title: CD8+T-cell recognition of a synthetic epitope formed by t-butyl modification.
abstract: We set out to clone Bax-specific CD8+T-cells from peripheral blood samples of primary chronic lymphocytic leukemia patients. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-aa sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (>95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild type (wt) sequence to generate functionally active peptides. Computer modeling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the TCR. This modified product formed <1% of the original P603 crude peptide preparation and <0.05% of the original 23 peptide mixture used for T-cell stimulation. The data presented here, illustrates the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlights the exquisite capacity of TCR to discriminate between structurally similar peptide sequences. Furthermore this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses. This article is protected by copyright. All rights reserved.
authors.affiliation: Institute of Cancer and Genetics, Cardiff University, School of Medicine, Cancer and Genetics Building, Heath Park, Cardiff, U.K.
authors.forename: Reiss A
authors.initials: RA
authors.lastname: Reid
authors.forename: James E
authors.initials: JE
authors.lastname: Redman
authors.forename: Pierre
authors.initials: P
authors.lastname: Rizkallah
authors.forename: Chris
authors.initials: C
authors.lastname: Fegan
authors.forename: Chris
authors.initials: C
authors.lastname: Pepper
authors.forename: Stephen
authors.initials: S
authors.lastname: Man
keywords: CD8+ T-cells; Mass spectrometry; Peptides