The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Proteasome activity and epitope production are altered by IFN-γ treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro. However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported. Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days. Proteasome replacements were markedly reduced in IFN-γ−/− mice, but were only slightly affected in IFN-αR−/− and perforin−/− mice. The proteasome regulator PA28α/β was up-regulated, whereas PA28γ was reduced in the liver of lymphocytic choriomeningitis virus-infected mice. Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver. Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines.

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam 3 CysSK 4 (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpGϳ conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pamϳconjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin-or caveolindependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpGϳconjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8؉ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Two activators, named PA700 and PA28, are known to bind to 20 S proteasomes, forming two different complexes. The PA700-proteasome complex, also known as the 26 S proteasome, can degrade intact proteins, whereas complexes with PA28 can degrade only peptides. Monoclonal antibodies to 20 S proteasomes or the p45 ATPase subunit (Trip1, Sug1) of PA700 precipitated the same set of proteins from HeLa extracts, including six different ATPase subunits of PA700. This shows that p45 is not present in other protein complexes and suggests that all 26 S proteasome particles contain the same set of ATPase subunits. Interferons alpha and gamma had no effect on the composition of the 26 S proteasome, except for the replacement of subunits delta, MB1 and Z with Lmp2, Lmp7 and MECL1 respectively. Surprisingly, antibodies to PA28 precipitated p42, a component of PA700. Conversely, anti-p45 antibodies precipitated not only 26 S proteasomes but also PA28 alpha, beta and gamma, indicating that 20 S proteasomes can simultaneously bind both PA700 and PA28. PA28 alpha beta is known to be involved in antigen presentation. Conceivably, intact substrate proteins are recognized by PA700 and fed into proteasomes whose cleavage specificity is optimized for antigen presentation on MHC class I by PA28 and three interferon inducible proteasome subunits.

Dendritic cells (DCs) are crucial for priming of naive CD8 ؉ T lymphocytes to exogenous antigens, so-called ''cross-priming.'' We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte crosspriming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained crosspresentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.antigen processing ͉ cross-presentation ͉ endocytosis ͉ Fc receptor ͉ MHC class I D endritic cells (DCs) are crucial in the initiation and orchestration of the T cell immune response (1-3). DCs operate as sentinels of an infection in the periphery and subsequently as conductors of the T cell response in the lymph nodes. To exert these functions, DCs are specialized in the ingestion, processing, and presentation of antigens acquired by receptor-independent pinocytosis or by receptor-mediated endocytosis (4-6). To this end, they are equipped with a diverse set of receptors for uptake of antigen, such as scavenger receptors, lectin receptors, or IgG (Fc␥) receptors (7).Immature DCs have the capacity to efficiently acquire antigen, but a poor capacity to migrate and to stimulate T cells. Proper T cell response initiation requires maturation of the DCs, a process that is triggered by contact with infectious or inflammatory signals. Mature DCs characteristically show enhanced migratory capacity, up-regulation of the MHC class I processing machinery, and enhanced expression of MHC I and II and costimulatory molecules.DCs present antigenic peptides in either MHC class I to induce CD8 T cell responses and in MHC class II to induce CD4 T cell responses. Whereas MHC class I ligands are commonly derived from breakdown products of endogenous proteins that are degraded by the proteasome (8), DCs also have the unique capacity to present peptides derived from exogenous antigens in MHC class I to CD8 ϩ T cells, a process called ''crosspresentation.'' This process is crucial for induction of effective cytotoxic T lymphocyte (CTL) immunity against tumors, which lack direct priming capacity themselves, but also against microorganisms including viruses (1).DCs have dedicated organelles to facilitate efficient loading of antigenic peptides in MHC class II molecules. These MHC class II compartments (MIIC) are multivesicular endosomes that express high levels of MHC class II and invariant chain and are abundantly present in immature DCs (9). On maturation of the DC, rapid reorganization of the MIIC takes place that facilitates transport of MHC class II...

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4 + and CD8 + T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasomeand transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8 + T-cell activation.Keywords: Antigen presentation/processing r Cellular immunology r CD8 + T cells r Dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site lower efficiency compared to SLP-loaded DCs (Fig. 1B). Prestimulation of DCs with the TLR4 ligand LPS had no effect on the MHC class I presentation of OVA-protein but improved Ag presentation of SSP-OVA 8aa (data not shown) and long peptide Ag (Fig. 1C). HLA-B7-restricted presentation by human monocyte-derived DCs (MoDCs) of HIV-derived protein and SLPs was also studied. We were unable to detect cytokine production by CD8 + T cells cocultured with GAG-protein-loaded DCs. In contrast, SLP-GAG 22aa induced significant CD8 + T-cell activation (see Fig. 2 and below).Together, these data show that cross-presentation of SLPs is superior to that of proteins as examined with both mouse and human DCs. Rapid Ag presentation of SLPs by murine and human DCsThe efficiency of SLP-processing was assessed by studying the time required for DCs to present Ag on MHC class I (H2-K b )molecules. Murine DCs were incubated with a single concentration of SLPs, synthetic short peptides (SSPs) or protein for the indicated time periods. The minimal peptide, SSPs, was rapidly presented to CD8 + T cells resulting in strong activation already after 1 h. DCs loaded with SLP also activated CD8 + T cells 1 h after Ag loading but with lower potency. We excluded that SLPs were cleaved extracellularly, processed, and loaded on MHC class I and II molecules by incubating paraformaldehyde (PFA) fixed cells with the peptide Ag and observed no cross-presentation (data not shown). DCs loaded with 10 μM OVA-protein failed to induce significant CD8 + T-ce...