Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo

Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors.     

A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.     

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.     

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.     

Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli

Dendritic cells (DCs)-based immunotherapy represents an approach to the prevention and treatment of cancers. Targeting antigens to receptors on DCs can be expected to enhance immune response. We have constructed an expression vector pET32a(+)-ScFv(CD11c)-TRP2 based on a single-chain antibody fragment (ScFv) that targets the high affinity receptor CD11c which is expressed on murine DCs. The 3'-terminal end of the ScFv was ligated to the gene for MHC class I molecule-recognized peptide from mouse tyrosine-related protein 2 (TRP2). Using this vector, we have expressed and purified ScFv(CD11c)-TRP2, a fusion protein that could target TRP2 peptide to CD11c on DCs in vivo to elicit anti-tumor responses. This fusion protein was expressed in inclusion bodies in Escherichia coli BL21(DE3) and was refolded and purified on-column effectively by immobilized metal affinity chromatography using His-tag. Flow cytometry assays showed the specific binding ability of ScFv(CD11c)-TRP2 to DCs, which could be blocked by a hamster anti-mouse CD11c produced by N418 hybridoma. Further studies demonstrated that ScFv(CD11c)-targeted TRP2 peptide processed by DCs was capable of stimulating T cells proliferation. Thus, this fusion protein provides a basis for further research in cancer therapy in vivo.     

Rapid activation of spleen dendritic cell subsets following lymphocytic choriomeningitis virus infection of mice: analysis of the involvement of type 1 IFN

In this study, we report the dynamic changes in activation and functions that occur in spleen dendritic cell (sDC) subsets following infection of mice with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV). Within 24 h postinfection (pi), sDCs acquired the ability to stimulate naive LCMV-specific CD8+ T cells ex vivo. Conventional (CD11chigh CD8+ and CD4+) sDC subsets rapidly up-regulated expression of costimulatory molecules and began to produce proinflammatory cytokines. Their tendency to undergo apoptosis ex vivo simultaneously increased, and in vivo the number of conventional DCs in the spleen decreased markedly, dropping approximately 2-fold by day 3 pi. Conversely, the number of plasmacytoid (CD11clowB220+) DCs in the spleen increased, so that they constituted almost 40% of sDCs by day 3 pi. Type 1 IFN production was up-regulated in plasmacytoid DCs by 24 h pi. Analysis of DC activation and maturation in mice unable to respond to type 1 IFNs implicated these cytokines in driving infection-associated phenotypic activation of conventional DCs and their enhanced tendency to undergo apoptosis, but also indicated the existence of type 1 IFN-independent pathways for the functional maturation of DCs during LCMV infection.     

The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells

Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4+ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.     

FasL基因重组慢病毒载体感染SD大鼠树突状细胞的研究

目的 探讨FasL基因重组慢病毒载体感染SD大鼠树突状细胞的效率和FasI 蛋白的表达情况,为进一步研究转FasL基因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.方法 将培养一周的细胞重铺于六孔板中,每孔细胞数量为5×105,24 h后观察,细胞适合感染,按照MOI=10感染细胞,使用GFP阳性对照质粒作对照实验,感染24 h后,培养皿中添加1 ml新鲜培养基,每隔1 d加细胞因子继续培养,荧光显微镜观察荧光强度和数量,添加病毒液后10 d收集细胞进行实时定量检测和WB检测.结果 FasL基困重组慢病毒载体感染DC 8 d后,细胞开始出现荧光,10 d感染效率为100%;实时定量PCR检测瞬时转染后目的 基因的表达显示以细胞的1.00%为参照,Cell＋FasL质粒为167.03%;免疫印迹检测转染后FasL蛋白的表达显示以细胞的1.00%为参照,细胞＋FasL质粒为34.15%.结论 FasL基因重组慢病毒载体成功感染DC,实时定量PCR及Western印迹证实感染的Dc表达FasL明显提高.为进一步研究转FasL摹因在同种异体器官移植中诱导免疫耐受和保护移植物打下基础.     

Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8alpha, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.     

Cutting edge: mTORC1 in intestinal CD11c+ CD11b+ dendritic cells regulates intestinal homeostasis by promoting IL-10 production

The mammalian target of rapamycin (mTOR) controls cell growth and survival through two distinct complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although several reports have suggested the involvement of mTORC1 in development and function of dendritic cells (DCs), its physiological roles remain obscure. We therefore established mTORC1 signal-deficient mice lacking Raptor, an essential component of mTORC1 signal, specifically in DC lineage (referred to here as Raptor(DC-/-)). Raptor(DC-/-) mice exhibited cell expansion in specific subsets of DCs such as splenic CD8(+) DCs and intestinal CD11c(+)CD11b(+) DCs. We also found that impaired mTORC1 signal resulted in the suppression of IL-10 production along with enhanced CD86 expression in intestinal CD11c(+)CD11b(+) DCs and that Raptor(DC-/-) mice were highly susceptible to dextran sodium sulfate-induced colitis. Our results uncover mTORC1-mediated anti-inflammatory programs in intestinal CD11c(+)CD11b(+) DCs to limit the intestinal inflammation.     

Peripheral tolerance to a nuclear autoantigen: dendritic cells expressing a nuclear autoantigen lead to persistent anergic state of CD4+ autoreactive T cells after proliferation.

It remains unknown why the T cell tolerance to nuclear autoantigens is impaired in systemic autoimmune diseases. To clarify this, we generated transgenic mice expressing OVA mainly in the nuclei (Ld-nOVA mice). When CD4+ T cells from DO11.10 mice expressing a TCR specific for OVA(323-339) were transferred into Ld-nOVA mice, they were rendered anergic, but persisted in vivo for at least 3 mo. These cells expressed CD44(high), CD45RB(low), and were generated after multiple cell divisions, suggesting that anergy is not the result of insufficient proliferative stimuli. Whereas dendritic cells (DCs) from Ld-nOVA (DCs derived from transgenic mice (TgDCs)), which present rather low amount of the self-peptide, efficiently induced proliferation of DO11.10 T cells, divided T cells stimulated in vivo by TgDCs exhibited a lower memory response than T cells stimulated in vitro by peptide-pulsed DCs. Furthermore, we found that repeated transfer of either TgDCs or DCs derived from wild-type mice pulsed with a lower concentration of OVA(323-339) induced a lower response of DO11.10 T cells in Ag-free wild-type recipients than DCs derived from wild-type mice. These results suggest that peripheral tolerance to a nuclear autoantigen is achieved by continuous presentation of the self-peptide by DCs, and that the low expression level of the peptide might also be involved in the induction of hyporesponsiveness.     

Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice

In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung dendritic cells (DCs) to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. Injection of Flt3L into mice resulted in an increased number of CD11c-expressing lung DCs, preferentially in the alveolar septa. FACS analysis allowed us to quantify a 19-fold increase in the absolute numbers of CD11c-positive, CD45R/B220 negative DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DCs (CD11c positive, CD45R/B220 negative, and CD11b positive) and only a 3-fold increase of lymphoid DCs (CD11c positive, CD45R/B220 negative, and CD11b negative) from the lungs of Flt3L-treated mice over vehicle-treated mice. Flt3L-treated lung DCs were more mature than vehicle-treated lung DCs as demonstrated by a significantly higher percentage of cells expressing MHC class II, CD86, and CD40. Freshly isolated Flt3L lung DCs were not fully mature, because after an overnight culture they continued to increase accessory molecule expression. Functionally, Flt3L-treated lung DCs were more efficient than vehicle-treated DCs at stimulating naive T cell proliferation. Our data show that administration of Flt3L favors the expansion of myeloid lung DCs over lymphoid DCs and enhanced their ability to stimulate naive lymphocytes.     

Chronic helminth infection promotes immune regulation in vivo through dominance of CD11cloCD103- dendritic cells

Gastrointestinal helminth infections are extremely prevalent in many human populations and are associated with downmodulated immune responsiveness. In the experimental model system of Heligmosomoides polygyrus, a chronic infection establishes in mice, accompanied by a modulated Th2 response and increased regulatory T cell (Treg) activity. To determine if dendritic cell (DC) populations in the lymph nodes draining the intestine are responsible for the regulatory effects of chronic infection, we first identified a population of CD11c(lo) nonplasmacytoid DCs that expand after chronic H. polygyrus infection. The CD11c(lo) DCs are underrepresented in magnetic bead-sorted preparations and spared from deletion in CD11c-diptheria toxin receptor mice. After infection, CD11c(lo) DCs did not express CD8, CD103, PDCA, or Siglec-H and were poorly responsive to TLR stimuli. In DC/T cell cocultures, CD11c(lo) DCs from naive and H. polygyrus-infected mice could process and present protein Ag, but induced lower levels of Ag-specific CD4(+) T cell proliferation and effector cytokine production, and generated higher percentages of Foxp3(+) T cells in the presence of TGF-β. Treg generation was also dependent on retinoic acid receptor signaling. In vivo, depletion of CD11c(hi) DCs further favored the dominance of the CD11c(lo) DC phenotype. After CD11c(hi) DC depletion, effector responses were inhibited dramatically, but the expansion in Treg numbers after H. polygyrus infection was barely compromised, showing a significantly higher regulatory/effector CD4(+) T cell ratio compared with that of CD11c(hi) DC-intact animals. Thus, the proregulatory environment of chronic intestinal helminth infection is associated with the in vivo predominance of a newly defined phenotype of CD11c(lo) tolerogenic DCs.     

Induction of rapid T cell activation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model

Dendritic cells (DCs) are thought to be responsible for sensitization to inhaled Ag and induction of adaptive immunity in the lung. The characteristics of T cell activation in the lung were studied after transfer of Ag-pulsed bone marrow-derived DCs into the airways of naive mice. Cell division of Ag-specific T cells in vivo was followed in a carboxyfluorescein diacetate succinimidyl ester-labeled cohort of naive moth cytochrome c-reactive TCR transgenic T cells. Our adoptive transfer system was such that transferred DCs were the only cells expressing the MHC molecule required for presentation of cytochrome c to transgenic T cells. Ag-specific T cell activation and proliferation occurred rapidly in the draining lymph nodes of the lung, but not in nondraining lymph nodes or spleen. No bystander activation of non-Ag-specific T cells was induced. Division of Ag-specific T cells was accompanied by transient expression of CD69, while up-regulation of CD44 increased with each cell division. Divided cells had recirculated to nondraining lymph nodes and spleen by day 4 of the response. In vitro restimulation with specific Ag revealed that T cells were primed to proliferate more strongly and to produce higher amounts of cytokines per cell. These data are consistent with the notion that DCs in the lung are extremely efficient in selecting Ag-reactive T cells from a diverse repertoire. The response is initially localized in the mediastinal lymph nodes, but subsequently spreads systemically. This system should allow us to study the early events leading to sensitization to inhaled Ag.     

The phenotype and function of naturally existing regulatory dendritic cells in nematode-infected mice

Immunosuppression associated with chronic helminth infections has been documented in many studies and regulatory T (Treg) cells have been shown to mediate the nematode-induced immunosuppression, but the role of dendritic cells (DCs) in the induction of Treg cell response and immunosuppression has not yet been fully determined. We analysed the response and function of DCs in mesenteric lymph node (MLNs) of mice infected with a gastrointestinal nematode, Heligmosomoides polygyrus, and observed a substantial expansion of DCs in MLNs following the infection. The CD11c⁺ DCs in MLNs of infected mice showed reduced expression of co-stimulatory molecules CD40, CD86 and MHC-II, and production of inflammatory cytokines IL-12 and IL-6. Analysis of MLN DC subsets defined by CD11c and CD45RB expression showed that the CD11c^lowCD45RB^mid subset increased rapidly following H. polygyrus infection and the CD11c^midCD45RB^high subset expanded from the third week after infection. In the co-culture of sorted DC subsets with ovalbumin-(OVA-)specific T cell receptor (TCR) transgenic CD4⁺ T cells, CD11c^lowCD45RB^mid DCs induced a low proliferation response and a high level of IL-10 production in CD4⁺ T cells, whereas CD11c^midCD45RB^high DCs induced more IFN-γ and IL-4 producing CD4⁺ T cells. Intracellular staining revealed that CD11c^lowCD45RB^mid DCs promoted CD4⁺ Foxp3⁺ differentiations. These results indicate that nematode infections selectively induce expansion of the CD11c^lowCD45RB^mid regulatory DC subset that promotes development of Foxp3⁺ and IL-10 producing Treg cells. The Treg cell responses and immunoregulatory cytokines induced by this regulatory DC subset in turn play an important role in mediation of the nematode-induced immunosuppression.     

Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.     

An engineered novel lentivector specifically transducing dendritic cells and eliciting robust HBV-specific CTL response by upregulating autophagy in T cells

Dendritic cells (DCs) play a predominant role in initiating cell immune responses. Here we generated a DC-targeting lentiviral vector (LVDC-UbHBcAg-LIGHT) and evaluated its capacity to elicit HBV-specific cytotoxic T lymphocyte (CTL) responses. DC-SIGN-mediated specific transduction using this construct was confirmed in DC-SIGN-expressing 293T cells and ex vivo-cultured bone marrow cells. LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in inducing HBV-specific CTLs. Mechanistic studies demonstrated autophagy blocking led to a significant increase in apoptosis and obvious inhibition of CD8 + T cells entry into S-phase, correspondingly attenuated LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses. This observation was supported by accumulation of pro-apoptotic proteins and the main negative cell cycle regulator-CDKN1B that otherwise would be degraded in activated T cells where autophagy preferentially occured. Our findings revealed an important role of autophagy in the activation of T cells and suggested LVDC-UbHBcAg-LIGHT may potentially be used as a therapeutic strategy to combat persistent HBV infection with higher security.     

Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.