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 containedmultiple 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.