCs respond to cyclic mechanical strain. In order to explore the hypothesis that DCs are responsive to mechanical strain, DCs were cultured in vitro on pre-adsorbed adhesive proteins (e.g., laminin, collagen, fibrinogen) and 1 Hz cyclic strain was applied for various durations and strain magnitudes. It was determined that a strain magnitude of 10% and 24 h duration adversely affected DC viability compared to no-strain controls, but culture on certain adhesive substrates provided modest protection of viability under this harsh strain regime. In contrast, application of 1 h of 1 Hz cyclic 3% strain did not affect DC viability and this strain regime was used for the remaining experiments for quantifying DC activation and T-cell priming capability. Application of 3% strain increased expression of stimulatory (MHC-II) and costimulatory molecules (CD86, CD40), and this effect was generally increased by culture on pre-coated adhesive substrates. Interestingly, the cytokine secretion profile of DCs was not significantly affected by strain. Lastly, strained DCs demonstrated increased stimulation of allogeneic T-cell proliferation, in a manner that was independent of the adhesive substrate. These observations indicate generation of a DC consistent with what has been described as a semi-mature phenotype. This work begins elucidating a potential role for DCs in tissue environments exposed to cyclic mechanical forces.

Protein Kinase C Inhibitor Generates Stable Human Tolerogenic Dendritic Cells

Tolerogenic dendritic cells (DCs) are a promising tool for a specific form of cellular therapy whereby immunological tolerance can be induced in the context of transplantation and autoimmunity. From libraries of bioactive lipids, nuclear receptor ligands, and kinase inhibitors, we screened conventional protein kinase C inhibitors (PKCIs) bisindolylmaleimide I, Gö6983, and Ro32-0432 with strong tolerogenic potential. PKCI-treated human DCs were generated by subjecting them to a maturation process after differentiation of immature DCs. The PKCI-treated DCs had a semimature phenotype, showing high production of IL-10, and efficiently induced IL-10-producing T cells and functional Foxp3(+) regulatory T cells from naive CD4(+) T cells, thus eliciting a strong immunosuppressive function. They also showed CCR7 expression and sufficient capacity for migration toward CCR7 ligands. Additionally, PKCI-treated DCs were highly stable when exposed to inflammatory stimuli such as proinflammatory cytokines or LPS. Conventional PKCIs inhibited NF-κB activation of both the canonical and noncanonical pathways of DC maturation, thus suppressing the expression of costimulatory molecules and IL-12 production. High production of IL-10 in PKCI-treated DCs was due to not only an increase of intracellular cAMP, but also a synergistic effect of increased cAMP and NF-κB inhibition. Moreover, PKCI-treated mouse DCs that had properties similar to PKCI-treated human DCs prevented graft-versus-host disease in a murine model of acute graft-versus-host disease. Conventional PKCI-treated DCs may be useful for tolerance-inducing therapy, as they satisfy the required functional characteristics for clinical-grade tolerogenic DCs.