RESUMO
Aminoglycosides have been proposed as therapies for genetic disorders caused by nonsense mutations, because of their capacity to enhance translational read-through of premature termination codons (PTCs), thereby permitting expression of functional full-length protein. However, a potential consequence of this strategy is the development of an autoimmune response to HLA-presented epitopes encoded downstream of the PTC or other stop codons. Using a recombinant virus-expression system in tissue culture and in mice, we demonstrate that gentamicin can induce expression and MHC class I presentation of a model epitope encoded downstream of a PTC at levels sufficient to activate CD8(+) T cells. The degree of read-through-derived peptide presentation varies with the sequence of the stop codon and +1 nucleotide. Additionally, we applied a mass spectrometry exploration of the HLA class I peptide repertoire of gentamicin-treated cells and identified multiple peptides derived from read-through of conventional stop codons. These results substantiate the possibility of self-reactivity to cryptic epitopes revealed by stop codon read-through therapies and potentially other therapeutic approaches involving compounds that alter translational fidelity.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Gentamicinas/farmacologia , Oligopeptídeos/genética , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Animais , Anticorpos Monoclonais , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Códon sem Sentido/genética , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica/imunologia , Imunoprecipitação , Luciferases , Camundongos , Oligonucleotídeos/genética , Oligopeptídeos/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Espectrometria de Massas em TandemRESUMO
Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.