Nature of the ligand recognized by a hapten- and carrier-specific, MHC-restricted T cell receptor.

The hapten- and carrier-specific T lymphocyte clone D5 and a T hybridoma (D5h) derived from D5 cells recognize several different protein Ag conjugated with p-azobenzenearsonate (arsonate) presented by the class II MHC protein I-Ad. We show here that the ligand recognized by the D5 TCR is a complex of a haptenated peptide bound to I-Ad. We have identified a peptide fragment generated by enzymatic cleavage of arsonate-conjugated OVA (Ars-OVA), which stimulates D5 cells when presented by I-Ad-bearing APC. A synthetic peptide corresponding to this fragment, OVA(36-50), forms a ligand for D5h cells when it is conjugated with arsonate and presented by cells bearing I-Ad. Paraformaldehyde-fixed, I-Ad-bearing cells present Ars-OVA(36-50), or the longer stimulatory peptide Ars-OVA(33-49), to D5h cells, demonstrating that haptenated synthetic peptides can substitute for naturally processed antigenic peptides. The peptide Ars-OVA(33-49) binds to the major peptide-binding site of I-Ad because it competitively inhibited presentation of the peptide OVA(323-339), previously demonstrated to bind to I-Ad directly in vitro, to the OVA/I-Ad-specific T cell hybridoma 3DO-54.8. The unconjugated OVA(33-49) peptide failed to inhibit the presentation of OVA(323-339), demonstrating that the hapten facilities binding of the peptide to I-Ad. Conversely, the peptide OVA(323-339) competitively inhibited the presentation of Ars-OVA(33-49) to D5h cells, indicating that the two peptides Ars-OVA(33-49) and OVA(323-339) bind to overlapping sites on I-Ad. Amino acid substitutions introduced into the beta 1 domain of I-Ad that affected recognition of OVA(323-339) by 3DO-54.8 cells also affected recognition of Ars-OVA(33-50) by D5h cells, demonstrating that similar regions on I-Ad are required for TCR recognition of conventional as well as haptenated peptides. These results represent the first demonstration that the ligand recognized by a hapten- and carrier-specific T cell clone restricted to an MHC class II protein is a haptenated peptide Ag bound to the MHC molecule.