Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.     

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.     

The P9 pocket of HLA-DQ2 (non-Aspbeta57) has no particular preference for negatively charged anchor residues found in other type 1 diabetes-predisposing non-Aspbeta57 MHC class II molecules

Susceptibility and resistance to type 1 diabetes are associated with MHC class II alleles that carry non-Asp and Asp at residue 57 of their beta chain respectively. The effect of Asp or non-Aspbeta57 may relate to a differential ability of distinct class II molecules to bind specific immuno-pathogenic peptides. Recent studies in man and mouse have revealed that some type 1 diabetes-predisposing non-Aspbeta57 class II molecules (i.e. DQ8, DR4Dw15 and I-Ag7) preferentially bind peptides with a negatively charged anchor residue at P9. It has been suggested that this is a common feature of type 1 diabetes-predisposing class II molecules. The molecular explanation for such a phenomenon could be that class II beta chains with Aspbeta57 form a salt bridge between Aspbeta57 and a conserved Arg of the a chain, whereas in non-Aspbeta57 molecules the Arg is unopposed and free to interact with negatively charged P9 peptide anchor residues. We have investigated the specificity of the P9 pocket of the type 1 diabetes-associated DQ2 molecule and in particular examined for charge effects at this anchor position. Different approaches were undertaken. We analyzed binding of a high-affinity binding ligand and P9-substituted variants of this peptide, and we analyzed the binding of a set of synthetic random peptide libraries. The binding analyses were performed with wild-type DQ2 and a mutated DQ2 with Ala at beta57 substituted with Asp. Our results indicate that the wild-type DQ2 (non-Aspbeta57) prefers large hydrophobic residues at P9 and that there is no particular preference for binding peptides with negatively charged residues at this position. The specificity of the P9 pocket in the mutated DQ molecule is altered, indicating that the beta57 residue contributes to determining the specificity of the P9 pocket. Our data do not lend support to the hypothesis that all non-Asp beta57 class II molecules predispose to development of disease by binding peptides with negatively charged P9 anchor residues.     

Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

Thymic positive selection and peripheral activation of islet antigen-specific T cells: separation of two diabetogenic steps by an I-A(g7) class II MHC beta-chain mutant

The diabetes-susceptible class II MHC genes (in human and mouse) share unique nonaspartic acid residues at position 57 of the class II beta-chain. Transgenic expression of a mutant I-A(g7), substituting histidine and serine at position 56 and 57 of beta-chain with proline and aspartic acid (I-A(g7)PD), respectively, inhibits diabetes development in the nonobese diabetic mouse model. Here, we demonstrate that immature thymocytes expressing a diabetogenic islet Ag-specific transgenic TCR are positively selected by I-A(g7)PD class II MHC to give rise to mature CD4+ T cells. However, splenic APCs expressing the same I-A(g7)PD fail to present pancreatic islet Ag to mature T cells bearing this diabetogenic TCR. These results indicate that nonaspartic acid residues at position 57 of class II MHC beta-chain is important for diabetogenic CD4+ T cell activation in the periphery but is not essential for the formation of a diabetogenic T cell repertoire in the thymus.     

In vitro and in vivo evidence for high frequency of I-Ab-reactive CD4+ T cells in HLA-DQ or HLA-DRA transgenic mice lacking endogenous MHC class I and/or class II expression

Although T cells are educated to recognize foreign antigenic peptides in the context of self MHC molecules during their development in the thymus, peripheral T cells also recognize allo- and xeno-MHC molecules. The lower frequency of xeno-MHC-reactive T cells than that of allo-MHC-reactive T cells is often explained by the difference in the degree of homology between xeno- or allo-MHC and self MHC molecules, as well as by the species barrier of the molecules involved in immune recognition. To distinguish these two possibilities, we estimated the frequency of I-Ab-reactive CD4+ T cells selected by HLA-DQ or DR alpha E beta b molecules, using HLA-DQ6 and HLA-DRA transgenic C57BL/6 (B6) mice lacking endogenous MHC class I and/or class II molecules (DQ6A0/0 and DR alpha 30A0/0 beta 20/0). CD4+ lymph node T cells from DQ6A0/0 and DR alpha 30A0/0 beta 20/0 showed the strong proliferative response to I-Ab molecules. In addition, DQ6A0/0 and DR alpha 30A0/0 beta 20/0 rejected the skin graft from mice expressing I-Ab molecules irrespective of MHC class I expression, indicating that the CD4+ T cells recognizing I-Ab molecules are directly involved in this rejection. The estimated frequency of I-Ab-reactive CD4(+)CD8- thymocytes in DR alpha 30A0/0 beta 20/0 and DQ6A0/0 was comparable with that observed in the MHC class II-disparate strains. Our findings thus indicate that CD4+ T cells selected to mature on xeno-MHC class II molecules such as HLA-DQ6 or DR alpha E beta b, when these molecules are expressed in mice, recognize I-Ab molecules as allo-MHC class II, despite the less structural homology.     

Effects of residues of deltamethrin in cattle faeces on the development and survival of three species of dung-breeding insect

We report on the role of HLA-DQA1 and DQB1 alleles in determining susceptibility to insulin-dependent diabetes mellitus (IDDM) in Hong Kong Chinese and investigate whether these alleles affect the age of onset of the disease. We studied 76 unrelated Chinese patients and 250 controls. There was no apparent predisposing effect of non-aspartic acid residues at position 57 of the DQ beta chain (Asp57-) but there was an excess of homozygous genotypes containing arginine at position 52 of the DQ alpha chain (Arg52+). This excess was mainly attributable to the genotype DQA1*0301/DQA1*05011 in early-onset disease. There was a significant excess of heterodimers of DQ alpha and DQ beta carrying Arg52+ and Asp57- in both early-onset and late-onset disease, but the excess in early-onset disease was mainly attributable to a single heterodimer formed by DQA1*05011 and DQB1*0201. Of three DQA1/DQB1 genotypes containing a double dose of Arg52+ and Asp57-, only one had a strong association with both early-onset and late-onset disease. We show that early-onset IDDM and late-onset IDDM in Chinese may be separated on the basis of their associated DQA1 and DQB1 genotypes and we conclude that previously reported associations of IDDM with Arg52+ and Asp57- residues in Chinese are secondary to specific combinations of DQA1 and DQB1 alleles. We also show that DRB1 molecules play a distinct role in determining susceptibility to early-onset IDDM but the greatest effect is exerted by specific DR/DQ genotypic combinations.     

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.     

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.     

The role of I-Ag7 beta chain in peptide binding and antigen recognition by T cells

We examine here how the beta chain of the class II MHC molecule I-Ag7 influences T cell recognition. Three sets of T cell clones were identified. The first set recognizes peptides bound to I-Ag7, I-Ad and I-Ag7 mutant in which the allele-specific residues His and Ser at position 56 and 57 were changed to the Pro at residue 56 and to non-polymorphic Asp at residue 57. The second set responds to the antigen presented only by I-Ag7 and does not recognize the peptides bound to the other class II molecules. The third set is also specific for I-Ag7 as a result of the poor binding of the peptide to I-Ad and the mutant I-Ag7. These results indicate that positions 56 and 57 of the I-Ag7 class II MHC beta chain play a role in both T cell recognition of the MHC-peptide complex and peptide binding to MHC. These two different functions may be involved in I-Ag7-restricted beta cell antigen recognition by diabetogenic T cell clones.     

A phase I/II study to evaluate the effect of fractionated hemibody irradiation in the treatment of osseous metastases--RTOG 88-22

Previous studies have indicated that certain alleles of HLA-DR and -DQ genes were strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM), and the role of DQ molecule in IDDM has been suggested. To further clarify the association of DQ alleles with IDDM, we determined the nucleotide sequences of full-length cDNA from 13 DQA1 alleles and 14 DQB1 alleles. The sequencing analysis revealed sequence polymorphisms outside the hypervariable region of DQ genes. We then analyzed the DQA1 and DQB1 polymorphisms along with that of DRB genes in 86 B-lymphoblastoid cell lines (B-LCLs) from various ethnic groups and in healthy unrelated Japanese and Norwegian individuals. The allelic and haplotypic distributions in each population revealed the characteristic haplotypic formation in the HLA class II region. HLA genes in 139 Japanese and 100 Norwegian IDDM patients were analyzed. DQB1*0301 was negatively associated with IDDM in both ethnic groups, irrespective of associated DRB1 and DQA1 alleles. In DQB1*0302 positive populations, which represented a positive association with IDDM in both ethnic groups, DRB1*0401, *0404, *0802 haplotypes increased in the patients, whereas DRB1*0406 haplotype decreased. Considering about the hierarchy in DRB1 alleles with IDDM susceptibility (DRB1*0401>*0404>*0403 in Norwegian and DRB1*0802>*0403>*0406 in Japanese), the genetic predisposition to IDDM is suggested to be defined by the combination of DR-associated susceptibility and DQ-associated susceptibility and by the DQ-associated resistance which is a dominant genetic trait.     

Polymorphism at the HLA-DQ locus determines susceptibility to experimental autoimmune myasthenia gravis

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-A beta0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-A beta0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.     

The orientation of a T cell receptor to its MHC class II:peptide ligands

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.     

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.     

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.     

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.     

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.     

How HLA-DM affects the peptide repertoire bound to HLA-DR molecules

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.     

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.