Impact of Cysteine Residues on MHC Binding Predictions and Recognition by Tumor-Reactive T Cells

The availability of MHC-binding prediction tools has been useful in guiding studies aimed at identifying candidate target Ags to generate reactive T cells and to characterize viral and tumor-reactive T cells. Nevertheless, prediction algorithms appear to function poorly for epitopes containing cysteine (Cys) residues, which can oxidize and form disulfide bonds with other Cys residues under oxidizing conditions, thus potentially interfering with their ability to bind to MHC molecules. Analysis of the results of HLA-A*02:01 class I binding assays carried out in the presence and absence of the reducing agent 2-ME indicated that the predicted affinity for 25% of Cys-containing epitopes was underestimated by a factor of 3 or more. Additional analyses were undertaken to evaluate the responses of human CD8⁺ tumor-reactive T cells against 10 Cys-containing HLA class I-restricted minimal determinants containing substitutions of α-aminobutyric acid (AABA), a cysteine analogue containing a methyl group in place of the sulfhydryl group present in Cys, for the native Cys residues. Substitutions of AABA for Cys at putative MHC anchor positions often significantly enhanced T cell recognition, whereas substitutions at non-MHC anchor positions were neutral, except for one epitope where this modification abolished T cell recognition. These findings demonstrate the need to evaluate MHC binding and T cell recognition of Cys-containing peptides under conditions that prevent Cys oxidation, and to adjust current prediction binding algorithms for HLA-A*02:01 and potentially additional class I alleles to more accurately rank peptides containing Cys anchor residues.