Ii Processing Research Articles

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

SummaryAntigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

Identification of cathepsin B from large yellow croaker (Pseudosciaena crocea) and its role in the processing of MHC class II-associated invariant chain

In teleost, cathepsin B has been identified from several species and shown to play roles in the host immune response during pathogen challenge. However, the mechanism of how cathepsin B modulates the immune response in teleosts remains poorly understood. In this study, we identified and characterized cathepsin B (LycCatB) and invariant chain (LycIi) from the large yellow croaker (Pseudosciaena crocea). Sequence comparison and phylogenetic analysis indicated that LycCatB and LycIi are highly conserved within teleosts. Quantitative RT-PCR analysis showed that LycCatB mRNA was widely expressed in all examined tissues. We then recombinantly expressed LycCatB and Lyc-TR-Ii (transmembrane domain removed Ii chain) in Pichia pastoris and Escherichiacoli, respectively. The recombinant LycCatB (rLycCatB) can hydrolyze the substrate Z-FR-AMC with a Km value of 40.68μM. Furthermore, co-incubation of rLycCatB with rLyc-TR-Ii led to an efficient cleavage of rLyc-TR-Ii in a time-dependant manner. These results indicated that cathepsin B may be involved in MHC class II-associated Ii processing in large yellow croaker, and provide new information helping to elucidate the immunological functions of teleost cathepsin B.

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.

Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.

Inhibition of Invariant Chain Processing, Antigen-Induced Proliferative Responses, and the Development of Collagen-Induced Arthritis and Experimental Autoimmune Encephalomyelitis by a Small Molecule Cysteine Protease Inhibitor

Members of the papain family of cysteine proteases (cathepsins) mediate late stage processing of MHC class II-bound invariant chain (Ii), enabling dissociation of Ii, and binding of antigenic peptide to class II molecules. Recognition of cell surface class II/Ag complexes by CD4(+) T cells then leads to T cell activation. Herein, we demonstrate that a pan-active cathepsin inhibitor, SB-331750, attenuated the processing of whole cell Ii p10 to CLIP by Raji cells, and DBA/1, SJL/J, and C57BL/6 splenocytes. In Raji cells and C57BL/6 splenocytes, SB-331750 inhibited class II-associated Ii processing and reduced surface class II/CLIP expression, whereas in SB-331750-treated DBA/1 and SJL/J splenocytes, class II-associated Ii processing intermediates were undetectable. Incubation of lymph node cells/splenocytes from collagen-primed DBA/1 mice and myelin basic protein-primed SJL/J mice with Ag in the presence of SB-331750 resulted in concentration-dependent inhibition of Ag-induced proliferation. In vivo administration of SB-331750 to DBA/1, SJL/J, and C57BL/6 mice inhibited splenocyte processing of whole cell Ii p10 to CLIP. Prophylactic administration of SB-331750 to collagen-immunized/boosted DBA/1 mice delayed the onset and reduced the severity of collagen-induced arthritis (CIA), and reduced paw tissue levels of IL-1beta and TNF-alpha. Similarly, treatment of myelin basic protein-primed SJL/J lymph node cells with SB-331750 delayed the onset and reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis (EAE). Therapeutic administration of SB-331750 reduced the severity of mild/moderate CIA and EAE. These results indicate that pharmacological inhibition of cathepsins attenuates CIA and EAE, potentially via inhibition of Ii processing, and subsequent Ag-induced T cell activation.

Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation

Presentation of Ag by class II MHC is regulated by lysosomal proteases that not only destroy the class II invariant chain (Ii) chaperone but also generate the peptide Ag that is loaded onto the class II MHC dimer. We sought to determine the extent to which asparagine endopeptidase (AEP) influences human Ag and Ii processing. Our data confirm the constructive function of AEP in tetanus toxoid processing, but they are discordant with findings that suggest a destructive role for AEP in processing of the immunodominant myelin basic protein epitope. Furthermore, we observed no effect on invariant chain processing following AEP inhibition for several distinct allelic variants of human class II MHC products. We find that cysteine and aspartic proteases, as well as AEP, can act redundantly to initiate Ii processing. We detected considerable variation in lysosomal activity between different EBV-transformed B cell lines, but these differences do not result in altered regulation of invariant chain catabolism. We propose that, as for bound peptide Ag, the identity of the lysosomal enzyme that initiates invariant chain cleavage is dependent on the class II MHC allelic variants expressed.

Cathepsin S is not crucial to TSHR processing and presentation in a murine model of Graves' disease

By regulating invariant (Ii) chain processing and MHC class II peptide loading, the endosomal protease cathepsin S (Cat S) has a potential role in autoimmune susceptibility. Indeed, Cat S null mice are resistant to I-Ab-restricted experimental myasthenia gravis due to inadequate peptide presentation. To explore the role of Cat S in a Graves' disease model, I-Ad-restricted wild-type (WT) and Cat S(-/-) mice were immunized with adenovirus encoding the A subunit of thyroid stimulating hormone receptor (TSHR). TSHR adenovirus immunized mice develop Th1 T cells, TSHR antibodies, and a proportion become overtly hyperthyroid. Although TSHR presentation in vitro was initially impaired in Cat S(-/-) mice, subsequent TSHR presentation in vitro and disease development were similar in both groups but with higher antibody responses in Cat S null mice. WT and Cat S(-/-) mice recognized similar T cell epitopes from a panel of overlapping TSHR peptides. TSHR responses were found to be I-Ad-restricted and Cat S(-/-) I-Ad B cells had marked defects in Ii processing. These data imply that loading of TSHR peptides critical to TSHR antibody responses becomes Ii-independent. Contrasting findings among organ-specific murine autoimmune models imply that potential uses of Cat S inhibitors to ameliorate autoimmunity must be determined empirically.

Mycobacterium bovis BCG Attenuates Surface Expression of Mature Class II Molecules through IL-10-Dependent Inhibition of Cathepsin S

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.

Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation.

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class II (MHC II) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC II chaperone Ii, and hence in the formation of MHC II-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, Ii processing and MHC II peptide loading proceeded similarly in all three DC populations. We then analyzed MHC II localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Antigen processing and presentation in human muscle: cathepsin S is critical for MHC class II expression and upregulated in inflammatory myopathies

The immunological properties of muscle cells are of critical importance for both the pathogenesis of inflammatory muscle disorders as well as for understanding and controlling novel therapeutic strategies. Muscle cells can present antigens to both CD4 and CD8 cells. However, the cellular biochemistry of antigen processing and presentation by muscle cells is not clear. Cathepsins play a central role in the generation of antigenic peptide and control transport and maturation of MHC class II molecules. To further elucidate the molecular basis for the MHC class II-mediated antigen presentation by muscle cells, we here analyzed cultured human myoblasts and biopsies from inflammatory myopathies with respect to the expression and function of the constituents of the MHC class II antigen presentation machinery. We identified cathepsin S (CatS) as the dominant endocytic protease that is specifically upregulated under inflammatory conditions to significant mRNA levels, synchronously with HLA-DR, -DM and the class II invariant chain (Ii), both in muscle biopsies from affected individuals with inflammatory myopathies and in human myoblasts cultured in the presence of IFN-γ. This led to translation of the mature CatS polypeptide that was enzymatically active in human myoblasts under inflammatory conditions. By contrast, expression of CatL and CatB was unaffected by IFN-γ at both the expression and activity levels. CatS activity is required for efficient surface display of MHC class II in this cell type: functional inhibition of CatS using a CatS-selective inhibitor reduced the levels of surface class II αβ:peptide complexes on stimulated myoblasts by almost 50%. Surprisingly, and in contrast to B cells and dendritic cells, this was not due to inefficient processing of Ii in the absence of CatS, which was unaffected by the elimination of CatS activity. We therefore conclude that CatS is involved in the regulation of class II expression in human myoblasts independently from Ii processing.

Asparagine Endopeptidase Can Initiate the Removal of the MHC Class II Invariant Chain Chaperone

The invariant chain (Ii) chaperone for MHC class II molecules is crucial for their effective function. Equally important is its removal. Cathepsins S or L are known to be required for the final stages of Ii removal in different APCs, but the enzymes which initiate Ii processing have not been identified. Here we show that this step can be performed in B lymphocytes by asparagine endopeptidase (AEP), which targets different asparagine residues in the lumenal domain of human and mouse invariant chain. Inhibition of AEP activity slows invariant chain processing and hinders the expression of an antigenic peptide engineered to replace the groove binding region of Ii (CLIP). However, the initiation of Ii removal can also be performed by other proteases, reflecting the importance of this step.

Involvement of Cathepsin E in Exogenous Antigen Processing in Primary Cultured Murine Microglia

We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-gamma. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266-281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-gamma-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266-281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.

Regulation of CD1 Function and NK1.1 + T Cell Selection and Maturation by Cathepsin S

NK1.1 + T cells develop and function through interactions with cell surface CD1 complexes. In I-A b mice lacking the invariant chain (Ii) processing enzyme, cathepsin S, NK1.1 + T cell selection and function are impaired. In vitro, thymic dendritic cells (DCs) from cathepsin S −/− mice exhibit defective presentation of the CD1-restricted antigen, α-galactosylceramide (α-GalCer). CD1 dysfunction is secondary to defective trafficking of CD1, which colocalizes with Ii fragments and accumulates within endocytic compartments of cathepsin S −/− DCs. I-A k , cathepsin S −/− mice do not accumulate class II-associated Ii fragments and accordingly do not display CD1 abnormalities. Thus, function of CD1 is critically linked to processing of Ii, revealing MHC class II haplotype and cathepsin S activity as regulators of NK T cells.

Functional Early Endosomes Are Required for Maturation of Major Histocompatibility Complex Class II Molecules in Human B Lymphoblastoid Cells

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.

Cathepsin S activity regulates antigen presentation and immunity.

MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.

Ii chain controls the transport of major histocompatibility complex class II molecules to and from lysosomes.

Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting alpha beta dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A(b) alpha beta dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A(b) class II molecules in lysosomes. In leupeptin, I-A(b) dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A(b) dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

Selective Modulation of the Major Histocompatibility Complex Class II Antigen Presentation Pathway following B Cell Receptor Ligation and Protein Kinase C Activation

We noticed that B cell receptor ligation or phorbol 12-myristate 13-acetate treatment induced intracellular vesicles containing major histocompatibility complex (MHC) class II and invariant chain (Ii), and increased the amount of transmembrane p12 Ii fragments coimmunoprecipitated with class II molecules. To determine the influence of protein kinase C activation on the MHC class II presentation pathway, we analyzed the subcellular distribution of Ii, the induction of SDS-stable forms of class II molecules, and their ability to present different antigens. Ii chains visualized with luminal and cytoplasmic directed antibodies appeared in early endosomal compartments accessible to transferrin in response to phorbol 12-myristate 13-acetate treatment, whereas transmembrane Ii degradation products equivalent to the p12 Ii fragments were colocalized with the B cell receptors internalized after cross-linking. Protein kinase C activation delayed in parallel the formation of SDS-stable forms of class II molecules and reduced the presentation of antigenic determinants requiring newly synthesized class II alphabeta-Ii complexes. These data indicate that B cell activation affects Ii processing and MHC class II peptide loading in endosomal compartments intersecting the biosynthetic pathway.

DM-mediated release of a naturally occurring invariant chain degradation intermediate from MHC class II molecules.

The nonpolymorphic, nonclassical class II molecule DM has been shown to be important in the processing and presentation of antigenic epitopes by MHC class II molecules. The dependence of class II molecules on the coexpression of DM varies with the particular allele or epitope studied. In an effort to resolve disparities that exist for some alleles of class II in their requirements for DM, we have constructed a species-matched murine transfection model with which we can test both the functional and biochemical consequences of DM expression for different alleles of murine class II. When we evaluated the ability of class II molecules to form SDS-stable dimer and release CLIP, we find that while I-A(d) requires DM for the formation of SDS-stable dimers and the release of CLIP, I-A(k) is capable of both SDS-stable dimer formation and CLIP release in the absence of DM. Despite the apparent differences in the biochemical consequences of DM expression for I-A(d) and I-A(k), we find that Ag presentation by both alleles can be enhanced by the expression of DM. Interestingly, we find that DM can facilitate the removal of a natural intermediate in Ii processing (p12). The ability of DM to catalyze the dissociation of p12 and possibly larger Ii fragments from class II in vivo offers a possible mechanism to account for the observed DM enhancement of Ag presentation for alleles that have a low affinity for CLIP. Our findings indicate that DM may function in multiple compartments and on multiple class II/Ii substrates.

T cell recognition of major histocompatibility complex class II complexes with invariant chain processing intermediates.

Peptides from the lumenal portion of invariant chain (Ii) spanning residues 80-106 (class II-associated Ii peptide [CLIP]) are found in association with several mouse and human major histocompatibility complex (MHC) class II allelic variants in wild-type and presentation-deficient mutant cells. The ready detection of these complexes suggests that such an intermediate is essential to the MHC class II processing pathway. In this study, we demonstrate that T cells recognize CLIP/MHC class II complexes on the surface of normal and mutant cells in a manner indistinguishable from that of nominal antigenic peptides. Surprisingly, T cell hybrids specific for human CLIP bound to murine MHC class II molecule I-Ab and a new monoclonal antibody 30-2 with the same specificity, recognize two independent epitopes expressed on this peptide/class II complex. T cell recognition is dependent on a Gln residue (position 100) in CLIP, whereas the 30-2 antibody recognizes a Lys residue-at position 90. These two residues flank the 91-99 sequence that is conserved among human, mouse, and rat Ii, potentially representing an MHC class II-binding site. Our results suggest that the COOH-terminal portion of CLIP that includes TCR contact residue Gln 100 binds in the groove of I-Ab molecule. Moreover, both T cells and the antibody recognize I-Ab complexed with larger Ii processing intermediates such as the approximately 12-kD small leupeptin-induced protein (SLIP) fragments. Thus, SLIP fragments contain a CLIP region bound to MHC class II molecule in a conformation identical to that of a free CLIP peptide. Finally, our data suggest that SLIP/MHC class II complexes are precursors of CLIP/MHC class II complexes.