MHC class I-associated peptides produced from endogenous gene products with vastly different efficiencies

Abstract

We compared the efficiency of generating antigenic peptides from various polypeptide contexts expressed by recombinant vaccinia viruses. These included full-length influenza virus nucleoprotein (NP(1-498)), two truncated forms, and cytosolic and endoplasmic reticulum-targeted minimal peptides. Two peptides were studied, NP(50-57) (Kk-restricted) and NP(147-155) (Kd-restricted). The efficiency of peptide generation was measured in cytotoxicity assays by determining 1) the kinetics of presentation following infection using brefeldin A to block additional presentation and 2) the concentration of anti-class I mAbs required to block presentation. The two determinants behaved similarly, being presented most efficiently from minigene products, with intermediate efficiency from fragments, and least efficiently from NP(1-498). Direct quantitation of HPLC-purified peptides supported the validity of these simple methods to roughly estimate the efficiency of class I Ag presentation. It also surprisingly revealed that 60- to 90-fold more NP(50-57) than NP(147-155) peptide was present in cells expressing NP(1-498) or a rapidly degraded fragment (for NP(1-498), 1800 peptides/cell of NP(50-57) vs 30 peptides/cell of NP(147-155)). By contrast, nearly identical (and much greater) amounts of peptides were recovered from cells expressing minigene products (55,000 copies of either peptide/cell). These findings demonstrate 1) that immunodominant peptides from the same protein can be generated with vastly different efficiencies, and 2) that cytosolic or endoplasmic reticulum-targeted minigene products are presented far more efficiently than longer polypeptides.