CARBOHYDRATE AND PEPTIDE SPECIFICITY OF MHC CLASS II-RESTRICTED T-CELL HYBRIDOMAS RAISED AGAINST AN O-GLYCOSYLATED SELF-PEPTIDE

MHC class II E(k)-restricted, IL-2 secreting T cell hybridomas were ra ised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T-72( Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas a gainst the glycan moiety was investigated by testing their response ag ainst a panel of Hb(67-76)-derived glycopeptides, all with a glycan at tached to serine or threonine at the position 72 in the peptide, but w ith different glycans. The hybridomas showed a high degree of specific ity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of th ese were structurally very similar to alpha-GalNAc. The fine specifici ty of the hybridomas for the peptide moiety was investigated by testin g their responses to a panel of Hb(67-76)-alpha-GalNAc glycopeptides w ith alanine substitutions at all positions except at the two MHC bindi ng anchor positions, I-68 and K-76 and the T-72 to which the alpha-Gal NAc was attached. Glycopeptides substituted with alanine at positions where the amino acid side chain pointed toward the TCR did not stimula te the hybridomas, whereas glycopeptides substituted with alanine at p ositions orientated down into the MHC binding groove stimulated many o f the hybridomas. These results indicate that the glycan attached to t he peptide as well as solvent-accessible parts of the peptide are reco gnized with a high degree of specificity by the T cells, whereas the p arts of the peptide buried in the MHC binding site are less important or totally ignored by the T cells.