Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.

The expression of housekeeping and/or immunoproteasomes in isolated thymic stroma subsets has so far not been analyzed but may have important consequences for self peptide repertoires presented by MHC class I molecules during positive and negative thymic selection. Here we determined the expression of housekeeping and immunoproteasome b subunits and of PA28 in positively and negatively selecting stroma subsets. Positively selecting cortical thymic epithelial cells (cTEC) expressed only housekeeping but no immunoproteasome b subunit mRNA and proteins. However, immunoproteasome b subunits could be induced in cTEC by infection with Listeria monocytogenes or injection of IFN-c. In negatively selecting stroma including medullary epithelial cells and dendritic cells, incomplete and low representation of housekeeping b subunit proteins but high and complete expression of immunoproteasome b subunit proteins suggests absence of proper housekeeping proteasomes and predominance of immunoproteasomes. Expression of immunoproteasome b subunits in negatively selecting stroma was independent of IFN-c receptor as shown in knockout (KO) mice. Absence of LMP2 altered thymic selection of the MHC class I-restricted transgenic P14 TCR in KO mice. The data suggest that negative selection may primarily involve immunoproteasome peptide repertoires and that peripheral infection may influence peptide repertoires involved in positive selection.

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8 ؉ T cells. This coincides with up-regulation of p53 and dysregulation of NF-B. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors.apoptosis ͉ senescence ͉ T lymphocytes ͉ tripeptidyl peptidase II T ripeptidyl peptidase II (TPPII) forms the largest known protease complex (Ϸ6 MDa) in eukaryotic cells. TPPII operates mostly downstream of proteasomes in cytosolic proteolysis (1-4). Several lines of evidence suggest that TPPII is important for proliferation and survival of malignant lymphoid cells, particularly under conditions of cellular stress. TPPII is up-regulated in EL4 lymphoma cells adapted to grow in the presence of proteasome inhibitors (3, 5). Conversely, overexpression of TPPII increases resistance of lymphoma cells against proteasome inhibitors (6). TPPII is up-regulated by c-Myc, as observed in Burkitt's lymphoma cells expressing high amounts of c-Myc. The semispecific T PPII inhibitor Ala-Ala-Phechloromethylketone (AAF-CMK) reduces proliferation and initiates apoptosis in Burkitt's lymphoma cells (7). TPPII is upregulated in nutritionally starved lymphoma cells in vitro, as well as in vivo in advanced lymphomas (8). In addition to TPPII up-regulation, such cellular stress conditions are commonly associated with decreased proteasomal activity and upregulation of deubiquitinating enzymes (7,8). Under these conditions tumor cells share an antiapoptotic phenotype, to which TPPII may contribute (8). TPPII-transfected and TPPIIsiRNA-treated tumor cells show accelerated and decelerated proliferation, respectively, and both show mitotic aberrations. TPPII overexpression permits tumor cell survival in the presence of mitotic inhibitors (9, 10). Together, these results provide evidence for the importance of TPPII in tumor cell proliferation and survival. In contrast, only scattered information exists on the role of TPPII in nonmalignant cells. For example, a role in the regulation of apoptosis in differentiated macrophages has been proposed (11). TPPII has also been implicated in MHC class I antigen processing (12).We have generated TPPII-deficient mice and provide here genetic evidence that TPPII is important for proliferative survival of nonm...

The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta VDJ rearrangements and pre-T alpha mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3 zeta/Lck double-deficient mice fail to express TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCR beta VDJ genes but before expression of TCR beta proteins. Most NKT cells of TCR alpha-deficient mice exhibit cell surface gamma delta TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.