No abstract

CTL are important in combating cancer and viruses. Therefore, triggering the complete potential of CTL effector functions by new vaccination strategies will not only improve prophylaxis of tumor or virus-related diseases, but also open opportunities for effective therapeutic immunizations. Using transcutaneous immunization, we show that epicutaneous (e.c.)4 application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity, and the production of IFN-γ that is completely restricted to the epitope used for vaccination. Our results obtained by simple e.c. application of an ointment, without further skin irritating procedures, provide the basis for the development of new, easy to use vaccines against cancer or virus-associated diseases.

Influenza virus infection induces maturation of murine dendritic cells (DCs), which is most important for the initiation of an immune response. However, in contrast to EL-4 and MC57 cells, DCs present viral CTL epitopes with a delay of up to 10 h. This delay in Ag presentation coincides with the up-regulation of MHC class I molecules as well as costimulatory molecules on the cell surface and the accumulation of newly synthesized ubiquitinated proteins in large cytosolic structures, called DC aggresome-like-induced structures (DALIS). These structures were observed previously after LPS-induced maturation of DCs, and it was speculated that they play a role in the regulation of MHC class I Ag presentation. Our findings provide the first evidence for a connection between DC maturation, MHC class I-restricted Ag presentation, and DALIS formation, which is further supported by the observation that DALIS contain ubiquitinated influenza nucleoprotein.

Differences in the cleavage specificities of constitutive proteasomes and immunoproteasomes significantly affect the generation of MHC class I ligands and therefore the activation of CD8-positive T cells. Based on these findings, we investigated whether proteasomal specificity also influences CD8-positive T cells during thymic selection by peptides derived from self proteins. We find that one of the self peptides responsible for positive selection of ovalbuminspecific OT-1 T cells, which is derived from the f-actin capping protein (Cp␣1), is efficiently generated only by immunoproteasomes. Furthermore, OT-1 mice backcrossed onto low molecular mass protein 7 (LMP7)-deficient mice show a 50% reduction of OT-1 cells. This deficiency is also observed after transfer of BM from OT-1 mice in LMP7-deficient mice and can be corrected by the injection of the Cp␣1 peptide. Interestingly, WT and LMP7-deficient mice mount comparable immune responses to the ovalbumin-derived epitope SIINFEKL. However, their cytotoxic T lymphocytes (CTL) differ in the use of T cell receptor V␤ genes. CTL derived from WT mice use V␤8 or V␤5 (the latter is also used by OT-1 cells), whereas SIINFEKL-specific CTL from LMP7-deficient mice are exclusively V␤8-positive. Taken together, our experiments provide strong evidence that proteasomal specificity shapes the repertoire of T cells participating in antigen-specific immune responses. selection ͉ T cell repertoire C ytotoxic T lymphocytes (CTL) recognize a complex of MHC class I molecules and peptides derived mainly from intracellular proteins. The generation of these peptides crucially depends on the activity of proteasomes, which represent the main proteolytic activity present in the cytoplasm and are responsible for the generation of the C termini of most peptides presented by MHC class I molecules (1-5). The 20S proteasome represents the proteolytic core complex, which is composed of seven different ␣-and seven different ␤-subunits organized in a complex of four stacked rings with ␣ 7 ␤ 7 ␤ 7 ␣ 7 stoichiometry (6-8). The specificity of this protease is influenced by the incorporation of three IFN-inducible ␤-subunits (␤1i, ␤2i, and ␤5i), which replace the three proteolytically active constitutive subunits (␤1, ␤2, and ␤5) during proteasome assembly, resulting in the formation of so-called immunoproteasomes (9, 10). This type of proteasome generates reduced numbers of peptides with acid C termini and increased numbers of peptides with hydrophobic C termini (11,12), which is in favor of a more efficient transporter associated with antigen-presentation transport and binding to MHC class I molecules (13). In addition, a change in proteolytic specificity is apparent from the numerous examples reporting that either constitutive proteasomes or immunoproteasomes are required for the efficient generation of certain MHC class I ligands and consequently for the activation of CTL (14-19). As a result, proteasomal specificities provide a major contribution to the hierarchies of epitopes recognized by pathog...