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The precise role of human epidermal Langerhans cells (LCs) in immune response is highly controversial. While studying the gene expression profile of these cells, we were intrigued to identify the HLA-DQB2 gene as potentially expressed in LCs. Despite a strong evolutionary conservation of their sequences, the concomitant expression of the poorly polymorphic HLA-DQA2/HLA-DQB2 genes, paralogous to the HLA-DQA1/HLA-DQB1 genes, has never been detected in any cell type. We confirmed by RT-PCR that the HLA-DQA2 and -DQB2 genes are both expressed in LCs, but not in monocyte-derived dendritic cells, or in blood CD1c+ or plasmacytoid dendritic cells. The presence of the HLA-DQβ2 chain in LCs could be demonstrated by Western blotting, whereas immunofluorescence revealed its localization in early endosomes. As in the case of other HLA class II molecules, the HLA-DQα2 and -DQβ2 chains formed heterodimers that had to associate with the invariant chain to reach endosomal compartments. HLA-DQα2/β2 heterodimers were expressed at the cell surface, where they could mediate staphylococcal superantigen stimulation of T cells. Interestingly, HLA-DQα2 and HLA-DQβ1 chains formed mixed heterodimers which efficiently left the endoplasmic reticulum. These observations strongly suggest that the poorly polymorphic HLA-DQA2 and -DQB2 genes should be considered to be of immunological importance. The HLA-DQα2/β2 molecules could influence the complexity of the repertoire of Ags presented by LCs.

International audience; Recent reports have evidenced a role for the Myocyte Enhancer Factor 2C (MEF2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show here that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A/MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunopoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of Trichostatin A, a histone deacetylase inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D:HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D:HDAC1 complexes, we analyzed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is upregulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D:p300 activator complex. The targets of MEF2A/D:HDAC1:HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D human macrophages, as activator or as repressor of gene transcription.

Self-glycosphingolipids bind to surface CD1 molecules and are readily displaced by other CD1 ligands. This capacity to exchange antigens at the cell surface is not common to other antigen-presenting molecules and its physiological importance is unclear. Here we show that a large pool of cell-surface CD1a, but not CD1b molecules, is stabilized by exogenous lipids present in serum. Under serum deprivation CD1a molecules are altered and functionally inactive, as they are unable to present lipid antigens to T cells. Glycosphingolipids and phospholipids bind to, and restore functionality to CD1a without the contribution of newly synthesized and recycling CD1a molecules. The dependence of CD1a stability on exogenous lipids is not related to its intracellular traffic and rather to its antigen-binding pockets. These results indicate a functional dichotomy between CD1a and CD1b molecules and provide new information on how the lipid antigenic repertoire is immunologically sampled.

Birbeck granules (BG) are organelles specific to Langerhans cells (LCs), which form where the C-type lectin Langerin accumulates. Their function remains obscure due to morphologic and dynamic alterations induced by maturation of isolated LC. In this study, we attempted to reconstitute Langerin traffic and BG formation in the endosomal pathway of a human melanoma cell line. In the selected Langerin-transfected cell line, M10-22E, Langerin is distributed between the early recycling endosomal compartment and the plasma membrane, as in LC. Whereas mainly concentrated in membranes related to the Rab11(+) endosomal recycling compartment at the steady state, Langerin also recycles in M10-22E cells and drives BG biogenesis in the endosomal recycling compartment. Interruption of endocytosis or recycling induces redistribution of intracellular Langerin with an associated alteration in BG location and morphology. We have, therefore, generated a stable, Langerin-transfected cell line in which Langerin traffic and distribution and BG morphology replicate that seen in freshly isolated LC. This practical model can now be used to further delineate the nature and function of BG.

Previous work has suggested that the peptide-carrier, heat-shock protein (hsp)70, could directly activate APC. Here we show that this ability is related to endotoxin contamination of the human rhsp70 produced in Escherichia coli. Hence, the ability of 1-3 microg/ml of rhsp70 to induce the maturation of human monocyte-derived DC is abrogated in the presence of the LPS-antagonist polymyxin B or when the rhsp70 contains less than 60 IU/mg endotoxin. Such a level of contamination of the rhsp70 is, however, sufficient - in the presence of soluble rCD14, the LPS co-receptor - to induce cytokine secretion from monocytes and DC, despite the presence of polymyxin B. However, when endotoxin contamination is below 10 IU/mg, rhsp70 does not induce cytokine secretion - even in the presence of soluble rCD14 - or activate p38 mitogen-activated protein kinase signaling pathways, thus showing that an "endotoxin free" hsp70 does not activate APC.

Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-α or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1α/β and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA-A24 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA-A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA-A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.

Immature dendritic cells are antigen presenting cells highly specialized for capturing and processing foreign protein antigens. These cells express Fc gamma RII and Fc epsilon RI which, by their ability to internalize and use the endocytic pathway, increase their capacity to process antigens. Immature dendritic cells, such as epidermal Langerhans cells, also release soluble forms of Fc gamma RII. These latter molecules are likely to compete with the membrane-associated Fc gamma R to diminish or abrogate the capacity of dendritic cells to present immune complexes, as suggested by our in vitro experiments using both human and mouse epidermal Langerhans cells. However, when dendritic cells mature in vitro and become efficient stimulators of resting T cells, they rapidly down-regulate and sometimes completely abolish the expression of their membrane-associated Fc gamma R and Fc epsilon RI. Consequently, they lose or at least strongly diminish their capacity to capture immune complexes. At this stage, the release of soluble Fc gamma R by dendritic cells is also markedly diminished. One can hypothesize that the membrane-associated Fc gamma RII and the soluble Fc gamma RII are molecules expressed when dendritic cells are potent capturing and processing cells, the soluble Fc gamma RII molecule acting by competition as a negative regulatory element on the Fc gamma RII-mediated internalization of IgG-containing immune complexes. Thus, the expression of membrane-associated Fc gamma R and Fc epsilon RI, as well as the release of soluble Fc gamma R, would seem to characterize the immature stage of dendritic cells.

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A–RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

Langerhans cells (LC) are a special subset of dendritic cells integrating cutaneous immunity. The study of LC function is of major interest not only for efforts of vaccine design and immunotherapy but also for gaining an insight into the pathogenesis of immune-mediated cutaneous diseases and neoplasias. Recently, defined antigen-presenting cells were described that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro and in vivo. Here, we show that stimulation with interferon-gamma (IFN-gamma) induces the expression of functionally active IDO in highly purified human epidermal LC. The induction of IDO after stimulation of LC with IFN-gamma seems to follow a defined kinetic with rapid upregulation followed by a downregulation after about 24 h of culture. Accordingly, proliferation of T cells induced by anti-CD3 antibodies was modulated by supernatants of IFN-gamma-activated human epidermal LC. Importantly, downregulation of T cell proliferation by supernatants of 24 h IFN-gamma-activated LC was prevented by inhibition of IDO. These results indicate that LC not only have the capacity to stimulate but also to inhibit T cells, and suggest that LC possess an immunoregulatory function in promoting T cell tolerance by production of IDO.

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9 x 10(4) M(-1) s(-1) and 0.07 s(-1) respectively, yielding a binding constant of 4.2 x 10(5) M(-1). Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin-6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS-rLBP-rsCD14 complex.

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary., website publisher: http://www.springerlink.com/content/ha0d2c351qhjhjdm/

Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.