Generation of MHC class I ligands in the secretory and vesicular pathways

CD8⁺ T lymphocytes screen the surface of all cells in the body to detect pathogen infection or oncogenic transformation. They recognize peptides derived from cellular proteins displayed at the plasma membrane by major histocompatibility complex (MHC) class I molecules. Peptides are mostly by-products of cytosolic proteolytic enzymes. Peptidic ligands of MHC class I molecules are also generated in the secretory and vesicular pathways. Features of protein substrates, of proteases and of available MHC class I molecules for loading peptides in these compartments shape a singular collection of ligands that also contain different, longer, and lower affinity peptides than ligands produced in the cytosol. Especially in individuals who lack the transporters associated with antigen processing, TAP, and in infected and tumor cells where TAP is blocked, which thus have no supply of peptides derived from the cytosol, MHC class I ligands generated in the secretory and vesicular pathways contribute to shaping the CD8⁺ T lymphocyte response.     

Evolutionary and functional perspectives of the major histocompatibility complex class I antigen-processing machinery

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8+ T cells, providing the basis for immune recognition of pathogen-infected cells. Peptides generated mainly by proteasomes in the cytosol are transported into the lumen of the endoplasmic reticulum by transporters associated with antigen processing (TAP). The maturation of MHC class I molecules is controlled by a number of accessory proteins and chaperones that are to a varying degree dedicated to the assembly of MHC class I. Several newly characterised proteins have been demonstrated to play important roles in this process. This review focuses on the functional relationship and evolutionary history of the antigen-processing machinery (APM) components and MHC class I itself. These are of great interest for further elucidating the origin of the immune system and understanding the mechanisms of antigen presentation and immunology in general.     

Modeling the MHC class I pathway by combining predictions of proteasomal cleavage,TAP transport and MHC class I binding

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).Received 26 November 2004; received after revision 4 February 2005; accepted 4 March 2005S. Tenzer and B. Peters contributed equally to this work.     

Assembly of MHC class I peptide complexes from the perspective of disulfide bond formation

Assembly of functional major histocompatibility complex (MHC) class I peptide complexes within the endoplasmic reticulum is critically important for the development of an adaptive immune response. The highly regulated loading of peptides onto MHC class I molecules is controlled by a multi-component chaperone system called the MHC class I peptide loading complex. The recent identification of the thioredoxin family member ERp57 as a component of the loading complex led to an interesting question: Why is there a thiol-disulfide oxidoreductase inside a complex dedicated to inserting peptides into a receptor binding site? Most recently, specific ERp57-mediated disulfide bond rearrangements have been identified inside the loading complex. What these biochemical events mean for the peptide loading process remains a matter of conjecture. While several important questions wait to be answered, this review intends to summarize our current view of the oxidative folding of MHC class I molecules and addresses the question of how the receptor ligand interaction might be regulated by thiol-based redox reactions.     

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host. Received 4 October 2005; received after revision 19 November 2005; accepted 24 November 2005     

The role of the proteasome in the generation of MHC class I ligands and immune responses

The ubiquitin–proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses.     

Cross-presentation of IgG-containing immune complexes

IgG is a molecule that functionally combines facets of both innate and adaptive immunity and therefore bridges both arms of the immune system. On the one hand, IgG is created by adaptive immune cells, but can be generated by B cells independently of T cell help. On the other hand, once secreted, IgG can rapidly deliver antigens into intracellular processing pathways, which enable efficient priming of T cell responses towards epitopes from the cognate antigen initially bound by the IgG. While this process has long been known to participate in CD4⁺ T cell activation, IgG-mediated delivery of exogenous antigens into a major histocompatibility complex (MHC) class I processing pathway has received less attention. The coordinated engagement of IgG with IgG receptors expressed on the cell-surface (FcγR) and within the endolysosomal system (FcRn) is a highly potent means to deliver antigen into processing pathways that promote cross-presentation of MHC class I and presentation of MHC class II-restricted epitopes within the same dendritic cell. This review focuses on the mechanisms by which IgG-containing immune complexes mediate such cross-presentation and the implications that this understanding has for manipulation of immune-mediated diseases that depend upon or are due to the activities of CD8⁺ T cells.     

Legionella pneumophila down-regulates MHC class I expression of human monocytic host cells and thereby inhibits T cell activation

Legionella (L.) pneumophila, the causative agent of Legionnaires disease, is an intracellular pathogen of alveolar macrophages that resides in a compartment displaying features of endoplasmatic reticulum (ER). In this study, we show that intracellular multiplication of L. pneumophila results in a remarkable decrease in MHC class I expression by the infected monocytes. During intracellular multiplication, L. pneumophila absorbs ER-resident chaperons such as calnexin and BiP, molecules that are required for the correct formation of the MHC class I complex. Due to reduced MHC class I expression, stimulation of allogeneic blood mononuclear cells was severely inhibited by infected host cells but cytotoxicity of autologous natural killer cells against Legionella-infected monocytes was not enhanced. Thus, reduced expression of MHC class I in infected monocytes may resemble a new immune escape mechanism induced by L. pneumophila.Received 22 November 2004; received after revision 27 December 2004; accepted 5 January 2005     

Molecular chaperones in the processing and presentation of antigen to helper T cells

Helper T lymphocytes recognize peptide fragments of antigen bound to Major Histocompatibility Complex (MHC) class II molecules on the surfaces of antigen presenting cells (APC). Antigen processing involves internalization of the antigen into an acidic compartment where the antigen is degraded and the resulting peptide fragments of the antigen are bound to MHC class II molecules and the complexes subsequently displayed at the APC surface. Thus, antigen processing represents a complex, intracellular assembly process which may, like many intracellular protein folding and assembly processes, require the function of molecular chaperones. This contribution focuses on the evidence which suggests that members of the heat shock protein family of molecular chaperones play a role in this pathway.     

Endothelial cells as antigen-presenting cells: role in human transplant rejection

The immunological properties of human endothelial cells suggest they perform a pivotal role in acute and chronic rejection following solid organ transplantation. In this review the basic features of acute and chronic rejection are described as are the cellular and molecular requirements for antigen presentation. Traditionally, antigen-presenting cells are considered to be bone marrow-derived cells. However, these conclusions have been derived from rodent models of allograft rejection where bone marrow-derived passenger leukocytes are the only source of donor major histocompatibility complex (MHC) class II in the grafted organ. In contrast, in humans, virtually all the microvascular and small vessel endothelial cells are ‘constitutively’ positive for MHC class II antigens. The phenotypic properties of human endothelial cells, their response to cytokines and their ability to stimulate resting T cells are described. Unlike bone marrow-derived antigen presenting cells (APCs), which utilise B7/CD28 interactions, human endothelial cells utilise lymphocyte function antigen 3 (LFA3)/CD2 pathways to stimulate T cells. They activate a CD45RO + B7-independent subpopulation of T cells. Their effect on allogeneic T cells is compared with other non-bone marrow-derived cells such as fibroblasts, epithelial cells and smooth muscle cells, which are unable to stimulate resting T cells. Evidence is presented suggesting that release of MHC and non-human leukocyte antigens (HLA) from endothelial cells stimulates an alloantibody and autoimmune response leading to chronic rejection. Received 30 March 1998; received after revision 4 May 1998; accepted 4 May 1998     

Generation of cellular immune responses to antigenic tumor peptides

Tumor immunotherapy is currently receiving close scrutiny. However, with the identification of tumor antigens and their production by recombinant means, the use of cytokines and knowledge of major histocompatibility complex (MHC) class I and class II presentation has provided ample reagents for use and clear indications of how they should be used. At this time, much attention is focused on using peptides to be presented by MHC class I molecules to both induce and be targets for CD8+ cytolytic T cells. Many peptides generated endogenously or given exogenously can enter the class I pathway, but a number of other methods of entering this pathway are also known and are discussed in detail herein. While the review concentrates on inducing cytotoxic T cells (CTLs), it is becoming increasingly apparent that other modes of immunotherapy would be desirable, such as class II presentation to induce increased helper activity (for CTL), but also activating macrophages to be effective against tumor cells.     

Memory T lymphocytes

Immunological memory protects organisms from recurrent challenge by pathogens. The persistence of a heightened reactive state initiated by antigenic challenge is mediated by long-lived memory lymphocytes. The survival of memory T cells is thought to require stimulation through the T cell receptor (TCR), sometimes by persistent antigen. However, memory T cells can survive in the absence of antigen, in which case TCR stimulation provided by cell surface self-peptide/ major histocompatibility complex (MHC) molecules and cytokines are required to sustain memory T cells. Recent work using mouse models has provided insights into the origin of memory T cells. Understanding the mechanisms that underlie the differentiation and persistence of memory T cells may improve the effectiveness of vaccines through the induction of T cell memory.     

Conformational dynamics of the beta2-microglobulin C terminal in the cell-membrane-anchored major histocompatibility complex type I

We have recently described an anti-beta2-microglobulin (beta2-m) monoclonal antibody (mAb 14H3) capable of recognizing the epitope 92-99 of the protein in the monomeric native state as well as in the fibrillar polymeric state, but not in the major histocompatibility complex type I (MHCI) anchored to the cell membrane. In the present study, we investigated the molecular basis for the inaccessibility of the C-terminal end of beta2-m in the MHCI complex, and demonstrated that mAb 14H3 binds the soluble fraction of the MHCI complex with a Kd of 0.3 microM. An interaction between the complex and the membrane protects beta2-m from immunological recognition at the MHCI level. This protection from antibody recognition can be weakened by procedures such as heat shock or gamma irradiation that perturb the membrane structure and commit the cell to the apoptotic pathway. mAb 14H3 can recognize MHCI in a transient state that most likely precedes beta2-m shedding and may be proposed as a useful tool for dynamic analysis of MHCI conformational modifications.     

Non-conventional sources of peptides presented by MHC class I

Effectiveness of immune surveillance of intracellular viruses and bacteria depends upon a functioning antigen presentation pathway that allows infected cells to reveal the presence of an intracellular pathogen. The antigen presentation pathway uses virtually all endogenous polypeptides as a source to produce antigenic peptides that are eventually chaperoned to the cell surface by MHC class I molecules. Intriguingly, MHC I molecules present peptides encoded not only in the primary open reading frames but also those encoded in alternate reading frames. Here, we review recent studies on the generation of cryptic pMHC I. We focus on the immunological significance of cryptic pMHC I, and the novel translational mechanisms that allow production of these antigenic peptides from unconventional sources.     

Post-proteasomal and proteasome-independent generation of MHC class I ligands

Peptide ligands presented by MHC class I molecules are produced by intracellular proteolysis, which often involves multiple steps. Initial antigen degradation seems to rely almost invariably on the proteasome, although tripeptidyl peptidase II (TPP II) and insulin-degrading enzyme (IDE) may be able to substitute for the proteasome in rare cases. Recent evidence suggests that the net effect of cytosolic aminopeptidases is destruction of potential class I ligands, although a positive role in selected cases has been documented. This may apply particularly to the trimming of long precursors by TPP II. In contrast, trimming of ligand precursors in the endoplasmic reticulum is essential for the generation of suitable peptides and has a substantial impact on the repertoire of ligands presented. Trimming by the ER aminopeptidase (ERAP) enzymes most likely acts on free precursors and is adapted to the needs of class I molecules by way of a molecular ruler mechanism. Trimming by ERAP enzymes also occurs for cross-presented ligands, which can alternatively be processed in a special endosomal compartment by insulin-regulated aminopeptidase.     

Polypeptide translocation machinery of the yeast endoplasmic reticulum

Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. InSaccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of theEscherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK andS. cerevisiae Ssa1p, resepectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of 70 residue similarity to the J box, the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61 p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.     

Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein

Three isoforms of plasma membrane Ca²⁺-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca²⁺ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.     

Intestinal epithelial barrier and mucosal immunity

Intestinal mucosa integrates primary digestive functions with immune functions such as pathogen surveillance, antigen transport and induction of mucosal immunity and tolerance. Intestinal adaptive immunity is elicited in organized mucosa-associated lymphoid tissue (O-MALT) that is composed of antigen-presenting cells and lymphocytes and achieved by effector cells widely distributed in mucosa (diffuse MALT or D-MALT). Interaction between the intestinal epithelium, the O-MALT and the diffuse MALT plays a critical role in establishing an adequate immune response. In regions associated to O-MALT, lympho-epithelial cross-talks lead to acquisition of a specific epithelial phenotype that contributes to O-MALT organization and functionality. Beyond the expression of several innate immune functions, the intestinal epithelium may directly take up and present antigens due to the expression of major histocompatibility complex (MHC) and MHC-related molecules. A complex genetic program that will be outlined in the present review controls the development of immune functions of the intestinal epithelium. The effect of environmental signals on the modulation of this ontogenetic program during development and neonatal life, from bioactive components of amniotic fluid to lactation and bacterial colonization, will be discussed.