T-Cell Recognition of Tumor-associated Carbohydrates: The Nature of the Glycan Moiety Plays a Decisive Role in Determining Glycopeptide Immunogenicity¹

Aberrant glycosylation is one of the most constant traits of the malignant cell phenotype. To study T-cell responses to tumor-associated glycans, the mouse hemoglobin-derived decapeptide Hb(67–76), which binds well to the MHC class II molecule E^k and is nonimmunogenic in CBA/J mice, was either O- or N-glycosylated at its primary T-cell receptor contact residue, position 72, with different glycans attached to either threonine, serine, or asparagine. The carbohydrate moieties included tumor-associated mucins, i.e., the Tn and T antigens, mucin-related glycans, and mucin-unrelated glycans. The side chain of the amino acid in position 72 points away from the MHC binding site when the Hb(67–76) peptide is bound to E^k, so the assumption was that this was also the case for glycans attached to this position. The glycosylated Hb(67–76) peptide analogues were then studied for binding to E^k and for immunogenicity in CBA/J mice. All 16 glycopeptides bound well to E^k, although those with the more complex carbohydrates bound more weakly than those with monosaccharides. Six of 12 O-glycosylated and 0 of 4 N-glycosylated glycopeptides were able to induce a T-cell proliferative response with a stimulation index above 3.0. Some glycopeptides were not immunogenic, suggesting that there may be holes in the T-cell repertoire due to a lack of T-cell receptor regions accommodating certain glycan structures. The four strongest immunogenic glycopeptides were all O-glycosylated, and interestingly, three of them carried the tumor-associated Tn or T antigen. On the other hand, the Hb(67–76) peptide analogue with the natural mucin Core2 structure attached did not elicit any T-cell response. T cells primed to a glycopeptide with a simple glycan structure such as Tn did not cross-respond significantly to other glycopeptides, indicating a high degree of carbohydrate specificity in T-cell recognition. T cells primed to a glycopeptide carrying the more complex T antigen showed a complicated pattern of cross-responses to glycopeptides with simpler glycan moieties. The fact that it is possible to raise MHC class II-restricted T-cell responses against tumor-associated carbohydrate structures opens new perspectives for the designing of cancer vaccines.