IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences

Immunostimulatory sequences (ISS) are short oligonucleotides containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that stimulate innate immune responses through Toll-like receptor-9 on B cells and plasmacytoid dendritic cell (PDC) precursors. The anti-inflammatory cytokine interleukin (IL)-10 is predicted to be a potent inhibitor of many of the activities described for ISS, and this may impact the use of ISS in disease states characterized by elevated IL-10. As the activities of ISS on PDCs are central to many clinical applications of ISS, we have studied the effects of IL-10 on PDC stimulation by 3 distinct classes of ISS. IL-10 inhibited cytokine production and survival of ISS-activated PDCs; however, IL-12 induction was much more sensitive to inhibition than interferon (IFN)-alpha induction. Within the PDC population are cells that respond to ISS by producing either IL-12 or IFN-alpha but not both cytokines. IL-12-producing PDCs require costimulation through CD40 and appear more mature than IFN-alpha-producing PDCs. The 3 distinct classes of ISS differed with respect to induction of PDC maturation and T-cell priming capacity. IL-10 regulated PDC activation but did not inhibit the subsequent T-cell-priming ability of PDCs already activated by ISS.     

Murine plasmacytoid dendritic cells induce effector/memory CD8+ T-cell responses in vivo after viral stimulation

Like their human counterparts, mouse plasmacytoid dendritic cells (pDCs) play a central role in innate immunity against viral infections, but their capacity to prime T cells in vivo remains unknown. We show here that virus-activated pDCs differentiate into antigen-presenting cells able to induce effector/memory CD8(+) T-cell responses in vivo against both epitopic peptides and endogenous antigen, whereas pDCs activated by synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG) acquire only the ability to recall antigen-experienced T-cell responses. We also show that immature pDCs are unable to induce effector or regulatory CD8(+) T-cell responses. Thus, murine pDCs take part in both innate and adaptive immune responses by directly priming naive CD8(+) T cells during viral infection.     

Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice.

Interferon alpha/beta plays an important role in the first-line defense against viral infections and can modulate cytokine responses by T-helper cells. Type 1 interferons (IFNs) are clinically important in infectious diseases and in the treatment of leukemia and lymphomas. Many different cell types have the capacity to produce IFN-alpha after encounter with virus and bacteria. The major, natural type 1 IFN-producing cell in humans was recently described as the plasmacytoid T cell, or pDC2, and it can differentiate into dendritic cells (DCs) on culture. This study describes the murine natural IFN-alpha-producing cell, or pDC2, that shares morphologic features with its human counterpart but has some distinct phenotypical characteristics. Murine plasmacytoid DCs can be differentially isolated based on their expression of CD11c, B220 (CD45R), and Thy1.2 (CD90). They lack expression of myeloid (eg, CD11b) antigens and CD8 alpha, a marker used to isolate lymphoid DCs. Like human pDC2, murine plasmacytoid DCs exhibit their maximal type 1 IFN-producing capacity at a precursor stage; pDCs isolated from bone marrow responded to viral stimulation with higher IFN-alpha production than cells of the same phenotype isolated from spleen. Mobilization of mice with Flt3 ligand (Flt3L) or Flt3L and granulocyte-macrophage colony-stimulating factor, hematopoietic factors that specifically enhance DC growth, resulted in strikingly increased numbers of pDC in bone marrow and spleen. The isolation of this novel murine DC subset may serve as a useful tool in the study of viral immunobiology and for the design of treatments for murine malignancies.     

The Notch ligand delta-1 is a hematopoietic development cofactor for plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity, prompting interest in mechanisms controlling the production of this lineage of cells. Notch signaling via one of the Notch ligands, delta-like 1 (delta-1), influences the hematopoietic development of several lymphoid and myeloid lineages, but whether or not delta-1 affects the formation of pDCs is unknown and was tested here. Human CD34+ progenitor cells were cultured onto delta-1-expressing OP9 stroma in the presence of flt-3 ligand and IL-7, and this efficiently generated BDCA-2+ CD123+ CD4+ CD11c- cells with the characteristic morphology of pDCs, expressing toll-like receptor-9 (TLR9), pre-Talpha mRNAs, and secreting CpG-induced IFN-alpha. Delta-1 augmented the numbers of BDCA-2+ cells produced without affecting their proliferation, and the effect was blocked by gamma-secretase inhibition. The development of pDCs was stroma-, delta-1-, and cytokine-dependent and could be induced from committed lymphoid progenitor cells, which responded to delta-1 by opposite changes in pDC- and B-cell production. Our results identify delta-1 as a novel factor enhancing pDC hematopoiesis and delineate a new role for Notch signaling in lymphopoiesis by showing its opposite effect on pDC and B lineage determination.     

Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand

The objective of this study was to investigate whether the immunostimulatory properties of human monocyte-derived dendritic cells (DCs) could be enhanced by triggering OX40/OX40L signaling. Since monocyte-derived DCs possess only low-cell surface levels of OX40L in the absence of CD40 signaling, OX40L was expressed by transfection of DCs with the corresponding mRNA. We show that OX40L mRNA transfection effectively enhanced the immunostimulatory function of DCs at multiple levels: OX40L mRNA transfection augmented allogeneic and HLA class II epitope-specific CD4+ T-cell responses, improved the stimulation of antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without interfering with the prostaglandin E2 (PGE2)-mediated migratory function of the DCs, and facilitated interleukin 12 p70 (IL-12p70)-independent T helper type 1 (Th1) polarization of naive CD4+ T-helper cells. Furthermore, vaccination of tumor-bearing mice using OX40L mRNA-cotransfected DCs resulted in significant enhancement of therapeutic antitumor immunity due to in vivo priming of Th1-type T-cell responses. Our data suggest that transfection of DCs with OX40L mRNA may represent a promising strategy that could be applied in clinical immunotherapy protocols, while circumventing the current unavailability of reagents facilitating OX40 ligation.     

Role of hematopoietic growth factors/flt3 ligand in expansion and regulation of dendritic cells

Dendritic cells (DCs) are hematopoietic cells that initiate immune responses by presenting antigen to T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a primary growth factor for DCs in vitro, but recently it was recognized that other factors including flt3 ligand (FL) and G-CSF expand various DC subsets in vivo. DCs undergo a complex series of maturation and activation steps after they acquire antigen and before they can activate resting T cells. In addition, they must traffic to T-cell-rich areas of lymph nodes (LN) to achieve this. Each of these steps is tightly regulated, and in the last year progress has been made in identifying some of the key molecules involved in each of these steps. This progress will further the efforts underway to develop DCs as vaccine adjuvants.     

Stimulated plasmacytoid dendritic cells impair human T-cell development

Thymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into the thymus, allowing for interactions between thymic pDCs and precursor cells. We demonstrate that in vitro-generated pDCs stimulated with CpG or virus impaired the development of human autologous CD34(+)CD1a(-) thymic progenitor cells into the T-cell lineage. Rescue by addition of neutralizing type I interferon (IFN) antibodies strongly implies that endogenously produced IFN-alpha/beta is responsible for this inhibitory effect. Consistent with this notion, we show that exogenously added IFN-alpha had a similar impact on IL-7- and Notch ligand-induced development of thymic CD34(+)CD1a(-) progenitor cells into T cells, because induction of CD1a, CD4, CD8, and TCR/CD3 surface expression and rearrangements of TCRbeta V-DJ gene segments were severely impaired. In addition, IL-7-induced proliferation but not survival of the developing thymic progenitor cells was strongly inhibited by IFN-alpha. It is evident from our data that IFN-alpha inhibits the IL-7R signal transduction pathway, although this could not be attributed to interference with either IL-7R proximal (STAT5, Akt/PKB, Erk1/2) or distal (p27(kip1), pRb) events.     

Human Flt-3-ligand-mobilized dendritic cells require additional activation to drive effective immune responses

OBJECTIVE: Dendritic cells (DCs) play a pivotal role in the induction of immunity in response to pathogenic challenge or vaccination. As such, the fms-like tyrosine kinase 3-ligand (Flt-3L) has been used to increase DC populations in vivo, with contrasting outcomes, which include an increase in immunity, tolerance induction, or expansion of regulatory cells. This study examines the adjuvant role that human Flt-3L (hFL) administration has in generating immune responses upon immunization with a poorly immunogenic and soluble protein antigen. MATERIALS AND METHODS: Mice were immunized with the nominal antigen, ovalbumin, alone or with antigen emulsified in complete Freund's adjuvant (CFA), with or without prior hFL-mediated expansion of DC subsets. The maturation of DC subsets and activation status of antigen-specific T cells were analyzed by flow cytometry, with effector function assessed in cytolytic T-lymphocyte assays. RESULTS: hFL treatment expanded both conventional DC and plasmacytoid DC in vivo, resulting in increased antigen presentation by both direct and cross-presentation pathways. However, it was only in the context of CFA that antigen immunization could mature DCs and subsequently fully activate antigen-specific T cells with enhanced cytolytic activity. CONCLUSIONS: Our studies reveal that hFL essentially acts as a coadjuvant, as hFL augments the size of an immune response but requires further adjuvant activation to alter the quality of the response.     

Interleukin-2 inhibits FMS-like tyrosine kinase 3 receptor ligand (flt3L)-dependent development and function of conventional and plasmacytoid dendritic cells

Steady-state development of plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) requires the ligand for FMS-like tyrosine kinase 3 receptor (flt3L), but little is known about how other cytokines may also control this process. In this study, we show that IL-2 inhibits the development of both pDCs and cDCs from bone marrow cells under flt3L stimulation, by acting on lineage(-) flt3(+) precursors. This inhibition of DC development by IL-2 requires IL-2Rα and IL2Rβ. IL-2Rα is specifically expressed in one stage of the DC precursor: the monocyte and DC progenitors (MDPs). Furthermore, more MDPs are found in flt3L-stimulated bone marrow cultures when IL-2 is present, suggesting that IL-2 may be inhibiting DC development at the MDP stage. Consistent with our in vitro findings, we observe that nonobese diabetic (NOD) mice, which express less IL-2 compared with diabetes-resistant NOD.Idd3/5 mice, have more splenic pDCs. Additionally, DCs developed in vitro in the presence of flt3L and IL-2 display reduced ability to stimulate T-cell proliferation compared with DCs developed in the presence of flt3L alone. Although the addition of IL-2 does not increase the apoptosis of DCs during their development, DCs developed in the presence of IL-2 are more prone to apoptosis upon interaction with T cells. Together our data show that IL-2 can inhibit both the development and the function of DCs. This pathway may have implications for the loss of immune tolerance: Reduced IL-2 signaling may lead to increased DC number and T-cell stimulatory capacity.     

Imaging of plasmacytoid dendritic cell interactions with T cells

Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4(+) T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC-T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.     

Mechanisms of TRAIL-induced apoptosis in leukemic plasmacytoid dendritic cells

OBJECTIVE: Dendritic cells play a central role in regulating the innate and adaptive immune responses. Plasmacytoid dendritic cells (PDC) represent a newly identified kind of DC with specialized functions aimed at fighting against viral infections. Recently, we have shown that CD4+CD56+ malignancies were leukemia arising from PDC, with a particularly aggressive clinical course. Hence, we asked whether these malignant PDC could be killed via TRAIL, a death-inducing ligand that belongs to a new class of anticancer drugs currently under development. MATERIALS AND METHODS: In this study we used a PDC line (GEN2.2) we recently developed from leukemic PDC as a model. RESULTS: We show that GEN2.2 PDC are sensitive to TRAIL-induced apoptosis and can be killed in vitro by TRAIL-expressing NK cells. Our results suggest that TRAIL binds to Death Receptor 5 (DR5) expressed by GEN2.2 and induces apoptosis mainly via caspases 10, 8, and 3. Interestingly, during infection with influenza, DR5 decreases on GEN2.2 cell surface, which consequently become resistant to TRAIL-induced apoptosis. Moreover, we confirmed the expression of DR5 or DR4 on half of LPDC tested, suggesting the possibility to kill these cells via TRAIL. Hopefully, normal PDC expressed neither DR4 nor DR5. CONCLUSION: These results suggest that TRAIL agonists represent a therapeutic alternative for the treatment of LPDC.     

Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells

The migratory properties of dendritic cells (DCs) are important for their functions. Although several chemokines and their receptors have been implicated in DC migration, the downstream signaling molecules are largely unknown. Here we show that DOCK2, a hematopoietic cell-specific CDM family protein, is indispensable for migration of plasmacytoid DCs (pDCs), but not myeloid DCs (mDCs). Although DOCK2-deficiency did not affect development of pDCs, DOCK2-deficient (DOCK2^–/–) mice exhibited a severe reduction of pDCs in the spleen and lymph nodes. Adoptive transfer experiments revealed that DOCK2^–/– pDCs failed to migrate into the periarteriolar lymphoid sheaths of the spleen. In DOCK2^–/– pDCs, chemokine-induced Rac activation was severely impaired, resulting in the reduction of motility and the loss of polarity during chemotaxis. In contrast, DOCK2^–/– mDCs did not show any defects in Rac activation and migration. These results indicate that pDCs and mDCs use distinct molecules to activate Rac during chemotaxis.     

Regulation of dendritic cell expansion in aged athymic nude mice by FLT3 ligand

This report describes age-related alterations of dendritic cells (DC) distribution in nude athymic mice in vivo and reversal of certain age-dependent defects by an in vivo administration of hematopoietic growth factor FLT3 ligand (FLT3L). There are decreased percentages of CD11c(+) DC in the bone marrow and spleen and a reduced expression of MHC class II and CD86 molecules on DC in old nude mice. The decreased levels of CD11c(+) DC were due to the CD8alpha(-) DC subset. The distribution of CD11c(+) CD8alpha(+) DC in the lymphoid tissues was not different in young and old mice. The effect of in vivo administration of FLT3L on the generation and distribution of DC in the lymphoid tissues in young and old nude mice was also evaluated. Although, FLT3L had a higher inductive potential on the expansion of DC from the bone marrow in the elderly mice, the total level of CD11c(+) DC in the young animals was still significantly higher as compared to the old animals. Interestingly, FLT3L induced a pronounced redistribution and accumulation of MHC class II(+) DC in the lymphoid tissues in old mice, markedly increased the accumulation of CD8alpha(-) DC in the bone marrow in both young and old nude mice, and elevated both CD8alpha(-) and CD8alpha(+) DC in the spleen in young mice. However, only the level of CD8alpha(+) DC was up regulated in the spleen in old athymic mice after FLT3L-based therapy. In summary, abnormalities in DC generation and distribution in old athymic mice could be, in part, circumvented by the in vivo administration of FLT3L.     

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA(+)CD27(-)CCR7(-)). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer(+) T cells, confirming the role of CD8 T cells in this treatment strategy.     

Extensive flow cytometric characterization of plasmacytoid dendritic cell leukemia cells

OBJECTIVES: Accumulating evidence suggests that non-T, non-B cell CD4+CD56+ neoplasms with lymphoblastic morphology include clinically and immunophenotypically diverse entities. Although their cells of origin or classification are still controversial several entities clearly represent a distinct type of neoplasms that are clinically aggressive. METHODS: In this work we present the immunophenotypic and genotypic features of bone marrow (BM), peripheral blood (PB), lymph node and skin lymphocytes from a patient diagnosed as plasmacytoid dendritic cell leukemia involving the skin, BM, PB, lymph nodes, liver and spleen. For determination of immunophenotypic characteristics of malignant plasmacytoid dendritic cells 73 monoclonal antibodies detecting lineage markers, chemokine receptors, cytokine receptors, activation, and co-stimulatory molecules were used. RESULTS AND CONCLUSION: The malignant cells proved to express CD4+, CD56+ lineage negative leukemia phenotype characteristically positive for CD36, CD38, CD40, CD45, CD45RA, CD68, CD123, CD184, HLA-DR, BDCA2, and granzyme-B corresponding to the preplasmacytoid dendritic cell developmental stage. The presence of CD11a/CD18, CD84, CD91, CD95, alphavbeta5, CDw197, and the absence of CD52 and CD133 in this case can be regarded as additional features of malignant cells. Completing the immunophenotypes with multidrug resistance function can provide additional information for characterizing pDC leukemia.     

CpG ODN2216刺激慢性乙型肝炎患者浆样树突状细胞表型和功能的变化

目的观察慢性乙型肝炎患者外周血浆样树突状细胞(pDCs)的特点,并探讨pDCs在乙型肝炎病毒(HBV)感染发病机制中的作用。方法收集20例慢性乙型肝炎患者和15名健康人外周抗凝血,应用流式细胞仪分析pDCs的频率；同时检测CpG ODN2216刺激后pDCs表型及分泌干扰素(IFN)α功能的变化；并分析HBV慢性感染患者外周血pDCs频率、数量和功能的变化及其与临床病情的关系。结果应用免疫磁珠分选技术可得到纯度大于95%的pDCs。经CpG ODN2216刺激后纯化的pDCs能高表达共刺激分子CD80、CD86、CD40和CD83,并分泌大量的IFNα。与健康人比较,HBV感染患者外周血中pDCs频率明显下降,分别为0．287%±0．142%和0．192%±0．110%；经CpG ODN2216刺激后,其表面共刺激分子CD80和CD40表达明显下降,产生IFNα的能力也显著降低,分别为(3142．9±1292．3) pg/ml和(972．6±705．5)pg/ml。相关性分析表明,患者pDCs产生的IFNα水平与丙氨酸氨基转移酶水平呈明显负相关,但与血清病毒载量无明显相关性。结论CpG ODN2216可以刺激pDCs成熟并分泌大量IFNα。慢性乙型肝炎患者外周血pDCs频率、表型和功能明显下降,这可能是HBV持续感染的重要致病机制。