Summary: MHC class I molecules function to present peptides comprised of eight to I 1 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory lo understand bow cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and nun‐proteasomal proteases lo peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.

TAP can efficiently transport peptides up to twice as long as those bound to MHC class I molecules, suggesting a role for endoplasmic reticulum (ER) proteases in the trimming of TAP-transported peptides. To better define ER processing of antigenic peptides, we examined the capacity of TAP-deficient cells to present determinants derived from ER-targeted proteins encoded by recombinant vaccinia viruses. TAP-deficient cells failed to present antigenic peptides from internal locations in secreted proteins to MHC class I-restricted T lymphocytes. The same peptides were liberated from the C termini of a secreted protein and the lumenal domains of two membrane proteins delivered to the ER via different routes. These findings suggest that proteases in the secretory compartment can liberate C-terminal antigenic peptides from virtually any context. We propose that this activity often participates in the removal of N-terminal extensions from TAP-transported peptides, thereby creating optimally sized products for MHC class I binding. We further demonstrate that ER trimming of C termini can occur if we express an appropriate carboxypeptidase in the secretory pathway. The absence of such trimming under normal circumstances suggests that carboxypeptidase activity is generally deficient in the ER, consistent with the concordance between the specificity of TAP and MHC class I molecules for the same types of C-terminal residues.

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor polypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II. membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.

Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.

We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

Major histocompatibility complex (MHC) class I molecules bind peptides of 8-10 residues in the endoplasmic reticulum (ER) and convey them to the cell surface for inspection by CD8-expressing T cells (TCD8+). Antigenic peptides are predominantly derived from a cytosolic pool of polypeptides. The proteolytic generation of peptides from polypeptides clearly begins in the cytosol, but it is uncertain whether the final proteolytic steps occur before or after peptides are transported into the ER by the MHC-encoded peptide transporter (TAP). To study the trimming of antigenic peptides in the secretory pathway in the absence of cytosolic processing, we used an NH2-terminal signal sequence to target to the ER of TAP-deficient cells, "tandem" peptides consisting of two defined TCD8+ determinants arranged from head to tail. We find that in contrast to cytosolic proteases in TAP-expressing cells, which are able to liberate antigenic peptides from either end of a tandem peptide, proteases (probably aminopeptidases) present in an early secretory compartment preferentially liberate the COOH-terminal determinant. These findings demonstrate that proteolytic activities associated with antigen processing are not limited to the cytosol, but that they also exist in an early secretory compartment. Such secretory aminopeptidases may function to trim TAP-transported peptides to the optimal size for binding to class I molecules.