The generation of pathogen-specific immune responses is dependent on the signaling capabilities of pathogen-recognition receptors. DC-SIGN is a C-type lectin that mediates capture and internalization of viral, bacterial, and fungal pathogens by myeloid dendritic cells. DC-SIGN–interacting pathogens are thought to modulate dendritic cell maturation by interfering with intracellular signaling from Toll-like receptor molecules. We report that engagement of DC-SIGN by specific antibodies does not promote dendritic cell maturation but induces ERK1/2 and Akt phosphorylation without concomitant p38MAPK activation. DC-SIGN ligation also triggers PLCγ phosphorylation and transient increases in intracellular calcium in dendritic cells. In agreement with its signaling capabilities, a fraction of DC-SIGN molecules partitions within lipid raft–enriched membrane fractions both in DC-SIGN–transfected and dendritic cells. Moreover, DC-SIGN in dendritic cells coprecipitates with the tyrosine kinases Lyn and Syk. The relevance of the DC-SIGN–initiated signals was demonstrated in monocyte-derived dendritic cells, as DC-SIGN cross-linking synergizes with TNF-α for IL-10 release and enhances the production of LPS-induced IL-10. These results demonstrate that DC-SIGN–triggered intracellular signals modulate dendritic cell maturation. Since pathogens stimulate Th2 responses via preferential activation of ERK1/2, these results provide a molecular explanation for the ability of DC-SIGN–interacting pathogens to preferentially evoke Th2-type immune responses.

CD38, a surface glycoprotein of unrestricted lineage, is an ectoenzyme (adenosine diphosphate [ADP] ribosyl cyclase/cyclic ADP-ribose hydrolase) that regulates cytoplasmic calcium. The molecule also performs as a receptor, modulating cell-cell interactions and delivering transmembrane signals, despite showing a structural ineptitude to the scope. CD38 ligation by agonistic monoclonal antibodies induced signals leading to activation of the lytic machinery of natural killer (NK) cells from adults; similar signals could not be reproduced in YT and NKL, 2 CD16⁻ human NK-like lines. It was hypothesized that CD38 establishes a functional cooperation with professional signaling molecules of the NK cell surface. The present work answers the question about the molecule exploited by CD38 for signaling in NK cells, using as a model CD16⁻ NK lines genetically corrected for CD16 expression. Our results indicate that a functional CD16 molecule is a necessary and sufficient requisite for CD38 to control an activation pathway, which includes calcium fluxes, tyrosine phosphorylation of ZAP70 and mitogen-activated protein kinase, secretion of interferon-γ, and cytotoxic responses. Fluorescence resonance energy transfer and cocapping experiments also showed a surface proximity between CD38 and CD16. These results were confirmed by using the NKL cell line, in which CD16⁺ and CD16⁻ variants were obtained without genetic manipulation. Together, our findings show CD38 to be a unique receptor molecule that cannot signal by itself but whose receptor function is rescued by functional and physical associations with a professional signaling structure that varies according to lineage and environment. This molecule is CD16 in NK cells.

Liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin [CLEC4G]) is a C-type lectin encoded within the liver/lymph node–specific intercellular adhesion molecule-3–grabbing nonintegrin (L-SIGN)/dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN)/CD23 gene cluster. LSECtin expression has been previously described as restricted to sinusoidal endothelial cells of the liver and lymph node. We now report LSECtin expression in human peripheral blood and thymic dendritic cells isolated ex vivo. LSECtin is also detected in monocyte-derived macrophages and dendritic cells at the RNA and protein level. In vitro, interleukin-4 (IL-4) induces the expression of 3 LSECtin alternatively spliced isoforms, including a potentially soluble form (Δ2 isoform) and a shorter version of the prototypic molecule (Δ3/4 isoform). LSECtin functions as a pathogen receptor, because its expression confers Ebola virus–binding capacity to leukemic cells. Sugar-binding studies indicate that LSECtin specifically recognizes N-acetyl-glucosamine, whereas no LSECtin binding to Mannan- or N-acetyl-galactosamine–containing matrices are observed. Antibody or ligand-mediated engagement triggers a rapid internalization of LSECtin,which is dependent on tyrosine and diglutamic-containing motifs within the cytoplasmic tail. Therefore, LSECtin is a pathogen-associated molecular pattern receptor in human myeloid cells. In addition, our results suggest that LSECtin participates in antigen uptake and internalization, and might be a suitable target molecule in vaccination strategies.

Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca²⁺ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca²⁺, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-ζ and FcεRIγ chains, ζ-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-γ and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (FcγRIIIA); moreover, Ca²⁺ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.

During immunologic synapse (IS) formation, human CD38 redistributes to the contact area of T cell–antigen-presenting cell (APC) conjugates in an antigen-dependent manner. Confocal microscopy showed that CD38 preferentially accumulated along the contact zone, whereas CD3-ζ redistributed toward the central zone of the IS. APC conjugates with human T cells or B cells transiently expressing CD38–green fluorescent protein revealed the presence of 2 distinct pools of CD38, one localized at the cell membrane and the other in recycling endosomes. Both pools were recruited to the T/APC contact sites and required antigen-pulsed APCs. The process appeared more efficient in T cells than in APCs. CD38 was actively recruited at the IS of T cells by means of Lck-mediated signals. Overexpression of CD38 in T cells increased the levels of antigen-induced intracellular calcium release. Opposite results were obtained by down-regulating surface CD38 expression by means of CD38 siRNA. CD38 blockade in influenza HA-specific T cells inhibited IL-2 and IFN-γ production, PKCθ phosphorylation at Thr538, and PKCθ recruitment to the IS induced by antigen-pulsed APCs. These results reveal a new role for CD38 in modulating antigen-mediated T-cell responses during IS formation.