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The major histocompatibility complex (MHC) is a term for all gene groups of a major histocompatibility antigen. It binds to peptide chains derived from pathogens and displays pathogens on the cell surface to facilitate T-cell recognition and perform a series of immune functions. MHC molecules are critical in transplantation, autoimmunity, infection, and tumor immunotherapy. Combining machine learning algorithms and making full use of bioinformatics analysis technology, more accurate recognition of MHC is an important task. The paper proposed a new MHC recognition method compared with traditional biological methods and used the built classifier to classify and identify MHC I and MHC II. The classifier used the SVMProt 188D, bag-of-ngrams (BonG), and information theory (IT) mixed feature representation methods and used the extreme learning machine (ELM), which selects lin-kernel as the activation function and used 10-fold cross-validation and the independent test set validation to verify the accuracy of the constructed classifier and simultaneously identify the MHC and identify the MHC I and MHC II, respectively. Through the 10-fold cross-validation, the proposed algorithm obtained 91.66% accuracy when identifying MHC and 94.442% accuracy when identifying MHC categories. Furthermore, an online identification Web site named ELM-MHC was constructed with the following URL: http://server.malab.cn/ELM-MHC/ . more...

The large-scale and in-depth identification of MHC class I- and II-presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as for developing innovative immunotherapeutic approaches against cancer and other diseases. In this chapter we briefly review the existing strategies for the isolation of MHC-restricted peptides and provide a detailed protocol for the immunoaffinity purification of MHC class I- and II-presented peptides from primary tissues or cells. more...

Comprehensive knowledge of the HLA class I and class II peptides presented to T cells is crucial for designing innovative therapeutics against cancer and other diseases. So far, methodologies for recovery of HLA class I and II peptides for subsequent mass spectrometry-based analysis have been a major limitation. In this chapter we describe a detailed protocol for a high-throughput, reproducible, and sensitive immunoaffinity-purification of HLA-I and HLA-II peptides from up to 96 samples in a plate format, suitable for tissue samples and cell lines. Our methodology reduces sample handling, has a competitive peptide yield, and can be completed within 5 h. This simplified pipeline is applicable for basic and clinical applications. more...

Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry. more...

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.) more...

The major histocompatibility complex of class II (MHCII) immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4 + T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU)] allowing the identification and label-free quantification (LFQ) of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAtEAU algorithm in facilitating robust and accessible quantitative analysis of immunopeptidomes across cellular contexts. In silico analysis of the peptides enriched for each HLA-DM expression conditions suggests a higher affinity of the pool of peptides isolated from the high DM expression samples. Interestingly, our analysis reveals that while for certain autoimmune-relevant epitopes their presentation increases upon DM expression others are clearly edited out from the peptidome. more...

The major histocompatibility complex (MHC) with its three classes represents a cluster of tightly linked genes with defined immunological and non-immunological functions. The DM, a MHC class II molecule is formed by the non-covalent association of DMα and DMβ chains. It binds with the processed peptide antigens and presents them to T lymphocytes, thereby triggering the immune responses. Startlingly, the expression pattern and structural organization of DMα and DMβ proteins in buffalo remains undefined. We isolated and purified the DMα and DMβ proteins from Bubalus bubalis using gel filtration chromatography. Employing western blotting and immunohistochemistry, highest expression of these proteins was observed in spleen and were later localized in the cytoplasm. We modelled 3D structures of the proteins and assessed the binding interface of BuLA-DM docked complex. In the process, we uncovered 9 DMα and 8 DMβ specific residues participating in the formation of BuLA-DM complex. Our work demonstrated active participation of the critical amino acid residues engaged in the formation of BuLA-DM complex facilitating deeper understanding on the structure-function relationship of these proteins. J. Cell. Biochem. 118: 4254-4266, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.) more...

Major histocompatibility complex (MHC) is the best-characterized genetic region associated with resistance and susceptibility to a wide range of diseases. In cattle, the most important example of the relationship between the MHC and infectious diseases has been established by the resistance to Bovine leukemia virus (BLV) infection. The association of the bovine MHC class II BoLA-DRB3.2 alleles with BLV infection profiles was examined. BoLA-DRB3.2 allelic diversity was determined in 190 Iranian Holstein cattle using direct sequencing method. Association of the DRB3.2 alleles with BLV infection profiles was found as the odds ratio. Effects of the alleles on lymphocyte subsets were also evaluated by multivariate regression analysis and GLM procedures. The studied cattle were categorized into three groups: BLV seronegative, BLV seropositive with persistent lymphocytosis (PL), and BLV seropositive with lymphosarcoma (LS). The PL profile was significantly associated with the BoLA-DRB3.2*0101, *1101 and *4201 alleles, although the *3202 allele mediating resistance to PL was observed. Significant association was found between the BoLA-DRB3.2*1802, *3202, and *0901 alleles and susceptibility to LS, while the *0101 and *1101 alleles were associated with resistance to LS. BoLA-DRB3.2 alleles also showed a significant correlation with CD4, CD8, CD21 cells and CD4/CD8 ratio. Allelic differences influence the immune response to BLV infection and developing the disease profile. These differences also have important consequences for tumor resistance. more...

Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses. more...

There are different degrees of similarity among vertebrate invariant chains (Ii). The aim of this study was to determine the relationship between quail and other vertebrate Ii MHC class II molecules. The two quail Ii isoforms (qIi-1, qIi-2) were cloned by RACE, and qRT-PCR analysis of different organs showed that their expression levels were positively correlated with MHC II gene (B-LB) transcription levels. Confocal microscopy indicated that quail full-length Ii co-localized with MHC II of quail, chicken or mouse in 293FT cells co-transfected with both genes. Immunoprecipitation and western blotting further indicated that these aggregates corresponded to polymers of Ii and MHC class II molecules. This cross-species molecular association of quail Ii with chicken and mouse MHC II suggests that Ii molecules have a high structural and functional similarity and may thereby be used as potential immune carriers across species., (Copyright © 2013 Elsevier Ltd. All rights reserved.) more...

Decellularized biologic matrices are plausible biomedical materials for the bioengineering in liver transplantation. However, one of the concerns for safe medical application is the lack of objective assessment of the immunogen within the materials and the in vivo immune responses to the matrices. The purpose of this study was the production of immunogen-reduced and biocompatible matrices from porcine liver. In the present study, 0.1% SDS solution was effective for removing DNA fragments and sequences encoding possible immunogenic and viral antigens within the matrices. The PCR analysis showed that galactose-α-1,3 galactose β-1,4-N-acetylglucosamine (1,3 gal), swine leukocyte antigen (SLA), and porcine endogenous retrovirus (PERV) were completely removed in the matrices. Collagen and glycosaminoglycans (GAGs) were preserved over 63%-71%, respectively, compared to those of native liver. The implanted decellularized tissues showed minimal host responses and naturally degraded within 10 weeks. In this study, we produced immunogen-reduced and biocompatible extracellular matrices from porcine liver. Although future investigations would be required to determine the mechanism of the host reaction, this study could provide useful information of porcine liver-derived biologic matrices for liver researches., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.) more...

Ubiquitinated protein detection is often troublesome since in most cases this modification reduces the half-life of targeted proteins, inducing their degradation. Furthermore, ubiquitination is reversible thanks to the action of highly specific deubiquitinases present in all eukaryotic cells. MHC molecules ubiquitination has been demonstrated to be a key event in the regulation of the potent immunostimulatory properties of activated human dendritic cells. more...

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions. more...

Gamma interferon Inducible Lysosomal Thiol reductase (GILT) is a unique lysosomal reductase that reduces disulfide bonds of endocytosed proteins. Lack of GILT clearly decreases CD4 T cell-antigen specific responses against some epitopes of antigens containing disulfide bonds, but not to proteins with few or no disulfide bridges. Hence, global impact of GILT on antigen presentation is currently not well understood. We used Nano-LC-ESI-MS/MS to investigate how GILT affects diversity of self-peptides presented by MHC class II molecules. Surprisingly, the repertoire of self-peptides in the absence of GILT does not appear to be significantly different, as only few peptide species (approximately 2%) were found to be the unique indicators of GILT's presence or absence. In the absence of GILT about thirty peptide species (approximately 5%) were found either uniquely or fourteen to hundred fold more abundantly expressed than in the presence of GILT. Our data indicate that GILT has limited yet unexpected effect on self-peptide species presented by MHC class II antigens. more...

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study. more...

This unit describes a method of measuring the kinetic stability of complexes formed between purified MHC class II molecules and antigenic N-terminal fluorescein-labeled peptides. An HPLC-SEC allows for the separation of fluoresceinated MHC class II:peptide complexes from excess free dissociating peptides, while the fluorescence detector allows for the quantification of the remaining complexes throughout the dissociation time-course. more...

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity. more...

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus. more...

We dissected the requirements for disease induction of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in MHC (RT1 in rat) congenic rats with overlapping MOG peptides. Immunodominance with regard to peptide-specific T cell responses was purely MHC class II dependent, varied between different MHC haplotypes, and was linked to encephalitogenicity only in RT1.B(a)/D(a) rats. Peptides derived from the MOG sequence 91-114 were able to induce overt clinical signs of disease accompanied by demyelinated CNS lesions in the RT1.B(a)/D(a) and RT1(n) haplotypes. Notably, there was no detectable T cell response against this encephalitogenic MOG sequence in the RT1(n) haplotype in peripheral lymphoid tissue. However, CNS-infiltrating lymphoid cells displayed high IFN-gamma, TNF-alpha, and IL-4 mRNA expression suggesting a localization of peptide-specific reactivated T cells in this compartment. Despite the presence of MOG-specific T and B cell responses, no disease could be induced in resistant RT1(l) and RT1(u) haplotypes. Comparison of the number of different MOG peptides binding to MHC class II molecules from the different RT1 haplotypes suggested that susceptibility to MOG-experimental autoimmune encephalomyelitis correlated with promiscuous peptide binding to RT1.B and RT1.D molecules. This may suggest possibilities for a broader repertoire of peptide-specific T cells to participate in disease induction. We demonstrate a powerful MHC class II regulation of autoaggression in which MHC class II peptide binding and peripheral T cell immunodominance fail to predict autoantigenic peptides relevant for an autoaggressive response. Instead, target organ responses may be decisive and should be further explored. more...

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively. more...

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus. more...

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection. more...

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacterium Listeria monocytogenes into human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytose L. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using an L. monocytogenes strain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. Although L. monocytogenes infection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria. more...

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice. more...

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone. more...

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery. more...

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine. more...

There is a growing body of information about the soluble forms of HLA in serum but there are only a few reports discussing sHLA in other body fluids. We quantitated sHLA-I and sHLA-II concentrations in sweat, saliva and tear samples from five normal individuals with known HLA-phenotypes. We also studied sweat samples from an additional 12 normal nonphenotyped subjects, as well as in CSF of 20 subjects with different illnesses, using solid phase enzyme linked immunoassay. Sweat, saliva and tears from normal subjects were found to contain very low or nondetectable amounts of sHLA-I. In contrast, sHLA-II molecules were found in each of these body fluids, although, with considerable variation between individuals. The presence of sHLA-II in saliva was further confirmed by Western-blotting. It was observed that sHLA-II having molecular mass of 43,900 and 18,100 daltons was comparable with that found in serum from normal individuals. In addition, no association of sHLA-II levels with allospecificities in either body fluid or in serum was apparent. The results of CSF sHLA concentrations in different diseases were as follows: (1) High CSF SHLA-I levels were measured during viral encephylitis (n = 3), while none of these patients contained sHLA-II in CSF; (2) The levels of sHLA-II, but not sHLA-I were elevated in CSF of patients during seizure (n = 6) and of patients with neonatal hepatitis (1 of 2) or with connective tissue disease accompanied with viral infection (n = 2); (3) No CSF sHLA-I or sHLA-II could be detected at polyneuropathy (n = 2), or in patients with syphilis (n = 3), or leukemia (n = 2) with evidence of neurologic involvement of central nervous system. Taken together, it may be concluded that the presence of sHLA in several body fluids is physiologically normal. It appears that sHLA-II is the predominant class of HLA molecules present in different body fluids. We propose that the system responsible for sHLA-II production in various body fluids must involve different mechanisms than those responsible for sHLA-I synthesis in serum. more...

A small but significant proportion of people who receive the hepatitis B vaccine do not produce anti-hepatitis B antibodies, a phenomenon associated with certain human leukocyte antigen (HLA) class II haplotypes. We were interested in determining whether natural allelic differences between two HLA-DR4 molecules associated with responder versus nonresponder subtypes differed with respect to binding of an immunodominant hepatitis B surface antigen (HBsAg) peptide as measured using a resonant mirror biosensor. In contrast to our original hypothesis, we found a ten-fold difference in the affinity in favor of the nonresponder DRB1*0401 allele, with a KD of 6.89 x 10(-8) M versus a KD of 6.71 x 10(-7) M for the responder DRB1*0404 allele. Half-times of dissociation were 1.3 min and 7.7 min, respectively, although association rate constants for both HLA class II molecules were similar (approximately 10(4) M(-1)s(-1)). Of particular interest was the observation of different on-rates during the association phase, suggesting that stoichiometry of binding was not 1:1 or that different structural forms of the HLA-peptide complex exist. Our observations indicate that whereas HBsAg peptide binding to HLA class II molecules is influenced by HLA polymorphism, the nonresponse to hepatitis B vaccine associated with this HLA-DR4 subtype is not a result of failure of processed HBsAg to bind HLA class II molecules. more...

X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (alpha beta)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple alpha beta heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the alpha beta and (alpha beta)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (alpha beta)2 complex displays thermal and pH stability similar to that of the alpha beta complex, both being denatured by SDS at temperatures above 50 degrees C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (alpha beta)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor. more...

MHC class II molecules present antigenic peptides to T cells. The sequence characteristics of peptides associated with various class II alleles have been examined by analysis of peptide mixtures extracted from purified class II molecules as well by direct binding assays with substituted synthetic peptides and purified class II molecules. Here, in vitro binding assays with purified DRB1*0101 and glycine substituted analogues of H gamma 321-340 and alanine substituted analogues of TT948-967, universal CD4+ epitopes of the gamma subunit of the human nicotinic acetylcholine receptor and tetanus toxin, respectively, were able to compete for binding to an extent similar to that of the unsubstituted peptides. Testing whether this is a property of promiscuous, but not allele-specific peptide epitopes, a designer peptide containing the proposed anchor residues for binding DRB1*0101 was used in similar binding assays. As expected, the binding capacity of this designer peptide was much higher than that of the universal epitope peptides. However, substitution of the anchor residues for alanine in the context of this designer peptide did not abrogate binding to DRB1*0101 and, in fact, enhanced it. Therefore, it appears that for both class II allele-specific and universal epitope peptides, binding is a result of the combinatory effects of a few residues. The individual replacement of these residues with the sterically and electrostatically neutral residue alanine does not negatively affect binding in the continued presence of other anchor residues. more...

The efficacy of a malaria peptide vaccine would be enhanced by the inclusion of a parasite-derived universal T cell epitope to ensure that all vaccinees develop parasite-specific cellular and humoral immunity. Two circumsporozoite (CS) protein T cell epitopes, previously identified by CD4+ T cell clones derived from Plasmodium falciparum sporozoite-immunized volunteers, were studied to determine their HLA class II binding potential. One epitope, located in amino acid (aa) 326-345 of the P. falciparum (NF54 strain) CS protein, was "universal" in that it could bind to multiple DR and DQ molecules in vitro. In contrast, the second epitope, T1, which is located in the CS repeat region, was recognized by T cells in the context of DQ6 (DQB1*0603) and did not bind with high affinity to any of the class II molecules tested in the peptide binding assays. The in vitro patterns of peptide/HLA interactions correlated with immunogenicity in vivo. A multiple antigen peptide (MAP) containing the aa 326-345 epitope elicited responses in eight inbred strains (H-2(a,b,d,k,p,q,r,s)), while the T1 MAP was recognized by only a single haplotype, H-2b. The combination of the universal aa 326-345 T cell epitope and the T1 repeat in a di-epitope MAP overcame the genetic restriction to the P. falciparum CS repeat region and elicited antisporozoite Ab responses in all of the MAP-immunized mice. Synthetic peptide malaria vaccines containing the aa 326-345 universal T cell epitope would be expected to elicit parasite-specific immune responses in both sporozoite-primed and naive individuals of diverse genetic backgrounds. more...

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the basolateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells. more...

We have previously identified an intracellular compartment involved in the association between processed lysozyme and IAk major histocompatibility complex class II molecules (called the lysozyme-loading compartment (LLC)). Here, we show that the LLC polypeptide composition analyzed by two-dimensional gel electrophoresis shares similarities with that of early endosomes, but not with that of late endosomes. The transferrin receptor, a well known marker for both early and recycling endosomes, colocalizes with IAk molecules in LLC. Moreover, both transferrin and fluid-phase markers have access to LLC after 15 min of internalization. In the presence of concanamycin B, SDS-stable dimer formation and transport of class II molecules out of LLC are impaired. In contrast, nocodazole treatment has no effect. These results suggest that LLC is a specialized compartment of the recycling pathway involved in lysozyme loading and in the targeting of lysozyme-major histocompatibility class II complexes toward the cell surface. more...

Synovial lymphocytes, from the site of disease, by their response to microbiological antigen stimulation as measured by the [3H]thymidine uptake method, indicate the microbiological causes of reactive arthritis and also oligoarthritis unassociated with enteric or genital symptoms. In the study of the etiology and pathogenesis of rheumatoid arthritis, the application of the same procedures gives an indication that the disease is an immune response to a variety of common infective agents, both viral and bacterial. The demonstration of antigens or nucleic acid of an infective agent at the site of disease, in association with a specific local immune response suggests the pathogenetic importance of the agent. Recent studies of relationships between epitopes of infective agents and MHC gene products suggest several ways in which infective agents can directly cause a disease such as rheumatoid arthritis without any requirement for autoimmune contributions. Because the infective agent may be the primary determining factor and the one most amenable to correction or eradication, the term "infective-immune" is suggested in preference to "autoimmune" for these immune-mediated diseases. more...

Standardized techniques that allow the direct identification of tumor antigens are now available. Several murine antigens recognized by T cells have already been identified. So far, the majority of these antigens derive from cellular proteins similar to those that give rise to human tumor antigens. While many of the known human tumor antigens are widely shared, most of the murine tumor antigens appear to be unique to the individual tumor from which they were isolated. Nonetheless, common features between murine and human tumor antigens are emerging, suggesting that these murine antigens will provide essential tools in the evaluation of antigen-based vaccines for the future treatment of cancer. more...

Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first stop of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA-DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+ progenitor cells, and HLA-DR+ breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage. more...

Structural studies of cellular receptor molecules involved in immune recognition require the production of large quantities of the extracellular domains of these glycoproteins. The murine major histocompatibility complex (MHC) class II-restricted response has been extensively studied by functional means, but the engineering and purification of the native, empty form of the most-studied murine MHC class II molecule, IA, has been difficult to achieve. IA molecules, which are the murine equivalent of human histocompatibility leukocyte antigen-DQ molecules, have a low efficiency of chain pairing, which results in poor transport to the cell surface and in the appearance of mixed isotype pairs. We have engineered soluble IA molecules whose pairing has been forced by the addition of leucine zipper peptide dimers at their COOH-terminus. The molecules are secreted "empty" into the extracellular medium and can be loaded with single peptide after purification. These IA molecules have been expressed in milligram quantity for crystallization as well as for activation of T cells and measurement of MHC class II-T cell receptor interactions. more...

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class. more...

We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands. more...

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process. more...

A complex between HLA-DR3 and a fragment of invariant chain called CLIP was isolated from a human cell line defective in antigen presentation and its X-ray crystal structure determined. Previous data indicate that this complex is an intermediate in class II histocompatibility maturation, occurring between invariant chain-DR3 and antigenic peptide-DR3 complexes. The structure shows that the CLIP fragment binds to DR3 in a way almost identical to that in which antigenic peptides bind class II histocompatibility glycoproteins. The structure is the substrate for the loading of antigenic peptides by an exchange process catalysed by DM. more...

We have exploited Drosophila melanogaster Schneider cells and compatible inducible expression vectors to produce large amounts of secreted major histocompatibility complex (MHC) class II molecules (I-Ed). A simple two-step purification protocol was developed. In the first step, recombinant molecules were enriched using a monoclonal anti-class II antibody column followed by a nickel chelate column which further purified and concentrated the recombinant protein to several mg/ml. Characterization of the purified material indicates that the molecules are correctly assembled into alpha beta heterodimers. Further analysis shows that the recombinant MHC class II molecules are devoid of endogenous peptides and, therefore, homogeneous peptide/MHC complexes could be prepared by adding exogenous I-Ed-specific peptides at slightly acidic pH. Upon peptide addition, molecules underwent a conformational change into a more compact form revealed by gel filtration analysis. In addition, the peptide/MHC complexes were biologically active. As little as 10 ng of these complexes coated on plastic from a 100 ng/ml solution were sufficient to trigger antigen-specific T cell hybridomas. These MHC class II molecules, together with various forms of soluble T cell receptor (TcR) proteins, provide valuable tools to analyze the molecular details of TcR/antigen recognition. more...

Minor histocompatibility (H) Ags, encoded by autosomal and sex-linked genes, have classically been identified by the cytolytic T lymphocyte response to class I-bound minor H Ags. A limited number of studies have identified minor H Ags (defined by congenic strains) that stimulate Th cells. We have selected a panel of minor H Ag-specific, CD4+ Th hybrids by the fusion of a BW5147 variant with a C57BL/6 anti-BALB.B T cell line. Studied hybrids secrete IL-2 following stimulation with BALB.B spleen cells and stimulation is blocked with anti-I-Ab Ab. These Th hybrids recognize a minimum of six different Ags (helper T target (HTT)) encoded by independently segregating genes as defined by stimulator cells from 11 CXB recombinant inbred strains. The CXB strain distribution patterns of detected Ags did not match the distribution pattern of the BALB/c MTV6 viral genome indicating that none of the detected Ags were known viral superantigens. Four tested HTT Ags appeared not to be acquired by stimulators as exogenous Ags as BALB.B spleen cells but not a mixture of BALB/c spleen cells and C57BL/6 APC stimulated IL-2 production by the respective Th hybrids. To determine whether Th hybrids recognized peptides bound to I-Ab, I-Ab molecules were immunoprecipitated from the LB27.4 cell line that stimulated the A6.5.5 hybrid (HTT-4 specific). Peptides were extracted from I-Ab molecules, ultrafiltered, and separated by HPLC. A single fraction (with flanking peaks of lower activity) sensitized syngeneic APC to stimulate A6.5.5 hybrid cells; sensitization was observed at pH 5.0 but not pH 7.0, consistent with HTT-4 peptide:I-Ab binding occurring in an endosomal compartment. These observations indicate that at least one class II-restricted minor H Ag is recognized by Th cells as a peptide bound to class II molecules. more...

Major histocompatibility complex expression of activated peripheral blood lymphocytes of captive African green monkeys from Barbados and from Africa were analyzed biochemically; class I molecules by one-dimensional isoelectric focusing and class II DR molecules by one-dimensional nonequilibrium pH gradient electrophoresis. Much less diversity was observed in the major histocompatability molecule expression of the African green monkeys of Barbados than in the African cohort. more...

Invariant chain (Ii) associates with major histocompatibility complex (MHC) class II molecules and is crucial for antigen presentation by class II molecules. The exact nature of Ii interaction with MHC class II molecules remains undefined. A nested set of Ii peptides, CLIPs (class II-associated Ii peptides), have been eluted from various MHC class II molecules, suggesting that CLIPs correspond, at least in part, to the Ii motif which blocks the conventional peptide binding site in MHC class II molecules. Here we report how CLIPs interact with class II MHC molecules, I-A. We have identified regions critical for binding of CLIPs and I-A class II molecules. In most cases, the binding of CLIPs to a number of I-A molecules is modulated by the steric bulk of methionine residues at positions 93 and 99. In addition, the binding of CLIPs to an I-A molecule, I-Au, is sensitive to substitutions at aspartic acid-59 in the alpha chain and threonine-86 in the beta chain, whereas the binding of an antigen-derived peptide is not. Taken together, these results provide an insight as to how CLIPs bind to MHC class II heterodimers. more...

HLA-DR molecules associated with class II-associated invariant chain peptides (CLIP) are generated in vivo as an intermediate in class II maturation. Such complexes can be produced in vitro by proteolytic digestion of DR alpha beta I complexes, suggesting that CLIP is a residual fragment that remains associated with class II molecules following I chain degradation. In vitro, CLIP dissociation from DR alpha beta dimers occurs at different rates depending on the allele, and is facilitated by low pH and by detergents containing 8-10 carbon unbranched hydrocarbons, or by primary aliphatic amines or carboxylic acids. The accumulation of DR alpha beta CLIP complexes in HLA-DM-negative antigen-processing mutant cells argues that a functionally similar mechanism, dependent on HLA-DM expression, catalyzes in vivo CLIP dissociation and generation of normal class II-peptide complexes. more...