Systematic P2Y receptor survey identifies P2Y11 as modulator of immune responses and virus replication in macrophages.

The immune system is in place to assist in ensuring tissue homeostasis, which can be easily perturbed by invading pathogens or nonpathogenic stressors causing tissue damage. Extracellular nucleotides are well known to contribute to innate immune signaling specificity and strength, but how their signaling is relayed downstream of cell surface receptors and how this translates into antiviral immunity is only partially understood. Here, we systematically investigated the responses of human macrophages to extracellular nucleotides, focusing on the nucleotide-sensing GPRC receptors of the P2Y family. Time-resolved transcriptomic analysis showed that adenine- and uridine-based nucleotides induce a specific, immediate, and transient cytokine response through the MAPK signaling pathway that regulates transcriptional activation by AP-1. Using receptor trans-complementation, we identified a subset of P2Ys (P2Y1, P2Y2, P2Y6, and P2Y11) that govern inflammatory responses via cytokine induction, while others (P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14) directly induce antiviral responses. Notably, P2Y11 combined both activities, and depletion or inhibition of this receptor in macrophages impaired both inflammatory and antiviral responses. Collectively, these results highlight the underappreciated functions of P2Y receptors in innate immune processes.

CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota.

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

Type I interferon mediated induction of somatostatin leads to suppression of ghrelin and appetite thereby promoting viral immunity in mice.

Loss of appetite (anorexia) is a typical behavioral response to infectious diseases that often reduces body weight. Also, anorexia can be observed in cancer and trauma patients, causing poor quality of life and reduced prospects of positive therapeutic outcomes. Although anorexia is an acute symptom, its initiation and endocrine regulation during antiviral immune responses are poorly understood. During viral infections, plasmacytoid dendritic cells (pDCs) produce abundant type I interferon (IFN-I) to initiate first-line defense mechanisms. Here, by targeted ablation of pDCs and various in vitro and in vivo mouse models of viral infection and inflammation, we identified that IFN-I is a significant driver of somatostatin (SST). Consequently, SST suppressed the hunger hormone ghrelin that led to severe metabolic changes, anorexia, and rapid body weight loss. Furthermore, during vaccination with Modified Vaccinia Ankara virus (MVA), the SST-mediated suppression of ghrelin was critical to viral immune response, as ghrelin restrained the production of early cytokines by natural killer (NK) cells and pDCs, and impaired the clonal expansion of CD8+ T cells. Thus, the hormonal modulation of ghrelin through SST and the cytokine IFN-I is fundamental for optimal antiviral immunity, which comes at the expense of calorie intake.

Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes.

Although plasmacytoid dendritic cells (pDCs) have been shown to play a critical role in generating viral immunity and promoting tolerance to suppress antitumor immunity, whether and how pDCs cross-prime CD8 T cells in vivo remain controversial. Using a pDC-targeted vaccine model to deliver antigens specifically to pDCs, we have demonstrated that pDC-targeted vaccination led to strong cross-priming and durable CD8 T cell immunity. Surprisingly, cross-presenting pDCs required conventional DCs (cDCs) to achieve cross-priming in vivo by transferring antigens to cDCs. Taking advantage of an in vitro system where only pDCs had access to antigens, we further demonstrated that cross-presenting pDCs were unable to efficiently prime CD8 T cells by themselves, but conferred antigen-naive cDCs the capability of cross-priming CD8 T cells by transferring antigens to cDCs. Although both cDC1s and cDC2s exhibited similar efficiency in acquiring antigens from pDCs, cDC1s but not cDC2s were required for cross-priming upon pDC-targeted vaccination, suggesting that cDC1s played a critical role in pDC-mediated cross-priming independent of their function in antigen presentation. Antigen transfer from pDCs to cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos), that were also produced by pDCs under various conditions. Importantly, all these pDCexos primed naive antigen-specific CD8 T cells only in the presence of bystander cDCs, similarly to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a mechanism for pDCs to achieve cross-priming. In summary, our data suggest that pDCs employ a unique mechanism of pDCexo-mediated antigen transfer to cDCs for cross-priming.

Increased Incidence of Colon Tumors in AOM-Treated Apc 1638N/+ Mice Reveals Higher Frequency of Tumor Associated Neutrophils in Colon Than Small Intestine.

Colorectal cancer (CRC) is one of the most common cancers and a major cause of mortality. Mice with truncating Apc germline mutations have been used as a standard model of CRC, but most of the Apc-mutated lines develop multiple tumors in the proximal small intestine and rarely in the colon precluding detailed analysis of colon tumor microenvironment. Our aim was to develop a model with higher resemblance to human CRC and to characterize tumor infiltrating immune cells in spontaneously developing colon tumors compared to small intestinal tumors. Therefore, the Apc 1638N/+ line was treated repeatedly with azoxymethane (AOM) and 90% colon tumor incidence and 4 to 5 colon tumors per mouse were achieved. Of note, AOM treatment specifically increased the tumor burden in the colon, but not in the small intestine. Histological grading and WNT-signaling activity did not differ significantly between small intestinal and colon tumors with some lesions progressing to invasive adenocarcinoma in both locations. However, characterization of the intratumoral myeloid cell compartment revealed a massive infiltration of colon tumors with neutrophils - 6-fold higher than in small intestinal tumors. Moreover, CCL17-expressing macrophages and dendritic cells accumulated in the tumors indicating the establishment of a tumor-promoting immunosuppressive environment. Thus, Apc 1638N/+ mice treated with AOM are a suitable and straightforward model to study the influence of immune cells and chemokines on colon carcinogenesis.

Dendritic Cell Accumulation in the Gut and Central Nervous System Is Differentially Dependent on α4 Integrins.

Homing of pathogenic CD4+ T cells to the CNS is dependent on α4 integrins. However, it is uncertain whether α4 integrins are also required for the migration of dendritic cell (DC) subsets, which sample Ags from nonlymphoid tissues to present it to T cells. In this study, after genetic ablation of Itga4 in DCs and monocytes in mice via the promoters of Cd11c and Lyz2 (also known as LysM), respectively, the recruitment of α4 integrin-deficient conventional and plasmacytoid DCs to the CNS was unaffected, whereas α4 integrin-deficient, monocyte-derived DCs accumulated less efficiently in the CNS during experimental autoimmune encephalomyelitis in a competitive setting than their wild-type counterparts. In a noncompetitive setting, α4 integrin deficiency on monocyte-derived DCs was fully compensated. In contrast, in small intestine and colon, the fraction of α4 integrin-deficient CD11b+CD103+ DCs was selectively reduced in steady-state. Yet, T cell-mediated inflammation and host defense against Citrobacter rodentium were not impaired in the absence of α4 integrins on DCs. Thus, inflammatory conditions can promote an environment that is indifferent to α4 integrin expression by DCs.

Comparison of iron-reduced and iron-supplemented semisynthetic diets in T cell transfer colitis.

Clinical observations in inflammatory bowel disease patients and experimental studies in rodents suggest that iron in the intestinal lumen derived from iron-rich food or oral iron supplementation could exacerbate inflammation and that iron depletion from the diet could be protective. To test the hypothesis that dietary iron reduction is protective against colitis development, the impact of iron reduction in the diet below 10 mg/kg on the course of CD4+ CD62L+ T cell transfer colitis was investigated in adult C57BL/6 mice. Weight loss as well as clinical and histological signs of inflammation were comparable between mice pretreated with semisynthetic diets with either < 10mg/kg iron content or supplemented with 180 mg/kg iron in the form of ferrous sulfate or hemin. Accumulation and activation of Ly6Chigh monocytes, changes in dendritic cell subset composition and induction of proinflammatory Th1/Th17 cells in the inflamed colon were not affected by the iron content of the diets. Thus, dietary iron reduction did not protect adult mice against severe intestinal inflammation in T cell transfer induced colitis.

What Makes a pDC: Recent Advances in Understanding Plasmacytoid DC Development and Heterogeneity.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that originate in the bone marrow and are continuously replenished from hematopoietic progenitor cells. Conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are distinguished by morphology and function, and can be easily discriminated by surface marker expression, both in mouse and man. Classification of DCs based on their ontology takes into account their origin as well as their requirements for transcription factor (TF) expression. cDCs and pDCs of myeloid origin differentiate from a common DC progenitor (CDP) through committed pre-DC stages. pDCs have also been shown to originate from a lymphoid progenitor derived IL-7R+ FLT3+ precursor population containing cells with pDC or B cell potential. Technological advancements in recent years have allowed unprecedented resolution in the analysis of cell states, down to the single cell level, providing valuable information on the commitment, and dynamics of differentiation of all DC subsets. However, the heterogeneity and functional diversification of pDCs still raises the question whether different ontogenies generate restricted pDC subsets, or fully differentiated pDCs retain plasticity in response to challenges. The emergence of novel techniques for the integration of high-resolution data in individual cells promises interesting discoveries regarding DC development and plasticity in the near future.

Clustering of samples with a tree-shaped dependence structure, with an application to microscopic time lapse imaging.

MOTIVATION: Recent imaging technologies allow for high-throughput tracking of cells as they migrate, divide, express fluorescent markers and change their morphology. The interpretation of these data requires unbiased, efficient statistical methods that model the dynamics of cell phenotypes. RESULTS: We introduce treeHFM, a probabilistic model which generalizes the theory of hidden Markov models to tree structured data. While accounting for the entire genealogy of a cell, treeHFM categorizes cells according to their primary phenotypic features. It models all relevant events in a cell's life, including cell division, and thereby enables the analysis of event order and cell fate heterogeneity. Simulations show higher accuracy in predicting correct state labels when modeling the more complex, tree-shaped dependency of samples over standard HMM modeling. Applying treeHFM to time lapse images of hematopoietic progenitor cell differentiation, we demonstrate that progenitor cells undergo a well-ordered sequence of differentiation events. AVAILABILITY AND IMPLEMENTATION: The treeHFM is implemented in C++. We provide wrapper functions for the programming languages R (CRAN package, https://CRAN.R-project.org/package=treeHFM) and Matlab (available at Mathworks Central, http://se.mathworks.com/matlabcentral/fileexchange/57575-treehfml). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exosome-Based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients.

Purpose: About 60% of non-small cell lung cancer (NSCLC) patients develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy through the EGFR T790M mutation. Patients with this mutation respond well to third-generation tyrosine kinase inhibitors, but obtaining a tissue biopsy to confirm the mutation poses risks and is often not feasible. Liquid biopsies using circulating free tumor DNA (cfDNA) have emerged as a noninvasive option to detect the mutation; however, sensitivity is low as many patients have too few detectable copies in circulation. Here, we have developed and validated a novel test that overcomes the limited abundance of the mutation by simultaneously capturing and interrogating exosomal RNA/DNA and cfDNA (exoNA) in a single step followed by a sensitive allele-specific qPCR.Experimental Design: ExoNA was extracted from the plasma of NSCLC patients with biopsy-confirmed T790M-positive (N = 102) and T790M-negative (N = 108) samples. The T790M mutation status was determined using an analytically validated allele-specific qPCR assay in a Clinical Laboratory Improvement Amendment laboratory.Results: Detection of the T790M mutation on exoNA achieved 92% sensitivity and 89% specificity using tumor biopsy results as gold standard. We also obtained high sensitivity (88%) in patients with intrathoracic disease (M0/M1a), for whom detection by liquid biopsy has been particularly challenging.Conclusions: The combination of exoRNA/DNA and cfDNA for T790M detection has higher sensitivity and specificity compared with historical cohorts using cfDNA alone. This could further help avoid unnecessary tumor biopsies for T790M mutation testing. Clin Cancer Res; 24(12); 2944-50. ©2018 AACR.

Liquid Biopsies Using Plasma Exosomal Nucleic Acids and Plasma Cell-Free DNA Compared with Clinical Outcomes of Patients with Advanced Cancers.

Purpose: Blood-based liquid biopsies offer easy access to genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. Exosomal nucleic acids (exoNAs) originate from living cells, which can better reflect underlying cancer biology.Experimental Design: Next-generation sequencing (NGS) was used to test exoNA, and droplet digital PCR (ddPCR) and BEAMing PCR were used to test cfDNA for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations in 43 patients with progressing advanced cancers. Results were compared with clinical testing of archival tumor tissue and clinical outcomes.Results: Forty-one patients had BRAF, KRAS, or EGFR mutations in tumor tissue. These mutations were detected by NGS in 95% of plasma exoNA samples, by ddPCR in 92% of cfDNA samples, and by BEAMing in 97% cfDNA samples. NGS of exoNA did not detect any mutations not present in tumor, whereas ddPCR and BEAMing detected one and two such mutations, respectively. Compared with patients with high exoNA mutation allelic frequency (MAF), patients with low MAF had longer median survival (11.8 vs. 5.9 months; P = 0.006) and time to treatment failure (7.4 vs. 2.3 months; P = 0.009). A low amount of exoNA was associated with partial response and stable disease ≥6 months (P = 0.006).Conclusions: NGS of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared with clinical testing of archival tumor and testing of plasma cfDNA. Low exoNA MAF is an independent prognostic factor for longer survival. Clin Cancer Res; 24(1); 181-8. ©2017 AACR.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Human Plasmacytoid Dendritic Cells Display and Shed B Cell Maturation Antigen upon TLR Engagement.

The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs.

Continuous single cell imaging reveals sequential steps of plasmacytoid dendritic cell development from common dendritic cell progenitors.

Functionally distinct plasmacytoid and conventional dendritic cells (pDC and cDC) shape innate and adaptive immunity. They are derived from common dendritic cell progenitors (CDPs) in the murine bone marrow, which give rise to CD11c+ MHCII- precursors with early commitment to DC subpopulations. In this study, we dissect pDC development from CDP into an ordered sequence of differentiation events by monitoring the expression of CD11c, MHC class II, Siglec H and CCR9 in CDP cultures by continuous single cell imaging and tracking. Analysis of CDP genealogies revealed a stepwise differentiation of CDPs into pDCs in a part of the CDP colonies. This developmental pathway involved an early CD11c+ SiglecH- pre-DC stage and a Siglec H+ CCR9low precursor stage, which was followed rapidly by upregulation of CCR9 indicating final pDC differentiation. In the majority of the remaining CDP pedigrees however the Siglec H+ CCR9low precursor state was maintained for several generations. Thus, although a fraction of CDPs transits through precursor stages rapidly to give rise to a first wave of pDCs, the majority of CDP progeny differentiate more slowly and give rise to longer lived precursor cells which are poised to differentiate on demand.

Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells.

Plasmacytoid dendritic cells (pDCs) are unique bone-marrow-derived cells that produce large amounts of type I interferon in response to microbial stimulation. Furthermore, pDCs also promote T cell tolerance in sterile-inflammation conditions. However, the immunomodulatory role of aortic pDCs in atherosclerosis has been poorly understood. Here, we identified functional mouse and human pDCs in the aortic intima and showed that selective, inducible pDC depletion in mice exacerbates atherosclerosis. Aortic pDCs expressed CCR9 and indoleamine 2,3-dioxygenase 1 (IDO-1), an enzyme involved in driving the generation of regulatory T cells (Tregs). As a consequence, loss of pDCs resulted in decreased numbers of Tregs and reduced IL-10 levels in the aorta. Moreover, antigen presentation by pDCs expanded antigen-specific Tregs in the atherosclerotic aorta. Notably, Tregs ablation affected pDC homeostasis in diseased aorta. Accordingly, pDCs in human atherosclerotic aortas colocalized with Tregs. Collectively, we identified a mechanism of atheroprotection mediated by tolerogenic aortic pDCs.

A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer.

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.

IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.

Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24(+)CD64(-) DCs and contaminating CSF-1R-dependent CD24(-)CD64(+) macrophages. Functionally, loss of CD24(+)CD11b(+) DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24(+)CD11b(+) DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies.

Regulation of RLR-mediated innate immune signaling--it is all about keeping the balance.

The current view of cytoplasmic RNA-mediated innate immune signaling involves the differential activation of the RNA helicases retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology-2 (LGP2) by distinct RNA viruses. RIG-I, MDA5 and LGP2 form the RIG-I like receptor family (RLR). Since the initial characterization of the RLRs rapid progress has been made in the understanding of the molecular mechanisms that upon virus infection lead to the activation of downstream signaling cascades and the subsequent induction of type I interferon (IFN) and proinflammatory cytokines by these receptors. However, antiviral responses must be tightly regulated in order to prevent uncontrolled production of type I IFN that might have deleterious effects on the host. Exploring the structural and molecular mechanisms that underlie RLR signaling thus was accompanied by the discovery of how RLR-dependent antiviral responses are modulated. This article summarizes the current understanding of endogenous regulation in RLR signaling by various intrinsic molecules that exert their regulatory function in both the steady state or upon viral infection by targeting multiple steps of the signaling cascade.

Antigen delivery to plasmacytoid dendritic cells -induction of tolerance and immunity.

Dendritic cells are professional antigen presenting cells linking the innate and the adaptive immune system. Antibody mediated antigen delivery to functionally distinct DC subpopulations is a promising approach to induce or inhibit antigen specific immune responses in vivo and is useful for designing therapeutic vaccination strategies. This review focuses on the state of the art of this technique and describes recent findings of antigen targeting to the subpopulation of plasmacytoid dendritic cells and the possibilities to induce immunity or attenuate autoimmunity by delivering antigens specifically to this cell type.