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Epidermal Langerhans cells (LCs) are the first dendritic cells to encounter skin pathogens. However, their function has recently been challenged, especially in the initiation of T-cell responses to viral antigens. We have previously reported that fresh immature human LCs express mRNA encoding TLR3. Here we analyze the response of highly purified human LCs to poly(I:C), a synthetic mimetic of viral dsRNA recognized by TLR3. We show that LCs exposed for 2 days to poly(I:C) under serum-free conditions up-regulated co-stimulatory molecules, a process associated with increased allostimulatory capacity. Furthermore, poly(I:C) significantly enhanced LC survival and induced them to produce CXCL10, IL-6, and IL-12 p40. Bioactive IL-12 p70, IL-1beta, IL-15, IL-18, and IL-23 were never detected, even after CD40 ligation. LC incubation in the presence of bafilomycin completely reversed the effect of poly(I:C) on LC phenotypic activation and survival, indicating that endosomal TLR3 is involved in this process. Most interestingly, we report here that poly(I:C)-treated LCs favored alloreactive CD4(+) T-cell differentiation toward a Th1 profile and concomitant differentiation of IL-10-producing CD4(+) T cells that might limit, at another time, the inflammatory response and subsequent tissue damage.

Dendritic cells, a sentinel immunity cell lineage, include different cell subsets that express various C-type lectins. For example, epidermal Langerhans cells express langerin, and some dermal dendritic cells express DC-SIGN. Langerin is a crucial component of Birbeck granules, the Langerhans cell hallmark organelle, and may have a preventive role toward HIV, by its internalization into Birbeck granules. Since langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis gp120 high-mannose oligosaccharide binding has been evaluated by molecular modeling. Hydrodynamic studies reveal a very elongated shape of recombinant langerin extracellular domain (ECD). A molecular model of the langerin ECD, integrating the CRD structure, has been generated and validated by comparison with hydrodynamic parameters. In parallel, Langerhans cells were isolated from human skin. From their analysis by electron microscopy and the langerin ECD model, an ultrastructural organization is proposed for Birbeck granules. To delineate the role of the different langerin domains in Birbeck granule formation, we generated truncated and mutated langerin constructs. After transfection into a fibroblastic cell line, we highlighted, in accordance with our model, the role of the CRD in the membrane zipping occurring in BG formation as well as some contribution of the cytoplasmic domain. Finally, we have shown that langerin ECD triggering with a specific mAb promotes global rearrangements of LC morphology. Our results open the way to the definition of a new membrane deformation mechanism.

Objective: The early steps of HIV entry into intact vaginal mucosa still need to be clarified. Here we investigated how HIV translocated across the vaginal pluristratified epithelium, either by transcytosis or by uptake in Langerhans cells., Methods: Using human primary fibroblasts and vaginal epithelial cells, we developed an in-vitro model of vaginal mucosa in which Langerhans cells could also be integrated. Owing to the absence of T lymphocytes and macrophages, we specifically studied the role of Langerhans cells in HIV transmission and the transcytosis of cell-associated HIV., Results: Our model has a normal mucosal tissue architecture and Langerhans cells were efficiently integrated within the pluristratified epithelium. In addition, tight junction proteins' expression, high transepithelium resistance and low fluorescein isothiocyanate-BSA passage confirmed the integrity and impermeability of the reconstruction. Furthermore, we showed that human Langerhans cells also expressed tight junction proteins. Then, we demonstrated that neither transcellular nor intercellular transport of free infectious virus released by R5-infected or X4-infected peripheral blood mononuclear cells inoculated apically occured in the vaginal mucosa, irrespective to the presence of Langerhans cells., Conclusion: For the first time, we documented that, within 4 h following contact with HIV-infected cells, translocation of free HIV particles across a pluristratified mucosa is not detectable and that, in this context, it seemed that Langerhans cells do not increase HIV transmission. Moreover, we provided a useful model for the development of strategies preventing HIV entry into the female genital tract, especially for testing the efficiency of various microbicides.

Dendritic cells (DC) are APCs essential for the development of primary immune responses. In pluristratified epithelia, Langerhans cells (LC) are a critical subset of DC which take up Ags and migrate toward lymph nodes upon inflammatory stimuli. TLR allow detection of pathogen-associated molecular patterns (PAMP) by different DC subsets. The repertoire of TLR expressed by human LC is uncharacterized and their ability to directly respond to PAMP has not been systematically investigated. In this study, we show for the first time that freshly purified LC from human skin express mRNA encoding TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10. In addition, keratinocytes ex vivo display TLR1-5, TLR7, and TLR10. Accordingly, highly enriched immature LC efficiently respond to TLR2 agonists peptidoglycan and lipoteichoic acid from Gram-positive bacteria, and to dsRNA which engages TLR3. In contrast, LC do not directly sense TLR7/8 ligands and LPS from Gram-negative bacteria, which signals through TLR4. TLR engagement also results in cytokine production, with marked differences depending on the PAMP detected. TLR2 and TLR3 ligands increase IL-6 and IL-8 production, while dsRNA alone stimulates TNF-alpha release. Strikingly, only peptidoglycan triggers IL-10 secretion, thereby suggesting a specific function in tolerance to commensal Gram-positive bacteria. However, LC do not produce IL-12p70 or type I IFNs. In conclusion, human LC are equipped with TLR that enable direct detection of PAMP from viruses and Gram-positive bacteria, subsequent phenotypic maturation, and differential cytokine production. This implies a significant role for LC in the control of skin immune responses.

Objective: Coinfection of rhesus macaques with human/simian immunodeficiency virus chimeras harbouring the minimal core-promoter/enhancer elements from HIV-1 clade B, C and E viral prototypes (STR-B, STR-C and STR-E) revealed a remarkable dichotomy in terms of spatio-temporal viral replication. The clade C chimera (STR-C) predominated in primary infection. The present study was aimed at identifying the origin of STR-C plasma viraemia at this infection phase., Design: By competing isogenic viruses differing only in their promoters, it was possible to identify subtle phenotypical differences in viral replication kinetics and compartmentalization in vivo., Methods: Two rhesus macaques were coinfected by the three STR chimeras and the relative colonization of different compartments, particularly blood and stool, was determined for each chimera. Moreover, growth competition experiments in thymic histocultures enriched in interleukin (IL)-7 were performed and relative percentages of chimeras were estimated in supernatants and thymocytes lysates at different time points., Results: It is demonstrated here that at the peak of primary infection, preferential replication of STR-C was supported by the gut-associated lymphoid tissue (GALT), an IL-7 rich microenvironment. This was shown by the correlation of the RNA viral genotype in blood and stools, compartments directly draining virions from the GALT. Thymic histocultures confirmed that replication of STR-C is particularly susceptible to this cytokine, compared to its STR-B and STR-E counterparts., Conclusions: These data show that the GALT cytokine network may well favour HIV-1 clade C replication during primary infection. This could result in enhanced transmission.

Epidermal Langerhans cells, a constituent of the skin immune system, have a spectrum of different functions with implications that extend far beyond the skin. They have the potential to internalize particulate agents and macromolecules, and display migratory properties that endow them with the unique capacity to journey between skin and draining lymph nodes where they encounter antigen-specific T lymphocytes. In addition, LC are considered to play a pivotal role in infectious disease such as Aids, allergy, chronic inflammatory reactions, tumor rejections or transplantation. Herein, we will review the features of Langerhans cells, emphasizing characteristics representative of their life-cycle stages that occur within the skin.

Cutaneous dendritic cells (DC) include epidermal Langerhans cells (LC), interstitial/dermal dendritic cells (DDC), as well as plasmacytoid DC (pDC) that occur under pathological conditions. These immune cells have a spectrum of different functions with implications that extend far beyond the skin. They have the potential to internalize particulate agents and macromolecules, and display migratory properties that endow them with the unique capacity to journey between skin and draining lymph nodes where they encounter antigen-specific T lymphocytes. Herein, we will review the features of human and mouse cutaneous DC, emphasizing characteristics representative of their life-cycle stages that occur within the skin.

Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC). The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage. Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown. Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro. We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors. These cells were either integrated in SEMLE or maintained in suspension. Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)). Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media. Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo. Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets. Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE. These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation. In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.

Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.

Langerhans cells (LCs) are immature dendritic cells of epidermis and epithelia, playing a sentinel role through their specialized function in antigen capture, and their capacity to migrate to secondary lymphoid tissue to initiate specific immunity. A unique feature of LCs is the presence of Birbeck granules (BGs), which are disks of two limiting membranes, separated by leaflets with periodic "zipperlike" striations. The recent identification of Langerin/CD207 has allowed researchers to decipher the mechanism of BG formation and approach an understanding of their function. Langerin is a type II lectin with mannose specificity expressed by LCs in epidermis and epithelia. Remarkably, transfection of Langerin cDNA into fibroblasts creates a dense network of membrane structures with features typical of BGs. Furthermore, mutated and deleted forms of Langerin have been engineered to map the functional domains essential for BG formation. Langerin is a potent LC-specific regulator of membrane superimposition and zippering, representing a key molecule to trace LCs and to probe BG function.

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Dendritic cells take up antigens in peripheral tissues, process them into proteolytic peptides, and load these peptides onto major histocompatibility complex (MHC) class I and II molecules. Dendritic cells then migrate to secondary lymphoid organs and become competent to present antigens to T lymphocytes, thus initiating antigen-specific immune responses, or immunological tolerance. Antigen presentation in dendritic cells is finely regulated: antigen uptake, intracellular transport and degradation, and the traffic of MHC molecules are different in dendritic cells as compared to other antigen-presenting cells. These specializations account for dendritic cells' unique role in the initiation of immune responses and the induction of tolerance.

In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca(2+)-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34(+) progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37 degrees C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.

Generation of monoclonal antibodies restricted to human dendritic cells generated from CD34+ hematopoietic precursors has enabled the identification of Langerin, a Ca(++)-dependent type II lectin. Only expressed by Langerhans cells, Langerin is responsible for Birbeck granule formation by membrane superimposition and zippering. Furthermore, cell-surface Langerin is rapidly internalized into Birbeck granules, and does not colocalize with MHC class II rich compartments. Langerin gene transfected into mouse fibroblasts induces the formation of Birbeck granule-like structures, that would permit a better understanding of the function of Birbeck granules.

Langerhans cell histiocytosis (LCH) consists of lesions composed of cells with a dendritic Langerhans cell (LC) phenotype. The clinical course of LCH ranges from spontaneous resolution to a chronic and sometimes lethal disease. We studied 25 patients with various clinical forms of the disease. In bone and chronic lesions, LCH cells had immature phenotype and function. They coexpressed LC antigens CD1a and Langerin together with monocyte antigens CD68 and CD14. Class II antigens were intracellular and LCH cells almost never expressed CD83 or CD86 or dendritic cell (DC)-Lamp, despite their CD40 expression. Consistently, LCH cells sorted from bone lesions (eosinophilic granuloma) poorly stimulated allogeneic T-cell proliferation in vitro. Strikingly, however, in vitro treatment with CD40L induced the expression of membrane class II and CD86 and strongly increased LCH cell allostimulatory activity to a level similar to that of mature DCs. Numerous interleukin-10-positive (IL-10(+)), Langerin(-), and CD68(+) macrophages were found within bone and lymph node lesions. In patients with self-healing and/or isolated cutaneous disease, LCH cells had a more mature phenotype. LCH cells were frequently CD14(-) and CD86(+), and macrophages were rare or absent, as were IL-10-expressing cells. We conclude that LCH cells in the bone and/or chronic forms of the disease accumulate within the tissues in an immature state and that most probably result from extrinsic signals and may be induced to differentiate toward mature DCs after CD40 triggering. Drugs that enhance the in vivo maturation of these immature DCs, or that induce their death, may be of therapeutic benefit.

We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.

We have analyzed the presence of immature and mature dendritic cells (DCs) within adenocarcinoma of the breast using immunohistochemistry. Immature DCs were defined by expression of CD1a-, Langerin-, and intracellular major histocompatibility complex class II-rich vesicles. Mature DCs were defined by expression of CD83 and DC-Lamp. Breast carcinoma cells were defined by morphology and/or cytokeratin expression. We demonstrate two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature CD1a(+) DCs, mostly of the Langerhans cell type (Langerin(+)), were retained within the tumor bed in 32/32 samples and (b) mature DCs, CD83(+)DC-Lamp(+), present in 20/32 samples, are confined to peritumoral areas. The high numbers of immature DCs found in the tumor may be best explained by high levels of macrophage inflammatory protein 3alpha expression by virtually all tumor cells. Confirming the immature/mature DC compartmentalization pattern, in vitro-generated immature DCs adhere to the tumor cells, whereas mature DCs adhere selectively to peritumoral areas. In some cases, T cells are clustering around the mature DCs in peritumoral areas, thus resembling the DC-T cell clusters of secondary lymphoid organs, which are characteristic of ongoing immune reactions.

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor beta(TGF-beta) for its development. In this study, we show that endogenous TGF-beta is required for the development of both LC and non-LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF-beta polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E-cadherin+, Lag+CD68-, and Factor XIIIa-LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF-beta displayed overlapping functions with CD1a+ precursor-derived DC. In particular, unlike CD14(+)-derived DC obtained in the absence of TGF-beta, they neither secreted interleukin-10 (IL-10) on CD40 triggering nor stimulated the differentiation of CD40-activated naive B cells. Finally, IL-4, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF), induced TGF-beta-independent development of non-LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM-CSF and IL-4 was TGF-beta independent. Collectively these results show that TGF-beta polarized CD34+ HPC differentiation toward LC, whereas IL-4 induced non-LC DC development independently of TGF-beta.

We generated monoclonal antibody (mAb) DCGM4 by immunization with human dendritic cells (DC) from CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor and TNF-alpha. mAb DCGM4 was selected for its reactivity with a cell surface epitope present only on a subset of DC. Reactivity was strongly enhanced by the Langerhans cell (LC) differentiation factor TGF-beta and down-regulated by CD40 ligation. mAb DCGM4 selectively stained LC, hence we propose that the antigen be termed Langerin. mAb DCGM4 also stained intracytoplasmically, but neither colocalized with MHC class II nor with lysosomal LAMP-1 markers. Notably, mAb DCGM4 was rapidly internalized at 37 degrees C, but did not gain access to MHC class II compartments. Finally, Langerin was immunoprecipitated as a 40-kDa protein with a pI of 5.2 - 5.5. mAb DCGM4 will be useful to further characterize Langerin, an LC-restricted molecule involved in routing of cell surface material in immature DC.

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-alpha. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.

Based on the relative expression of CD11c and CD1a, we have identified three fractions of dendritic cells (DCs) in human peripheral blood, including a direct precursor of Langerhans cells (LCs). The first two fractions were CD11c+ DCs, comprised of a major CD1a+/CD11c+ population (fraction 1), and a minor CD1a-/CD11c+ component (fraction 2). Both CD11c+ fractions displayed a monocyte-like morphology, endocytosed FITC-dextran, expressed CD45RO and myeloid markers such as CD13 and CD33, and possessed the receptor for GM-CSF. The third fraction was comprised of CD1a-/CD11c- DCs (fraction 3) and resembled plasmacytoid T cells. These did not uptake FITC-dextran, were negative for myeloid markers (CD13/CD33), and expressed CD45RA and a high level of IL-3Ralpha, but not GM-CSF receptors. After culture with IL-3, fraction 3 acquired the characteristics of mature DCs; however, the expression of CD62L (lymph node-homing molecules) remained unchanged, indicating that fraction 3 can be a precursor pool for previously described plasmacytoid T cells in lymphoid organs. Strikingly, the CD1a+/CD11c+ DCs (fraction 1) quickly acquired LC characteristics when cultured in the presence of GM-CSF + IL-4 + TGF-beta1. Thus, E-cadherin, Langerin, and Lag Ag were expressed within 1 day of culture, and typical Birbeck granules were observed. In contrast, neither CD1a-/CD11c+ (fraction 2) nor CD1a-/CD11c- (fraction 3) cells had the capacity to differentiate into LCs. Furthermore, CD14+ monocytes only expressed E-cadherin, but lacked the other LC markers after culture in these cytokines. Therefore, CD1a+/CD11c+ DCs are the direct precursors of LCs in peripheral blood.