In the absence of aminopeptidase ERAAP, MHC class I molecules present many unstable and highly immunogenic peptides

Immunosurveillance by cytotoxic T cells requires that cells generate a diverse spectrum of peptides for presentation by major histocompatibility complex (MHC) class I molecules. Those peptides are generated by proteolysis, which begins in the cytoplasm and continues in the endoplasmic reticulum by the unique aminopeptidase ERAAP. The overall extent to which trimming by ERAAP modifies the peptide pool and the immunological consequences of ERAAP deficiency are unknown. Here we show that the peptide-MHC repertoire of ERAAP-deficient mice was missing many peptides. Furthermore, ERAAP-deficient cells presented many unstable and structurally unique peptide-MHC complexes, which elicited potent CD8+ T cell and B cell responses. Thus, ERAAP is a 'quintessential editor' of the peptide-MHC repertoire and, paradoxically, its absence enhances immunogenicity.     

Sheep MHC class II molecules. II. Identification and characterization of four distinct subsets of sheep MHC class II molecules

A panel of seven monoclonal antibodies, sequential immunoprecipitation and two-dimensional NEPHGE/SDS-PAGE analyses were used to identify and characterize subsets of sheep MHC class II molecules. Using sequential immunoprecipitation four distinct subsets of class II molecules were identified by the monoclonal antibodies SBU.II 28-1, 37-68, 38-27 and 42-20, while another monoclonal antibody, SBU.II 49-1, recognized all four subsets of class II molecules. These four subpopulations of sheep class II molecules displayed different two-dimensional gel profiles and, using splenocytes from four outbred sheep, the class II molecules recognized by SBU.II 28-1, 37-68 and 42-20 showed structurally detectable allelic polymorphism in their beta polypeptides, but no detectable variation in their alpha polypeptides. In contrast, the class II molecules recognized by SBU.II 38-27 showed allelic variation in both their alpha and beta polypeptides. Two-dimensional (2-D) gel analyses of non-glycosylated class II molecules immunoprecipitated by SBU.II 49-1 suggested that approximately 10-12 different class II molecules were expressed by a single sheep. The results of this study show that sheep express class II molecules that can be divided into four structurally and serologically distinct subsets, and provide additional evidence for the subdivision of the sheep MHC class II genetic region into at least three distinct subregions.     

NK cells, MHC class I molecules and the missing self

This article is based on a lecture presented at the Novartis Prize ceremony at the International Congress of Immunology in July 2001. It gives a personal and historical perspective on the research performed by the author and his colleagues during the development and pursuit of the model of 'missing-self recognition' for natural killer (NK) cells. This model is based on the idea that one important function of NK cells is to detect and eliminate cells because they fail to express normal self markers. Further mechanistic models predicted the existence of inhibitory major histocompatibility complex (MHC) class I specific receptors, later identified by the fellow Novartis laureates contributing in this issue. The article covers the first decade (1980-1990) of research on this concept. It discusses factors contributing to the formulation of a hypothesis, the use of predictions and experimental test models, the importance of international collaborations and reagent exchange, and several other aspects that allowed the progression of this research project. Finally, the perspective of today's knowledge is used to discuss some surprising findings where the missing-self hypothesis made the wrong predictions, or at least failed to make the correct ones.     

Mutations of class II MHC molecules

It remains unclear how the tertiary interaction of T-cell receptor, la molecule and foreign antigen results in the extensive diversity of the helper T cell repertoire. Here Laurie Glimcher and Irwin Griffith focus on what has been learned about the relationship between structure and function of the la molecule from the use of mouse strains with mutations in the genes coding for these glycoproteins.     

One of two unstructured domains of Ii becomes ordered in complexes with MHC class II molecules.

We studied the role of the invariant chain (Ii) protein's structure in its ability to form complexes with major histocompatibility complex class II molecules. Multidimensional nuclear magnetic resonance experiments demonstrated that Ii contains two unstructured, flexible domains: a 39 residue sequence that contains a region (CLIP) critical for Ii/class II complex formation and becomes rapidly ordered when Ii/class II complexes are assembled, and a 30 residue sequence that contains the insertion point for a protease inhibitor domain included in an alternative splice form of Ii. Mobility of these domains guarantees accessibility to CLIP and the inhibitor insert, and ordering of the CLIP-containing domain may provide protection against proteolysis and contribute, along with Ii's compact 118-192 domain, to allotype-independent class II binding.     

The wide diversity and complexity of peptides bound to class II MHC molecules

The identification and quantitation of peptides selected by class II MHC molecules during natural processing of proteins is of key importance in understanding the repertoire and distribution of T cells. The examination of peptides selected by class II MHC molecules has depended greatly on mass spectrometry, a powerful technique that identifies and sequences peptides in complex mixtures with great sensitivity and precision. Such analysis has resulted in the identification of several factors, including the repertoire of peptides selected by MHC molecules during natural processing of proteins, motifs important for selection of processed peptides, conformational isomers of peptide-MHC complexes, and post-translational changes to the peptides.     

Modeling MHC class II molecules and their bound peptides as expressed at the cell surface

A detailed insight to the structure of a given major histocompatibility complex (MHC)-peptide complex can strongly support and also improve the analysis of the peptide binding capabilities of the MHC molecule and the characterization of the developing T cell response. The number of MHC class II-peptide crystal structures is limited, therefore constructing and analyzing computer models can serve as efficient complementary tools when someone deals with experimentally determined binding and/or functional data. Commercial programs are available for modeling protein and protein-protein complexes, in general. However, more accurate results can be obtained if the parameters are directly optimized to a given complex, especially in the case of special proteins as MHC class II, an integral membrane protein, whose functional parts behave like regular globular proteins. Here, we present the optimization of an approach used for modeling MHC class II molecules complexed with various peptides fitting into the binding groove and several ways to analyze them with the help of experimental data.     

Intracellular transport of MHC class II molecules.

MHC class II molecules associate, during biosynthesis, with peptides derived from endocytosed antigen. Here, Jacques Neefjes and Hidde Ploegh describe the intracellular transport of MHC class II molecules and its relationship to the binding of peptides in endosomal compartments. They discuss alternative routes for the delivery of antigen to sites at which peptides associate with MHC class II molecules and raise the possibility of cell type-specific differences in the handling of MHC class II molecules, and hence in antigen presentation.     

Endogenous antigen presentation by MHC class II molecules

T cell recognition of antigen requires that a complex form between peptides derived from the protein antigen and cell surface glycoproteins encoded by genes within the major histocompatibility complex (MHC). MHC class II molecules present both extracellular (exogenous) and internally synthesized (endogenous) antigens to the CD4 T cell subset of lymphocytes. The mechanisms of endogenous antigen presentation are the subject of this review. Isolation and amino acid sequencing of peptides bound to the class II molecule indicate that a very high proportion (70–90%) of the total peptides presented by the class II molecule are in fact derived from the pool of proteins that are synthetized within the antigen-presenting cell (APC). This type of sequence information as well as the study of model antigens has indicated that proteins expressed in a diversity of intracellular sites, including the cell surface, endoplasmic reticulum and cytosol can gain access to the class II molecule, albeit with different efficiencies. The main questions that remain to be answered are the intracellular trafficking patterns that allow colocalization of internally synthesized antigens with the class II molecule, the site(s) within the cell where peptide: class II molecule complex formation can take place and whether presentation of ‘foreign’ as well as ‘self’ antigens takes place by mechanisms that vary from one cell type to another or that vary with the metabolic state of the APC. If such variability exists, is would imply that the array of peptides displayed by class II molecules at the cell surface has similar variability, a possibility that would impact on self tolerance and autoimmunity.     

Accessory molecules for MHC class II peptide loading

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.     

The peptide-binding strategy of the MHC class II I-A molecules

Despite the importance of murine major histocompatibility complex (MHC) class II I-A molecules for immunological research, the overall peptide-binding specificities of I-A and the homologous human HLA-DQ molecules remain unresolved. Here, Boris Reizis and colleagues review current evidence suggesting that DQ/I-A molecules bind peptides with a different hierarchy of anchor positions than has been found in the well-characterized DR/I-E proteins.     

Presentation of cytosolic antigens via MHC class II molecules

Major histocompatibility (MHC) class II molecules function to present antigenic peptides to CD4 T lymphocytes. The pathways by which these molecules present exogenous antigens have been extensively studied. However by contrast, far less is known about the processing and trafficking of cytosolic antigens, which can also serve as an alternative source of ligands for MHC class II molecules. Self-proteins, tumor antigens, as well as viral proteins found within the cytosol of cells, can be presented via MHC class II molecules, resulting in the activation of specific CD4 T cells. Studies have begun to reveal unique steps as well as some similarities in the pathways for cytosolic and exogenous antigen presentation. Recent developments in this area are summarized here.     

Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4⁺ T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4⁺ T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II–peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II–peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4⁺ T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II–peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide–MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.     

Introducing endogenous antigens into the major histocompatibility complex (MHC) class II presentation pathway. Both Ii mediated inhibition and enhancement of endogenous peptide/MHC class II presentation require the same Ii domains

The invariant chain (Ii) plays a key role in regulating the antigen presentation function of major histocompatibility complex (MHC) class II molecules. Ii also influences the presentation of usually excluded endogenously synthesized proteins into the MHC class II presentation pathway. To evaluate the role of Ii in the generation of peptide-MHC class II complexes derived from endogenously synthesized proteins, we tested mutant Ii constructs in two model systems. Co-expression of wild-type Ii inhibits the presentation of hen-egg lysozyme (HEL) 35-45/Ak complex, but enhances the presentation of ovalbumin (OVA) 247-265/Ak complex from endogenously synthesized HEL or OVA precursors. The differential sensitivity of these antigens to chloroquine was consistent with their being processed in distinct compartments. Nevertheless, with a panel of Ii deletion constructs we show here that both the Ii-mediated inhibition and enhancement functions require the endosomal targeting and CLIP residues. Surprisingly, the Ii mutant lacking the endoplasmic reticulum lumenal residues 126-215, despite apparently lower expression, was at least as effective as full-length Ii in antigen presentation assays. Thus, alternative pathways exist for processing endogenously expressed antigens, and Ii-mediated inhibition and enhancement of peptide/MHC class II expression depend upon the same regions, with neither requiring the 89 C-terminal, lumenal Ii residues.     

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.