The ubiquitin-editing protein A20 prevents dendritic cell activation, recognition of apoptotic cells, and systemic autoimmunity.

Dendritic cells (DCs) regulate both immunity and tolerance. Here we have shown that the ubiquitin editing enzyme A20 (Tnfaip3) determines the activation threshold of DCs, via control of canonical NF-κB activation. Tnfaip3(fl/fl)Cd11c-cre(+) mice lacking A20 in DCs demonstrated spontaneous proliferation of conventional and double-negative T cells, their conversion to interferon-γ (IFN-γ)-producing effector cells, and expansion of plasma cells. They developed ds-DNA antibodies, nephritis, the antiphospholipid syndrome, and lymphosplenomegaly-features of systemic lupus erythematosus-and extramedullary hematopoiesis. A20-deficient DCs were resistant to apoptosis, caused by increased sensitivity to CD40L and RANKL prosurvival signals and upregulation of antiapoptotic proteins Bcl-2 and Bcl-x. They captured injected apoptotic cells more efficiently, resisted the inhibitory effects of apoptotic cells, and induced self-reactive effector lymphocytes. Because genetic polymorphisms in TNFAIP3 are associated with human autoimmune disorders, these findings identify A20-mediated control of DC activation as a crucial checkpoint in the development of systemic autoimmunity.

Impaired survival of regulatory T cells in pulmonary sarcoidosis.

BACKGROUND: Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis. METHODS: Patients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC). RESULTS: In sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO(+) Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs--but not Th cells--showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs. CONCLUSION: In untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells.

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.

Alternatively activated macrophages inhibit T-cell proliferation by Stat6-dependent expression of PD-L2.

Alternatively activated macrophages (AAM) accumulate in tissues during Th2-associated immune responses like helminth infections and allergic disorders. These cells differentiate in response to interleukin 4 (IL-4)/IL-13-mediated activation of Stat6 and possess potent inhibitory activity against T cells. The molecular mechanism that leads to T-cell suppression remains unclear and could involve soluble factors or inhibitory ligands. Microarray analysis revealed that the inhibitory ligand, programmed death ligand 2 (PD-L2) was strongly induced by IL-4 in macrophages from wild-type but not Stat6-deficient mice. PD-L2 expression correlated with other established markers for AAM-like Relm-α/Fizz1, arginase1, or Ym1 and thereby serves as useful surface marker to identify and isolate AAM from tissues. Antibodies against PD-L2 blocked the inhibitory activity of AAM and retroviral expression of PD-L2 in macrophages from Stat6(-/-) mice was sufficient to inhibit T-cell proliferation, which demonstrates that PD-L2 mediates potent and nonredundant inhibition of T cells independently of other Stat6-regulated genes. Infection of conditional IL-4/IL-13-deficient mice with the helminth Nippostrongylus brasiliensis further showed that PD-L2 expression was dependent on IL-4/IL-13 from Th2 cells. In vivo blockade of PD-L2 during N brasiliensis infection caused an enhanced Th2 response in the lung, indicating that AAM inhibit Th2 cells by expression of PD-L2.

Evidence for local dendritic cell activation in pulmonary sarcoidosis.

BACKGROUND: Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation. METHODS: We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c+DCs was assessed in mucosal airway biopsies. RESULTS: mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4+ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86+ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients. CONCLUSION: Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.

Persistent activation of dendritic cells after resolution of allergic airway inflammation breaks tolerance to inhaled allergens in mice.

RATIONALE: Polysensitization of patients who are allergic is a common feature. The underlying immunologic mechanism is not clear. The maturation status of dendritic cells (DCs) is considered to be important for priming naive T cells in the draining lymph nodes. We hypothesized that chronic airway inflammation can induce an enhanced maturation of airway DCs and facilitate subsequent priming to neoallergens. OBJECTIVES: To investigate whether chronic airway inflammation could induce an altered activation of airway DCs in mice and whether this influences the development of allergic sensitization. METHODS: Balb/c mice were repeatedly challenged with DCs to induce a chronic airway inflammation. We evaluated (1) the induction of the main characteristic features of human asthma including persistent remodeling, (2) the maturation status of airway DCs 1 month after inflammation resolved, (3) whether this influences tolerance to inhaled neoallergen, and (4) what type of T helper response would be induced by DCs. MEASUREMENTS AND MAIN RESULTS: Airway DCs displayed a mature phenotype after complete resolution of airway eosinophilia. Inhalation of a neoallergen without any adjuvant was able to induce airway inflammation in postinflammation lungs but not in control lungs. One month after inflammation, airway DCs were able to induce Th2 polarization in naive T cells consistent with the up-regulation of the Th2 skewing molecules Ym1/2 and OX-40L compared with DCs of control airways. CONCLUSIONS: This study provides evidence that sustained maturation of DCs after resolution of Th2-mediated inflammation can contribute to polysensitization.

An anti-inflammatory role for plasmacytoid dendritic cells in allergic airway inflammation.

It was previously shown that administration of recombinant human Fms-like tyrosine kinase receptor-3 ligand (Flt3L) before allergen challenge of sensitized mice suppresses the cardinal features of asthma through unclear mechanisms. Here, we show that Flt3L dramatically alters the balance of conventional to plasmacytoid dendritic cells (pDCs) in the lung favoring the accumulation of pDCs. Selective removal of pDCs abolished the antiinflammatory effect of Flt3L, suggesting a regulatory role for these cells in ongoing asthmatic inflammation. In support, we found that immature pDCs are recruited to the lungs of allergen-challenged mice irrespective of Flt3L treatment. Selective removal of pDCs during allergen challenge enhanced airway inflammation, whereas adoptive transfer of cultured pDCs before allergen challenge suppressed inflammation. Experiments in which TLR9 agonist CpG motifs were administered in vitro or in vivo demonstrated that pDCs were antiinflammatory irrespective of their maturation state. These effects were mediated through programmed death-1/programmed death ligand 1 interactions, but not through ICOS ligand, IDO, or IFN-alpha. These findings suggest a specialized immunoregulatory role for pDCs in airway inflammation. Enhancing the antiinflammatory properties of pDCs could be employed as a novel strategy in asthma treatment.

Prevalence of Micronutrient Deficiencies and Relationship with Clinical and Patient-Related Outcomes in Pulmonary Hypertension Types I and IV.

BACKGROUND: Pulmonary hypertension (PH) is a rare progressive and lethal disease affecting pulmonary arteries and heart function. The disease may compromise the nutritional status of the patient, which impairs their physical performance. This study aimed to determine the prevalence of micronutrient deficiencies in pulmonary arterial hypertension (PAH) and chronic thrombo-embolic pulmonary hypertension (CTEPH) patients. METHODS: Eighty-one blood samples from a prospective observational cohort study were analyzed for concentrations of micronutrients and inflammation-related factors. The samples consisted of newly diagnosed (treatment-naive) PAH and CTEPH patients and patients treated for 1.5 years according to ERS/ESC guidelines. RESULTS: In the newly diagnosed group, 42% of PAH patients and 21% of CTEPH patients were iron deficient compared to 29% of PAH patients and 20% of CTEPH patients in the treatment group. Vitamin D deficiency occurred in 42% of the newly diagnosed PAH patients, 71% of the newly diagnosed CTEPH patients, 68% of the treated PAH patients, and 70% of the treated CTEPH patients. Iron levels correlated with the 6 min walking distance (6MWD). CONCLUSIONS: Iron and vitamin D deficiencies are highly prevalent in PAH and CTEPH patients, underlining the need for monitoring their status. Studies evaluating the effects of supplementation strategies for iron and vitamin D are necessary.

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization.

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.

Colony lift assay using cell-coated filters: a fast and efficient method to screen phage libraries for cell-binding clones.

For efficient screening of phage antibody libraries obtained by selection on whole cells, we have developed a modified colony lift assay using cell-coated filters. Both cells growing in suspension as well as adherent cells can be coated onto nitrocellulose filters and used to detect bacterial colonies responsible for the production of cell-binding (specific) single chain variable fragment (scFv) antibodies. We demonstrate, using a selected library developed in our laboratory (named "AB" library) as a model system, that the frequency of specific clones as detected by colony lift assay using cell-coated filter is comparable to the frequency of positive clones as detected by the "classical" method (i.e. random picking and flow cytometric analysis). However, the colony lift assay enables detection and isolation of a higher number of specific clones as compared to the random pick. This is due to screening of a much higher number of clones simultaneously (it is possible to screen at least 1000 clones plated on one 9-cm agar dish). Using this method, clones occurring at a low frequency (such as present in early selection rounds) can be detected and isolated efficiently. We clearly demonstrate the usefulness of the colony lift assay with cell-coated filter by applying it to screen the head-and-neck carcinoma (HN) library (a selected library generated in our laboratory). Using the assay, but not the random picking, we were able to isolate specific clones from 2nd to 3rd selection rounds of the HN library.

Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearman's rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

Facilitated antigen uptake and timed exposure to TLR ligands dictate the antigen-presenting potential of plasmacytoid DCs.

Subsets of antigen-presenting cDCs have a differential capacity to present exogenous and endogenous protein antigens to CD4(+) and/or CD8(+) T lymphocytes, depending on expression of antigen-uptake receptors, processing machinery, and microbial instruction. pDCs are also capable of antigen presentation, but the conditions under which they do this have not been systematically addressed. Highly purified cDCs and pDCs were exposed to exogenous, soluble OVA peptide or whole protein. Alternatively, they were made to express cytoplasmic or endosomal OVA by retroviral transduction or by infection with influenza virus containing OVA epitopes. Like cDCs, pDCs expressed the MHC I processing machinery and could present endogenous or cross-present exogenous OVA to CD8(+) T cells, provided they had been stimulated by CpG motif TLR9 ligands or by influenza. Unlike cDCs, the cross-priming activity of pDCs was enhanced, not decreased, by simultaneous TLR stimulation. Processing and presentation of exogenous OVA to CD4(+) T cells required TLR9 ligation prior to antigen encounter and addition of OVA-specific Igs. These stimuli up-regulated critical MHC II processing machinery and enhanced routing to acidic endosomal organelles in a FcγRII-dependent manner. Endogenous antigen was not presented to CD4(+) T cells when expressed in the cytoplasm of pDCs by retrovirus or contained in influenza, unless an Ii-chain-derived endosomal routing signal was present. Thus, timing of TLR ligation and facilitated antigen uptake dictate the potential of pDCs to present endogenous or exogenous antigen by influencing endosomal traffic and antigen-processing machinery.

Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.

It is unclear how Th2 immunity is induced in response to allergens like house dust mite (HDM). Here, we show that HDM inhalation leads to the TLR4/MyD88-dependent recruitment of IL-4 competent basophils and eosinophils, and of inflammatory DCs to the draining mediastinal nodes. Depletion of basophils only partially reduced Th2 immunity, and depletion of eosinophils had no effect on the Th2 response. Basophils did not take up inhaled antigen, present it to T cells, or express antigen presentation machinery, whereas a population of FceRI(+) DCs readily did. Inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required. We favor a model whereby DCs initiate and basophils amplify Th2 immunity to HDM allergen.

Both conventional and interferon killer dendritic cells have antigen-presenting capacity during influenza virus infection.

Natural killer cells are innate effector cells known for their potential to produce interferon-gamma and kill tumour and virus-infected cells. Recently, B220(+)CD11c(int)NK1.1(+) NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice.

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11c(hi) DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) beta and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTbeta receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c(+) monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells.

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b- and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b-CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b-CD8+ DCs presented to CD8 cells, and migratory CD11b-CD8- DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b-CD11chi DCs were depleted using either CD11c-diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.

Respiratory syncytial virus differentially activates murine myeloid and plasmacytoid dendritic cells.

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis in young children. Upon infection both T helper 1 (Th1) and Th2 cytokines are produced. Because RSV-induced Th2 responses have been associated with severe immunopathology and aggravation of allergic reactions, the regulation of the immune response following RSV infection is crucial. In this study we examined the influence of RSV on the activation and function of murine bone marrow-derived dendritic cells (DCs). RSV induced the expression of maturation markers on myeloid DCs (mDCs) in vitro. The mDCs stimulated with RSV and ovalbumin (OVA) enhanced proliferation of OVA-specific T cells, which produced both Th1 and Th2 cytokines. In contrast to mDCs, RSV did not induce the expression of maturation markers on plasmacytoid DCs (pDCs), not did it enhance the proliferation of OVA-specific T cells that were cocultured with pDCs. However, RSV stimulated the production of interferon-alpha (IFN-alpha) by pDCs. Our findings indicate a clear difference in the functional activation of DC subsets. RSV-stimulated mDCs may have immunostimulatory effects on both Th1 and Th2 responses, while RSV-stimulated pDCs have direct antiviral activity through the release of IFN-alpha.

Contribution of the PD-1 ligands/PD-1 signaling pathway to dendritic cell-mediated CD4+ T cell activation.

Dendritic cells (DC) are extremely proficient inducers of naïve CD4+ T cell activation due to their high expression level of peptide-MHC and an array of accessory molecules involved in cell migration, adhesion and co-signaling, including PD-1 ligand 1 (PD-L1) and PD-1 ligand 2 (PD-L2). Whether PD-L1 and PD-L2 have a stimulatory or inhibitory function is a matter of debate, and could be partially dependent on the model system used. In this study we examined the role of PD-L1 and PD-L2 expressed by DC in naïve CD4+ T cell activation in a more physiologically relevant model system, using OVA-specific T cells in combination with various levels of TCR stimulation. Overexpression of PD-L1 or PD-L2 by DC did not inhibit T cell proliferation, even when B7-1 and B7-2 mediated costimulation was absent, although IL-2 production was consistently decreased. Surprisingly, blocking PD-L1 and PD-L2 with soluble programmed death-1 (sPD-1) also inhibited T cell activation, probably via reverse signaling via PD-L1 and/or PD-L2 into DC, leading to reduced DC maturation. This study suggests a relatively minor contribution of PD-1 ligands in DC-driven CD4+ T cell activation and provides evidence for reverse signaling by PD-L1 and PD-L2 into DC, resulting in a suppressive DC phenotype.