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 raised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T72(Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas against the glycan moiety was investigated by testing their response against a panel of Hb(67-76)-derived glycopeptides, all with a glycan attached to serine or threonine at the position 72 in the peptide, but with different glycans. The hybridomas showed a high degree of specificity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of these were structurally very similar to alpha-GalNAc. The fine specificity of the hybridomas for the peptide moiety was investigated by testing their responses to a panel of Hb(67-76)-alpha-GalNAc glycopeptides with alanine substitutions at all positions except at the two MHC binding anchor positions, I68 and K76, and the T72 to which the alpha-GalNAc was attached. Glycopeptides substituted with alanine at positions where the amino acid side chain pointed toward the TCR did not stimulate the hybridomas, whereas glycopeptides substituted with alanine at positions orientated down into the MHC binding groove stimulated many of the hybridomas. These results indicate that the glycan attached to the peptide as well as solvent-accessible parts of the peptide are recognized with a high degree of specificity by the T cells, whereas the parts of the peptide buried in the MHC binding site are less important or totally ignored by the T cells.
Jensen
T
T
Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark.
Hansen
P
P
Galli-Stampino
L
L
Mouritsen
S
S
Frische
K
K
Meinjohanns
E
E
Meldal
M
M
Werdelin
O
O
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D005260:Female; D006020:Glycopeptides / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology*; D051379:Mice; D008808:Mice, Inbred CBA; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D000949:Histocompatibility Antigens Class II