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Neurturin (NTN) was previously described for its neuronal activities, but recently, we have shown that this factor is also involved in asthma physiopathology. However, the underlying mechanisms of NTN are unclear. The aim of this study was to investigate NTN involvement in acute bronchial Th2 responses, to analyze its interaction with airway structural cells, and to study its implication in remodeling during acute and chronic bronchial inflammation in C57BL/6 mice. We analyzed the features of allergic airway inflammation in wild-type and NTN−/− mice after sensitization with two different allergens, OVA and house dust mite. We showed that NTN−/− dendritic cells and T cells had a stronger tendency to activate the Th2 pathway in vitro than similar wild-type cells. Furthermore, NTN−/− mice had significantly increased markers of airway remodeling like collagen deposition. NTN−/− lung tissues showed higher levels of neutrophils, cytokine-induced neutrophil chemoattractant, matrix metalloproteinase 9, TNF-α, and IL-6. Finally, NTN had the capacity to decrease IL-6 and TNF-α production by immune and epithelial cells, showing a direct anti-inflammatory activity on these cells. Our findings support the hypothesis that NTN could modulate the allergic inflammation in different mouse asthma models.

International audience; Graft failure is a major complication after unrelated cord blood transplantation. Presence of HLA-antibodies before cord blood transplantation may impact graft failure. To analyze the effect of anti-HLA antibodies on unrelated cord blood transplantation outcomes, we analyzed 294 unrelated cord blood transplant recipients after reduced intensity conditioning regimen. The majority of the patients (82%) were transplanted for malignancies, 60% with double-unrelated cord blood transplant, 63% were HLA mismatched. Retrospectively, pre-unrelated cord blood transplant serum was tested for HLA-Ab using Luminex™ platform. Results were interpreted as mean fluorescence intensity (MFI) against donor-specific mismatch. Among 62 recipients (23%) who had anti-HLA antibodies before unrelated cord blood transplant, 14 patients had donor specific anti-HLA antibodies (DSA) (7 were donor-specific anti-HLA antibodies for single unrelated cord blood transplant and 7 for double unrelated cord blood transplant). Donor specific anti-HLA antibodies threshold ranged from 1620-17629 of mean fluorescence intensity (MFI). Cumulative incidence of Day-60 neutrophil engraftment was 76%: 44% for recipients with donor specific anti-HLA antibodies and 81% in those without donor specific anti-HLA antibodies (P=0.006). The cumulative incidence of 1-year transplant related mortality was 46% in patients with donor specific anti-HLA antibodies and 32% in those without antibodies (P=0.06). The presence of donor specific anti-HLA antibodies was associated with a trend for decreased survival rate (42% vs. 29%; P=0.07). Donor specific anti-HLA antibody in recipients of unrelated cord blood transplant is associated with graft failure and decreased survival. Patient's screening for donor specific anti-HLA antibodies before unrelated cord blood transplantation is recommended before choosing an HLA mismatched cord blood unit. Whenever possible it is important to avoid selecting a unit for which the patient has donor specific anti-HLA antibodies.

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.

The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.

International audience; CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombi-nant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spec-trometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human den-dritic cells might be expanded.

A 56 year-old, multiparous woman suffering from a myeloproliferative syndrome, who had received multiple red blood cell and platelet transfusions, was the recipient of an allograft of peripheral blood stem cells derived from her HLA-A, B, DR, DQ and DP and ABO identical sister, following myeloablative conditioning. The persistence of severe, isolated thrombopenia resistant to platelet transfusions led to the discovery of anti-HLA class I allo-immunisation. As HLA compatible platelet transfusions did not result in satisfactory platelet increments, we then discovered the simultaneous presence of anti-HPA-1a allo-immunisation. Genotyping of the HPA-1 systems of the patient (HPA-1B/B) and her sister (HPA-1A/B) enabled us to elucidate the mechanism underlying the persistent thrombopenia and the inefficacy of transfusion. In fact, only transfusion of HPA-1B/B platelets (HLA compatible or incompatible) proved to be efficacious. To reduce the level of anti-HPA-1a antibodies, we performed plasmapheresis sessions and used an anti-CD20 monoclonal antibody. It was only on achieving total haematopoietic chimerism, through rapid interruption of the immunosuppression, that we obtained spontaneous normalisation of the platelet count. The present case emphasises the necessity, before undertaking any allograft of haematopoietic stem cells - even if the latter come from a strictly HLA identical member of the family - of performing a search for eventual anti-HPA allo-immunisation.

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.

CD1e displays unique features in comparison with other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is transported directly to lysosomes, where it is cleaved into a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. In the present study, we show that the N-terminal end of the membrane-associated molecule begins at amino acid 20, whereas the soluble molecule consists of amino acids 32–333. Thus immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartments and so is absent from its mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e α-chain with β2-microglobulin, whereas propeptide-deleted CD1e molecules are immunologically active. Comparison of CD1e cDNAs from different mammalian species indicates that the CD1e propeptide is conserved during evolution, suggesting that it may also optimize the generation of CD1e molecules in other species.

CD1e is a membrane-associated protein predominantly detected in the Golgi compartments of immature human dendritic cells. Without transiting through the plasma membrane, it is targeted to lysosomes (Ls) where it remains as a cleaved and soluble form and participates in the processing of glycolipidic antigens. The role of the cytoplasmic tail of CD1e in the control of its intracellular pathway was studied. Experiments with chimeric molecules demonstrated that the cytoplasmic domain determines a cellular pathway that conditions the endosomal cleavage of these molecules. Other experiments showed that the C-terminal half of the cytoplasmic tail mediates the accumulation of CD1e in Golgi compartments. The cytoplasmic domain of CD1e undergoes monoubiquitinations, and its ubiquitination profile is maintained when its N- or C-terminal half is deleted. Replacement of the eight cytoplasmic lysines by arginines results in a marked accumulation of CD1e in trans Golgi network 46+ compartments, its expression on the plasma membrane and a moderate slowing of its transport to Ls. Fusion of this mutated form with ubiquitin abolishes the accumulation of CD1e molecules in the Golgi compartments and restores the kinetics of their transport to Ls. Thus, ubiquitination of CD1e appears to trigger its exit from Golgi compartments and its transport to endosomes. This ubiquitin-dependent pathway may explain several features of the very particular intracellular traffic of CD1e in dendritic cells compared with other CD1 molecules.

The human CD1a–d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM6. CD1e is formed by the association of β2-microglobulin with an α-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM6 presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.

International audience; Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2-12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-alpha, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells "foamy DCs" and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A.

International audience; We retrospectively analyzed the impact of HLA-DPB1 mismatches in a large cohort of 1342 French patients who underwent 10/10 HLA-matched unrelated HSCT. A significant impact of HLA-DPB1 allelic mismatches (2 vs 0) was observed in severe acute GVHD (aGVHDIII-IV) (risk ratio (RR)=1.73, confidence interval (CI) 95% 1.09-2.73, P=0.019) without impact on OS, TRM, relapse and chronic GVHD (cGVHD). According to the T-cell epitope 3 (TCE3)/TCE4 HLA-DPB1 disparity algorithm, 37.6% and 58.4% pairs had nonpermissive HLA-DPB1, respectively. TCE3 and TCE4 disparities had no statistical impact on OS, TRM, relapse, aGVHD and cGVHD. When TCE3/TCE4 disparities were analyzed in the graft-vs-host or host-vs-graft (HVG) direction, only a significant impact of TCE4 nonpermissive disparities in the HVG direction was observed on relapse (RR=1.34, CI 95% 1.00-1.80, P=0.048). In conclusion, this French retrospective study shows an adverse prognosis of HLA-DPB1 mismatches (2 vs 0) on severe aGVHD and of nonpermissive TCE4 HVG disparities on relapse after HLA-matched 10/10 unrelated HSCT.Bone Marrow Transplantation advance online publication, 3 November 2014; doi:10.1038/bmt.2014.253.

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.

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Dendritic cells (DCs) present antigens to T cells via CD1, HLA class I or class II molecules. During maturation, HLA class II-restricted presentation is optimized. The relocalization of CD1e from Golgi to endosomal compartments during DC maturation suggests also an optimization of the antigen-presentation pathway via CD1 molecules. We here detail the biosynthesis and cellular pathway of CD1e in immature and maturing DCs. Unlike the other CD1 molecules, CD1e was found to reach late endosomes through sorting endosomes, without passing through the plasma membrane in either immature or maturing cells. After induction of DC maturation, CD1e disappeared rapidly from the Golgi and was transiently localized in HLA-DR+ vesicles, while the number of CD1e+/CD1b+ compartments increased for at least 20 h. High-resolution light microscopy showed that, in immature DCs, CD1e+ vesicles were often in close apposition to EEA1+ or HLA-DR+ compartments, while CD1e displayed a nearly exclusive distribution in the lysosomes of mature DCs, a finding corroborated by immunoelectron microscopy. During maturation, CD1e synthesis progressively declined, while the endosomal cleavage of CD1e still occurred. Thus, CD1e displays peculiar properties, suggesting an unexpected role among the family of CD1 antigen-presenting molecules.

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

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.

A better understanding of the antigen presentation pathways that lead to CD8 + T cell recognition of HIV epitopes in vivo is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4 + T lymphocytes by dendritic cells to CD8 + T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4 + T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4 + T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFNα immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.

In addition to their essential role in adaptive immunity, dendritic cells (DCs) participate in innate immunity. In the context of measles virus (MV) or cytomegalovirus infections, they develop cytotoxic functions that may contribute in vivo to the elimination of virus-infected cells, but that also kill infected and noninfected T lymphocytes. Because the human immunodeficiency virus (HIV) induces T cell depletion through mechanisms that are still obscure, we investigated its ability to trigger DC cytotoxicity. When incubated with HIV, monocyte-derived DCs induced apoptosis in MDA-231 cells, which are sensitive to MV-induced DC cytotoxicity, and in uninfected as well as HIV-infected H9 CD4+ T cell lines. This apoptosis was inhibited by a mixture of FasL, TRAIL, TNF-alpha, and TWEAK inhibitors. Indeed, HIV infection induced or enhanced sensitivity to TRAIL, TNF-alpha, and TWEAK in H9 cells. Moreover, dendritic cells incubated with HIV-1 BAL or a wildtype HIV-1 isolate induced apoptosis in autologous primary CD4+ T lymphocytes, infected or not with a wild-type HIV-1 isolate. Therefore, induction of DC cytotoxicity by HIV may be relevant to in vivo HIV infection. Induction of cytotoxicity in DCs by HIV might contribute to HIV-associated T cell depletion through induction of apoptosis, especially in the early stages of infection. It may also contribute to elimination of infected cells in vivo, thereby enhancing cross-presentation of HIV by DCs. Therefore this new cytotoxic function of DCs may play an important role in innate and adaptive immunity during HIV infection.

There is growing evidence that dendritic cells, the major antigen-presenting cells and T-cell activators, have a broad effect on peripheral T-cell tolerance and regulation of immunity. Very recently, a new feature of regulatory antigen-presenting cells was observed. Certain dendritic cells, monocytes, and macrophages express the enzyme indoleamine 2,3-dioxygenase, and thus because of enhanced degradation of the essential amino acid tryptophan, they modulate T-cell activity in specific local tissue environments. In this review we discuss the various and apparently disparate effects of indoleamine 2,3-dioxygenase induction in cells of the immune system. We place current knowledge about this mechanism in the context of atopy. We introduce the hypothesis that tryptophan degradation might add to the ability to control and downregulate allergen-specific T-cell responses in atopic individuals.

In humans, a family of five genes encodes the CD1 molecules. Four of these proteins, CD1a, b, c, and d, are expressed on the plasma membrane and traffic between the cell surface and endocytic compartments, where they are loaded with antigenic glycolipids. The existence of human CD1e was demonstrated recently. This molecule surprisingly remains inside the cell, accumulating mainly in the Golgi compartments of immature dendritic cells and in the late endosomes of mature dendritic cells. In the latter compartments, CD1e is cleaved and becomes soluble. To determine whether these properties were specific to human CD1e, we investigated the presence and characteristics of CD1e in the rhesus macaque, an evolutionarily distant species of the primate lineage. Our results show that the cellular and biochemical properties of the human and simian CD1e molecules are similar, suggesting that the particular intracellular distribution of CD1e is important for its physiological and/or immunological function.

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.

FcεRI is suspected to play a pivotal role in the pathophysiology of atopic disorders such as atopic dermatitis. In search for genes differentially regulated by FcεRI on APCs, a differential cDNA bank of receptor-stimulated and unstimulated monocytes was established. By means of suppression subtractive hybridization, we identified kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase (IDO) to be overexpressed in FcεRI-activated monocytes. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. We show that cross-linking of FcεRI on monocytes results in low tryptophan concentrations associated with impaired T cell stimulatory capacity. Importantly, T cell suppression could be prevented by the addition of tryptophan or inhibition of IDO. Moreover, stimulation of T cells by FcεRI-activated monocytes was increased compared with T cell stimulation by nonactivated monocytes if exogenous supply of tryptophan was available. We speculate that the expression of IDO by FcεRI+ APCs in vivo allows these cells to regulate T cell responses in atopic disorders by inhibiting or stimulating T cell proliferation, depending on the metabolic environment.

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.

International audience; Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e,g, phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

Background: Graft-versus-host disease (GVHD) is a major cause of mortality after unrelated hematopoietic stem cell transplantations (HSCT). Despite the development of modern immunosuppressive strategies, a nearly perfectly controlled compatibility of the classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) and availability of numerous so-called minor histocompatibility antigens (e.g. HY or HA-1), its incidence remains largely unexplained to date. MIC genes (MHC class I chain-related) - a distinct lineage of MHC class I genes – are promising candidates to explain, at least partially, the incidence of GVHD in HLA-matched transplantations. MICA and MICB are highly polymorphic (100 alleles for MICA and 40 for MICB) and encode functional cell-surface glycoproteins up-regulated by cell stress. They interact with NKG2D, an activating receptor expressed on the surface of cytotoxic αβ CD8+ and γδ T lymphocytes and natural killer cells. MIC genes are already known to have a HLA-independent effect on solid graft outcomes and may play a similar role in HSCT by triggering GVHD. Objective: The objective of the present study was to determine the impact of donor/patient matching at the MICA and MICB loci on the incidence of GVHD in patients undergoing unrelated HSCT. Methods: We retrospectively analyzed a multicenter cohort of 1072 unrelated transplantations performed between 1996 and 2013. All donor-recipient pairs were fully typed at high resolution for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 and were matched for ten of ten HLA alleles (HLA 10/10 matched). High resolution genotyping of MICA and MICB was performed by sequenced-based typing in order to define matching grades between donors and patients. The endpoints of the study were acute and chronic GVHD. Apart from HLA-DPB1 matching, statistical models were adjusted for major clinical variables which have been shown to be associated with outcome (patient’s age, patient’s and donor’s sex, patient’s and donor’s serological status for cytomegalovirus, year of transplantation, time to transplantation, transplantation center, source of stem cells, conditioning regimen, GVHD prophylaxis, treatment with anti-thymocyte globulin, disease category and severity at transplantation). Results: Of the 1072 transplantations, 134 (12.5 %) and 380 (35.4 %) were mismatched at the MICA and MICB locus, respectively. Both MICA and MICB mismatches were significantly associated with an increased incidence of severe acute GVHD (grades III-IV) in univariate and multivariate models (multivariate model: HR = 2.32, 95 % CI = 1.84-2.92; p=0.0003 for MICA and HR = 1.49, 95 % CI = 1.24-1.79; p=0.03 for MICB). At day 100 post-HSCT severe acute GVHD incidences in mismatched vs. matched transplantations were 19.62 % vs. 15.08 % and 20.00 % vs. 14.84 % for MICA and MICB, respectively (Figure 1). Chronic GVHD was associated with MICA and MICB mismatches in univariate analysis (HR = 1.55, 95 % CI = 1.27-1.89; p=0.029 for MICA and HR=1.38, 95 % CI = 1.19-1.62; p=0.03 for MICB), but showed only a trend for association in multivariate models. Figure 1 Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Figure 1. Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Conclusion: To date this is the largest reported MICA and MICB sequence analysis whether in HSCT or solid organ transplantation. Inclusion of MICA and MICB typing in the donor selection process may be a practical clinical strategy for lowering the risks of severe acute GVHD after unrelated HSCT. Disclosures No relevant conflicts of interest to declare.

The HIV early regulatory Nef protein downregulates surface expression of major histocompatibility class I (MHC I) molecules on various immortalized cell lines and on T lymphocytes. MHC I-restricted presentation induces CD8+ T cell responses, which have a major role in limiting HIV infection. Induction of primary immune responses requires dendritic cells, which are major candidates as the first cells that can internalize the virus and present it to T cells in mucosal contamination. To test the effect of Nef on MHC I-restricted antigen presentation by dendritic cells, we used recombinant vaccinia viruses. Flow cytometric analysis of double labeling for a vaccinia protein and MHC I showed that HIV-1 Lai Nef indeed downregulated MHC I surface expression on dendritic cells. MHC I-restricted presentation to a Nef-specific CD8+ cell clone from an infected patient was decreased in an interferon γ ELISpot assay. Presentation of a reverse transcriptase epitopic peptide on sorted Nef-infected cells was decreased in ...

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.

CD8(+) T lymphocytes, which are major immune effectors, require primary stimulation by dendritic cells (DC) presenting MHC class I molecule-bound epitopes. Sensitization to exogenous protein epitopes that are not synthesized in DC, such as cross-priming, is obtained through pathways leading to their association with MHC class I. To follow class I-restricted pathways in human DC, we have tracked a lipopeptide derived from the conserved HLA-A*0201-restricted HIV-1 reverse transcriptase 476-484 epitope, by N-terminal addition of an Nepsilon-palmytoyl-lysine. Indeed, lipopeptides elicit cytotoxic responses from CD8(+) T lymphocytes, whereas peptides without a lipid moiety do not. The lipopeptide and its parent peptide were labeled unequivocally by rhodamine to study their entry into immature monocyte-derived human DC by confocal microscopy. The lipid moiety induced endocytosis of the lipopeptide, assessed by rapid entry into vesicles, colocalization with Dextran-FITC and dependence on energy. Internalization occurred even when actin filaments were depolymerized by Cytochalasin B. This internalization induced functional stimulation of specific CD8(+) T lymphocytes in IFN-gamma ELISPOT assays. The peptide alone was not visualized inside the DC and was presented through direct surface association to HLA-A*0201. Therefore, lipopeptides are a unique opportunity to define precisely the pathways that lead exogenous proteins to associate with MHC class I molecules in DC. The results will also be useful to design lipopeptide vaccines.

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.

Thrombocytopenia in newborns is often due to maternal alloimmunization against platelet alloantigens of the foetus which have been inherited from the father and are absent in the mother. Our aim was to develop a "ready-to-use" typing kit based on polymerase chain reactions, using sequence-specific primers for rapid and simultaneous genotyping of the eight principal human platelet alloantigens. The typing technique uses two specific primer pairs for each bi-allelic system and a monomorphic primer pair as amplification control, with a single temperature cycle programme and identical PCR stringency conditions for all pairs of primers. This kit allows typing of blood samples of small volume within three hours after reception. Validation criteria are essential to check the reliability and specificity of the test, and DNA controls carrying the targeted HPA alleles must be obtained from typed individuals or created in vitro by PCR.

Background: Recent findings have shown that the surface expression of the high-affinity receptor for IgE (FcϵRI) on human CD1a + Langerhans cells (LC) and related dendritic cells (DC) in the skin, despite a constant intracellular expression of its α chain (FcϵRIα), is highly up-regulated in atopic dermatitis. Moreover, this surface expression correlates with the IgE serum level, strongly suggesting yet-to-be-defined common signals in the regulation of FcϵRI display on LC/DC and IgE synthesis. Objectives: In this study we examined the influence of different cytokines on the expression of FcϵRI on in vitro–generated CD1a + LC/DC. Methods: CD34 + precursor cells were isolated from cord blood with use of high-gradient magnetic cell sorting, cultured with GM-CSF, TNF-α, IL-4, or IFN-γ, and surface and cytoplasmic staining for flow cytometry were performed. Results: IL-4 strongly enhanced the generation of CD1a + LC/DC and also up-regulated the expression of the skin-homing structures E-cadherin and cutaneous lymphocyte antigen. In contrast, IFN-γ was found to suppress the E-cadherin expression and to be a strong antagonist of IL-4 by inhibiting the production of CD1a + cells. Most important, IL-4 induced the cytoplasmic expression of FcϵRIα in CD1a + LC/DC but not its surface expression. This up-regulation was antagonized by IFN-γ. Conclusion: IL-4 is not only a key cytokine in the regulation of IgE but also induces the expression of its receptor binding chain as well as up-regulation of skin homing molecules on LC/DC. Expression of these structures during generation of LC/DC reflects the in vivo situation encountered in LC. (J Allergy Clin Immunol 2000;105:150-6.)

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.

The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I–deficient patients.

The HIV-1 Nef protein down-modulates surface expression of MHC class I proteins. Primary infected T lymphocytes thus escape lysis by cytotoxic T lymphocytes (CTL). In contrast, during HIV-1 infection there are strong CTL responses to several HIV proteins, and there is mounting evidence that CTL are critical for controlling the virus. The present study was carried out to assess Nef protein-cell interaction as it occurs in naturally infected antigen-presenting cells. To evaluate the presentation of peptides derived from viral antigen to CTL, we transfected nef genes obtained from peripheral blood mononuclear cells of HIV-1-seropositive subjects into dendritic cells isolated from monocytes of healthy donors. We demonstrate that expression and subsequent processing of Nef by transfected dendritic cells did not alter the presentation of an immunodominant epitope of Nef to CTL of HIV+ subjects. However, mutations in nef gene sequences from primary isolates may abolish this presentation by a mechanism that probably interferes with protein processing.

We have recently described a system for the generation of dendritic cells (DC) and Langerhans cells (LC) from defined CD34+ precursors purified from peripheral blood of healthy adult volunteers (1). This study has now been extended by the characterization of two distinct subpopulations of CD34+ cells in normal human peripheral blood as defined by the expression of the skin homing receptor cutaneous lymphocyte-associated antigen (CLA). CD34+/CLA+ cells from normal peripheral blood were found to be CD71LOW/CD11a+/CD11b+/CD49d+/ CD45RA+ whereas CD34+/CLA− cells displayed the CD71+/CD11aLOW/CD11bLOW/CD49d(+)/ CD45RALOW phenotype. To determine the differentiation pathways of these two cell populations, CD34+ cells were sorted into CLA+ and CLA− fractions, stimulated with GM-CSF and TNF-α in vitro, and then were cultured for 10 to 18 d. Similar to unfractionated CD34+ cells, the progeny of both cell populations contained sizable numbers (12–22%) of dendritically shaped, CD1a+/HLA-DR+++ cells. In addition to differences in their motility, the two dendritic cell populations generated differed from each other by the expression of LC-specific structures. Only the precursors expressing the skin homing receptor were found to differentiate into LC as evidenced by the presence of Birbeck granules. In contrast, CLA− precursor cells generated a CD1a+ DC population devoid of Birbeck granule–containing LC. Provided that comparable mechanisms as found in this study are also operative in vivo, we postulate that the topographic organization of the DC system is already determined, at least in part, at the progenitor level.

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.

International audience; The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents.

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 recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.

Two siblings with pansinusitis, nasal polyps, and bronchiectasis were found to have histocompatibility lymphocyte antigen (HLA) class I antigen deficiency ("bare lymphocyte syndrome") and dysfunction of natural killer cells. Reduced class I cell surface expression resulted from a single mutation in the TAP2 gene, which is located in the class II region of the major histocompatibility complex and encodes subunit 2 of the class I peptide transporter. The defect was transmitted in an autosomal recessive manner. This deficiency did not lead to severe viral infections but was apparently associated with susceptibility to bacterial infections of the respiratory mucosae. We suggest that class I HLA typing should be systematically performed in children with unexplained bronchiectasis. (J PEDIATR 1995;127:895-900)

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO+/RA-/RB-. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-alpha to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fc epsilon receptor type I (Fc epsilon RI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their Fc epsilon RI-mediated activation.

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

International audience; Lipids are important antigens that induce T cell-mediated specific immune responses. They are presented to T lymphocytes by a specific class of MHC-I like proteins, termed CD1. The majority of the described CD1-presented mycobacterial antigens are presented by the CD1b isoform. We previously demonstrated that the stimulation of CD1b-restricted T cells by the hexamannosylated phosphatidyl-myo-inositol (PIM(6)), a family of mycobacterial antigens, requires a prior partial digestion of the antigen oligomannoside moiety by α-mannosidase and that CD1e is an accessory protein absolutely required for the generation of the lipid immunogenic form. Here, we show that CD1e behaves as a lipid transfer protein influencing lipid immunoediting and membrane transfer of PIM lipids. CD1e selectively assists the α-mannosidase-dependent digestion of PIM(6) species according to their degree of acylation. Moreover, CD1e transfers only diacylated PIM from donor to acceptor liposomes and also from membranes to CD1b. This study provides new insight into the molecular mechanisms by which CD1e contributes to lipid immunoediting and CD1-restricted presentation to T cells.