The CD4+ T-cell epitope-binding register is a critical parameter when generating functional HLA-DR tetramers with promiscuous peptides.

Detection of CD4(+) T cells specific for tumor-associated antigens is critical to investigate the spontaneous tumor immunosurveillance and to monitor immunotherapy protocols in patients. We investigated the ability of HLA-DR 1101 multimers to detect CD4(+) T cells specific for three highly promiscuous MAGE-A3 derived peptides: MAGE-A3(191-205) (p39), MAGE-A3(281-295) (p57) and MAGE-A3(286-300) (p58). Tetramers stained specific CD4(+) T cells only when loaded with p39, although all peptides activated the specific T cells when presented by plastic-bound HLA-DR 1101 monomers. This suggested that tetramer staining ability was determined by the mode rather than the affinity of peptide binding to HLA-DR 1101. We hypothesized that peptides should bear a single P1 anchor residue to bind all arms of the multimer in a homogeneous register to generate peptide-HLA-DR conformers with maximal avidity. Bioinformatics analysis indicated that p39 contained one putative P1 anchor residue, whereas the other two peptides contained multiple ones. Designing p57 and p58 analogues containing a single anchor residue generated HLA-DR 1101 tetramers that stained specific CD4(+) T cells. Producing HLA-DR 1101 monomers linked with the optimized MAGE-A3 analogues, but not with the original epitopes, further improved tetramer efficiency. Optimization of CD4(+) T-cell epitope-binding registers is thus critical to generate functional HLA-DR tetramers.