Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised th at prevent/suppress an anti-RTA immune response in patients treated with RT A-based immunotoxins. RTA-specific human T-cell lines and T-cell clones wer e produced by in vitro priming of PBMC. The T-cell clones used a limited se t of V beta chains (V beta1, V beta2 and V beta8) to recognize RTA epitopes . The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The resp onse to RTA peptides of T-cell lines and T-cell clones from two donors allo wed the identification of immunogenic segments (D124-G140 and L161-T190) re cognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and H LA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was in vestigated in greater detail. We found that the HLA-DRB1*03011 allele prese nts a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA pept ides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showin g sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1 *11011/03011 alleles.