Figure 3. TCR recognition of the PIK3CA neoantigen presented by HLA-A3 is critically dependent on the flip of the tryptophan at position 6.
A) The tryptophan analog Bta replaces the indole NH with a sulfur atom, removing the capacity of the tryptophan side chain to serve as a hydrogen bond donor. B) In the structure of the free neoantigen/HLA-A3 complex, the pTrp6 side chain remains accessible to solvent. The accessible surface of the indole nitrogen is blue; the surface of the carbon atoms is cyan. C) Substitution of pTrp6 with Bta does not alter peptide binding to HLA-A3 as indicated by differential scanning fluorimetry. Datapoints indicate the temperature derivative of the fluorescence ratio; only every 5th datapoint is shown for clarity. Tm and error values are the average and standard deviation of four replicates. D) Substitution of pTrp6 with Bta does not alter the structural properties of the neoantigen in the HLA-A3 binding groove as shown by the crystallographic structure of the Bta6-neoantigen/HLA-A3 complex. The Bta-substituted peptide is superimposed on the unsubstituted neoantigen, with an all-common atom RMSD of 0.7 Å. The orange surface shows the solvent accessibility of the Bta sulfur atom to compare with that of the indole nitrogen in panel B. Electron density of the Bta-substituted neoantigen is from a 2Fo-Fc composite OMIT map calculated with simulated annealing, contoured at 1σ. E) SPR experiments show no detectable binding of TCR4 or TCR3 to the Bta-substituted neoantigen/HLA-A3 complex, although binding to the non-substituted neoantigen complex was quantifiable. Experiments were performed at 25 °C; KD and error values are the average and standard deviation of three replicates. F) SPR experiments confirming binding of the Bta-substiuted and non-substituted neoantigen/HLA-A3 complexes to the peptide-independent s3–4 scTv. Experiments were performed at 25 °C; KD and error values are the average and standard deviation of four replicates. G) Measurement of T cell function via production of the cytokine TNF-α. T cells expressing either TCR4 or TCR3 were co-cultured with HLA-A3+ antigen presenting cells in the presence of increasing amounts of Bta6-modified or unmodified neoantigen. Although unmodified neoantigen recognition is clear, there is no recognition of the Bta6-modified version. The unmodified and Bta6-modified WT peptides at the highest concentration were included as controls. Data shown are absolute frequencies derived from the averages of two independent experiments with three biological replicates per experiment. Points are means and error bars are standard deviations.