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Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients., Competing Interests: The authors have declared that no competing interests exist.

Plasmacytoid dendritic cells (PDCs) play powerful regulatory roles in innate and adaptive immune responses and are a major source of type I interferon (IFN) following viral infection. During inflammation and mechanical stress, cells release nucleotides into the extracellular space where they act as signaling molecules via G protein-coupled P2Y receptors. We have previously reported on the regulation of myeloid dendritic cell (DC) function by nucleotides. Here, we report that human PDCs express several subtypes of P2Y receptors and mobilize intracellular calcium in response to nucleotide exposure. As a functional consequence, PDCs acquire a mature phenotype that is further enhanced in the context of CD40 ligation. Strikingly, nucleotides strongly inhibit IFN-alpha secretion induced by influenza virus or CpG-A. This effect is most pronounced for the uridine nucleotides UDP and UTP and the sugar nucleotide UDP-glucose, ligands of P2Y(6), P2Y(4), and P2Y(14), respectively. Nucleotide-induced inhibition of IFN-alpha production is blocked by suramin, a P2Y receptor antagonist. Pharmacological data point toward a role of protein kinase C in the negative regulation of type I IFN. Manipulating PDC function with P2Y receptor agonists may offer novel therapeutic strategies for autoimmune diseases or cancer.

The role of plasmacytoid dendritic cells (PDC), the major producers of alpha interferon upon viral infection, in the nasal mucosa is largely unknown. Here we examined the presence of PDC together with myeloid dendritic cells (MDC) in the nasal epithelia of healthy individuals, of asymptomatic patients with chronic nasal allergy, of patients undergoing steroid therapy, and of patients with infectious rhinitis or rhinosinusitis. Considerable numbers of PDC and MDC could be detected in the nasal epithelium. Furthermore, we demonstrate the expression of SDF-1, the major chemoattractant for PDC, in the nasal epithelium. PDC levels were significantly lower for patients with allergies than for healthy individuals. Interestingly, PDC and MDC were almost absent from patients who received treatment with glucocorticoids, while very high numbers of PDC were found for patients with recent upper respiratory tract infections. Our results demonstrate for the first time a quantitative analysis of PDC and MDC in the healthy nasal epithelium and in nasal epithelia from patients with different pathological conditions. With the identification of PDC, the major target cell for CpG DNA or immunostimulatory RNA, in the nasal epithelium, this study forms the basis for a local nasal application of such oligonucleotides for the treatment of viral infection and allergy.

Human Valpha24+ Vbeta11+ natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as alpha-galactosylceramide (alpha-GalCer) presented on CD1d. In the present study we found that highly purified Valpha24+ NKT cells lack TLR9 mRNA, and thus are not sensitive towards stimulation with CpG oligodeoxynucleotides (ODN). Within PBMC, however, CpG ODN synergistically activated NKT cells stimulated with their cognate antigen alpha-GalCer. Depletion of plasmacytoid dendritic cells (PDC) or myeloid dendritic cells (MDC) revealed that both DC subsets were necessary for the synergistic activation of NKT cells by alpha-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented alpha-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-alpha, whereas full activation of NKT cells as indicated by IFN-gamma production required cell-to-cell contact of PDC and NKT cells in addition to IFN-alpha; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance alpha-GalCer-specific NKT cell activation, and bias activated NKT cells towards a Th1 phenotype. Our results lead to a novel concept of PDC function: to regulate effector activity of antigen-stimulated T cells in a cell contact-dependent manner without the need of simultaneous presentation of the cognate T cell antigen.

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-alpha and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-alpha (IFN-alpha) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and 3' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-alpha (CpG-B) gained the potency of CpG-A to induce IFN-alpha. Higher ODN uptake in PDC was not responsible for the higher IFN-alpha-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-alpha in PDC.

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-alpha induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-alpha induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-alpha induction in PDC. HRSV-induced IFN-alpha production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-alpha by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-alpha induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.

It has been reported that interferon alpha (IFN-alpha) enhances humoral immunity and that dendritic cells of the myeloid lineage promote B-cell differentiation. Here we studied whether the plasmacytoid dendritic cell (PDC), a subset of dendritic cells specialized for the production of IFN-alpha, is involved in regulating B-cell differentiation and immunoglobulin production. The recently identified class of CpG oligonucleotides (CpG-C) was used to activate both B cells and PDCs via Toll-like receptor 9 (TLR9). The presence of PDCs synergistically enhanced CD86 expression, cytokine production (interleukin 6 [IL-6], tumor necrosis factor alpha, and IL-10) and plasma cell differentiation of isolated human peripheral blood B cells stimulated through CpG-C and B-cell antigen receptor (BCR) ligation. This stimulation protocol was sufficient to drive purified naive B cells into IgM-producing plasma cells and to trigger IgG synthesis in memory B cells. PDCs contributed to B-cell activation via IFN-alpha secretion. Up-regulation of TLR9 on B cells was not involved. These results demonstrate that CpG-stimulated PDCs induce plasma cell differentiation in naive and memory B cells in the absence of T-cell help, providing an explanation for the excellent activity of CpG oligonucleotides as a humoral vaccine adjuvant.

Plasmacytoid dendritic cells (PDCs) are potent regulators of immune function and the major source of type I interferon (IFN) following viral infection. PDCs are found at sites of inflammation in allergic reactions, autoimmune disorders, and cancer, but the mechanisms leading to the recruitment of PDCs to these sites remain elusive. During inflammation, adenosine is released and functions as a signaling molecule via adenosine receptors. This study analyzes adenosine receptor expression and function in human PDCs. Adenosine was found to be a potent chemotactic stimulus for immature PDCs via an A(1) receptor-mediated mechanism. The migratory response toward adenosine was comparable to that seen with CXCL12 (stromal-derived factor-1 alpha [SDF-1 alpha), the most potent chemotactic stimulus identified thus far for immature PDCs. Upon maturation, PDCs down-regulate the A(1) receptor, resulting in a loss of migratory function. In contrast, mature PDCs up-regulate the A(2a) receptor, which is positively coupled to adenylyl cyclase and has been implicated in the down-regulation of DC cytokine-producing capacity. We show that in mature PDCs adenosine reduces interleukin-6 (IL-6), IL-12, and IFN-alpha production in response to CpG oligodeoxynucleotides (ODN). These findings indicate that adenosine may play a dual role in PDC-mediated immunity by initially recruiting immature PDCs to sites of inflammation and by subsequently limiting the extent of the inflammatory response induced by mature PDCs by inhibiting their cytokine-producing capacity.

The antitumor activity of IFN-alpha is well established. However, the role of the plasmacytoid dendritic cell (PDC), the major producer of IFN-alpha upon viral infection, in tumor biology is unknown. We sought to study the presence and function of PDC in a human solid tumor. Here, we demonstrate that PDCs infiltrate tumor tissue of patients with head and neck squamous cell carcinoma (HNSCC). Functional activity of PDC was examined by using CpG motif containing oligonucleotides, a defined microbial stimulus for PDCs (recognized via toll-like receptor 9). We found that HNSCC diminished the ability of PDC to produce IFN-alpha in response to CpG motif containing oligonucleotide. Tumor-induced down-regulation of toll-like receptor 9 was identified as one mechanism likely contributing to impaired PDC function within the tumor environment. In tumor-draining lymph nodes, suppression of CpG-induced IFN-alpha production was less pronounced than in single-cell suspensions of primary tumor tissue. In these lymph nodes, CpG-induced IFN-alpha production was associated with increased levels of interferon-induced protein 10 and IFN-gamma and activation of CD4 and CD8 T cells. These results show for the first time the presence of PDCs in human solid tumor tissue and that tumors suppress the capacity of PDCs to produce IFN-alpha. PDCs, which in the absence of appropriate stimulation are reported to promote regulatory CD8 T cells, may contribute to an impaired T-cell-mediated immune response in HNSCC.

Two different types of CpG motif-containing oligonucleotides (CpG ODN) have been described: CpG-A with high induction of IFN-alpha in plasmacytoid dendritic cells; and CpG-B with little induction of IFN-alpha, but potent activation of B cells. In this study, we demonstrate that CpG-A fail to activate B cells unless plasmacytoid dendritic cells are present. We identified a new set of CpG ODN sequences which induces high levels of IFN-alpha in plasmacytoid dendritic cells but remains capable of directly activating B cells. These new CpG ODN (termed CpG-C) are more potent stimulants of B cells than CpG-B due to their ability of directly and indirectly (via plasmacytoid dendritic cells) activating B cells. The sequence of CpG-C combines structural elements of both CpG-A and CpG-B. The most potent sequence, M362, contains a 5'-end 'TCGTCG-motif' and a 'GTCGTT-motif', both of which are present in CpG-B (ODN 2006); a palindromic sequence characteristic for CpG-A (ODN 2216); but no poly G motif required for CpG-A. In conclusion, we defined the first CpG-containing sequences that potently activate both TLR9-expressing immune cell subsets in humans, the plasmacytoid dendritic cell and the B cell. CpG-C may allow for improved therapeutic immuno-modulation in vivo.

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-alpha and IFN-beta) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7(-)/CD45RA(-) CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-gamma production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.

The vertebrate immune system has established TLR9 to detect microbial DNA based on unmethylated CG dinucleotides within certain sequence contexts (CpG motifs). In humans, the expression of toll-like receptor 9 (TLR9) is restricted to B cells and plasmacytoid dendritic cells (PDC). The PDC is characterized by the ability to rapidly synthesize large amounts of type I IFN (IFN-alpha and IFN-beta) in response to viral infection. In contrast to other dendritic cell subsets which express a broad profile of TLRs, the TLR profile in PDC is restricted to TLR7 and TLR9. So far, CpG DNA is the only defined microbial molecule recognized by PDC. An intriguing feature of PDC is its ability to simultaneously produce the two major Th1-inducing cytokines in humans, IFN-alpha and IL-12, both at high levels. The ratio of IFN-alpha versus IL-12 and the quantity of these cytokines are regulated by T helper cell-mediated costimulation via CD40 ligation. The ratio also depends on the differentiation stage of the PDC at the time of stimulation and the type of CpG ODN used. We propose a model in which the establishment of Th1 responses in vivo is improved by appropriately stimulated PDC that otherwise - in the absence of CpG DNA--support Th2 or Th0 responses and thus have been called DC2.

Epidermal dendritic cells found in inflamed skin include Langerhans cells and the recently identified population of inflammatory dendritic epidermal cells. Another subset of dendritic cells in humans is the plasmacytoid dendritic cell in peripheral blood, which is characterized by the production of large amounts of type I interferon (interferon-alpha and interferon-beta) upon viral infection. We hypothesized that plasmacytoid dendritic cells might be involved in anti-viral defense mechanisms of the skin. Here we investigated plasmacytoid dendritic cells, inflammatory dendritic epidermal cells, and Langerhans cells in epidermal single cell suspensions of normal looking skin from healthy volunteers and of lesional skin from patients with different inflammatory skin diseases. Langerhans cells were found in normal and in inflamed skin samples. In normal skin, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells were low or absent. Lesional skin samples from patients with psoriasis vulgaris and contact dermatitis contained relatively high numbers of both inflammatory dendritic epidermal cells and plasmacytoid dendritic cells. In contrast, many inflammatory dendritic epidermal cells but only very few plasmacytoid dendritic cells could be detected in atopic dermatitis lesions. Lupus erythematosus was characterized by high numbers of plasmacytoid dendritic cells but low numbers of inflammatory dendritic epidermal cells. These results demonstrate that in addition to resident Langerhans cells, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells are selectively recruited to the skin lesions depending on the type of skin disease. The lack of plasmacytoid dendritic cells in atopic dermatitis may predispose atopic dermatitis patients to viral infections such as eczema herpeticum, a secondary infection of atopic dermatitis lesions with herpes simplex virus. The composition of dendritic cell subsets may help to clarify the etiology of inflammatory skin diseases and forms the basis for therapeutic intervention with selective microbial molecules such as immunostimulatory CpG oligonucleotides.

The use of dendritic cells (DC) loaded with tumor antigen is one of the most advanced approaches in cancer immunotherapy. CpG motifs within microbial DNA detected by toll-like receptor 9 are responsible for the favorable properties of CpG oligodeoxynucleotides (ODN) as immune modulators. In this study, mature antigen-pulsed DC or peritumoral injections of CpG ODN, both effective for the treatment of small established tumors, were almost ineffective against large established tumors (1-cm diameter) in a syngeneic murine colon carcinoma model. For large tumors, the antitumor activity of mature antigen-pulsed DC was strongly increased by coinjection of CpG ODN, resulting in a transient control of tumor growth. Rejection of large tumors and long-term cure of mice was achieved by combining injection of antigen-pulsed DC plus CpG ODN at a site distant to the tumor with peritumoral injections of CpG ODN. Depletion of CD8 T cells abrogated the therapeutic activity. Large numbers of DEC-205-positive DC infiltrated the tumor in treated mice. Therapy with 5-fluorouracil and leucovorin was unable to control tumors of the same size. In conclusion, we demonstrate that the immune system, provided that appropriate stimulation with DC and CpG ODN is given, has the potential to cure animals of large solid tumors in situations where even chemotherapy is not efficient.

Background: Dendritic cells form a link between innate and acquired immunity. They are capable to detect pathogens based on the recognition of pathogen-associated microbial molecules and trigger the appropriate type of immune responses. In humans, three major subsets of dendritic cells can be distinguished, Langerhans cells of the skin, myeloid DC (MDC) and plasmacytoid DC (PDC). It was reported that PDC infiltrate nasal mucosa in allergen-induced rhinitis. Information about the role of MDC in nasal mucosa and the corresponding mucosa-associated lymphoid tissue, the nasopharyngeal adenoids, is limited., Patients and Methods: : Here we examined the presence of MDC in adenoids and in nasal mucosa of healthy individuals (n = 9) and in patients with allergic rhinitis. MDC were detected by flow cytometry by positive staining for MHC II and CD11c and the lack of lineage markers. Dead cells were excluded from analysis., Results: In adenoids, 0.4 % of all cells were MDC. Considerable numbers of MDC could also be detected in nasal mucosa. No difference was found between healthy individuals and patients with allergies (0.3 % vs. 0.45 % MDC; p = 0.12). Interestingly, MDC were absent in patients who received treatment with glucocorticoids, while very high numbers of MDC were found in patients who recently had upper respiratory tract infections., Conclusion: Our results demonstrate for the first time the presence of MDC in nasal mucosa. MDC numbers were similar in healthy individuals and in patients with allergy. This study forms the basis for examining the role of MDC in the pathogenesis of allergic rhinitis, and for the modulation of MDC functional activity with microbial molecules such as CpG oligonucleotides.

Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll-like receptor (TLR) expression. TLR1-TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDC but not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL-8, IP-10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN-alpha and IL-12 including bioactive IL-12 p70. Previous incubation of PDC with IL-3 decreased the amount of CpG-induced IFN-alpha and shifted the cytokine response in favor of IL-12. CpG ODN-activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN-stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL-12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activated T cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell-controlled restriction to foreign antigens is maintained.

The immature plasmacytoid dendritic cell (PDC) is identical with the principal type I IFN-producing cell upon viral infection. Oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN) are recognized by the vertebrate immune system. Previously, we described CpG ODN that strongly activate human B cells and human blood dendritic cells. Here we describe distinct CpG-containing oligonucleotide sequences which, in contrast to previously described CpG ODN, induced high amounts of IFN-alpha and IFN-beta in peripheral blood mononuclear cells (PBMC). Intracellular staining for IFN-alpha revealed that within PBMC CpG ODN-induced IFN-alpha is produced exclusively by PDC. Unlike IFN-alpha, TNF-alpha is up-regulated in PDC by all CpG ODN tested. Purified PDC responded to CpG ODN, demonstrating direct activation of PDC by CpG ODN. The most active sequence induced the production of up to 5 pg IFN-alpha per single PDC, resulting in more than 400 ng/ml IFN-alpha in the supernatant of PBMC enriched for PDC. The potency of CpG ODN to stimulate IFN-alpha correlated with their ability to stimulate NK cell lytic activity, while purified NK cells did not respond to CpG ODN. IFNgamma production in PBMC was dependent on CpG ODN-induced IFN-alpha/beta as demonstrated by IFN-alpha/beta blocking antibodies. IFN-alpha-inducing CpG ODN strongly supported IFN-gamma production of TCR-triggered CD4 T cells but were less active than other CpG ODN in stimulating B cells. In conclusion our results demonstrate that particular CpG ODN sequences exist which, due to high IFN-alpha/beta induction in PDC, induce a set of immune responses typical for viral infection.