Dendritic cells (DC) that express the type II C-type lectin DC-SIGN (CD209) are located in the submucosa of tissues, where they mediate HIV-1 entry. Interestingly, the pathogen Candida albicans, the major cause of hospital-acquired fungal infections, penetrates at similar submucosal sites. Here we demonstrate that DC-SIGN is able to bind C. albicans both in DC-SIGN-transfected cell lines and in human monocyte-derived DC. The binding was shown to be time-as well as concentration-dependent, and live as well as heat-inactivated C. albicans were bound to the same extent. Moreover, in immature DC, DC-SIGN was able to internalize C. albicans in specific DC-SIGN-enriched vesicles, distinct from those containing the mannose receptor, the other known C. albicans receptor expressed by DC. Together, these results demonstrate that DC-SIGN is an exquisite pathogen-uptake receptor that captures not only viruses but also fungi.

The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLHpulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1- KLH-targeted IntroductionDendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a key role in regulating antigen-specific immunity. DCs capture antigens, process them into peptides, and present these to T cells. 1 The interaction between DC and T-cell controls the type and magnitude of the resulting immune response. Recently, preclinical and clinical studies have exploited DCs in an attempt to improve vaccine efficacy. 2 Most of these studies involve ex vivo antigen loading of autologous monocyte-derived DCs that are readministrated to the patient, a laborious and costly procedure. A more direct strategy involves targeting of antigens specifically to antigen uptake receptors on the DC in vivo. Potential candidate receptors highly expressed by DCs include Fc receptors [3][4][5] and members of the C-type lectin family. 6,7 Whereas Fc receptors are expressed by many different cell types, the expression of some members of the C-type lectin family are more DC restricted. 8 C-type lectins bind sugar residues in a calcium-dependent manner via a highly conserved carbohydrate recognition domain. C-type lectin receptors expressed by DCs are implicated in immunoregulatory processes, such as antigen capture, DC trafficking, and DC-T-cell interactions. 8 Based on the location of the amino (N) terminus, 2 types of membrane-bound C-type lectins can be distinguished on DCs. Type I C-type lectins have their N terminus located outside, while type II C-type lectins have their N terminus located inside the cell. Several studies have been conducted on antigen targeting to C-type lectin receptors for vaccination purposes, mainly focusing on the type I C-type lectins mannose receptor (MR) 9 and DEC-205. 6,10,11 Vaccines based on natural MR ligands have been shown to effectively induce humoral and cellular responses. 9 However, these ligands lack specificity for the MR, and may target multiple lectins with overlapping binding sp...

IntroductionThe interaction between a T cell and an antigen-presenting cell (APC) is the central event in the induction of an adaptive immune response and involves different and sequential cellular events. Initially, the T cell transiently adheres to the APC and scans its surface for the presence of specific peptide-major histocompatibility (MHC) complexes in an antigen-independent manner. Several receptor-ligand pairs such as CD2/lymphocyte function-associated antigen-3 (LFA-3), LFA-1/intercellular adhesion molecule-1 (ICAM-1) and ICAM-3, and ICAM-3/dendritic cell (DC)-specific ICAM-3-grabbing nonintegrin (DC-SIGN) have been implicated in these early T-cell-APC interactions. 1 In particular, ICAM-1 and -3 play a key role in mediating the initial, antigen-independent adhesion of T cells and APC. 1,2 Once the initial contact has been generated, the contact interface is stabilized by molecular reorganization of antigen receptors, adhesion molecules, costimulatory molecules, and the actin cytoskeleton. This highly organized supramolecular structure is known as the immunological synapse. 3 Adhesion molecules and T-cell-receptor (TCR)-associated components are segregated into 2 major areas within the immunological synapse: the central supramolecular activation cluster (SMAC), which is enriched in TCR/CD3 complexes, costimulatory molecules (CD4, CD2, CD28), and kinases (protein kinase C-[PKC-], Lck, and Fyn), and the peripheral SMAC, including LFA-1 and talin. 4,5 Synapse formation is accompanied by cytoskeletal rearrangements, induction of tyrosine phosphorylation and an increase in intracellular free Ca 2ϩ . Upon Ca 2ϩ mobilization, the nuclear factor of activated T cells (NFAT) is dephosphorylated and translocates to the nucleus, where it acts as a transcriptional regulator of interleukin-2 (IL-2) expression. 6 Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin (Ig) superfamily of proteins. 7 Although ALCAM is expressed on a wide variety of cells, within the leukocyte population its expression is particularly high on DC. In addition, monocytic cells in synovium from patients with rheumatoid arthritis show strongly increased ALCAM levels compared with resting monocytes, suggesting that ALCAM is involved in regulating immunologic processes such as inflammation. 8 However, the precise role of ALCAM in the immune system is as yet unclear. Similar to several other Ig-like adhesion molecules (NCAM, CEA), ALCAM mediates homotypic ALCAM-ALCAM interactions, 7,9,10 but also heterotypic interactions with the T-cell antigen CD6 have been described. 7 CD6 is a surface receptor expressed by T lymphocytes, thymocytes, and a subset of B cells. [11][12][13] 18 It has been suggested that CD6 fine-tunes CD5 tyrosine phosphorylation by recruiting specific kinases of different families, such as Itk and Lck. 19 CD6 physically associates with the TCR/CD3 complex, it relocalizes upon T-cell activation at the central SMAC (cSMAC) and it modulates immunological synapse maturation in a Jurkat-Raji mo...