Mature proteins derived from Epstein–Barr virus fail to feed into the MHC class I antigenic pool

Abstract

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein-Barr virus (EBV) antigens was induced were rapidly recognized by antigen-specific CD8 + T cells in a time-and dosage-dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half-lives and are hence overexpressed in tumors. Immunol. 2012. 42: 3167-3173 antigenic peptides originate from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are defective in some manner and rapidly degraded [3,4]. Evidence in support of the DRiP hypothesis has been provided by functional studies that linked peptide generation with protein translation [5][6][7][8], and by the finding that a significant proportion of all newly synthesized proteins is rapidly degraded [9]. Following these seminal observations, a number of ensuing studies performed with multiple antigens and in different cell types with different allomorphs pointed to DRiPs as the major, if not the sole source of antigenic peptides [4,10,11]. However, the contribution of DRiPs versus mature proteins to the antigenic peptide pool is typically assessed by abrogating protein synthesis with translation-inhibiting drugs such as cycloheximide (CHX), that might not only affect precursor pool size but also other features of the antigen presentation pathway, for example, protein degradation as well as synthesis, loading, and transport of MHC molecules [5,12]. Also, the use of chimeric proteins as the source of antigen and viruses for antigen expression may have led to an overestimation of the contribution of DRiPs to the immunopeptidome; the fusion of genes may decrease the overall fidelity of protein synthesis or protein folding [13] and differences in viral translation mechanisms can greatly increase the fraction of DRiPs [14]. Likewise, studies performed with antigens with very long half-lives and hence, very slow turnover, might have led to an underestimation of the contribution of mature proteins to the antigenic pool [7,10,15]. Moreove...

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

“…1E). We and others have previously observed this phenomenon, and the data suggest that some inherently destabilized form of newly synthesized SCRAP-SVG is degraded and presented, despite the presence of a saturating dose of Shield-1 (17,21,26). This meets the definition of a defective ribosomal product (DRiP) (27), which are important sources of peptides for the antigen presentation pathway (28,29).…”

Re-examining class-I presentation and the DRiP hypothesis

“…In these experiments, terminating the synthesis of several EBV antigens (including EBV nuclear antigens), and the LCMV nucleoprotein, resulted in a partial or complete loss of antigen presentation, even when the cells contained pools of mature protein [40,55–57]. While these results appear consistent with a major contribution from newly synthesized proteins, there are some caveats.…”

Substrate-Induced Protein Stabilization Reveals a Predominant Contribution from Mature Proteins to Peptides Presented on MHC Class I

“…For LCMV (nucleoprotein) and EBV (EBNA1) viral antigens it was reported that when Tet was removed and antigen synthesis was terminated, the presentation of some peptides decreased at times when there was still a substantial pool of mature protein (46, 47). Similarly, while this manuscript was in preparation, it was reported for a tet-inducible FKBP-EBV antigen system that mature antigen did not contribute to antigen presentation (25). It is possible that these antigens are ones where the contribution from DRiPs is more substantial.…”

“…In order to bind Shield these constructs must be properly folded and therefore by definition are not DRiPs (11, 19). Shield was shown to selectively stabilize FKBP fusions without affecting other cellular processes (19, 23, 24), and has been used by others to study antigen presentation (11, 12, 25). We fused FKBP to the copepod green fluorescent protein (copGFP).…”

“…In contrast we and others detect no substantial ubiquitin laddering of the Shield stabilized constructs (34). In fact, proponents of the DRiPs hypothesis agree that such Shield-stabilized proteins are not DRiPs (11, 25). …”