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Complex carbohydrates are not removed during processing of glycoproteins by dendritic cells: processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T cells.

Vlad AM, Muller S, Cudic M, Paulsen H, Otvos L, Hanisch FG, Finn OJ - J. Exp. Med. (2002)

Bottom Line: MUC1 is expressed on the surface as well as secreted by human adenocarcinomas.Glycopeptides that are presented on DCs are recognized by T cells.This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Pittsburgh School of Medicine, Biomedical Science Tower, Terrace & DeSoto Streets, Pittsburgh, PA 15261, USA.

ABSTRACT
In contrast to protein antigens, processing of glycoproteins by dendritic cells (DCs) for presentation to T cells has not been well studied. We developed mouse T cell hybridomas to study processing and presentation of the tumor antigen MUC1 as a model glycoprotein. MUC1 is expressed on the surface as well as secreted by human adenocarcinomas. Circulating soluble MUC1 is available for uptake, processing, and presentation by DCs in vivo and better understanding of how that process functions in the case of glycosylated antigens may shed light on antitumor immune responses that could be initiated against this glycoprotein. We show that DCs endocytose MUC1 glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on major histocompatability complex (MHC) class II molecules without removing the carbohydrates. Glycopeptides that are presented on DCs are recognized by T cells. This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

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Related in: MedlinePlus

Inhibition of uptake and processing in endocytic compartments of both long and short MUC1 peptides and glycopeptides. DCs were either briefly fixed in 1% PFA (A, white bars), pretreated with 2 mM sodium azide/100 μM 2 deoxyglucose (B, white bars) or with a protease inhibitors cocktail (C) 100-fold (white bars) or 25-fold diluted (gray bars), 30 min before addition of 20 μg/ml MUC1 peptides and glycopeptides. No inhibitor was added to control DCs (black bars in A–C). In panel B, DCs were pulsed with antigen on ice (gray bars). In panel C, hatched bars represent DMSO control treated DCs. After 6 h of pulse, treated and untreated DCs were incubated with the VF5 cells and IL-2 was measured by ELISA. (D–F) Confocal micrographs of DCs exogenously pulsed with Cy3-labeled (red) 100mer (D), 19mer (E), and 13mer (F) MUC1 peptides for 2 h. During the last 30 min of the pulse period, DCs were also fed BODIPY FL Pepstatin A (green). Surface staining for MHC class II is shown in blue. Overlay sections show area of red-green colocalization (yellow) marked with arrows. (G–I). Confocal micrograph of DCs pulsed with Cy5-labeled 19mer peptide for 2 h and stained after the pulse for extracellular and intracellular I-Ab (green, G). Colocalization of antigen (red, H) and MHC class II in yellow (I).
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fig4: Inhibition of uptake and processing in endocytic compartments of both long and short MUC1 peptides and glycopeptides. DCs were either briefly fixed in 1% PFA (A, white bars), pretreated with 2 mM sodium azide/100 μM 2 deoxyglucose (B, white bars) or with a protease inhibitors cocktail (C) 100-fold (white bars) or 25-fold diluted (gray bars), 30 min before addition of 20 μg/ml MUC1 peptides and glycopeptides. No inhibitor was added to control DCs (black bars in A–C). In panel B, DCs were pulsed with antigen on ice (gray bars). In panel C, hatched bars represent DMSO control treated DCs. After 6 h of pulse, treated and untreated DCs were incubated with the VF5 cells and IL-2 was measured by ELISA. (D–F) Confocal micrographs of DCs exogenously pulsed with Cy3-labeled (red) 100mer (D), 19mer (E), and 13mer (F) MUC1 peptides for 2 h. During the last 30 min of the pulse period, DCs were also fed BODIPY FL Pepstatin A (green). Surface staining for MHC class II is shown in blue. Overlay sections show area of red-green colocalization (yellow) marked with arrows. (G–I). Confocal micrograph of DCs pulsed with Cy5-labeled 19mer peptide for 2 h and stained after the pulse for extracellular and intracellular I-Ab (green, G). Colocalization of antigen (red, H) and MHC class II in yellow (I).

Mentions: While complex protein antigens and long synthetic peptides are expected to require intracellular processing before their presentation on MHC class II, small peptides derived from some antigens (but not others) could directly bind to cell surface MHC class II molecules (34–36) and be presented equally well by fixed and nonfixed APCs (37, 38). We showed earlier in Fig. 2 that MUC1 peptides, regardless of their length, are endocytosed by DCs. In Fig. 4 A we confirm that metabolically active DCs are required for MUC1 processing and presentation to the VF5 hybridoma since DCs fixed before the addition of peptides are not able to elicit a response. Fixing cells after incubation with the peptides did not affect peptide presentation, suggesting that the ability of the DCs to successfully activate T hybridomas was preserved. Similarly, DCs pulsed on ice or pretreated with sodium azide/2 deoxyglucose before antigen pulse to disrupt active endocytosis (34), generated significantly lower IL-2 responses (Fig. 4 B) suggesting that endocytosis is necessary for VF5 epitope presentation.


Complex carbohydrates are not removed during processing of glycoproteins by dendritic cells: processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T cells.

Vlad AM, Muller S, Cudic M, Paulsen H, Otvos L, Hanisch FG, Finn OJ - J. Exp. Med. (2002)

Inhibition of uptake and processing in endocytic compartments of both long and short MUC1 peptides and glycopeptides. DCs were either briefly fixed in 1% PFA (A, white bars), pretreated with 2 mM sodium azide/100 μM 2 deoxyglucose (B, white bars) or with a protease inhibitors cocktail (C) 100-fold (white bars) or 25-fold diluted (gray bars), 30 min before addition of 20 μg/ml MUC1 peptides and glycopeptides. No inhibitor was added to control DCs (black bars in A–C). In panel B, DCs were pulsed with antigen on ice (gray bars). In panel C, hatched bars represent DMSO control treated DCs. After 6 h of pulse, treated and untreated DCs were incubated with the VF5 cells and IL-2 was measured by ELISA. (D–F) Confocal micrographs of DCs exogenously pulsed with Cy3-labeled (red) 100mer (D), 19mer (E), and 13mer (F) MUC1 peptides for 2 h. During the last 30 min of the pulse period, DCs were also fed BODIPY FL Pepstatin A (green). Surface staining for MHC class II is shown in blue. Overlay sections show area of red-green colocalization (yellow) marked with arrows. (G–I). Confocal micrograph of DCs pulsed with Cy5-labeled 19mer peptide for 2 h and stained after the pulse for extracellular and intracellular I-Ab (green, G). Colocalization of antigen (red, H) and MHC class II in yellow (I).
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fig4: Inhibition of uptake and processing in endocytic compartments of both long and short MUC1 peptides and glycopeptides. DCs were either briefly fixed in 1% PFA (A, white bars), pretreated with 2 mM sodium azide/100 μM 2 deoxyglucose (B, white bars) or with a protease inhibitors cocktail (C) 100-fold (white bars) or 25-fold diluted (gray bars), 30 min before addition of 20 μg/ml MUC1 peptides and glycopeptides. No inhibitor was added to control DCs (black bars in A–C). In panel B, DCs were pulsed with antigen on ice (gray bars). In panel C, hatched bars represent DMSO control treated DCs. After 6 h of pulse, treated and untreated DCs were incubated with the VF5 cells and IL-2 was measured by ELISA. (D–F) Confocal micrographs of DCs exogenously pulsed with Cy3-labeled (red) 100mer (D), 19mer (E), and 13mer (F) MUC1 peptides for 2 h. During the last 30 min of the pulse period, DCs were also fed BODIPY FL Pepstatin A (green). Surface staining for MHC class II is shown in blue. Overlay sections show area of red-green colocalization (yellow) marked with arrows. (G–I). Confocal micrograph of DCs pulsed with Cy5-labeled 19mer peptide for 2 h and stained after the pulse for extracellular and intracellular I-Ab (green, G). Colocalization of antigen (red, H) and MHC class II in yellow (I).
Mentions: While complex protein antigens and long synthetic peptides are expected to require intracellular processing before their presentation on MHC class II, small peptides derived from some antigens (but not others) could directly bind to cell surface MHC class II molecules (34–36) and be presented equally well by fixed and nonfixed APCs (37, 38). We showed earlier in Fig. 2 that MUC1 peptides, regardless of their length, are endocytosed by DCs. In Fig. 4 A we confirm that metabolically active DCs are required for MUC1 processing and presentation to the VF5 hybridoma since DCs fixed before the addition of peptides are not able to elicit a response. Fixing cells after incubation with the peptides did not affect peptide presentation, suggesting that the ability of the DCs to successfully activate T hybridomas was preserved. Similarly, DCs pulsed on ice or pretreated with sodium azide/2 deoxyglucose before antigen pulse to disrupt active endocytosis (34), generated significantly lower IL-2 responses (Fig. 4 B) suggesting that endocytosis is necessary for VF5 epitope presentation.

Bottom Line: MUC1 is expressed on the surface as well as secreted by human adenocarcinomas.Glycopeptides that are presented on DCs are recognized by T cells.This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Pittsburgh School of Medicine, Biomedical Science Tower, Terrace & DeSoto Streets, Pittsburgh, PA 15261, USA.

ABSTRACT
In contrast to protein antigens, processing of glycoproteins by dendritic cells (DCs) for presentation to T cells has not been well studied. We developed mouse T cell hybridomas to study processing and presentation of the tumor antigen MUC1 as a model glycoprotein. MUC1 is expressed on the surface as well as secreted by human adenocarcinomas. Circulating soluble MUC1 is available for uptake, processing, and presentation by DCs in vivo and better understanding of how that process functions in the case of glycosylated antigens may shed light on antitumor immune responses that could be initiated against this glycoprotein. We show that DCs endocytose MUC1 glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on major histocompatability complex (MHC) class II molecules without removing the carbohydrates. Glycopeptides that are presented on DCs are recognized by T cells. This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

Show MeSH
Related in: MedlinePlus