Human Leukocyte antigen-DM Research Articles

HLA-DM catalytically enhances peptide dissociation by sensing peptide–MHC class II interactions throughout the peptide-binding cleft

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Bile-Derived Organoids From Patients With Primary Sclerosing Cholangitis Recapitulate Their Inflammatory Immune Profile.

Primary sclerosing cholangitis (PSC) is a heterogeneous and progressive fibroinflammatory cholangiopathy with no known etiology or effective treatment. Studies of PSC are limited due to difficulty in accessing the cholangiocyte, the small percentage of these cells in the liver, instability of in vitro culture systems, and reliance on samples from end-stage disease. Here, we demonstrate that stem cells can be isolated from the bile of PSC patients undergoing endoscopic retrograde cholangiopancreatography earlier in their clinical course and maintained long term in vitro as three-dimensional (3D) organoids that express a biliary genetic phenotype. Additionally, bile-derived organoids (BDOs) can be biobanked and samples obtained longitudinally over the course of the disease. These BDOs express known cholangiocyte markers including gamma glutamyl transferase, cytokeratin 19, epithelial cellular adhesion molecule, cystic fibrosis transmembrane conductance regulator, and anion exchanger 2. RNA sequence analysis identified 39 genes whose expression differed in organoids from PSC patients compared to non-PSC controls, including human leukocyte antigen DM alpha chain and chemokine (C-C motif) ligand 20 (CCL20), immune-related genes previously described in genome-wide association studies of PSC. Incubation of these BDOs with interleukin 17A or tumor necrosis factor alpha led to an immune-reactive phenotype with a significant increase in secretion of proinflammatory mediators, including CCL20, a T-cell chemoattractant. Conclusion: This study demonstrates that bile can be used as a source of biliary-like cells that can be maintained long term in vitro as 3D organoids; these BDOs retain features of cholangiopathies, including the ability to react to inflammatory stimuli by secreting chemokines and propagating an immune-reactive phenotype reflective of the pathogenesis of these diseases; thus, BDOs represent a platform for the study of the pathogenesis and therapy of cholangiopathies, particularly PSC.

HLA–DM Senses Peptide‐MHC Class II Interactions Throughout the Peptide Binding Groove

The major histocompatibility complex class II (MHCII) antigen‐processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T‐cells. Within the pathway, human leukocyte antigen DM (HLA‐DM) edits the repertoire of peptides that are presented to CD4 T‐cells by catalyzing multiple rounds of peptide exchange and accelerating peptide loading on MHCII molecules. In endosomes, HLA‐DM first catalyzes the dissociation and exchange of the proteolytically processed Ii derived Class II‐associated invariant chain peptide (CLIP) bound to MHCII molecules through a mechanism that follows Michaelis‐Menten kinetics and involves a transient interaction with the peptide‐MHCII complex. HLA‐DM then continues to catalyze the rate of dissociation and exchange of all other peptides. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide‐MHCII complexes and not others. This capacity to differentially edit peptide‐MHCII complexes has important biological implications through its effect of skewing the repertoire of foreign and self‐peptide complexes available for activation or tolerance induction of CD4 T‐cells. Despite the large body of evidence indicating the central role of HLA‐DM in the MHCII antigen‐processing pathway, the catalytic mechanism and the rules that govern the susceptibility of peptide‐MHCII complexes to DM mediated editing are not completely understood. Evidence suggests that unstable interactions at the N‐terminus of bound peptides is the key determinant of peptide‐MHCII complex sensitivity to HLA‐DM and that partial dissociation of a peptides N‐terminus is necessary for this sensitivity. Herein, we employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover, components of the catalytic mechanism, in real‐time kinetic measurements of DM activity. Using a series of amino acid substituted peptides, we have analyzed the effects of disfavored interactions on the kinetics of DM editing. We find that disruptions throughout the peptide binding groove contribute to the effective binding affinity of DM for a given peptide complex and to the rate of peptide dissociation from the DM–peptide‐MHCII catalytic complex. Experiments with mutant peptide‐MHCII complexes that disrupt elements in the conserved hydrogen bond network show that disruption near the C‐terminus results in increased DM affinity and catalytic turnover. Furthermore, we found that complexes with an N‐terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H‐bonds, showed an unexpected increase in catalytic turnover when compared to the unmodified HA‐MHCII complex. This result was even more pronounced when a conserved bidentate H‐bond forming residue near the P1 anchor of the HA‐MHCII complex was disrupted by site‐directed mutagenesis. Together, this implies that peptide‐MHCII interactions near the N‐terminal segment of a peptide are intact during the functionally active DM‐peptide‐MHCII catalytic complex and that interactions at the DM‐MHCII contact interface are intimately linked to unstable interactions throughout the peptide‐binding groove.Support or Funding InformationSupported in part by NIH grants AI30554 and AI33614

Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.

Sibling rivalry: competition between MHC class II family members inhibits immunity

Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

Cross-recognition of HLA DR4 alloantigen by virus-specific CD8+ T cells: a new paradigm for self-/nonself-recognition

AbstractThe ability of CD8+ T cells to engage a diverse range of peptide–major histocompatibility complex (MHC) complexes can also lead to cross-recognition of self and nonself peptide-MHC complexes and thus directly contribute toward allograft rejection or autoimmunity. Here we present a novel form of cross-recognition by herpes virus–specific CD8+ cytotoxic T cells that challenges the current paradigm of self/non-self recognition. Functional characterization of a human leukocyte antigen (HLA) Cw*0602-restricted cytomegalovirus-specific CD8+ T-cell response revealed an unusual dual specificity toward a pp65 epitope and the alloantigen HLA DR4. This cross-recognition of HLA DR4 alloantigen was critically dependent on the coexpression of HLA DM and was preferentially directed toward the B-cell lineage. Furthermore, allostimulation of peripheral blood lymphocytes with HLA DRB*0401-expressing cells rapidly expanded CD8+ T cells, which recognized the pp65 epitope in the context of HLA Cw*0602. T-cell repertoire analysis revealed 2 dominant populations expressing T-cell receptor beta variable (TRBV)4-3 or TRBV13, with cross-reactivity exclusively mediated by the TRBV13+ clonotypes. More importantly, cross-reactive TRBV13+ clonotypes displayed markedly lower T-cell receptor binding affinity and a distinct pattern of peptide recognition, presumably mimicking a structure presented on the HLA DR4 allotype. These results illustrate a novel mechanism whereby virus-specific CD8+ T cells can cross-recognize HLA class II molecules and may contribute toward allograft rejection and/or autoimmunity.

Differential signal transduction, membrane trafficking, and immune effector functions mediated by FcγRI versus FcγRIIa

AbstractReceptors for the fragment crystallizable region of immunoglobulin-G (FcγRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcγRI and FcγRIIa. Activation of FcγRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II–mediated antigen presentation to T lymphocytes. In contrast, activation of FcγRIIa elicits signaling through phospholipase Cγ1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

In antigen-presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen-binding sites in pre-endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii-degrading proteases, the peptide exchange factor, human leukocyte antigen-DM (HLA-DM), and its modulator, HLA-DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen-binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post-Golgi mechanisms; these factors include MHC class II-associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.

The optimization of peptide cargo bound to MHC class I molecules by the peptide‐loading complex

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

Class II-associated invariant chain peptide expression on myeloid leukemic blasts predicts poor clinical outcome.

Effective antitumor responses need the activation of CD4+ T cells. MHC class II antigen presentation requires the release of class II-associated invariant chain peptide (CLIP) from the antigen-binding site. In antigen-presenting cells, human leukocyte antigen DM (HLA-DM; abbreviated DM in this article) catalyzes CLIP dissociation. In B cells, HLA-DO (DO) down-modulates DM function. Cell surface CLIP:HLA-DR (DR) ratio correlates to DO:DM ratio and the efficacy of antigen presentation. We examined 111 blood and bone marrow samples of patients with newly diagnosed acute myeloid leukemia (AML) for the expression of CLIP, DR, DM, and DO by flow cytometry. Patients with DR+/CLIP- blasts had a significant longer disease-free survival than patients with DR+/CLIP+ blasts. DO, until now believed to be restricted to lymphoid cells, could be demonstrated at protein level as well as by reverse transcription-PCR. DO:DM ratio correlated to CLIP:DR ratio, suggesting that, unlike in other antigen-presenting cells of the nonlymphoid cell type, both DO and DM mediate regulation of CLIP expression in AML blasts. We hypothesize that DR+/CLIP- AML blasts are able to present leukemia-specific antigens to CD4+ T helper cells initiating an effective and long-lasting antitumor response resulting in a prolonged disease-free survival.

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

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

Lack of association between transporter associated with antigen processing (TAP) and HLA-DM gene polymorphisms and antibody levels following measles vaccination.

The transporter associated with antigen processing (TAP) and human leukocyte antigen-DM (HLA-DM) genes are involved in the antigen-processing pathway of both HLA class I and class II-restricted antigen presentation. We hypothesized that polymorphisms within the TAP and DM genes may influence antibody levels following measles vaccination. We examined TAP and DM polymorphisms in 242 school children from Olmsted County, Minnesota, USA who received one dose of measles-mumps-rubella-II (MMR-II) vaccine at the age of 15 months. Based on the level of serum measles-specific immunoglobulin G (IgG) antibodies, subjects were classified as seronegatives (n = 72) or seropositives (n = 170). We determined TAP1 and TAP2 allele types by polymerase chain reaction (PCR) amplification of specific alleles (PASA) and determined DM allele type by PCR amplification followed by direct sequencing of the polymorphic sites. We analysed the data for any TAP or DM allelic association with antibody levels post measles vaccination using the chi-square test and univariate linear regression analysis. We found no trend in the overall distribution of TAP and DM genotype frequencies between seronegative and seropositive subjects, suggesting that TAP and DM polymorphism and antibody levels following measles vaccination are not directly associated. In addition, we did not find an association between TAP (TAP1, P = 0.71; TAP2, P = 0.87) or DM (DMA, P = 0.42; DMB, P = 0.71) homozygosity and seronegativity to measles vaccine in this study group. Our study suggests that TAP and DM gene polymorphisms do not influence antibody levels post measles vaccination.

Typing alleles of HLA-DM.

Human leukocyte antigen DM (HLA-DM) is a major histocompatibility complex (MHC) class II-like molecule that facilitates antigen processing by catalyzing the exchange of invariant chain-derived peptides (CLIP) from class II molecules for antigenic peptides. The genes encoding HLA-DM, HLA-DMA and -DMB, are located in tandem in the class II region of the MHC, between HLA-DP and HLA-DQ loci (). HLA-DMA and -DMB products co-localize with HLA-DR in the specialized acidic MHC class II compartment (MIIC) of antigen presenting cells, but no expression of these molecules is detectable at the cell surface. The interaction of DM with class II also aids in the subsequent rapid loading of high-affinity antigen-derived peptides into the MHC class II groove. There is now accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands and skews the class II peptide repertoire toward high-stability ligands (, , ). Because of their role in antigen processing and their location within the class II region of the MHC, these genes have attracted attention as possible disease susceptibility loci.

Expression of HLA-DR and its enhancing molecules in muscle fibers in polymyositis.

Polymyositis (PM) is an autoimmune inflammatory muscle disease of unknown cause in which cellular immunity is thought to play an important pathogenic role. Class II major histocompatibility complex (class II MHC: human leukocyte antigen (HLA)-DR operates as a cofactor of antigen presentation in immunological responses. There has been a major debate over whether muscle fibers themselves synthesize and express HLA-DR molecules and play a role in antigen presentation in PM pathogenesis. In this study, we demonstrated that most muscle fibers from patients with PM synthesized and expressed HLA-DR molecules on their surface. Human leukocyte antigen-DR expression was highly specific to PM. In addition, class II transactivator (CIITA), human leukocyte antigen DM (HLA-DM), and invariant chain (Ii), which are indispensable for expression of mature HLA-DR molecules and for antigen processing and presentation, were co-expressed. One of the cytokines that could induce this expression is interferon-gamma (IFN-gamma), released by activated lymphocytes. Our results indicate that in PM muscle fibers synthesize and express HLA-DR molecules and may contribute to the inflammatory responses together with lymphocytes.

Distinct intracellular compartments involved in invariant chain degradation and antigenic peptide loading of major histocompatibility complex (MHC) class II molecules.

Major histocompatibility complex (MHC) class II molecules are transported to intracellular MHC class II compartments via a transient association with the invariant chain (Ii). After removal of the invariant chain, peptides can be loaded onto class II molecules, a process catalyzed by human leukocyte antigen-DM (HLA-DM) molecules. Here we show that MHC class II compartments consist of two physically and functionally distinct organelles. Newly synthesized MHC class II/Ii complexes were targeted to endocytic organelles lacking HLA-DM molecules, where Ii degradation occurred. From these organelles, class II molecules were transported to a distinct organelle containing HLA-DM, in which peptides were loaded onto class II molecules. This latter organelle was not directly accessible via fluid phase endocytosis, suggesting that it is not part of the endosomal pathway. Uptake via antigen-specific membrane immunoglobulin resulted however in small amounts of antigen in the HLA-DM positive organelles. From this peptide-loading compartment, class II-peptide complexes were transported to the plasma membrane, in part after transit through endocytic organelles. The existence of two separate compartments, one involved in Ii removal and the other functioning in HLA-DM-dependent peptide loading of class II molecules, may contribute to the efficiency of antigen presentation by the selective recruitment of peptide-receptive MHC class II molecules and HLA-DM to the same subcellular location.

A novel antigen-processing-defective phenotype in major histocompatibility complex class II-positive CIITA transfectants is corrected by interferon-gamma.

Presentation of exogenous protein antigens to T lymphocytes is based on the intersection of two complex pathways: (a) synthesis, assembly, and transport of major histocompatibility complex (MHC) class II-invariant chain complexes from the endoplasmic reticulum to a specialized endosomal compartment, and (b) endocytosis, denaturation, and proteolysis of antigens followed by loading of antigenic peptides onto newly synthesized MHC class II molecules. It is believed that expression of MHC class II heterodimers, invariant chain and human leukocyte antigen-DM is both necessary and sufficient to reconstitute a functional MHC class II loading compartment in antigen-presenting cells. Expression of each of these essential molecules is under the control of the MHC class II transactivator CIITA. Unexpectedly, however, whereas interferon gamma stimulation does confer effective antigen-processing function to nonprofessional antigen presenting cells, such as melanoma cells, expression of the CIITA transactivator alone is not sufficient. Activation of antigen-specific T cells thus requires additional CIITA-independent factor(s), and such factor(s) can be induced by interferon gamma.

HLA-DM induces clip dissociation from MHC class II αβ dimers and facilitates peptide loading

Human leukocyte antigen DM (HLA-DM) molecules are structurally related to classical MHC class II molecules and reside in the lysosome-like compartment where class II-restricted antigen processing Is thought to occur. Mutant cell lines lacking HLA-DM are defective in antigen processing and accumulate class II molecules associated with a nested set of invariant chain-derived peptides (class II-associated Invariant chain peptides, CLIP). Here we show that HLA-DM catalyzes the dissociation of CLIP from MHC class II-CLIP complexes In vitro and facilitates the binding of antigenic peptides. The reaction has an acidic pH optimum, consistent with its occurrence in a lysosome-like compartment in vivo. Antibody blocking experiments suggest that a transient Interaction between HLA-DM and the MHC class II-CLIP complex is required.