MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Of the many thousands of peptides encoded by a complex foreign antigen that can potentially be presented to CD8+ T cells (TCD8+), only a small fraction induce measurable responses in association with any given major histocompatibility complex class I allele. To design vaccines that elicit optimal TCD8+ responses, a thorough understanding of this phenomenon, known as immunodominance, is imperative. Here we review recent progress in unraveling the molecular and cellular basis for immunodominance. Of foremost importance is peptide binding to class I molecules; only approximately 1/200 of potential determinants bind at greater than the threshold affinity (Kd > 500 nM) associated with immunogenicity. Limitations in the TCD8+ repertoire render approximately half of these peptides nonimmunogenic, and inefficient antigen processing further thins the ranks by approximately four fifths. As a result, only approximately 1/2000 of the peptides in a foreign antigen expressed by an appropriate antigen presenting cell achieve immunodominant status with a given class I allele. A roughly equal fraction of peptides have subdominant status, i.e. they induce weak-to-nondetectable primary TCD8+ responses in the context of their natural antigen. Subdominant determinants may be expressed at or above levels of immunodominant determinants, at least on antigen presenting cells in vitro. The immunogenicity of subdominant determinants is often limited by immunodomination: suppression mediated by TCD8+ specific for immunodominant determinants. Immunodomination is a central feature of TCD8+ responses, as it even occurs among clones responding to the same immunodominant determinant. Little is known about how immunodominant and subdominant determinants are distinguished by the TCD8+ repertoire, or how (and why) immunodomination occurs, but new tools are available to address these questions.

Rare major histocompatibility complex (MHC) class I-like CD1-specific T cells have been isolated from human blood, but it has not been determined whether these clones are part of a defined subset of CD1-specific T cells selected during T cell development, or whether their recognition of CD1 is a fortuitous cross-reaction. In mice, an entire subset of alpha beta thymocytes with a unique phenotype was found to be CD1-specific. This particular subset, and its human counterpart, provide evidence that CD1 has a general role in selecting and interacting with specialized alpha beta T cells.

CD8+ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide-MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide-class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide-MHC class I interaction inside cells, and to visualize cells bearing specific peptide-MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Rapid antigenic evolution in the influenza A virus hemagglutinin precludes effective vaccination with existing vaccines. To understand this phenomenon, we passaged virus in mice immunized with influenza. Neutralizing antibodies selected mutants with single amino acid hemagglutinin substitutions that increased virus binding to cell surface glycan receptors. Passaging these high avidity-binding mutants in naïve mice, but not immune mice, selected for additional hemagglutinin substitutions that decreased cellular receptor binding avidity. Analyzing a panel of monoclonal antibody hemagglutinin escape mutants revealed a positive correlation between receptor binding avidity and escape from polyclonal antibodies. We propose that in response to variation in neutralizing antibody pressure between individuals, influenza A virus evolves by adjusting receptor binding avidity via amino acid substitutions throughout the hemagglutinin globular domain, many of which simultaneously alter antigenicity.Influenza A virus remains an important human pathogen due largely to its ability to evade antibodies specific for its attachment protein, the hemagglutinin (HA). This "antigenic drift" is due to accumulation of amino acid substitutions in HA epitopes recognized by antibodies that neutralize viral infectivity by blocking interaction of HA with sialic acid residues on hostcell membranes (1-3). The H1 subtype HA has four antigenic sites recognized by monoclonal antibodies with high neutralizing activity, designated Sa, Sb, Ca, and Cb (4). How can HA escape polyclonal antibodies given that the frequency of variants with simultaneous multiple point mutations is exceedingly low (5)? A popular model posits sequential selection by different individuals whose antibody responses focus on different individual antigenic sites (6,7).To better understand how antigenic drift occurs in human populations, we revisited classical experiments modeling drift in outbred Swiss mice (8). We generated three separate infectious stocks of the mouse-adapted strain A/Puerto Rico/8/34 (H1N1) (PR8) in MDCK cells using

Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.