Re-evaluating the Generation of a “Proteasome-Independent” MHC Class I-Restricted CD8 T Cell Epitope

Abstract

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP147–155 of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently “proteasome-independent” epitopes remain poorly defined. We have examined the generation of NP147–155 and a second proteasome-dependent NP epitope, NP50–57, using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP147–155, 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP50–57 or NP147–155, and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP147–155 epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.

Need for Tripeptidyl-peptidase II in Major Histocompatibility Complex Class I Viral Antigen Processing when Proteasomes are Detrimental

“…This publication (27) reported results that seem to conflict with those presented here. Whereas in both reports LC enhanced presentation and AAF-cmk blocked it, we reached apparently different conclusions based mainly on results obtained with the TPPII-specific inhibitor butabindide and with gene silencing of the protease.…”

“…Some differences in the experimental approach used in both reports may account for some of the discrepancies. First, Wherry et al (27) infected for longer periods and added the unstable butabindide less frequently than we did (as recommended (10)), which may leave a window of enzyme activity enough to allow for efficient presentation. Regarding gene silencing, Wherry et al (27) reported that only 50 -60% of cells were transfected, and thus, still many cells may present antigen, whereas we reached values of 95% leading to inhibition of antigen presentation.…”

“…The main processing activity for NP [147][148][149][150][151][152][153][154][155] , TPPII, which is also an endopeptidase, could perform this task as suggested (10). Indirect evidence from in vitro digestions of peptide substrates with purified and LC-inhibited 20 S proteasomes suggests that an altered proteasome activity (27) might also contribute to processing in living cells, but only secondarily, as this is strongly inhibited by AAF-cmk both in this report and in that of Wherry et al (27).…”

“…Even in the presence of LC, residual caspase-like proteasome activity (8) or altered proteasome activity (16,27) might be required for antigen processing, maybe in a sequential pathway involving proteasomes and TPPII (10) (Fig. 7).…”

“…This is true in the natural context of NP from two strains of influenza virus, A/Puerto Rico/8/34 (NP PR8 ) and A/NT/60/68 (NP NT60 ), as well as in a few mutated constructs of these proteins. Among several possible explanations (14) including the recent suggestion that an altered enzymatic activity of inhibitor-treated proteasomes might generate in vitro products compatible with peptide presentation (27), the current hypothesis derived from in vitro digestions (13,29) is that the naturally presented peptide or a precursor is destroyed by proteasomes.…”