Noble metals strip peptides from class II MHC proteins

Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a 'peptide-empty' conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.     

MHC class II proteins and disease: a structural perspective

MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.     

Zwitterionic capsular polysaccharides: the new MHCII-dependent antigens

The immune system has evolved the ability for T cells to recognize nearly any biological polymer, including peptides, protein superantigens, and glycolipids through presentation by the major histocompatibility complex (MHC) proteins such as MHC class I (MHCI), MHC class II (MHCII), and CD1. A recent and unexpected addition to this list is the zwitterionic capsular polysaccharide (ZPS). These bacterial molecules utilize MHCII presentation to activate T cells via recognition by alphabeta T cell receptor (alphabetaTCR) proteins. In this review, we explore what is currently known about ZPS processing and presentation within antigen-presenting cells (APCs) and the immune response that follows.     

Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells

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.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.     

NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

Background  

The major histocompatibility complex (MHC) molecule plays a central role in controlling the adaptive immune response to infections. MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells, whereas MHC class II molecules stimulate cellular and humoral immunity through presentation of extracellularly derived peptides to helper T cells. Identification of which peptides will bind a given MHC molecule is thus of great importance for the understanding of host-pathogen interactions, and large efforts have been placed in developing algorithms capable of predicting this binding event.     

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.     

Towards universal structure-based prediction of class II MHC epitopes for diverse allotypes

The binding of peptide fragments of antigens to class II MHC proteins is a crucial step in initiating a helper T cell immune response. The discovery of these peptide epitopes is important for understanding the normal immune response and its misregulation in autoimmunity and allergies and also for vaccine design. In spite of their biomedical importance, the high diversity of class II MHC proteins combined with the large number of possible peptide sequences make comprehensive experimental determination of epitopes for all MHC allotypes infeasible. Computational methods can address this need by predicting epitopes for a particular MHC allotype. We present a structure-based method for predicting class II epitopes that combines molecular mechanics docking of a fully flexible peptide into the MHC binding cleft followed by binding affinity prediction using a machine learning classifier trained on interaction energy components calculated from the docking solution. Although the primary advantage of structure-based prediction methods over the commonly employed sequence-based methods is their applicability to essentially any MHC allotype, this has not yet been convincingly demonstrated. In order to test the transferability of the prediction method to different MHC proteins, we trained the scoring method on binding data for DRB1*0101 and used it to make predictions for multiple MHC allotypes with distinct peptide binding specificities including representatives from the other human class II MHC loci, HLA-DP and HLA-DQ, as well as for two murine allotypes. The results showed that the prediction method was able to achieve significant discrimination between epitope and non-epitope peptides for all MHC allotypes examined, based on AUC values in the range 0.632-0.821. We also discuss how accounting for peptide binding in multiple registers to class II MHC largely explains the systematically worse performance of prediction methods for class II MHC compared with those for class I MHC based on quantitative prediction performance estimates for peptide binding to class II MHC in a fixed register.     

Monoclonal antibodies specific for the empty conformation of HLA-DR1 reveal aspects of the conformational change associated with peptide binding

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.     

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: Cancer cell expression of HLA-DR

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA–DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.     

Immune surveillance via self digestion

The adaptive immune system is orchestrated by CD4+ T cells. These cells detect peptides presented on Major Histocompatibility Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4+ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4+ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.     

DNase I hypersensitive sites flank the mouse class II major histocompatibility complex during B cell development

The mouse class II major histocompatibility complex (MHC) encodes a polymorphic, multigene family important in the immune response, and is expressed mainly on mature B cells, on certain types of dendritic cells and is also inducible by gamma-interferon on antigen presenting cells. To study the regulatory elements which control this expression pattern, we have examined the chromatin structure flanking the class II MHC region, in particular during B cell differentiation. Using a panel of well-characterised mouse cell lines specific for different stages of B cell development (pre-B, B, plasma cell) as well as non-B cell lines, we have mapped the DNase I hypersensitive (DHS) sites adjacent to the mouse MHC class II region. The results presented show, for the first time that there are specific hypersensitive sites flanking the class II MHC locus during pre B cell, B cell and plasma cell stages of B cell differentiation, irrespective of the status of class II MHC expression. These hypersensitive sites are not found in T cell, fibroblast or uninduced myelomonocytic cell lines. This suggests that these DHS sites define a developmentally stable, chromatin structure, which can be used as a marker of B cell lineage commitment and may indicate that a combination of these hypersensitive sites reflect regulatory proteins involved in the immediate expression of a particular class II MHC gene or possibly control of the entire locus.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.