Figure 4

Stabilization of pHLA complexes by combinational non-canonical short peptides. (a) Stabilization of pHLA complexes formed by HLA-A*11:01 and β2 m with combination epitopes (combitopes) of short C-terminal (C) and N-terminal (N) SSCSSCPLSK peptides. Refolding with both C- and N-terminal SSCSSCPLSK peptides (N + C) generates combination of contiguous peptides (Contiguous combitope) as well as combination of peptides with increasing number of overlapping amino acids (Overlapping combitope). Heavy chain (H), heavy and β2 m light chain (HL) served as the negative controls while pHLA complex with SSCSSCPLSK (pos) served as the positive control for each western blot panel. (b) Real time PCR thermofluor measurement for pHLA complexes formed by HLA-A*11:01 heavy chain and β2 m with SSCSSCPLSK (Tm = 68.1 °C), SCSSCPLSK (Tm = 69.7 °C), CSSCPLSK (Tm = 49.4 °C), SSCPLSK (Tm = 50.0 °C), SSCSSC + SSCPLSK (Tm = 49.4 °C), SSCSSC + SCPLSK (Tm = 48.7 °C), SSCSS + SCPLSK (Tm = 49.7 °C) and SSCS + SCPLSK (Tm = 50.3 °C). (c) Workflow for 14-day PBMC cultures following peptide pulsing and minigene electroporation is shown. Truncated peptides SSCSSC and SSCPLSK were introduced in combination or singly in the form of pulsed peptides or electroporated minigenes constructs. Recall CD8 T cell responses were detected by A*11:01/SSCSSCPLSK tetramer staining of 14-day PBMC cultures in two different HLA-A*11:01 donors.