Synthetic Peptides with Inadvertent Chemical Modifications Can Activate Potentially Autoreactive T Cells.

The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161-170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.

Binding interactions with sevelamer and polystyrene sulfonate in vitro.

This study explored the binding of 28 drugs, which were selected based on frequency of concomitant use and chemical properties, to sevelamer and polystyrene sulfonate in vitro. The relative binding was determined by dissolving the investigated drugs alone (=control), together with 800 mg of sevelamer and 15 g of polystyrene sulfonate at different pH levels (1.5, 5.5, and 7.4), respectively. After incubation at 37℃ and shaking for 60 min, the solutions were diluted and centrifuged, and the drug concentrations were quantified with validated analytical assays. The binding assays were performed in threefold. The mean relative binding (MRB) at each pH level was calculated, with a MRB >20% for at least one pH level to be considered as relevant binding. Fourteen and 23 potentially new binding interactions were identified with sevelamer and polystyrene sulfonate, respectively. These potentially new binding interactions have to be studied in vivo to assess their clinical relevance.