By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

A library of random 10 residue peptides fused to the N‐terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N‐end rule pathway, a ubiquitin (Ub)‐dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N‐terminal residues. One of the N‐terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub‐dependent proteolytic system distinct from the N‐end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co‐immunoprecipitation and two‐hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub‐dependent proteolysis of test substrates. The discovery of the Ufd3p–Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.

anti-microbial immunity ͉ antigen cross-presentation

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8+ T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deyiates from first-order kinetics. We consider this problem and discuss other applications of UPR.