The plastic role of dendritic cells (DCs) in the regulation of immune responses has made them interesting targets for immunotherapy, but also for pathogens or tumors to evade immunity. Functional alterations of DCs are often ascribed to manipulation of canonical NF-κB activity. However, though this pathway has been linked to murine myeloid DC biology, a detailed analysis of its importance in human myeloid DC differentiation, survival, maturation, and function is lacking. The myeloid DC subsets include interstitial DCs and Langerhans cells. In this study, we investigated the role of canonical NF-κB in human myeloid DCs generated from monocytes (monocyte-derived DCs [mo-DCs]) or CD34+ progenitors (CD34-derived myeloid DCs [CD34-mDCs]). Inhibition of NF-κB activation during and after mo-DC, CD34-interstitial DC, or CD34-Langerhans cell differentiation resulted in apoptosis induction associated with caspase 3 activation and loss of mitochondrial transmembrane potential. Besides regulating survival, canonical NF-κB activity was required for the acquisition of a DC phenotype. Despite phenotypic differences, however, Ag uptake, costimulatory molecule and CCR7 expression, as well as T cell stimulatory capacity of cells generated under NF-κB inhibition were comparable to control DCs, indicating that canonical NF-κB activity during differentiation is redundant for the development of functional APCs. However, both mo-DC and CD34-mDC functionality were reduced by NF-κB inhibition during activation. In conclusion, canonical NF-κB activity is essential for the development and function of mo-DCs as well as CD34-mDCs. Insight into the role of this pathway may help in understanding how pathogens and tumors escape immunity and aid in developing novel treatment strategies aiming to interfere with human immune responses.

Plasmacytoid dendritic cells (pDCs) are considered potential tools or targets for immunotherapy. However, current knowledge concerning methodologies to manipulate their development or function remains limited. Here, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mammalian target of rapamycin (mTOR) axis in human pDC development, survival, and function. In vitro pDC generation from human cord blood-derived CD34 ؉ hematopoietic progenitors was reduced by pharmacologic inhibition of PI3K, PKB, or mTOR activity, and peripheral blood pDCs required PI3K-PKB-mTOR signaling to survive. Accordingly, activity of this pathway in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active PKB or pharmacologic inhibition of negative regulator phosphatase and tensin homolog (PTEN) resulted in increased pDC numbers in vitro and in vivo. Furthermore, MHC class II and costimulatory molecule expression, and production of IFN-␣ and TNF-␣, were augmented, which could be explained by enhanced IRF7 and NF-B activation. Finally, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced PI3K-PKB-mTOR activity. In conclusion, intact PI3K-PKB-mTOR signaling regulates development, survival, and function of human pDCs, and pDC development and functionality can be promoted by PI3K-PKB hyperactivation. Manipulation of this pathway or its downstream targets could be used to improve the generation and function of pDCs to augment immunity. (Blood. 2012; 120(25):4982-4991) IntroductionPlasmacytoid dendritic cells (pDCs) represent a specialized DC subset that is recognized for its unique ability to rapidly produce large amounts of type 1 interferons (IFN-␣␤) on the interaction with viruses or nucleic acids of self or nonself origin. 1 Besides direct antiviral activity through the production of IFN-␣␤, pDCs have been implicated in playing a broader role in immunity. pDC activation results in the production of additional proinflammatory cytokines such as TNF-␣ and IL-6, and it stimulates the expression of MHC class I, MHC class II, and costimulatory molecules. These combined functions enable pDCs to activate T helper cells, cytotoxic T lymphocytes, natural killer cells, and plasma cell differentiation. Moreover, pDC-derived cytokines can act as adjuvants for other DC subsets such as myeloid or conventional DC. In contrast, tolerogenic functions of pDC, including the generation of regulatory T cells, have also been described. Similar to other DC subsets, pDC thus show the dual capability of being able to initiate as well as regulate immunity. [1][2][3] In accordance with their broad role in the regulation of immunity, the possibilities to use pDC as either tools or targets for immunotherapy are being increasingly explored. Activation of dysfunctional tumor-resident pDCs may be achieved by administration of TLR agonists, 4,5 and methods to induce antitumor responses by either targeting antigen to endogeno...

Hepatitis B virus (HBV) infection can cause chronic liver disease, which is associated with increased risk of liver cirrhosis, liver failure, and liver cancer. Clearance of HBV infection requires effective HBV-specific immunity; however, the immunological mechanisms that determine the development of effective HBV-specific immunity are poorly understood. Dendritic cells (DC) play a pivotal role in the regulation of antiviral immunity. Here, we investigated the interaction between HBV surface antigen (HBsAg), the main envelope glycoprotein of HBV, and BDCA1؉ myeloid dendritic cells (mDC). Exposure of peripheral bloodderived BDCA1؉ mDC to HBsAg resulted in strong DC maturation, cytokine production, and enhanced capacity to activate antigen-specific cytotoxic T cells (CTLs). By using neutralizing antibodies, crucial roles for CD14 and Toll-like receptor 4 (TLR4) in HBsAg-mediated BDCA1 ؉ mDC maturation were identified. Concordantly, HBsAg-mediated DC maturation required fetal calf serum (FCS) or human plasma, naturally containing soluble CD14 (sCD14). Intriguingly, HBsAg-induced DC maturation was significantly reduced in umbilical cord blood plasma, which contained less sCD14 than adult plasma, indicating that sCD14 is an important host factor for recognition of HBsAg by DC and subsequent DC activation. A direct interaction between sCD14 and HBsAg was demonstrated by using enzyme-linked immunosorbent assay (ELISA). Moreover, sCD14-HBsAg complexes were detected both in vitro and in sera of HBV-infected patients. The abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1 ؉ mDC in vivo. We conclude that HBsAg activates BDCA1 ؉ DC via an sCD14-dependent mechanism. These findings provide important novel insights into the initiation of HBV-specific immunity and facilitate development of effective immunotherapeutic interventions for HBV. Hepatitis B virus (HBV) is a double-stranded DNA (dsDNA) virus that is transmitted via blood and specifically infects hepatocytes. It can cause chronic liver disease and progressive liver injury leading to increased risk of liver cirrhosis, liver failure, and liver cancer (1). The current estimated prevalence of HBV infection is 248 million individuals globally (2). Although the initiation of an effective antiviral immune response is paramount for resolving HBV infection (3), the early steps in the recognition of the virus by immune cells and the functional consequences of this interaction remain to be resolved.A pivotal role for dendritic cells (DC) is anticipated, because these cells play a central role in the orchestration of antiviral immunity due to the expression of a wide variety of different pathogen recognition receptors (PRR) and their unique capacity to initiate virus-specific cytotoxic T cell (CTL) responses (4, 5). Among the different DC subsets, BDCA1 ϩ myeloid DC (mDC) are of particular interest for HBV-specific immunity, since hepatitis B surface antigen (HBsAg)-positive mDC were detected in liver (6) an...

Prototype hepatitis B virus (HBV)-derived synthetic long peptide (SLP) cross-presented by autologous dendritic cells boosted HBV-specific (CD4,CD8) T-cell responses in chronic HBV (CHB) patients ex vivo. Often, PD-L1 blockade improved SLP-responses. This supports therapeutic SLPbased vaccine development for CHB treatment.

Despite the crucial function of dendritic cells (DC) in immunity, the molecular mechanisms regulating human DC development remain poorly defined. STAT5 regulates various hematopoietic lineages and is activated by GM-CSF, a critical cytokine in DC development. In this study, we investigated the role of STAT5 during differentiation of human CD34+ hematopoietic progenitors into precursor DC (pre-DC) and their subsequent differentiation toward interstitial DC and Langerhans cells. Inhibiting STAT5 activity by dominant-negative STAT5 promoted Langerhans cell commitment of hematopoietic progenitors but resulted in loss of pre-interstitial DC development, showing subset-specific regulation. Increasing the low endogenous STAT5 activity by ectopic STAT5 activation downregulated expression of the critical DC transcription factor PU.1 and abrogated commitment to either DC lineage. In contrast, high STAT5 activity was beneficial in already committed pre-DC: terminal DC differentiation was associated with increased endogenous STAT5 phosphorylation levels, JAK2–STAT5 inhibition reduced terminal DC differentiation, and conditional STAT5 activation in pre-DC improved development of BDCA-1+, DC-SIGN+, and Langerin+ DC with normal maturation and T cell stimulation. These data show that STAT5 critically regulates human DC development, with specific requirements for the level of STAT5 activation at distinct differentiation stages. By regulating STAT5 activity, cytokines present at specific locations and under different pathophysiological conditions can determine the fate of DC precursors.

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Pegylated IFNα (PEG‐IFN) is one of the treatment options for chronic HBV (CHB) patients. However, the high patient treatment burden and limited response rate together clearly ask for biomarkers to predict PEG‐IFN response. Soluble CD14 (sCD14) is considered a marker for immune activation and has been shown to predict clinical outcome of HIV infection. However, studies on sCD14 in CHB infection are inconclusive, and its relationship with clinical outcome is largely unknown. Here, we measured sCD14 levels in CHB patients and investigated whether changes in sCD14 level related to PEG‐IFN response. Serum sCD14 levels were determined in 15 healthy controls, 15 acute self‐limited HBV, 60 CHB patients in different disease phases and 94 HBeAg+ CHB patients at week 0 and week 12 of a 52‐week PEG‐IFN treatment. Response to PEG‐IFN treatment was defined as HBeAg seroconversion or HBeAg loss at 26 weeks post‐treatment. The mean sCD14 level in acute HBV patients (3.0 µg/mL) was significantly higher than in CHB patients (2.4 µg/mL) and healthy controls (2.4 µg/mL). In CHB patients receiving PEG‐IFN, a significant increase in sCD14 was found after 12‐week treatment (median week 0:2.1 µg/mL; week 12:3.7 µg/mL). After 12‐week treatment, the fold change (FC = w12/w0) in sCD14 was significantly higher in responders compared to nonresponders (HBeAg seroconversion: median FCresponder = 2.1 vs FCnonresponder = 1.6; HBeAg loss: median FCresponder = 2.2 vs FCnonresponder = 1.5). Receiver operating characteristic curves demonstrated that FC‐sCD14wk12/wk0 levels can be of significant value as a stopping rule to select patients at week 12 who are not likely to benefit from further PEG‐IFN treatment.