Abstract
Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.
Highlights
From the Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
Two Distinct Anti-gp100 T-cell receptors (TCRs) Share Similar Binding Hot Spots—The CD8ϩ T-cell responses directed against gp100280–288 have been shown to be polyclonal in nature [16, 17]
In order to study the individual contribution of the peptide residues involved in TCR recognition of gp100280–288, in relation to peptide residues Glu3, an alanine scan mutagenesis was performed across the peptide backbone, and TCR binding affinity was evaluated by surface plasmon resonance (SPR) experiments
Summary
Generation of Expression Plasmids—The PMEL17 TCR (TRAV21 TRBV7-3) [12] and gp100 TCR (TRAV17 TRBV19) [11] are both specific for the human HLA-A*0201 restricted YLE epitope (gp100280–288, sequence YLEPGPVTA). For surface plasmon resonance (SPR) experiments, the soluble HLAA*0201 ␣-chain was tagged with a biotinylation sequence, as described previously [21]. Biotinylated pHLA-I complexes were prepared as described previously [21] and injected at a slow flow rate (10 l/min) to ensure a uniform distribution on the chip surface. The Kon and Koff values were calculated assuming 1:1 Langmuir binding, and the data were analyzed using a global fit algorithm (BIAevaluationTM version 3.1). Isothermal Titration Calorimetry (ITC)—ITC experiments were performed using a Microcal VP-ITC (GE Healthcare) as described previously [29], with 30 M pHLA-I in the calorimeter cell and 210 M soluble PMEL17 TCR in the syringe. Alignment of TCR CDR3 regions of PMEL17, gp100, MPD [16], and 296 gp100-specific TCRs [16]
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