Modulation of peptide specific T cell responses by non-native flanking regions

The deduced core (75RYPNVTI81) from a T-cell stimulatory epitope of the 38 kDa protein of M. tuberculosis was studied to identify the structural elements required for the creation of a synthetic peptide antigen from an epitope core, which alone was not capable of inducing CD4+ T-cell responses. Peptides were prepared with extensions composed of native and/or non-native sequences to clarify the role of the flanking regions adjacent to the epitope core. Their binding to isolated H-2-Ab MHC glycoprotein as well as T-cell stimulatory capacity were assayed using a specific murine hybridoma T-cell line [38.H6], lymph node cells from the native 20-mer peptide primed C57BL/10 mice and human PBMCs from sensitised individuals. Elongation of the epitope core by four alanines at both N- and C-terminals resulted in a 15-mer peptide A4-75-81-A4 which was stimulatory for hybridoma T-cells and showed a small decrease in H-2-Ab binding. Substitution of one Ala by Ser in the N-terminal flank had pronounced effect and peptide A2SA-75-81-A4 proved to be more effective than the native 20-mer sequence in the hybridoma as well as in the LN cell proliferation assays. The binding of this peptide and that of the native one were similar. Testing in human PBMC cultures from eight PPD positive individuals showed that in 50% of the donors' cells responded to the 'artificial' A2SA-75-81-A4 peptide. These results suggest that it is possible to construct simple, synthetic CD4+ T-cell stimulatory peptides of high potency from a non-stimulatory, 'silent' epitope core by addition of flanking residues not part of the native sequence.