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 Ek 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 Ek, 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 Ek and for immunogenicity in CBA/J mice. All 16 glycopeptides bound well to Ek, 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.
Supported by a grant from the Danish Cancer Society and in part by EC Science Program MM Grant SCI*-CT92-0765.