Cryptic MHC class I-binding peptides are revealed by aminoglycoside-induced stop codon read-through into the 3′ UTR

Abstract

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 fulllength 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.recoding | antigen presentation | autoimmunity

Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story

“…Although all termination events (and NMD) begin with a nonsense codon in the ribosomal A site, several observations indicate that the subsequent process appears to be mechanistically different for normal and premature translation termination (Figure 1 b ): ( a ) normal termination events do not trigger NMD; ( b ) yeast ribosomes recognizing normal termination codons (NTCs) in vitro do not yield toe prints unless eRF1 is inactivated by a temperature-sensitive lesion (4), but ribosomes at PTCs readily yield toe-print signals without eRF1 inactivation (4), and comparable results have been observed for termination at a PTC versus NTC of human β-globin mRNA (173); and ( c ) although some NTCs allow nonsense codon read-through (49, 72), PTCs are, in general, much more susceptible to read-through than NTCs (21, 48, 69, 75, 107, 120, 132, 133, 175, 223, 241). Thus, although translation termination at NTCs and PTCs are both triggered by the presence of a stop codon in the A site, the kinetics and the efficiency of the termination events at NTCs and PTCs are markedly different.…”

MHC class I–associated peptides derive from selective regions of the human genome

“…According to the dominant paradigm, MAPs preferentially originate from defective ribosomal products (DRiPs) which can be created by several mechanisms such as nonsense-mediated decay (NMD), mRNA destabilization, or noncanonical translation in the cytosol or the nucleus (16)(17)(18)(19)(20). Large-scale mass spectrometry (MS) offers the sole direct approach to analyzing the global molecular composition of the immunopeptidome.…”

The nature of self for T cells—a systems-level perspective

“…There is only a modest correlation between the amounts of MIPs and the relative abundance of their source protein: some MIPs derive from low abundance proteins whereas some highly abundant proteins do not generate MIPs [14,19,[23][24][25]. Two related factors explain why the immunopeptidome is not a mirror of the proteome.…”

“…Indeed, aminoglycosides such as gentamicin induce stop codon read-through at a level sufficient to generate immunogenic MIPs derived from 3 0 untranslated regions [25].…”

“…In addition, seminal observations provide compelling evidence that MIPs arising from 'untranslated regions' may derive from immature mRNAs translated in the nucleus [6,58 ,59 ,60]. A few of these so-called 'cryptic MIPs' were validated by MS sequencing [25,61]. However, since most putative cryptic MIPs have not been validated by MS, some of them must be considered with skepticism because their identification relied primarily on methods fraught with high false discovery rates [62,63 ,64]: bioinformatic predictions (that cannot predict key steps in MIP processing) and in vitro T-cell reactivity assays (which are tainted by T-cell cross-reactivity).…”