Diagnostic relevance of Langerin detection in cells from bronchoalveolar lavage of patients with pulmonary Langerhans cell histiocytosis,sarcoidosis and idiopathic pulmonary fibrosis

The diagnosis of pulmonary Langerhans cell histiocytosis might be refined by demonstrating reliability of a new cell marker, i.e., Langerin (CD207), used on bronchoalveolar lavage fluid. For this purpose, we collected material from patients with this disease and also with sarcoidosis and idiopathic pulmonary fibrosis as controls. In addition to the immunocytochemical detection of Langerin, we examined the expression profiles of CD1a and the macrophage tandem-repeat mannose receptor (CD206). To test accessibility of Langerin, a C-type lectin, for mannosides, we employed reverse lectin histochemistry using mannose-containing neoglycoproteins. The analysis revealed a significantly increased percentage of CD1a- and Langerin-positive cells in pulmonary Langerhans cell histiocytosis in comparison with both other studied diseases. No expression of the 175-kDa mannose-binding lectin (CD206) in Langerhans cells was observed. Evidently, binding sites on the cells were not accessible for the mannose-containing neoglycoligand. These results provide evidence for the usefulness of Langerin-directed immuno- and glycohistochemical monitoring of bronchoalveolar lavage fluid in the diagnosis of pulmonary Langerhans cell histiocytosis.     

Roles of lymphoid cells in the differentiation of Langerhans dendritic cells in mice

Langerhans dendritic cells are antigen presenting cells (APC) that reside within the epidermis and are capable of stimulating naive T cells. Reciprocally, lymphocytes may play a role in Langerhans cells (LC) differentiation. Our results show that the differentiation of skin LC is unaffected in the absence of lymphocytes and/or signaling through the common cytokine receptor gamma chain (gammac) required for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 signaling. Migration of LC and other dendritic cells (DC) from the skin to the draining lymph nodes (LNs) after FITC skin sensitization, is unaffected in the absence of lymphocytes or CD40. FITC+ LC/DC sorted from the LNs of lymphoid deficient or control mice stimulated naive T cells with similar efficiency. However, while the absence of lymphocytes did not appear to affect the phenotype or number of emigrating LN DC/LC, their persistence in the LN appears to depend on alphabeta T cells. Thus, DC are strikingly reduced in numbers in the peripheral LNs of T-cell deficient mice. Finally, CD8alpha expression on skin emigrants was low and dependent on the presence of CD8+ lymphocytes, while spleen CD8+ DC were present in the absence of lymphocytes. We conclude that the presence of T cells is not required for the differentiation and migration of resident skin DC but is critical for the maintenance of DC and LC migrating into the LNs.     

DC-SIGN specifically recognizes Streptococcus pneumoniae serotypes 3 and 14

The Gram-positive bacterium Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia. The ever-increasing frequency of antibiotic-resistant S. pneumoniae strains severely hampers effective treatments. Thus, a better understanding of the mechanisms involved in the pathogenesis of pneumococcal disease is needed; in particular, of the initial interactions that take place between the host and the bacterium. Recognition of pathogens by dendritic cells is one of the most crucial steps in the induction of an immune response. For efficient pathogen recognition, dendritic cells express various kinds of receptors, including the DC-specific C-type lectin DC-SIGN. Pathogens such as Mycobacterium tuberculosis and HIV target DC-SIGN to escape immunity. Here the in vitro binding of DC-SIGN with S. pneumoniae was investigated. DC-SIGN specifically interacts with S. pneumoniae serotype 3 and 14 in contrast to other serotypes such as 19F. While the data described here suggest that DC-SIGN interacts with S. pneumoniae serotype 14 through a ligand expressed by the capsular polysaccharide, the binding to S. pneumoniae serotype 3 appears to depend on an as yet unidentified ligand. Despite the binding capacity of the capsular polysaccharide of S. pneumoniae 14 to DC-SIGN, no immunomodulatory effects on the dendritic cells were observed. The immunological consequences of the serotype-specific capacity to interact with DC-SIGN should be further explored and might result in new insights in the development of new and more potent vaccines.     

Effects of recombinant human interleukin-3 on CD34-enriched normal hematopoietic progenitors and on myeloblastic leukemia cells

Induction of proliferation and differentiation in response to recombinant human interleukin-3 (hIL-3) was studied in liquid and semisolid cultures of umbilical cord blood and bone marrow cells that were fractionated by "panning" with anti-My10 antibody according to expression of CD34 antigen. Cells from enriched fractions (70% to 90% CD34+) were found to proliferate strongly in response to hIL-3. Phenotypic analysis and morphologic characterization of the proliferating cells demonstrated a rapid decrease in CD34+ cells and an exponential increase in the number of cells belonging to the neutrophilic, eosinophilic, monocyte/macrophage, and thrombocytic lineages. When combined with recombinant human erythropoietin, burst colonies and cells expressing glycophorin-A were detected, thereby demonstrating the effects of hIL-3 on erythroid progenitors. Further, the development of mixed-erythroid colonies indicated that multipotential cells within CD34-enriched fractions responded to hIL-3. In addition, we examined the effect of hIL-3 on the proliferation of primary acute myeloblastic leukemia cells in liquid culture. We found that hIL-3 was able to induce cell proliferation in a proportion of the cases tested. Heterogeneity of the responses to hIL-3 was in part related to French-American-British classification but could not be correlated with CD34 antigen expression by the leukemic cells. These results indicate that, although the effects of hIL-3 on proliferation and differentiation of cells obtained from normal hematopoietic specimens were primarily borne by CD34+ cells, expression of the CD34 molecule per se is an insufficient condition to determine a growth response to this lymphokine.     

Design of neo-glycoconjugates that target the mannose receptor and enhance TLR-independent cross-presentation and Th1 polarization

Cross-presentation is an important mechanism by which DCs present exogenous antigens on MHC-I molecules, and activate CD8(+) T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross-presentation of tumor antigens and Th polarization, processes that are pivotal for the anti-tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3-sulfo-Lewis(A) and tri-GlcNAc (N-acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3-sulfo-Lewis(A) or tri-GlcNAc specifically directs antigen to the MR. Both neo-glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross-presentation. Using MR(-/-) and MyD88-TRIFF(-/-) bone marrow-derived DCs (BMDCs), we show that the cross-presentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signaling. Whereas proliferation of antigen-specific CD4(+) T cells was unchanged, stimulation with neo-glycoconjugate-loaded DCs enhanced the generation of IFN-γ-producing T cells. We conclude that modification of antigen with either 3-sulfo-Lewis(A) or tri-GlcNAc enhances cross-presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC-based vaccination strategies to treat cancer.     

Vaginal epithelial dendritic cells renew from bone marrow precursors

Dendritic cells (DCs) represent key professional antigen-presenting cells capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by nonmonocyte bone-marrow-derived precursors, with a half-life of 13 days under steady-state conditions. Upon infection with HSV-2, the Gr-1(hi) monocytes were found to give rise to VEDCs. Furthermore, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b(+)F4/80(hi), CD11b(+)F4/80(int), and CD11b(-)F4/80(-). Importantly, these VEDC populations expressed CD207 at low levels and had a constitutively more activated phenotype compared with the skin LCs. Collectively, our results revealed mucosa-specific features of the VEDCs with respect to their phenotype, activation status, and homeostatic renewal potential.     

Long-term deposition of inhaled antigen in lung resident CD11b-CD11c+ cells

In this study we report the characterization of a population of lung resident CD11b(-)CD11c(+) cells that are able to take up inhaled antigen and retain it for extended periods of time. Ovalbumin conjugated to fluorescein-isothiocyanate (FITC-OVA) administered intranasally to mice was taken up by two main populations of cells in the lung, a migratory CD11c(+)CD11b(+) population consisting of dendritic cells (DC), which rapidly transported antigen to the draining lymph node (LN), and a resident CD11b(-)CD11c(+) population that retained engulfed antigen without apparently degrading it for up to 8 wk after administration. The FITC(+)CD11b(-)CD11c(+) cells did not migrate to draining LN at a detectable rate, and did not up-regulate expression of costimulatory molecules in response to LPS treatment. FITC(+)CD11b(-)CD11c(+) cells were found in the lung and bronchoalveolar lavage fluid, and their distribution was compatible with macrophages. Although FITC(+)CD11b(-)CD11c(+) cells expressed the DC marker DEC205 and other molecules associated with antigen-presenting cell function, they did not induce proliferation of antigen-specific CD4(+) T cells in vitro or acute cytokine production by activated CD4(+) T cells in vivo. Thus, FITC(+)CD11b(-)CD11c(+) cells appear to represent an intermediate cell type sharing properties with DC and macrophages. These cells may have a role in modulating the responses of lung resident T cells to inhaled antigens.     

Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti- CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.     

Human CD34+ CD11b- cord blood stem cells generate in vitro a CD34- CD11b+ subset that is enriched in langerin+ Langerhans dendritic cell precursors

OBJECTIVE: We investigated whether the expression of CD11b on precursors derived in vitro from CD34+ hematopoietic stem cells was related to their ability to generate CD11b- and CD11b+ Langerhans dendritic cells (LC). METHODS: Human CD34+ cells purified from cord blood were cultured with FLT3 ligand, thrombopoietin, and stem cell factor (FTS) for 2 weeks, analyzed, and sorted by FACS. Sorted fractions were cultured as above, or differentiated into LC with GM-CSF, IL-4, and TGF-beta1 (G4-TGF) for 6 days. The capacity of LC to internalize langerin and dextran was assessed. RESULTS: Ex vivo, human CD34+ cells were CD11b- and mostly CLA+. After 2 weeks of culture with FTS, CD34- CLA- CD11b- and CD34- CLA- CD11b+ cells emerged. CD11b- cells were the most ancestral because they were the only ones to proliferate with FTS, and constantly generated CD11b+ cells. Both CD11b- and CD11b+ sorted cells generated E-cadherin+ langerin+ LC after incubation with G4-TGF. The former fraction contained 46% +/- 15% of E-cadherin+ and 10% +/- 5% of langerin+ cells, whereas in the latter fraction these values reached respectively 66% +/- 23% and 30% +/- 16% (mean +/- SD, n = 7, p < 0.056). Looking at functional properties, CD11b- and CD11b+ LC were similar in terms of langerin and dextran endocytosis. By contrast, only CD11b+ LC internalized fluorescent LPS. CONCLUSION: Human CD34+ CD11b- cells differentiate in FTS culture into a CD34- CD11b- precursor that in turn generates CD34- CD11b+ cells. These cells are enriched in LC precursors compared to CD34- CD11b- cells. Both CD11b- and CD11b+ LC are generated in vitro, and each fraction may assume different functions in inflammatory situations.     

Langerin: a new lectin specific for Langerhans cells induces the formation of Birbeck granules

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.