Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.     

Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p

We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.     

Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endo. somal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DM, to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.     

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.     

Class II-associated invariant chain peptide-independent binding of invariant chain to class II MHC molecules

The class II-associated invariant chain peptide (CLIP) region of invariant chain (Ii) is believed to play a critical role in the assembly and transport of MHC class II alphabetaIi complexes through its interaction with the class II peptide-binding site. The role of the CLIP sequence was investigated by using mutant Ii molecules with altered affinity for the DR1 peptide-binding site. Both high- and low-affinity mutants were observed to efficiently assemble with DR1 and mediate transport to endosomal compartments in COS cell transfectants. Using N- and C-terminal truncations, a region adjacent to CLIP within Ii(103-118) was identified that can complement loss of affinity for the peptide-binding site in mediating efficient assembly of alphabetaIi. A C-terminal fragment completely lacking the CLIP region, Ii(103-216), was observed binding stably to class II molecules in immunoprecipitation studies and experiments with purified proteins. The Ii(103-118) region was required for this binding, which occurs through interactions outside of the alphabeta peptide-binding groove. We conclude that strong interactions involving Ii(103-118) and other regions of Ii cooperate in the assembly of functional alphabetaIi under conditions where CLIP has little or no affinity for the class II peptide-binding site. Our results support the hypothesis that the CLIP sequence has evolved to avoid high-stability interactions with the peptide-binding sites of MHC class II molecules rather than as a promiscuous binder with moderate affinity for all class II molecules.     

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.     

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.     

The inability of the nonobese diabetic class II molecule to form stable peptide complexes does not reflect a failure to interact productively with DM

Sequence variability in MHC class II molecules plays a major role in genetically determined susceptibility to insulin-dependent diabetes mellitus (IDDM). It is not yet clear whether MHC class II polymorphism allows selective binding of diabetogenic peptides or regulates some key intracellular events associated with class II-restricted Ag presentation. In this study, we have employed gene transfer techniques to analyze the intracellular events that control peptide acquisition by the unique class II molecule expressed by nonobese diabetic mice (I-Ag7). This structurally unique class II molecule fails to demonstrate stable binding to antigenic peptides and fails to undergo the conformational change associated with stable peptide binding to class II molecules. The experiments reported here demonstrate that I-Ag7 can productively associate with two protein cofactors important in class II-restricted Ag presentation, invariant chain (Ii) and DM. DM participates in the removal of the Ii-derived class II-associated Ii chain peptide and the p12 degradation product from the I-Ag7 molecule. In addition, I-Ag7 undergoes a conformational change when DM is expressed within the APC. Finally, DM can mediate accumulation of peptide/class II complexes on the surface of APCs. Collectively, our experiments indicate that the failure of the I-Ag7 molecule to stably bind peptide cannot be attributed to a failure to interact with the DM or Ii glycoproteins.     

S-2-amino-5-azolylpentanoic acids related to L-ornithine as inhibitors of the isoforms of nitric oxide synthase (NOS)

To characterize the importance of a highly conserved region of the class II beta chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated beta chain with a wild-type alpha chain in a murine L cell by gene transfection. The mutant class II molecule (81betaH-) assembles normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii) is coexpressed with 81betaH-, the class II-Ii complex is degraded in the endosomes. Expression of 81betaH- in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81betaH-. We propose that 81betaH- is protease sensitive because it is unable to productively associate with most peptides, including classII-associated invariant chain peptides. This model is supported by our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule and peptide are important for the integrity and stability of the complex, and that empty class II molecules are protease sensitive and degraded in endosomes. One function of DM may be to insure continuous groove occupancy of the class II molecule.     

Bacterial enteritides of poultry

We report an experimental system for abundant expression of specific peptide-class II complexes in vivo and in vitro. We have constructed a cassette which allows for the replacement of the CLIP region of invariant chain (Ii) with an antigenic peptide. In fibroblasts expressing an altered Ii protein, in which CLIP has been replaced with peptide 52-68 from the class II I-E alpha chain (pEalpha), pEalpha-I-Ab complexes are formed with high efficiency. This peptide loading occurs in the endoplasmic reticulum (ER) when the Ii:pEalpha fusion protein associates with the I-Ab alpha and beta chains. The trimeric complexes of Ii:pEalpha and I-Ab molecules are stable in SDS and can be detected by the pEalpha-I-Ab-specific mAb, YAe, indicating that pEalpha is bound in the class II groove in the context of full-length Ii. These data strongly suggest that the CLIP region of intact Ii prevents peptide loading in the ER by binding in the peptide binding groove of newly synthesized class II alphabeta dimers.     

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 +/- 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these alphabeta Ii complexes are converted into alphabeta-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the alphabeta Ii complexes. More than 80% of alphabeta dimers originating from internalized alphabeta Ii complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface alphbeta Ii into SDS-stable and -unstable alphabeta dimers. Altogether, these data suggest that newly liberated class II alphabeta heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.     

Exit of major histocompatibility complex class II-invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation

The Iip35 isoform of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains an endoplasmic reticulum (ER) targeting motif, but in B cell lines the ER retention is ineffective and a fraction of Iip35 is transported through the Golgi complex associated with class II molecules. We found Iip35 (but not Iip33, the major form of Ii) to be phosphorylated in B cell lines, as well as in transfected HeLa cells. The phosphorylation of Iip35 was found to be necessary for the exit of Iip35-class II complexes out of the ER. This requirement suggests that phosphorylation may change the interaction with factors responsible for ER retention/retrieval, and we did find that phosphorylated Iip35 associates with 14-3-3 proteins, a family of adaptor proteins that are involved in coordinating signal transduction pathways. This finding raises the intriguing possibility that the exit of Ii-class II complexes from the ER is regulated by intracellular signaling events.     

Ontogeny of Ia messenger RNA in the mouse small intestinal epithelium is modulated by age of weaning and diet

BACKGROUND: Exogenous antigenic peptides are presented to T cells by class II major histocompatibility complex (Ia) molecules on the surface of antigen-presenting cells. Class II-associated invariant chain (Ii) is also required for effective antigen presentation. Because messenger RNAs (mRNAs) for Ii chain and for class II I-A beta chain appear in the mouse intestinal epithelium after weaning, experiments were conducted to test the effect of age of weaning and diet on the appearance of Ia and Ii mRNA. METHODS: Four litters were split at day 17; one half was weaned and the other remained with the mother until day 24. On day 23, 25, 27, and 29, enterocytes were isolated from full-length small intestine by vascular perfusion with 30 mmol/L ethylenediaminetetraacetic acid, and the RNA was extracted. RESULTS: Appearance of Ii and I-A beta was significantly delayed by late weaning, as judged by RNA hybridization blots (Ii chain) and complementary DNA amplification (I-A beta chain). In mice on elemental diets, the appearance of Ii and I-A beta chain was delayed compared with littermates reared on standard chow. Ii mRNA failed to appear in mice maintained on the elemental diet by day 40, despite normal growth. CONCLUSIONS: Appearance of mRNA for both Ia and Ii depends on the introduction of a complex diet and not the "stress" of weaning or elimination of breast milk. Introduction of foreign dietary antigens or development of an altered intestinal flora may contribute to this process.     

Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles

B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers.     

Diminished class II-associated Ii peptide binding to the juvenile dermatomyositis HLA-DQ alpha 1*0501/DQ beta 1*0301 molecule

HLA class II molecules bind and present peptide Ags to T cells, binding specific sets of peptides due to polymorphism in the peptide binding groove. Class II proteins associate with the invariant chain (Ii chain) and its derived class II-associated Ii peptide (CLIP). Ii chain association is important for normal trafficking of class II proteins to the peptide loading vesicles and for blocking premature access of peptides to HLA class II molecules during maturation. We have previously shown that juvenile dermatomyositis is associated with the HLA-DQA1*0501 allele. There is limited information available about the interaction of any DQ molecule with the Ii chain and little information about binding of individual peptides to HLA-DQalpha1*0501/DQbeta1*0301. We sequenced peptides eluted from the juvenile dermatomyositis-associated class II allele HLA-DQalpha1*0501/DQbeta1*0301. Surprisingly, we found no Ii chain or CLIP. Further examination of peptide binding to the HLA-DQalpha1*0501/DQbeta1*0301 molecule demonstrated poor CLIP binding. However, newly synthesized HLA-DQalpha1*0501/DQbeta1*0301 molecules do associate with intact Ii chain. Molecular modeling suggests that CLIP binds differently to HLA-DQalpha1*0501/DQbeta1*0301 than to DR molecules. The lack of CLIP association suggests that HLA-DQalpha1*0501/DQbeta1*0301 has access to peptides earlier in the processing pathway and so might encounter novel peptides that induce autoimmunity.     

Difference in antigen presentation pathways between cortical and medullary thymic epithelial cells

Antigen presentation by thymic epithelial cells (TEC) to T cells that undergo maturation is one of the major events in the selection of the T cell repertoire. We have already reported that medullary TEC lines (mTEC) established from newborn C57BL/6 (H-2b) mice are able to present a soluble antigen, ovalbumin (OVA), to OVA-specific, I-Ab restricted helper T cell lines but cortical TEC (cTEC) lines are not (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601). In this report, to clarify the cause of this difference, we analyzed the biochemical nature as well as the distribution of both major histocompatibility complex (MHC) class II molecules and invariant chains (Ii) in both TEC by immunoprecipitation and laser confocal scanning microscopic analysis, as well as the expression of mRNA encoding H-2Ma or H-2Mb. Our results demonstrate that cTEC and mTEC are both able to present peptide antigens to peptide-specific, I-Ab-restricted helper T cell hybridoma and are able to present class II MHC alloantigens to an I-Ab-specific T cell line, that mRNA for H-2Ma and H-2Mb are expressed in both TEC, that cTEC and mTEC apparently incorporate tetramethylrhodamine isothiocyanate-labeled OVA in the same manner, and that the SDS-stable MHC class II molecules, onto which peptides were loaded, are formed in both cTEC and mTEC. However, these molecules were more rapidly degraded in mTEC than in cTEC. In addition, two Ii-derived polypeptides of approximately 21 kDa and 10 kDa were precipitated by the anti-class II monoclonal antibody Y3P; 10-kDa polypeptides were detected in the both TEC, while 21-kDa polypeptides were detected only in cTEC. Finally, beta chains of MHC class II with less sialylated oligosaccharides were precipitated from the cell surface of cTEC. Taken together, these results suggest that there are substantial differences in the antigen-presenting pathways of cTEC and mTEC, and these difference might be responsible for T cell selection events in the thymus.     

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.     

Major histocompatibility complex class II-dependent antigen presentation by human intestinal endothelial cells

BACKGROUND & AIMS: In the normal gut, human intestinal microvascular endothelial cells (HIMECs) express major histocompatibility complex (MHC) class II molecules. Enhanced expression is found in chronic inflammation. We examined the cytokine regulation of MHC class II molecules and the associated invariant chain (Ii) in HIMECs and investigated whether such cells can process and present a complex protein antigen to T cells. METHODS: Enzyme-linked immunosorbent assay, flow cytometry, immunoelectron microscopy, as well as T-cell activation assay with HIMECs and HLA-DR-restricted T-cell clones were employed. RESULTS: In unstimulated HIMEC monolayers, HLA-DR, -DP, and -DQ and Ii were undetectable at the protein level, but interferon gamma (IFN-gamma) (100 U/mL) induced expression that peaked for DR after 2-3 days, for DP after 4-6 days, for DQ after 10-12 days, and for Ii after 2-3 days. Tumor necrosis factor alpha had no effect alone but enhanced class II expression in combination with IFN-gamma, most notably for DQ and DP. HLA-DR3-restricted and Mycobacterium tuberculosis heat shock 65-kilodalton-specific T-cell clones were activated to produce IFN-gamma in response to relevant antigen presented by IFN-gamma-treated HIMECs. This response was inhibited by blocking monoclonal antibody to HLA-DR and by chloroquine when compared to professional antigen-presenting cells, HIMECs activated T-cell clones quite efficiently. CONCLUSIONS: These data suggest that microvascular endothelial cells can present complex protein antigens in the human gut.