Adjuvant dendritic cell therapy in stage IIIB/C melanoma: the MIND-DC randomized phase III trial.

Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88-1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73-2.38; p = 0.44). Grade 3-4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p < 0.001). In conclusion, while adjuvant nDC treatment in stage IIIB/C melanoma patients generated specific immune responses and was well tolerated, no benefit in RFS was observed.

T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

Heterologous prime-boost vaccination protects against EBV antigen-expressing lymphomas.

The Epstein-Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1), either targeted to the DEC205 receptor on DCs or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector, improved priming of antigen-specific CD4+ T cell help. This help supported the expansion and maintenance of EBNA1-specific CD8+ T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4+ and CD8+ T cell responses protected against EBNA1-expressing T and B cell lymphomas, including lymphoproliferations that emerged spontaneously after EBNA1 expression. In particular, the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as a prophylactic and therapeutic treatment for the respective lymphoma challenges. Our study shows that such heterologous prime-boost vaccinations against EBV-associated malignancies as well as symptomatic primary EBV infection should be further explored for clinical development.

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile.

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8+ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8+ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8+ T-cell expansion but are not required to control primary infection.

The clinical application of cancer immunotherapy based on naturally circulating dendritic cells.

Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy.

Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity.

Production of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4+ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer's patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c+ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c+ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.

Protein corona-mediated targeting of nanocarriers to B cells allows redirection of allergic immune responses.

BACKGROUND: Nanoparticle (NP)-based vaccines are attractive immunotherapy tools because of their capability to codeliver antigen and adjuvant to antigen-presenting cells. Their cellular distribution and serum protein interaction ("protein corona") after systemic administration and their effect on the functional properties of NPs is poorly understood. OBJECTIVES: We analyzed the relevance of the protein corona on cell type-selective uptake of dextran-coated NPs and determined the outcome of vaccination with NPs that codeliver antigen and adjuvant in disease models of allergy. METHODS: The role of protein corona constituents for cellular binding/uptake of dextran-coated ferrous nanoparticles (DEX-NPs) was analyzed both in vitro and in vivo. DEX-NPs conjugated with the model antigen ovalbumin (OVA) and immunostimulatory CpG-rich oligodeoxynucleotides were administered to monitor the induction of cellular and humoral immune responses. Therapeutic effects of this DEX-NP vaccine in mouse models of OVA-induced anaphylaxis and allergic asthma were assessed. RESULTS: DEX-NPs triggered lectin-induced complement activation, yielding deposition of activated complement factor 3 on the DEX-NP surface. In the spleen DEX-NPs targeted predominantly B cells through complement receptors 1 and 2. The DEX-NP vaccine elicited much stronger OVA-specific IgG2a production than coadministered soluble OVA plus CpG oligodeoxynucleotides. B-cell binding of the DEX-NP vaccine was critical for IgG2a production. Treatment of OVA-sensitized mice with the DEX-NP vaccine prevented induction of anaphylactic shock and allergic asthma accompanied by IgE inhibition. CONCLUSIONS: Opsonization of lectin-coated NPs by activated complement components results in selective B-cell targeting. The intrinsic B-cell targeting property of lectin-coated NPs can be exploited for treatment of allergic immune responses.

Vaccination with trifunctional nanoparticles that address CD8+ dendritic cells inhibits growth of established melanoma.

AIM: We wanted to assess the potency of a trifunctional nanoparticle (NP) that targeted and activated CD8+ dendritic cells (DC) and delivered an antigen to induce antitumor responses. MATERIALS & METHODS: The DC targeting and activating properties of ferrous NPs conjugated with immunostimulatory CpG-oligonucleotides, anti-DEC205 antibody and ovalbumin (OVA) as a model antigen to induce antigen-specific T-cell responses and antitumor responses were analyzed. RESULTS: OVA-loaded NP conjugated with immunostimulatory CpG-oligonucleotides and anti-DEC205 antibody efficiently targeted and activated CD8+ DC in vivo, and induced strong OVA-specific T-cell activation. Vaccination of B16/OVA tumor-burdened mice with this NP formulation resulted in tumor growth arrest. CONCLUSION: CD8+ DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.

Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus-induced interferon alpha responses of plasmacytoid dendritic cells.

UNLABELLED: Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV. IMPORTANCE: Although pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.

A multimerized form of recombinant human CD40 ligand supports long-term activation and proliferation of B cells.

BACKGROUND AIMS: CD40-activated B cells have long been studied as potent antigen-presenting cells that can potentially be used for cancer immunotherapy. Nevertheless, their use in human clinical trials has been limited by the lack of a Good Manufacturing Practice-grade soluble human CD40 ligand that is able to induce activation and proliferation of primary B cells. We describe an in vitro method to effectively generate and expand B cells through the use of a multimerized form of human recombinant CD40 ligand (rCD40L). METHODS: Human B cells were isolated from healthy donors and cultivated with either rCD40L or on a monolayer of murine NIH3T3 cells stably expressing human CD40L (NIH3T3/tCD40L) as a widely used standard method. Morphology, expansion rate, immune phenotype and antigen presentation function were assessed. RESULTS: B cells efficiently proliferated in response to rCD40L over 14 days of culture in comparable amounts to NIH3T3/tCD40L. B-cell division in response to CD40L was also confirmed by carboxyfluorescein succinimidyl ester dilution. Moreover, rCD40L induced on B cells upregulation of co-stimulatory molecules essential for antigen presentation. Additionally, proliferation of T cells from allogeneic healthy volunteers confirmed the immunostimulatory capacities of CD40-activated B cells. CONCLUSIONS: We demonstrated that B cells with potent antigen presentation capacity can be generated and expanded by use of a non-xenogeneic form of CD40L that could be implemented in future human clinical settings.

Sca-1 expression defines developmental stages of mouse pDCs that show functional heterogeneity in the endosomal but not lysosomal TLR9 response.

Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity and were shown to be identical to previously described natural interferon (IFN)-α-producing cells. Here, we describe two functionally distinct pDC subpopulations that are characterized by the differential expression of stem cell antigen-1 (Sca-1; Ly-6A/E). Sca-1(-) pDCs are mainly found in the BM, appear first during development, show a higher proliferative activity, and represent the more precursor phenotype. Sca-1(+) pDCs are mostly located in secondary lymphoid organs and represent a later developmental stage. Sca-1(-) pDCs give rise to an Sca-1(+) subset upon activation or in response to endogenous type I IFN. Interestingly, in contrast to Sca-1(-) pDCs, Sca-1(+) pDCs are defective in IFN-α production upon endosomal TLR9 stimulation, whereas lysosomal signaling via TLR9 is functional in both subsets. Gene expression analysis revealed that osteopontin is strongly upregulated in Sca-1(-) pDCs. These data provide evidence for the molecular basis of the observed functional heterogeneity, as the intracellular isoform of osteopontin couples TLR9 signaling to IFN-α expression. Taken together, our results indicate that Sca-1(-) pDCs are an early developmental stage of pDCs with distinct innate functions representing the true murine natural IFN-α-producing cells.

CD141+ dendritic cells produce prominent amounts of IFN-α after dsRNA recognition and can be targeted via DEC-205 in humanized mice.

Functional differences between human dendritic cell (DC) subsets and the potential benefits of targeting them with vaccines remain poorly defined. Here we describe that mice with reconstituted human immune system components (huNSG mice) develop all human conventional and plasmacytoid DC compartments in lymphoid organs. Testing different Toll-like receptor agonists for DC maturation in vivo, we found that IL-12p70 and interferon (IFN)-α production correlated with the maturation of CD141+ (BDCA3+) conventional DCs in huNSG mice. Furthermore, depletion of CD141+ DCs before stimulation significantly reduced IFN-α levels in vivo. This DC subset produced similar total amounts but different subtypes of IFN-α in response to synthetic double-stranded RNA compared with plasmacytoid DCs in response to a single-stranded RNA equivalent. Moreover, synthetic double-stranded RNA as adjuvant and antigen targeting to the endocytic receptor DEC-205, a combination that focuses antigen presentation for T-cell priming on CD141+ DCs, stimulated antigen-specific human CD4+ T-cell responses. Thus, the human CD141+ DC subset is a prominent source of IFN-α and interleukin-12 production and should be further evaluated for vaccine development.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA) in systemic lupus erythematosus (SLE). METHODS: IFNα (dissociation-enhanced lanthanide fluorescent immunoassay), IFNγ-inducible protein 10 (IP-10) (ELISA) and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) (flow cytometry) were measured in 79 accurately characterised patients with lupus and compared with serum titres of Anti-dsDNA (ELISA and radioimmunoassay), Anti-dsDNA-NcX ELISA, Anti-Nuc ELISA, and complement C3 and C4. DA was evaluated using the British Isles Lupus Assessment Group 2004 Index (BILAG-2004) and a modified SLE Disease Activity Index-2000 (mSLEDAI-2K). In addition, 31 clinically quiescent patients were monitored for flares over the course of 180 days. RESULTS: Increased levels of IFNα, IP-10 and SIGLEC-1 were found in 32%, 50% and 86%, respectively, of 66 patients with active SLE. IFNα (r=0.45; p<0.0001) and SIGLEC-1 (r=0.54; p<0.0001) correlated better with BILAG-2004 than did IP-10 (r=0.38; p=0.0002), Farr assay (r=0.40; p=0.0001), Anti-dsDNA-NcX ELISA (r=0.28; p=0.0061), Anti-dsDNA ELISA (r=0.31; p=0.0025), Anti-Nuc ELISA (r=0.25; p=0.0121), C3 (r=-0.43; p<0.0001) and C4 (r=-0.33; p=0.0013). Predictors of SLE flares were disease duration ≤92 months, mild clinical activity (in contrast with no activity), complement C3≤89 mg/dl and IFNα≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. CONCLUSIONS: IFNα, IP-10 and SIGLEC-1 emerged as beneficial biomarkers of DA in patients with SLE. Therefore the implementation of IFN biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-IFN-directed therapies.

BDCA-2 signaling inhibits TLR-9-agonist-induced plasmacytoid dendritic cell activation and antigen presentation.

Plasmacytoid dendritic cells (PDCs) express Toll-like receptor (TLR) 9, which mediates recognition of microbial DNA during infection or self-DNA in autoimmune diseases. Triggering TLR-9 in PDC induces either maturation (lysosomal TLR-9 triggering) or type I interferon (IFN-I) production (endosomal TLR-9 triggering). PDCs also express BDCA-2 (CD303), a C-type lectin receptor (CLR) unique to these cells. CLRs appear to function in innate immunity and microbial recognition, and may cooperate with TLRs to fine-tune inflammatory responses. It has been shown that anti-BDCA-2 monoclonal antibody is internalized by PDC for antigen presentation and inhibits TLR-9 induced IFN-I expression. Here we investigated the cross-talk between BDCA-2 and TLR-9-signaling during PDC maturation and antigen presentation. We found that BDCA-2-induced signaling in PDCs inhibits up-regulation of CD86 and CD40 molecules in CpG-activated PDCs, but not in CD40L-activated PDCs. Furthermore, triggering of BDCA-2 diminished the ability of CpG- and CD40L-stimulated PDCs to process and present antigen to antigen-specific autologous memory T cells. This study demonstrates that BDCA-2 represents an attractive target for clinical immunotherapy of IFN-I dependent autoimmune diseases influencing both, IFN-I production and antigen-specific T-cell stimulation by PDC.

Cutting edge: Plasmacytoid dendritic cells induce IL-10 production in T cells via the Delta-like-4/Notch axis.

Proinflammatory Th1 cells can produce large amounts of the immunosuppressive cytokine IL-10, thereby facilitating the self-limitation of inflammatory responses. Recently, we identified the Notch pathway as a main regulator of IL-10 production by Th1 cells. In this study, we show that plasmacytoid dendritic cells (pDCs), by means of their unique high-level expression of the Notch ligand Delta-like (Dll)-4, activate the Notch receptor on T cells to induce robust IL-10 production in vitro and in vivo. pDCs display a distinct pattern of Notch ligands compared with conventional dendritic cells, marked by the constitutive expression of Dll-4, the only Notch ligand to induce IL-10 expression in vivo, and Dll-1, while at the same time lacking the expression of Jagged. We provide a new mechanism for IL-10 induction by pDCs underlining the importance of the Dll-4/Notch axis in the regulation of inflammatory T cell responses.

Functional dichotomy of plasmacytoid dendritic cells: antigen-specific activation of T cells versus production of type I interferon.

Human plasmacytoid dendritic cells (PDC) are believed to link innate and adaptive immunity by producing type I interferon (IFN-I) and triggering adaptive T cell-mediated immunity. However, it remains elusive to which degree both PDC functions are linked. Here we show that CMV antigen targeted to PDC using a CD303 (blood dendritic cell antigen 2, BDCA-2) mAb is rapidly endocytosed and traffics via early sorting endosomes to emerging MHC-enriched compartments. Both processes occur independently of TLR ligand stimulation. Restimulation of CMV-specific CD4(+) effector-memory T helper cells by autologous PDC and induction of IFN-I production in PDC are dependent on appropriate stimulation. Type B CpG oligonucleotide (CpG-B)-stimulated PDC efficiently process and present CMV antigen and are thus capable of stimulating CMV-specific effector-memory T helper cells. CpG-A-stimulated PDC produce large amounts of IFN-I and express programmed death receptor-1 ligand 1. CpG-A plus CpG-B-co-stimulated PDC behave like CpG-B-stimulated PDC, suggesting that antigen processing and presentation in PDC is dependent on stimulation that concurrently inhibits IFN-I production. In vivo targeting of antigens to PDC via CD303 combined with appropriate PDC stimulation may allow induction of specific T cell activation.

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis.

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.