Improving T-cell antigens by altering MHC anchor residues is a common strategy used to enhance peptide vaccines but there has been little assessment of how such modifications affect TCR binding and T-cell recognition. Here, we use surface plasmon resonance and peptide-MHC tetramer binding at the cell surface to demonstrate that changes in primary peptide anchor residues can substantially and unpredictably alter TCR binding. We also demonstrate that the ability of TCRs to differentiate between natural and anchor-modified heteroclitic peptides distinguishes T-cells that exhibit a strong preference for either type of antigen. Furthermore, we show that anchor-modified heteroclitic peptides prime T-cells with different TCRs compared to those primed with natural antigen. Thus, vaccination with heteroclitic peptides may elicit T-cells that exhibit suboptimal recognition of the intended natural antigen and, consequently, impaired functional attributes in vivo. Heteroclitic peptide-based immune interventions therefore require careful evaluation to ensure efficacy in the clinic.

No abstract

CD8+ cytotoxic T lymphocytes (CTL) are essential for effective immune defence against intracellular microbes and neoplasia. CTL recognize short peptide fragments presented in association with major histocompatibility complex class I (MHCI) molecules on the surface of infected or dysregulated cells. Antigen recognition involves the binding of both T cell receptor (TCR) and CD8 co-receptor to a single ligand (pMHCI). The TCR/pMHCI interaction confers antigen specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to antigen. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. Here, we show that increasing the strength of the pMHCI/CD8 interaction by ~15-fold results in non-specific, cognate antigen-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with super-enhanced affinity for CD8 activate CTL in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL antigen specificity.

CD8+ cytotoxic T lymphocytes (CTLs) hold the key to the successful prevention and eradication of infectious and neoplastic disease. T-cells recognize ‘foreign’ peptide fragments often referred to as epitopes, in the context of ‘self’ major histocompatibility complex class I (MHCI) molecules. Epitope identification is essential to define targets for the effective control of disease. Peptides bind to MHCI molecules by anchoring at position 2 and the C-terminus of the peptide ligand. Techniques such as pool sequencing have been hugely instrumental in revealing HLA allele-specific peptide binding motifs. However, this approach only reveals the most dominant MHC anchor residues. The preferred binding motif identified by pool sequencing for the most common MHCI, HLA A*0201, is L/M at position 2 and V/I/L/A at the C-terminus. Here, we use an extremely sensitive combinatorial library screening approach to show that in the context of HLA A*0201, degeneracy at anchor residue positions is much higher than previously appreciated. We also examined the recognition of peptides with substitutions of all 20 amino acids at both anchor positions in three different clonal systems which confirm these observations. Indeed, in one clonal system, 9-mer peptides containing A, C, F, I, K, L, M, Q, S, T, V in position 2 and A, C, F, I, L, M, T, V at position 9 were recognized efficiently. These observations have implications for T-cell epitope prediction and future vaccine design.