Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells

Abstract

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcA1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines. [Cancer Res 2007;67(17):8358-67]

Targeting of macrophage galactose‐type C‐type lectin (MGL) induces DC signaling and activation

“…The use of synthetic Tn-TAAs for DC targeting represents a promising approach and our model of glycopeptide, MUC1 9Tn , represents here the "proof of concept" of this strategy. We believe and speculate that modifying Tn-density, the length and steric structure of the Tn-peptide, we can possibly obtain immunogens that can efficiently bind to MGL, strongly activate DCs, mimic the effects of a danger signal, and achieve an efficient presentation in HLA class I and II pathways as previously described [18]. Moreover, a wide variety of immunogens carrying Tn-epitopes could be envisaged, where the Tn-peptide stretch could only be a way to deliver other TAAs as well as other molecules.…”

“…Our previous study, conducted in human setting, demonstrated that the overall structure of Tn-carrying molecules internalized by this receptor highly influenced the antigen processing. In fact, recombinant Tnglycoproteins resembling those shed by tumors remained blocked in HLA class II compartment after internalization, while shorter Tn-antigen formulations were processed in HLA class I and II compartments [18].…”

“…We used two different types of ligands: an anti-MGL antibody as a classical model of specific receptor binder and MUC1 9Tn peptide as Tn-carrying TAA that we previously demonstrated to be cross-processed to HLA class I [18]. First, we analyzed DC phenotype changes induced by MGL engagement.…”

“…MUC1-TAAs carry shortened O-glycans such as GalNAcα1-O-Ser/Thr (Tn) or Galβ1-3GalNAcα1-O-Ser/Thr (T) which may be sialylated [20]. We previously demonstrated that DCs were able to bind to MUC1 9Tn glycopeptide (3TRs corresponding to 60 amino acids carrying nine Tn moles) through MGL and this interaction favored the Tn-antigen processing in human histocompatibility leukocyte Ag (HLA) class I and II compartments [18], suggesting its potential application in ex vivo DC-based vaccination.In this work, we have investigated the effects of MGL targeting on the phenotype and function of ex vivo-generated DC populations, in order to understand whether this receptor could be used as molecular target to activate a potent CD8 + T-cell activation for cell therapy. The ligands chosen for this study were the MUC1 9Tn peptide as a model of a Tn-carrying TAA and an anti-MGL antibody as a specific MGL binder.…”

“…Our previous study, conducted in human setting, demonstrated that the overall structure of Tn-carrying molecules internalized by this receptor highly influenced the antigen processing. In fact, recombinant Tnglycoproteins resembling those shed by tumors remained blocked in HLA class II compartment after internalization, while shorter Tn-antigen formulations were processed in HLA class I and II compartments [18].Since MGL is a strong binder for Tn-carrying molecular structures, such as Tn-TAAs, and is expressed on human DCs during differentiation, we were prompted to further investigate the role of this specific targeting receptor on DC performance, envisaging possible application in cancer vaccines.We used two different types of ligands: an anti-MGL antibody as a classical model of specific receptor binder and MUC1 9Tn peptide as Tn-carrying TAA that we previously demonstrated to be cross-processed to HLA class I [18]. First, we analyzed DC phenotype changes induced by MGL engagement.…”