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1.  Flt3 Ligand Generates Morphologically Distinct Semimature Dendritic Cells in Ovalbumin-sensitized Mice 
Dendritic cells (DCs) are unique antigen presenting cells that are immature prior to their encounter with an antigen. Exposure to allergens induces the maturation of DCs with changes in morphology and presence of dendrites. Here, we demonstrate that the DCs in the lungs of ovalbumin (OVA)-sensitized and challenged mice are more mature owing to their pronounced dendrites than the DCs in the lungs and spleen of PBS-treated mice, which are immature and possess cytoplasmic veils. Intermediate to these two groups are the DCs in the Flt3 ligand-treated group that exhibit comparatively fewer dendrites and cytoplasmic veils and hence are classified as semimature. Presence of large numbers of well developed mitochondria and rough endoplasmic reticulum in myeloid DCs from both lungs and spleen of OVA-sensitized and challenged mice indicate greater functional activity. Additionally, DCs from the OVA-sensitized and challenged mice also exhibit fat and glycogen stores, which are indicative of a mature population. In addition, treatment of the animals with Flt3 ligand attenuated airway hyperresponsiveness to methacholine in OVA-sensitized and challenged mice. These data suggest that morphological features could be indicative of the maturation and distinct functional state of DCs, and this could be associated with underlying mechanisms of Flt3 ligand-induced immunomodulation in allergic asthma.
PMCID: PMC2745173  PMID: 17182033
Airway hyperresponsiveness; Allergy; Asthma; Cytoplasmic veils; Dendritic cells; Flt3 ligand; Immunomodulation; Morphology of dendritic cells
2.  Negative Selection in the Thymus Includes Semimature T Cells 
The thymic medulla plays a key role in negative selection (self-tolerance induction) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+8− thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells reaching the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullary T cells displays unique requirements for tolerance induction; depending upon the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.
PMCID: PMC2196120  PMID: 9016875
3.  Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassical Macrophage Activation  
Infection and Immunity  2005;73(1):308-316.
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-κB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.
PMCID: PMC538969  PMID: 15618167
4.  In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates 
The Journal of Experimental Medicine  2011;208(12):2477-2488.
Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys.
Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.
PMCID: PMC3256968  PMID: 22025302
5.  Amplification of Autoimmune Response through Induction of Dendritic Cell Maturation in Inflamed Tissues1 
Dendritic cells (DCs) are essential in T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans in type 1 diabetes. In this study, we investigated T cell induction of intra-islet DC maturation during the progression of the disease in both autoimmune-prone NOD and resistant C57BL/6 mice. We demonstrated steady-state capture and retention of unprocessed β cell-derived proteins by semimature intra-islet DCs in both mouse strains. T cell-mediated intra-islet inflammation induced an increase in CD40 and CD80 expression and processing of captured Ag by resident DCs without inducing the expression of the p40 subunit of IL-12/23. Some of the CD40high intra-islet DCs up-regulated CCR7, and a small number of CD40high DCs bearing unprocessed islet Ags were detected in the pancreatic lymph nodes in mice with acute intra-islet inflammation, demonstrating that T cell-mediated tissue inflammation augments migration of mature resident DCs to draining lymph nodes. Our results identify an amplification loop during the progression of autoimmune diabetes, in which initial T cell infiltration leads to rapid maturation of intra-islet DCs, their migration to lymph nodes, and expanded priming of more autoreactive T cells. Therapeutic interventions that intercept this process may be effective at halting the progression of type 1 diabetes.
PMCID: PMC3057894  PMID: 19234153
6.  Systemic Administration of Tolerogenic Dendritic Cells Ameliorates Murine Inflammatory Arthritis 
The expression of various cell surface molecules and the production of certain cytokines are important mechanisms by which dendritic cells (DC) are able to bias immune responses. This paper describes the effects of the inflammatory cytokine tumor necrosis factor (TNF)-α on DC phenotype and function. TNF-α treatment resulted in upregulation of MHC class II and CD86 in the absence of increased cell surface CD40 and CD80 or the production of IL-12. Additionally TNF-α treated cells were able to bias T cell responses towards an anti-inflammatory profile. On a note of caution this tolerogenic phenotype of the DC was not stable upon subsequent TLR-4 ligation as a 4 hour pulse of the TNF-α treated DC with lipopolysaccharide (LPS) resulted in the restoration of IL-12 production and an enhancement of their T cell stimulatory capacity which resulted in an increased IFN-γ production. However, TNF-α treated DC, when administered in vivo, were shown to ameliorate disease in collagen induced arthritis, an experimental model of inflammatory joint disease. Mice receiving TNF-α treated DC but not LPS matured DC had a delayed onset, and significantly reduced severity, of arthritis. Disease suppression was associated with reduced levels of collagen specific IgG2a and decreased inflammatory cell infiltration into affected joints. In summary the treatment of DC with TNF-α generates an antigen presenting cell with a phenotype that can reduce the pro-inflammatory response and direct the immune system towards a disease modifying, anti-inflammatory state.
PMCID: PMC2627532  PMID: 19156221
7.  TLR9, TACI and CD40 synergize in causing B cell activation 
B cells receive activating signals from T cells via CD40, from microbial DNA via TLR9, and from dendritic cells (DCs) via transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). TLR9 and CD40 ligation augment TACI driven B cell activation, but only the mechanism of synergy between CD40 and TACI has been explored. Synergy between CD40 and TLR9 in B cell activation is controversial.
To examine the mechanisms by which TLR9 modulates CD40- and TACI-mediated activation of B cells, and to determine whether all three receptors synergize to activate B cells.
Naïve mouse B cells and human peripheral blood mononuclear cells (PBMCs) were stimulated with combinations of anti-CD40, CpG and APRIL in the presence of IL-4. Proliferation was measured by 3H-thymidine incorporation. Immuniglobulin production was measured by ELISA. Class switch recombination (CSR) was examined by measuring mRNA for germ line transcripts, activation-induced cytidine deaminase (AICDA) and mature Ig transcripts. Plasma cell differentiation was examined by syndecan-1/CD138 staining and mRNA expression of B lymphocyte-induced maturation protein 1 (Blimp-1).
TLR9 synergized with CD40 and TACI in driving CSR and inducing IgG1 and IgE secretion by naïve murine B cells, and synergized with TACI in driving B cell proliferation and plasma cell differentiation. All three receptors synergized together in driving mouse B cell proliferation, CSR, plasma cell differentiation and IgG1 and IgE secretion. TLR9 synergized with CD40 and TACI in driving IgG secretion in IL-4 stimulated human B cells.
Signals from TLR9, TACI and CD40 are integrated to promote B cell activation and differentiation.
Clinical Implications
Stimulation of B cells via CD40, TLR9 and TACI results in their optimal activation. Defects in these pathways may co-operate to impair B cell immunoglobulin production in patients with common variable immunodeficiency (CVID).
PMCID: PMC3164940  PMID: 21741080
TLR9; CD40; TACI; CSR; plasma cell; immunoglobulin; B cells
8.  TREM-2, triggering receptor expressed on myeloid cell-2, negatively regulates TLR responses in dendritic cells 
European journal of immunology  2011;42(1):176-185.
Dendritic cells (DCs) play a key role in defense against infections and also in preventing inflammatory and autoimmune diseases. The response of DCs to pathogens is tightly regulated by many mechanisms to allow for appropriate, but not pathogenic, responses. We previously showed that DCs with deficiencies for two ITAM-bearing signaling adapters, DAP12 and FcRγ, produce more inflammatory cytokines upon treatment with Toll-like receptor (TLR) agonists than wild-type (WT) DCs. Here, we investigated whether the TREM-2 receptor pairs with DAP12 to inhibit TLR responses in DCs. TREM-2-deficient BMDCs showed increased inflammatory cytokine and type 1 IFN production in response to TLR ligation. Additionally, TREM-2-deficient BMDCs had increased TLR-induced maturation and were more efficient at inducing antigen-specific T cell-proliferation upon CpG DNA stimulation compared with WT BMDCs. Finally, we showed that a TREM-2 ligand is expressed on the surface of BMDCs, suggesting that the TREM-2 receptor transduces inhibitory signals due to recognition of an endogenous ligand.
PMCID: PMC3444819  PMID: 21956652
TREM-2; TLR; dendritic cell; DAP12; ITAM signaling
9.  Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR 
PLoS ONE  2012;7(2):e31632.
The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR−/− mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR−/− mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.
PMCID: PMC3273462  PMID: 22328941
10.  Tim-1 Signaling Substitutes for Conventional Signal 1 and Requires Costimulation to Induce T Cell Proliferation1 
Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4+ T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1+ T cells and Tim-4+ dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.
PMCID: PMC3767978  PMID: 19155484
11.  Immuno-modulatory activity of Ganoderma lucidum-derived polysacharide on human monocytoid dendritic cells pulsed with Der p 1 allergen 
BMC Immunology  2011;12:31.
Ganoderma lucidum-derived polysaccharide (PS-G) can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs), suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs) from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10) were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile.
PS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1) cell cytokine (INF-γ) production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone.
The presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.
PMCID: PMC3127845  PMID: 21612588
dendritic cells; Th1/Th2 cells; PS-G; asthma
12.  Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells 
Journal of Leukocyte Biology  2008;84(1):124-133.
Promising immunotherapeutic tools for T cell-mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of naïve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1α,25-dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by naïve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Naïve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN-γ/high IL-10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL-2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL-12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in naïve T cells, indicating that the lack of IL-12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate naïve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.
PMCID: PMC2504714  PMID: 18430785
tolerance; cytokine deviation; hyporesponsiveness; immunotherapy
13.  TACI drives plasma cell differentiation in LPS activated B cells 
TACI expression on B cells is upregulated by TLR4.
To examine whether TACI synergizes with TLR4 in driving immunoglobulin (Ig) production by B cells and to examine the mechanism of this synergy.
Purified mouse naïve B cells were stimulated with the TACI ligand APRIL and with suboptimal concentrations of the TLR4 ligand LPS in the presence or absence of IL-4. Ig secretion was measured by ELISA. Surface IgG1+ (sIgG1+) B cells and CD138+ plasmacytoid cells were enumerated by FACS. Expression of γ1 and ε germ line transcripts (GLT), activation-induced cytidine deaminase (AICDA) and γ1 and ε mature transcripts was measured by RT-PCR.
APRIL synergized with LPS in driving B cell proliferation and IgM, IgG1, IgG3, IgE and IgA production. This was mediated by TACI as it was preserved in BCMA-/-, but not TACI-/-, B cells. APRIL and LPS synergized to promote isotype switching as evidenced by increased expression of AICDA and γ1 and ε mature transcripts, and generation of sIgG1+ cells. More importantly, APRIL and LPS strongly synergized to drive the plasma cell differentiation program, as evidenced by increase in CD138+ cells and expression of Blimp-1, IRF-4 and the spliced form of XBP-1. TACI-/- mice had impaired IgM and IgG1 antibody responses to immunization with a suboptimal dose of the type I T independent antigen TNP-LPS.
These observations suggest that TACI cooperates with TLR4 to drive B cell differentiation and immunoglobulin production in vitro and in vivo.
Clinical Implications
Impaired synergy of TACI with TLRs may contribute to low serum immunoglobulin levels in CVID (Common Variable Immune Deficiency) patients with TACI mutations.
Capsule Summary
This work shows that TACI drives plasma cell differentiation in LPS stimulated cells and increases immunoglobulin production in vitro and in vivo.
PMCID: PMC2693462  PMID: 19427685
B cells; TLR; LPS; TACI; APRIL; immunoglobulin
14.  Innate Pathways to B cell Activation and Tolerance 
B cells represent an important link between the adaptive and innate immune systems, as they express both antigen-specific B cell receptors (BCRs) as well as various toll-like receptors (TLRs). Several checkpoints in B cell development ensure that self-specific cells are eliminated from the mature B cell repertoire to avoid harmful autoreactive responses. These checkpoints are controlled by BCR-mediated events, but are also influenced by TLR-dependent signals from the innate immune system. Additionally, B cell-intrinsic and extrinsic TLR signaling are critical for inflammatory events required for the clearance of microbial infections. Factors secreted by TLR-activated macrophages or dendritic cells directly influence the fate of protective and autoreactive B cells. Additionally, naïve and memory B cells respond differentially to TLR ligands, as do different B cell subsets. We review here recent literature describing intrinsic and extrinsic effects of TLR stimulation on the fate of B cells, with particular attention to autoimmune diseases.
PMCID: PMC3422021  PMID: 20146708
B cells; Toll-like receptors; TLRs; autoimmunity; autoreactive; tolerance; innate; SLE; Lupus; BAFF; IFN-I
15.  G-CSF–stimulated Neutrophils Are a Prominent Source of Functional BLyS 
B lymphocyte stimulator (BLyS) is a novel member of the TNF ligand superfamily that is important in B cell maturation and survival. We demonstrate that human neutrophils, after incubation with G-CSF or, less efficiently, IFNγ, express high levels of BLyS mRNA and release elevated amounts of biologically active BLyS. In contrast, surface expression of the membrane-bound BLyS was not detected in activated neutrophils. Indeed, in neutrophils, uniquely among other myeloid cells, soluble BLyS is processed intracellularly by a furin-type convertase. Worthy of note, the absolute capacity of G-CSF–stimulated neutrophils to release BLyS was similar to that of activated monocytes or dendritic cells, suggesting that neutrophils might represent an important source of BLyS. In this regard, we show that BLyS serum levels as well as neutrophil-associated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. In addition, serum obtained from two of these patients induced a remarkable accumulation of neutrophil-associated BLyS in vitro. This effect was neutralized by anti–G-CSF antibodies, indicating that G-CSF, present in the serum, stimulated neutrophils to produce BLyS. Collectively, our findings suggest that neutrophils, through the production of BLyS, might play an unsuspected role in the regulation of B cell homeostasis.
PMCID: PMC2193843  PMID: 12566413
IFNγ; inflammation; B lymphocytes; monocytes; dendritic cells
16.  TLR-4 ligation of dendritic cells is sufficient to drive pathogenic T cell function in experimental autoimmune encephalomyelitis 
Experimental autoimmune encephalomyelitis (EAE) depends on the initial activation of CD4+ T cells responsive to myelin autoantigens. The key antigen presenting cell (APC) population that drives the activation of naïve T cells most efficiently is the dendritic cell (DC). As such, we should be able to trigger EAE by transfer of DC that can present the relevant autoantigen(s). Despite some sporadic reports, however, models of DC-driven EAE have not been widely adopted. We sought to test the feasibility of this approach and whether activation of the DC by toll-like receptor (TLR)-4 ligation was a sufficient stimulus to drive EAE.
Host mice were seeded with myelin basic protein (MBP)-reactive CD4+ T cells and then were injected with DC that could present the relevant MBP peptide which had been exposed to lipopolysaccharide as a TLR-4 agonist. We found that this approach induced robust clinical signs of EAE.
DC are sufficient as APC to effectively drive the differentiation of naïve myelin-responsive T cells into autoaggressive effector T cells. TLR-4-stimulation can activate the DC sufficiently to deliver the signals required to drive the pathogenic function of the T cell. These models will allow the dissection of the molecular requirements of the initial DC-T cell interaction in the lymphoid organs that ultimately leads to autoimmune pathology in the central nervous system.
PMCID: PMC3520704  PMID: 23111144
Multiple sclerosis; Experimental autoimmune encephalomyelitis; Dendritic cells; Myelin basic protein
17.  Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists 
PLoS ONE  2010;5(7):e11491.
Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC.
Principal Findings
In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation.
We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.
PMCID: PMC2901334  PMID: 20634889
18.  Non-canonical Notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli1 
Dendritic cell (DC) derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DC perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli. We demonstrate that concurrent Notch and TLR stimulation results in a unique cytokine profile in mouse bone-marrow derived DC characterized by enhanced IL-10 and IL-2 and reduced IL-12 expression compared to TLR ligation alone. Unexpectedly, modulation of cytokine production occurred through a non-canonical Notch signaling pathway, independent of γ-secretase activity. Modulation required de novo protein synthesis and PI3K, JNK and ERK activity were necessary for enhanced IL-2 expression while modulation of IL-10 only required PI3K activity. Further, we show that this γ-secretase independent Notch pathway can induce PI3K activity. In contrast, expression of the canonical Notch target gene Hes1 was suppressed in DC stimulated with Notch and TLR ligands simultaneously. Thus, our data suggest that Notch acts as an endogenous signal that modulates cytokine expression of DC through a non-canonical pathway and therefore has the potential to tailor the subsequent adaptive immune response in a tissue and/or stage dependent manner.
PMCID: PMC3442230  PMID: 22753939
19.  Isoflavones, Genistein and Daidzein, Regulate Mucosal Immune Response by Suppressing Dendritic Cell Function 
PLoS ONE  2012;7(10):e47979.
Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both in vitro and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/− isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/− isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4+ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ+ CD4+ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4+ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.
PMCID: PMC3478285  PMID: 23110148
20.  Human Endometrial Epithelial Cells Cyclically Express Toll-Like Receptor 3 (TLR3) and Exhibit TLR3-Dependent Responses to dsRNA 
Human immunology  2005;66(5):469-482.
Toll-like receptor 3 (TLR3) responds to dsRNA, a product of most viral life cycles, and initiates production of proinflammatory and antiviral cytokines. The role of TLR3 in human mucosal immunity of the endometrium has not been examined. The effects of TLR3 ligation in endometrial epithelium could be significant as the endometrium is a significant site for viral entry and infection. Additionally, the cytokine milieu plays an essential role in normal functions of the endometrium such as uterine cycle progression, epithelial proliferation and shedding, and embryo implantation. In this study, we demonstrated cycle dependent expression of functional TLR3 in primary endometrial epithelial tissue and expression of intracellular TLR3 in human endometrial epithelial cell lines. We established that stimulation of TLR3-positive cell lines and primary human endometrial epithelial cells with dsRNA leads to TLR3-dependent expression of interleukin (IL)-6, IL-8, interferon (IFN)-inducible protein 10, RANTES, and IFN-β. These results indicate that the cytokine profile of human endometrial epithelial cells can be modified through TLR3 stimulation. Our findings suggest that TLR3 is involved in the immune responses of endometrial epithelial cells after exposure to dsRNA and has the potential to alter the cytokine milieu and influence the outcome and consequences of infection.
PMCID: PMC1460969  PMID: 15935884
human; innate immunity; mucosa; reproductive immunology; Toll-like receptor
21.  Potent induction of antibody-secreting B-cells by human dermal-derived CD14+ dendritic cells triggered by dual Toll-like receptor ligation 
Targeting CD14+ dermal-derived dendritic cells (DDCs) is a rational approach for vaccination strategies aimed at improving humoral immune responses, because of their natural ability to stimulate naïve B-cells. Here, we show that CD14+ DDCs express mRNA for TLRs 1–9, but respond differentially to single or paired TLR ligands. Compared to single ligands, some combinations were particularly effective at activating CD14+ DDCs, as shown by enhanced expression of B-cell stimulatory cytokines (IL-6, IL-10 and TNF-α) and more pronounced phenotypic maturation. These combinations were Resiquimod (R-848) plus Polyinosinic:polycytidylic acid (Poly(I:C)); R-848 plus LPS; Pam3CSK4 plus Poly(I:C); LPS plus Poly(I:C). We also found that selected TLR ligand pairs (R-848 plus either LPS or Poly(I:C)) were superior to individual agents at boosting the inherent capacity of CD14+ DDCs to induce naïve B-cells to proliferate and differentiate into CD27+ CD38+ B-cells that secrete high levels of IgG and IgA. When treated with the same TLR ligand combinations, CD14+ DDCs also promoted the differentiation of Th1 (IFN-γ-secreting) CD4+ T-cells, but not of Th2 or Th17 CD4+ T-cells. These observations may help to identify adjuvant strategies aimed at inducing protective immune responses to various pathogens, including but not limited to HIV-1.
PMCID: PMC3951118  PMID: 23162132
Vaccination; TLR ligands; dendritic cells; B-cells
22.  An IFN-γ-IL-18 Signaling Loop Accelerates Memory CD8+ T Cell Proliferation 
PLoS ONE  2008;3(6):e2404.
Rapid proliferation is one of the important features of memory CD8+ T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than naïve T cells upon antigen stimulation. To examine antigen-specific CD8+ T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205+ dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8+ T cells, which showed rapid proliferation and multiple cytokine production (IFN-γ, IL-2, TNF-α) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-γ-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-γ receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-γ-receptor 1 also showed delayed expansion of memory CD8+ T cells in vivo. These results indicate that a positive regulatory loop involving IFN-γ and IL-18 signaling contributes to the accelerated memory CD8+ T cell proliferation during a recall response to antigen presented by DCs.
PMCID: PMC2408965  PMID: 18545704
23.  Naïve mouse macrophages become activated following recognition of L5178Y lymphoma cells via concurrent ligation of CD40, NKG2D, and CD18 molecules1 
Under different circumstances, tumors can inhibit or activate macrophage (Mϕ) effector functions. We studied the mechanisms of tumor-Mϕ interactions leading to Mϕ activation. The results show that L5178Y mouse T-cell lymphoma cells can prime naïve mouse Mϕ to subsequent LPS stimulation, resulting in increased NO production and anti-lymphoma effects in vitro. L5178Y cells, but not naïve splenocytes, primed Mϕ respond to ligation of TLR4 but not TLR9. L5178Y-primed Mϕ incubated with LPS showed down-regulation of CD40 and up-regulation of NKG2D expression. While L5178Y T cell lymphoma cells prime naïve mouse Mϕ, mouse A20 B cell lymphoma, B16 melanoma, or NIH-3T3 fibroblasts, and human Jurkat T cell lymphoma, Daudi B cell lymphoma, or M21 melanoma tumor cells lines all failed to prime mouse Mϕ. Neither L5178Y-conditioned supernatants nor co-culture of Mϕ and L5178Y cells in transwells resulted in priming, indicating that direct L5178Y cell-Mϕ contact was needed. Several receptor-ligand pairs are reciprocally expressed on Mϕ and L5178Y cell membranes and can be potentially involved in Mϕ priming. Of these, the CD40-CD154 pair played the most important role, as blocking the interaction of these molecules substantially reduced in vitro Mϕ priming. Furthermore, simultaneous blocking of interactions between CD40–CD154, NKG2D–H60, and CD18–ICAM-1/-2 led to complete abrogation of Mϕ-mediated NO secretion and complete inhibition of Mϕ-mediated tumor cell cytostasis. The priming of Mϕ to LPS with L5178Y cells was also observed in vivo. These results suggest that contact with certain tumor cells via CD40, NKG2D, and CD18 molecules on the Mϕ may facilitate Mϕ-mediated anti-tumor immune surveillance.
PMCID: PMC2757113  PMID: 19201847
Monocytes/macrophages; Innate immunity; Tumor recognition
24.  Toll-Like Receptor 3-Specific dsRNA Oligonucleotide Adjuvants Induce Dendritic Cell Cross-presentation, CTL Responses and Antiviral Protection 
Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activated DC and the expression patterns of TLR3 protein in DC subsets have not been established. Here we show that cross-priming CD8α+ and CD103+ DC subsets express much higher levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1+ endosomes upon stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of about 90 bp was sufficient to induce CD86, IL12p40, IFN-β, TNF-α, and IL-6 expression and to mature DC into APC that cross-presented exogenous antigens to CD8+ T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of antigen-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.
PMCID: PMC3078629  PMID: 21242525
25.  Skin Dendritic Cells in Burn Patients 
The body's immunological response to burn injury has been a subject of great inquiry in recent years. Burn injury disturbs the immune system, resulting in a progressive suppression of the immune response that is thought to contribute to the development of sepsis. Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells.
DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens, and their subsequent presentation on the cell surface is linked to major histocompatibility molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues, where they present antigen to T cells and induce an immune response. The purpose of this study was to determine the effects of burn injury on skin DCs in terms of percentage, HLA-DR, and Toll-like receptor-4 (TLR-4) expression. The skin DCs were isolated from burned skin and non-burned skin in the same patient at 7 days post-injury, and skin DCs were isolated from unburned healthy individuals as control. DCs from burned skin notably express low levels of HLA-DR and TLR-4 soon after cell isolation. In the post-burn period the ability of skin DCs to respond to bacterial stimuli is impaired. These changes in DC behaviour might contribute to the impaired host defences against bacteria during burn sepsis.
PMCID: PMC3188181  PMID: 21991177

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