Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously.We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

SummaryUsing monoclonal antibodies identifying all y/b and u//3 T cell receptors in cytofluorometric analysis, we have compared the composition of intestinal intraepithelial lymphocytes (i-IEL) in euthymic and athymic germ-free (GF) and conventional (SPF) mice . The results show a marked influence of microbial colonization in the numbers of single-positive (CD4+ or CD8+) a/(3 i-IEL, but little effect in the pool size or characteristics of y/6 MEL . In young athymic mice, virtually no oi/(31-IEL are detected, while considerable numbers of 'Y/b MEL remain, though reduced in GF animals. y/6 T cells are relatively rare in the lymphoid organs of adult mice (1-5), but well represented in epithelia (5-10) . The biological roles of these lymphocytes are unknown, but their preferential localization at the body surfaces has led to suggestions of a defensive role directed against microbial invasion (11). On the other hand, their skewed receptor repertoires and limited diversity at several anatomical sites (9,10,(12)(13)(14) indicate that specific ligands for y/b TCR are self components and that intraepithelial localization may be (external)antigen-independent (15). In apparent contrast, a/(3 T cells recirculate and are selectively retained in the sites where foreign antigen is presented (16,17) . The comparison of y/b and ot/a T cell populations in epithelia of animals that are either normal or free of microbial colonization should be informative in this regard . Intraepithelial lymphocytes (i-IEL)l in the mouse small intestine contain a subset of y/b cells (7, 18) with characteristic V gene usage and receptor diversity (10,12,13,19,20) . Using an anti-C6 monoclonal antibody identifying all y/b T cells (5), we have now compared i-IELs in germ-free (GF) and specific pathogen-free (SPF) adult mice, and found that colonization by the normal intestinal flora has little effect on either the numbers or the phenotype of y/b cells. In contrast, the MEL x/(3 T cell pool is sharply reduced in GF mice, particularly the single positive (CD4+ or CD8+) cells . These results indicate a predominant role of a/0 T cells in the response to microorganisms of the intestinal mucosa, and thus suggest other roles for y/b cells .1 Abbreviations used in this paper : CMF, Ca and Mg-free; GF, germfree; MEL, intraepithelial lymphocytes; SPF, specific pathogen-free. Materials and MethodsMice. GF BALB/c animals are from a stock maintained under germ-free conditions over 40 generations at Ciba-Geigy AG, Basel, Switzerland . All GF mice studied here were 7-14 wk old and devoid of macroscopically detectable lymph nodes and Peyer's patches. SPF BALB/c mice were from the same animal facilities, age and sex matched with GF animals.Monoclonal Antibodies. The following mAbs were used : 3A10, anti C6 (5) ; H57-597, anti C/3 (21) ; 145-2C11, anti-CD3 (22); J .1j, anti Thy-1 .2 (23); GK-1.5, anti-CD4 (24); 53 .6 .72, anti-CD8 (25).Lymphoid Cell Preparations. i-IEL cell suspensions were prepared according to standard procedures (26-28) with some minor modifications...

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A subset of CD8 + T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8 + T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7 + memory cells and CCR7 -effector cells. Since NKG2A can only be induced on naive CD8 + T cells while CD94 -memory cells are refractory, it is likely that commitment to the CD94:NKG2A + subset occurs during the first encounter with antigen. CCR7 + CD94:NKG2A + T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-c. These cells occur as a separate CD94:NKG2A + T cell lineage with a distinct TCR repertoire that differs from that of the other CD8 + CD94 -T cells activated in situ.

Autoimmune (type 1) diabetes mellitus in mouse, rat, and humans shares several features, including T lymphocyte infiltration into pancreatic islets and a dependence on permissive class II major histocompatibility complex (MHC) alleles. We report here on an experimental model involving mice that express influenza hemagglutinin (HA) under the control of the insulin promoter and, at the same time, a transgenic class II MHC-restricted T cell receptor (TcR) specific for an HA peptide. These mice spontaneously develop islet infiltrates resembling those found in NOD mice and most animals become diabetic within 8 weeks of age. Because of the availability of a clonotypic TcR antibody, we can be confident that the Ins-HA transgene does not induce any measurable alterations in the vast majority of T cells with the transgenic TcR in primary and secondary lymphoid organs. Continuous export of large numbers of HA-specific lymphocytes from the thymus was not required for the manifestation of the disease since mice thymectomized at 3 days after birth still developed the disease albeit with smaller infiltrates.