Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor α and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K δ subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor κB activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.

Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specifi c inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinfl ammatory responses of pDCs, including tumor necrosis factor ␣ and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K ␦ subunit, which was specifi cally involved in the control of type I IFN production. Although uptake and endosomal traffi cking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor B activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.

Interleukin-4 (IL-4), a major T-helper type 2 (Th2) cytokine, primes dendritic cells (DCs) for IL-12 production, suggesting a negative feedback loop to prevent dysregulated Th2 inflammation, such as allergy. We previously showed that human thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes of atopic dermatitis, activates CD11c ؉ DCs to induce the differentiation of naive CD4 ؉ IntroductionIn T-helper type 2 (Th2) responses, excessive production of interleukin-4 (IL-4), IL-13, and IL-5 should be regulated by the appropriate negative feedback loops to prevent allergic diseases. 1,2 One of the mechanisms that negatively regulate Th2 responses is IL-12 production by IL-4-or IL-13-primed dendritic cells (DCs), which subsequently switches the immune response toward a Th1 profile. 3,4 Quantitative 5-7 or qualitative 8 defectiveness of IL-12 production was suggested to be involved in dysregulated Th2 responses in allergic subjects or experimental models of allergy. However, extrinsic factors present in the cell microenvironment may lead to the maintenance of the Th2 phenotype even in the presence of physiologic levels of functional IL-12.Human thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, 9,10 strongly activates immature CD11c ϩ DCs. TSLPactivated DCs (TSLP DCs) induce a robust expansion of allogeneic and autologous T cells [11][12][13] and induce the differentiation of allogeneic naive CD4 ϩ and CD8 ϩ T cells into proallergic effectors. 11,12 TSLP is highly expressed by keratinocytes of atopic dermatitis, 11 making it a potential key player in the physiopathology of allergy.Because TSLP is an important cytokine for the induction of inflammatory Th2 responses, we have analyzed the ability of TSLP to prime DCs for the production of IL-12 and the consequences of such priming on the polarization of naive CD4 ϩ T cells. Study design DC purification, cultureCD11c ϩ DCs were purified from adult blood buffy coats to reach greater than 99% purity, as described. 11,13 DCs (0.5 ϫ 10 6 /mL in flat-bottom 96-well plates) were cultured in RPMI containing 10% fetal calf serum (FCS), 1% pyruvate, 1% HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid), and penicillin/streptomycin with TSLP (15 ng/mL), IL-4 (50 U/mL), IL-7 (50 ng/mL), lipopolysaccharide (LPS, 1 g/mL), poly(I:C) (100 g/mL), CD40 ligand (CD40L)-transfected L-fibroblasts (2.5 ϫ 10 4 /well), culture medium alone, or a combination of these conditions. DC IL-12 productionIL-12 production was measured in DC culture supernatants by high-sensitivity protein enzyme-linked immunosorbent assay (ELISA) for bioactive IL-12p70 or the IL-12p40 sub-unit (R&D Systems, Minneapolis, MN). A first set of supernatants was obtained after 24-hour culture of DCs. For a secondary stimulation, 24-hour cultured DCs were washed thoroughly to remove any remaining culture supernatant. Viable DCs were counted using trypan blue exclusion of dead cells and were reseeded in complete culture medium at equal cell numbers and concentrations with various DC activator...

Plasmacytoid predendritic cells (pDCs) play a key role in antiviral immunity through their capacity to produce large amounts of type I interferons in response to Toll-like receptor triggering, and to differentiate into dendritic cells (DCs). However, their antigen processing and presentation pathways remain poorly characterized. In this study, we analyzed major histocompatibility complex class II (MHC II) synthesis and transport in primary human pDCs. We show that stimulation of pDCs with influenza virus leads to a sustained neosynthesis of MHC II molecules, which rapidly accumulate in antigen loading compartments organized around the microtubule organization center. MHC II endocytosis as well as antigen internalization remain active during the entire process of pDC differentiation into DCs, suggesting a capacity to constantly renew surface peptide-MHC II complexes. IntroductionHuman peripheral blood contains 2 main populations of dendritic cells (DCs): the "conventional" DCs (cDCs), and the plasmacytoid predendritic cells (pDCs). 1 These 2 subsets express different sets of Toll-like receptors (TLR) and differentially respond to TLR ligands, indicating that they display nonredundant functions. 2 pDCs play a central role in the immune system by linking the innate with the adaptive immune responses. 3 First, pDCs rapidly produce large amounts of type I interferons (IFNs) after microbial stimulation. 4,5 pDC activation occurs through the recognition of viral nucleic acids (single-stranded RNA) by TLR7, and viral and bacterial DNA by TLR9. 6 Second, depending on the stimuli, pDCs can differentiate into cells with a dendritic phenotype. 3,7 This differentiation is accompanied by a strong up-regulation of the surface expression of major histocompatibility complex class I (MHC I) and class II (MHC II), as well as T-cell costimulatory molecules, suggesting that they become potent antigen-presenting cells. 3,7,8 However, the capacity of pDCs to act as antigenpresenting cells remains controversial. Depending on the stimuli, 3,9,10 the source of pDCs, 11,12 the way the antigen is internalized, 13,14 and the dose of antigen, 12 pDCs are able or not to activate naive CD4 T cells. It has also been proposed that pDCs are endowed with tolerogenic capacities by inducing regulatory T cells producing interleukin-10 (IL-10) both in vitro [15][16][17][18][19][20] and in vivo. 21,22 It is well established that antigen presentation by MHC II heavily depends on their intracellular traffic. The biosynthetic route followed by nascent MHC II has been well characterized in various antigen-presenting cells. 23 MHC II are synthesized in the endoplasmic reticulum (ER) where they associate with the chaperone invariant chain (Ii) to form ␣␤Ii complexes. Such complexes exit from the ER and traffic through the Golgi apparatus to most probably reach the plasma membrane where they are rapidly internalized into endosomes. There, Ii is degraded, allowing MHC II to acquire their peptide load. MHC II peptide-loaded complexes are then exported to the c...