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

• Published in: European journal of immunology Vol. 40; no. 6; pp. 1603 - 1616
• Main Authors: Cecconi, Virginia, Moro, Monica, Del Mare, Sara, Sidney, John, Bachi, Angela, Longhi, Renato, Sette, Alessandro, Protti, Maria Pia, Dellabona, Paolo, Casorati, Giulia
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.06.2010; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: Anchor residues; Antigens, Neoplasm - immunology; CD4-Positive T-Lymphocytes - immunology; Cell Line; Cell Separation; Epitopes, T-Lymphocyte - immunology; Flow Cytometry; HLA-DR Antigens - immunology; HLA‐DR1101; Humans; Lymphocytes; MAGE‐A3; MHC tetramers; Neoplasm Proteins - immunology; Peptide Fragments - immunology; Peptides; Polymerase Chain Reaction; Tumor antigens
• ISSN: 0014-2980; 1521-4141
• DOI: 10.1002/eji.200940123

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-A3₁₉₁₋₂₀₅ (p39), MAGE-A3₂₈₁₋₂₉₅ (p57) and MAGE-A3₂₈₆₋₃₀₀ (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.