RESUMEN
Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α-N-acetylgalactosamine (GalNAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal-regulated kinase 1,2 (ERK1,2) and nuclear factor-κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen-presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen-specific CD8(+) T-cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos/fisiología , Sistema de Señalización de MAP Quinasas/inmunología , Acetilglucosamina/inmunología , Acetilglucosamina/metabolismo , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/metabolismo , Calcio/inmunología , Calcio/metabolismo , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mucina-1/inmunología , Mucina-1/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.