Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of γ-interferon–inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

Immunogenic peptides are displayed in the context of class II histocompatibility proteins on the surface of antigen-presenting cells. Class H a and 13 subunits bind the invariant chain (I-chain), a transmembrane glycoprotein which must dissocite prior to peptide presentation. Proteolytic release of I-chain in an acidic compartment is followed by class II afi surface expression. Two distinct proteinases sequentially catalyze I-chain dissociation in B-lymphoblastoid cell lines. An aspartic proteinase initiates processing whereas a cysteine proteinase catalyzes the rmal stages of I-chain release. Inactivation of these enzymes prevents class II a1 maturation, demonstrating that acidic proteinases are essential for the generation of functional class II complexes. MATERIALS AND METHODSCells and Proteinase Inhibitors. The B-LCL Sweig (DR5) was cultured in Iscove's modification ofDulbecco's modified Eagle's medium plus 5% calf serum. Leupeptin (stock solution, 25 mg/ml in water) was used at a finial concentration of 5-50 jmg/ml. The aspartic proteinase inhibitors PD136517, PD136809, and PD134678 inactivate cathepsins D and E (8, 9). These inhibitors (50 mM stock solutions in dimethyl sulfoxide) were used at 25-50 ,uM, concentrations which reduced intracellular aspartic proteinase activity >75% after 24 hr.Antibodies. The antibody DA6.147 recognizes class II DR a chain, and L243 recognizes an epitope on mature DR5(wll) af3 (2, 10, 11). I-chain was detected with PIN1.1, an antibody specific for residues 12-28 (10).Immunoblotting and Metabolic Radiolabeling. Samples were normalized for protein and cell number before electrophoretic analysis and immunoblotting (10, 12). Class II a3 dimers were detected on immunoblots by using antibody L243 and sample buffer with 0.2% SDS (13). For metabolic radiolabeling, cells were pretreated with proteinase inhibitors for 1-2 hr, incubated with [35S]methionine for 30 min, and cultured in complete medium with or without inhibitors for 3.5 hr or 8 hr. In some experiments cells were radiolabeled for 4 hr. Cell lysates were immunoprecipitated for SDS/PAGE, and autoradiographs were quantitated by densitometry with the integrated area expressed in total intensity units. Resolution of glycosylated I-chain (Ip) from DR a was difficult on SDS/polyacrylamide gels. To calculate the accumulation of intact I-chain (It = I + Ip) with class II antigens, the formula (It + a)/, was used, assuming a constant a/l3 ratio. Subcellular Fractionation. Sweig cells (3 x 108) cultured with or without proteinase inhibitors were lysed in 0.5 mM CaCl2/1 mM NaHCO3/0.25 M sucrose/0.2 mM phenylmethanesulfonyl fluoride/0. 1 mM 7-amino-1-chloro-3-tosylamido-2-heptanone ("tosyllysine chloromethyl ketone") at pH 7.2 in a glass tissue homogenizer at 40C. The cell lysate was centrifuged at 900 x g for 10 min to pellet nuclei, and the supernatant was layered above 100 bil of 1.58 M sucrose and spun at 2000 x g for 15 min to remove mitochondria. The resulting supernatant was layered above continuous sucrose gradients (...

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IFN-γ-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal enzymatic activity at low pH. GILT plays a crucial role in unfolding the antigenic proteins in preparation for their proteolytic cleavage and presentation of resulting peptides by MHC class II. In this study, we demonstrate that GILT is expressed in T lymphocytes and that it has an APC-nonrelated role in the regulation of T cell activation. Surprisingly, comparison of wild-type and GILT-deficient T cell activation in vitro revealed stronger responsiveness in the absence of GILT. The effect of GILT in reducing the proliferative and cytotoxic responses was endogenous to T cells and resulted from decreased sensitivity at the individual cell level. Therefore, a molecule with primarily lysosomal localization suppresses T cell activation, a process characterized by signal transmission from plasma membrane to cytoplasm and nucleus.

Loading of antigenic peptide fragments on major histocompatibility complex class II molecules is essential for generation of CD4؉ T cell responses and occurs after cathepsin-mediated degradation of the invariant chain chaperone molecule. Cathepsins are expressed differentially in antigen presenting cells, and mice deficient in cathepsin S or cathepsin L exhibit severely impaired antigen presentation in peripheral lymphoid organs and the thymus, respectively. To determine whether these defects are due solely to the block in invariant chain cleavage, we used cathepsin-deficient B cells to examine the role of cathepsins S and B in the degradation of other molecules important in the class II presentation pathway. Our data indicate that neither cathepsin S nor B is critical for H-2M degradation or processing of precursor ␥-interferon-inducible lysosomal thiol reductase (GILT) to a mature thiol reductase, but suggest a role for cathepsin S in the turnover of mature GILT and in regulating levels of mature cathepsin L protein in B cells. Despite the presence of mature cathepsin L protein, no enzyme activity could be detected in B cells or dendritic cells. These experiments suggest a novel mechanism by which these functionally important enzymes may be regulated. Recognition of antigen (Ag) by CD4ϩ T cells requires presentation of short peptide fragments in the context of major histocompatibility complex (MHC) 1 class II molecules (1, 2). MHC class II molecules consist of an ␣/␤ heterodimer that is assembled in the endoplasmic reticulum in association with a third glycoprotein, invariant chain (Ii), which promotes the correct folding of the MHC class II molecules (3, 4). A region of Ii known as class II-associated Ii peptide (CLIP) occupies the MHC class II peptide-binding groove to prevent inappropriate peptide loading (5-7), and signals in the cytoplasmic domain of Ii target the MHC class II/Ii chain complex to MHC class II rich endosomal compartments (8 -11). Upon entering the endocytic pathway, Ii chain is cleaved in a defined stepwise manner by proteases known as cathepsins (12)(13)(14), leaving only CLIP associated with the MHC class II heterodimer. Removal of CLIP and subsequent loading of diverse peptides is mediated by the MHC class II-like molecule HLA-DM (H-2M in mice) prior to MHC class II transport to the cell surface (15, 16).The cathepsins are a large family of aspartyl (D and E) and cysteinal (B, S, and L) endosomal proteases that have been implicated as critical to MHC class II presentation (12-14, 17). However, recent studies using cathepsin-deficient mice have shown that cathepsin D (catD) and cathepsin B (catB) are unnecessary for MHC class II presentation (18, 19), whereas cathepsin S (catS) and cathepsin L (catL) are important for Ag presentation by discrete populations of cells (20 -23). Active catS was detected in B cells, dendritic cells (DCs), and peritoneal macrophages (pMs), where it was shown to be involved in the late stages of Ii degradation (21-23). CatL, however, was detected only in Ms an...

Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.Enzymes of the thiol reductase family carry out reduction, oxidation, and isomerization of protein disulfide bonds in cytosol (for example, thioredoxin) (1, 2), mitochondria (3), endoplasmic reticulum (protein-disulfide isomerase) (2), and lysosomes (gamma interferon-inducible thiol reductase, GILT).2 The majority of these enzymes are functional at neutral or slightly alkaline conditions (4), they have similar three-dimensional structures, and all feature a conservative active site loop containing two cysteines in the sequence -CGPC-(5). GILT is a unique and unusual member of the thiol reductase family because its optimal enzymatic activity is at a low pH (4.5-5.5) (6 -8) and has an atypical active site (-CGAC-).GILT is synthesized as a 35-kDa soluble glycoprotein precursor and is transported to the endosomal compartment via the mannose-6-P receptor pathway (9). It is processed into the mature form (30 kDa) by proteolytic removal of N-and C-terminal peptides. The protein has an approximate molecular mass of 30 kDa and was therefore initially named IP-30 (6). In addition to endosomal/lysosomal localization, GILT is secreted in the tissue culture medium of the GILT-expressing cell lines (7,10), and is present in mouse sera.3 GILT is constitutively expressed in professional antigen-presenting cells (APCs), but it is also inducible by pro-inflammatory cytokines such as interferon ␥, tumor necrosis factor ␣, and interleukin 1␤ (9).Using GILT Ϫ/Ϫ mice as a model, we have shown that GILT catalyzes initial unfolding of antigenic protein (protein becomes more accessible for further processing by cathepsins) and therefore facilitates protein/peptide binding to MHC class II molecules (10). By changing the redox state of exogenous antigenic proteins with disulfide bonds, GILT initiates the adaptive immune response. However, we have shown that GILT is constitutively expressed in T cells and has a role in the regulation of T cell activation. This is so far the only known GILT function not related to MHC class II processing (11). GILT Ϫ/Ϫ T cells show increased proliferation and cytotoxic T cell activity in response to anti-CD3 stimulation. This observation suggests that GILT has a more fundamental role in cellular processes than just reduction of antigens...