Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.     

On the complexity of self.

Self peptides bound to self major histocompatibility complex (MHC) molecules have been implicated both in positive and in negative selection of T cells during intrathymic development. We report here that the novel MHC-restricted monoclonal antibody Y-Ae detects the MHC class II bound form of a major self peptide. Y-Ae binds approximately 12% of the relevant MHC class II molecules on self antigen presenting cells. The peptide detected by Y-Ae is one of several major peptides eluted from the MHC molecule. These data suggest that self peptides presented by self MHC class II molecules at densities sufficient to signal a CD4 T cell are of very limited complexity. Furthermore, as Y-Ae stains antigen presenting cells that mediate negative selection but not thymic cortical epithelial cells that drive positive selection, differential expression of self peptide:self MHC class II complexes may be a key feature of intrathymic selection.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells

The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histocompatibility complex (MHC). These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments. Interestingly, in certain pathological conditions, for example in autoimmune diseases such as thyroiditis and diabetic insulitis, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+ thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.     

A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules

Class II and class I histocompatibility molecules allow T cells to recognize 'processed' polypeptide antigens. The two polypeptide chains of class II molecules, alpha and beta, are each composed of two domains (for review see ref. 6); the N-terminal domains of each, alpha 1 and beta 1, are highly polymorphic and appear responsible for binding peptides at what appears to be a single site and for being recognized by MHC-restricted antigen-specific T cells. Recently, the three-dimensional structure of the foreign antigen binding site of a class I histocompatibility antigen has been described. Because a crystal structure of a class II molecule is not available, we have sought evidence in class II molecules for the structural features observed in the class I binding site by comparing the patterns of conserved and polymorphic residues of twenty-six class I and fifty-four class II amino acid sequences. The hypothetical class II foreign-antigen binding site we present is consistent with mutation experiments and provides a structural framework for proposing peptide binding models to help understand recent peptide binding data.     

Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane

Central to the initiation of immune responses is recognition of peptide antigen by T lymphocytes. The cell biology of dendritic cells makes them ideally suited for the essential process of antigen presentation. Their life cycle includes several stages characterized by distinct functions and mechanisms of regulation. Immature dendritic cells synthesize large amounts of major histocompatibility complex class II molecules (MHC II), but the alpha beta-dimers are targeted to late endosomes and lysosomes (often referred to as MHC class II compartments) where they reside unproductively with internalized antigens. After exposure to microbial products or inflammatory mediators, endocytosis is downregulated, the expression of co-stimulatory molecules is enhanced, and newly formed immunogenic MHC II-peptide complexes are transported to the cell surface. That these MHC II molecules reach the surface is surprising, as the lysosomes comprise the terminal degradative compartment of the endocytic pathway from which exogenous components generally cannot be recovered intact. Here we have visualized this pathway in live dendritic cells by video microscopy, using cells expressing MHC II tagged with green fluorescent protein (GFP). We show that on stimulation, dendritic cells generate tubules from lysosomal compartments that go on to fuse directly with the plasma membrane.     

Antigen presentation enhanced by the alternatively spliced invariant chain gene product p41.

During biosynthesis, class II molecules of the major histocompatibility complex are associated with a nonpolymorphic protein called invariant chain, Ii, which facilitates folding of class II molecules and their exit from the endoplasmic reticulum, interferes with their association with peptide and directs their post-Golgi transport (refs 7-9). If Ii blocks class II loading with endogenous antigens in the endoplasmic reticulum and/or directs class II molecules to the exogenous antigen-loading compartment, then the co-expression of Ii should enhance the ability of class II molecules to present exogenous antigens to T cells. But data supporting a role for Ii in class II-restricted antigen presentation are controversial. Here we show that Ii can facilitate exogenous antigen presentation for a subset of antigens. Although all known functions of Ii have been ascribed to the principal form of Ii, p31, we find that in most cases antigen presentation is facilitated only by the alternatively spliced, minor form of Ii, p41.     

Sequence analysis of peptides bound to MHC class II molecules.

CD4 T cells recognize peptide fragments of foreign proteins bound to self class II molecules of the major histocompatibility complex (MHC). Naturally processed peptide fragments bound to MHC class II molecules are peptides of 13-17 amino acids which appear to be precessively truncated from the carboxy terminus, perhaps after binding to the MHC class II molecule. The finding of predominant self peptides has interesting implications for antigen processing and self-non-self discrimination.     

Excess beta 2 microglobulin promoting functional peptide association with purified soluble class I MHC molecules

T lymphocytes expressing alpha beta receptors recognize antigenic peptide fragments bound to major histocompatibility complex class I or class II molecules present on the surface membranes of other cells. Peptide fragments are present in the two available HLA crystal structures and recent data indicate that peptide is required for the stable folding of the class I heavy chain and maintenance of its association with the class I light chain, beta 2-microglobulin (beta 2m), at physiological temperature. To explain how the exogenous peptide used to create targets for cytotoxic cells bearing CD8 antigen could associate with apparently peptide-filled extracellular class I molecules, we hypothesized that stable binding of exogenous peptide to mature class I molecules reflects either the replacement of previously bound peptide during the well documented beta 2m exchange process or the loading of 'empty' class I heavy chains dependent on the availability of excess beta 2m. In either case, free beta 2m should enhance peptide/class I binding. Using either isolated soluble class I molecules or living cells, we show here that free purified beta 2m markedly augments the generation of antigenic complexes capable of T-cell stimulation.     

Structural and serological similarity of MHC-linked LMP and proteasome (multicatalytic proteinase) complexes

Major histocompatibility complex (MHC) class I molecules associate with peptides derived from endogenously synthesized antigens. Cytotoxic T-lymphocytes can thus scan class I molecules and bound peptide on the surface of cells for foreign antigenic determinants. Recent evidence demonstrates that the products of trans-acting, non-class I genes in the class II region of the MHC are required in the class I antigen-processing pathway. There are genes (called HAM1 and HAM2 in the mouse) in this region that encode proteins postulated to be involved in the transport of peptide fragments into the endoplasmic reticulum for association with newly synthesized class I molecules. But, the mechanism by which such peptide fragments are produced remains a mystery. At least two genes encoding subunits of the low-molecular mass polypeptide (LMP) complex are tightly linked to the HAM1 and HAM2 genes. We show that the LMP complex is closely related to the proteasome (multicatalytic proteinase complex), an intracellular protein complex that has multiple proteolytic activities. We speculate that the LMP complex may have a role in MHC class I antigen processing, and therefore that the MHC contains a cluster of genes required for distinct functions in the antigen processing pathway.     

T-cell engagement of dendritic cells rapidly rearranges MHC class II transport

Assembly of major histocompatibility complex (MHC) molecules, which present antigen in the form of short peptides to T lymphocytes, occurs in the endoplasmic reticulum; once assembled, these molecules travel from the endoplasmic reticulum to their final destination. MHC class II molecules follow a route that takes them by means of the endocytic pathway, where they acquire peptide, to the cell surface. The transport of MHC class II molecules in 'professional' antigen-presenting cells (APCs) is subject to tight control and responds to inflammatory stimuli such as lipopolysaccharide. To study class II transport in live APCs, we replaced the mouse MHC class II gene with a version that codes for a class II molecule tagged with enhanced green fluorescent protein (EGFP). The resulting mice are immunologically indistinguishable from wild type. In bone-marrow-derived dendritic cells, we observed class II molecules in late endocytic structures with transport patterns similar to those in Langerhans cells observed in situ. We show that tubular endosomes extend intracellularly and polarize towards the interacting T cell, but only when antigen-laden dendritic cells encounter T cells of the appropriate specificity. We propose that such tubulation serves to facilitate the ensuing T-cell response.     

Binding of labelled influenza matrix peptide to HLA DR in living B lymphoid cells

T cells recognize protein antigens as fragments (peptides) held in a defined binding site of class I or class II major histocompatibility (MHC) molecules. The formation of complexes between various immunologically active peptides and different MHC molecules has been demonstrated directly in binding studies between the peptides and solubilized, purified molecules of class II MHC. Studies with intact cells, living or fixed, have not directly demonstrated the binding of the peptides to MHC molecules on antigen-presenting cells, but the formation of such complexes has been shown indirectly through the capacity of antigen-presenting cells to stimulate specific T cells. Here we report evidence that supports directly the binding of radiolabelled influenza matrix peptide 17-29 to products of the human class II MHC locus HLA-DR, on living homozygous B-cell lines, and we show that the kinetics of such binding is much faster with living cells than with fixed cells. Furthermore, whereas the peptide reacts with HLA-DR molecules of all alleles, it binds preferentially to DR1, the restricting element in antigen presentation.     

Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding

Class II major histocompatibility complex (MHC) molecules are heterodimeric cell surface glycoproteins which bind and present immunogenic peptides to T lymphocytes. Such peptides are normally derived from protein antigens internalized and proteolytically degraded by the antigen-presenting cell. Class I MHC molecules also bind immunogenic peptides, but these are derived from proteins synthesized within the target cell. Whereas class I molecules seem to bind peptides in the endoplasmic reticulum, class II molecules are thought to bind peptides late in transport. Intracellular class II molecules associate in the endoplasmic reticulum with a third glycoprotein, the invariant (I) chain, which is proteolytically removed before cell surface expression of the alpha beta class II heterodimer. It has been suggested that the I chain prevents peptides from associating with class II molecules early in transport. Preventing such binding until the class II molecules enter an endosomal compartment could maintain the functional dichotomy between class I and class II MHC molecules. We have examined the ability of I chain-associated HLA-DR5 molecules to bind a well characterized influenza haemagglutinin-derived peptide (HAp). The results show that whereas mature HLA-DR alpha beta dimers effectively bind this peptide, the I chain-associated form does not.     

Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment

The pathways of intracellular traffic involved in antigen processing and presentation have been defined by immunoelectron microscopy. The export pathway for class II histocompatibility molecules and the antigen import pathway meet in a peripheral endocytic compartment having all the molecular machinery believed to be required for antigen processing and presentation, including internalized surface immunoglobulins, proteolytic enzymes and invariant chains. This compartment defines a site where peptides from endocytosed antigen can bind class II molecules en route to the cell surface for presentation to T cells.     

Irreversible association of peptides with class II MHC molecules in living cells.

Functional, morphological and biochemical evidence indicates that class II major histocompatibility complex (MHC) molecules associate with processed peptides during biosynthesis. Peptide/MHC complexes in living cells have been reported to be less stable than similar complexes generated in vitro, which has led to the suggestion that there may be a peptide exchange mechanism operating in vivo. Although this could increase the capacity for binding incoming antigens, it would reduce the efficacy of processed antigenic peptides by exchanging these for self peptides. Here we measure the half-life of peptide/class II complexes in human antigen-presenting cells and find that it is very similar to the half-life of class II molecules themselves, indicating that peptides are bound irreversibly under physiological conditions. Thus class II MHC retains long-term 'memory' of past encounters with antigen to maximize the opportunity for T cell/antigen-presenting cell interaction.     

The alpha 1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1

Several exoproteins from the bacterium Staphylococcus aureus are highly potent polyclonal activators of T cells in the presence of cells bearing class II antigens of the major histocompatibility complex (MHC). These toxins, including the toxic shock syndrome toxin (TSST-1), act at nanomolar concentrations, bind directly to class II molecules, and do not require the processing typical of nominal antigen. Each toxin is capable of stimulating a subpopulation of peripheral T lymphocytes bearing particular V beta sequences as part of their alpha beta T-cell receptors. It is not known how these so-called 'superantigens' bind to class II and how this binding stimulates T cells. In this study, the different affinities of TSST-1 for human class II molecules DR and DP were exploited to define the region of a class II molecule necessary for high-affinity binding. Using chimaeric alpha- and beta-chains of DR and DP expressed at the surface of transfected murine fibroblasts and a binding assay with TSST-1, it was shown that the alpha 1 domain of DR is essential for high-affinity binding, and further that TSST-1 binding did not prevent subsequent binding of a DR-restricted antigenic peptide. This is compatible with a model of superantigen making external contacts with both class II and T cell receptor, and suggests that the V beta portion of the T-cell receptor interacts with the nonpolymorphic alpha-chain of DR.     

Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes

Presentation of an exogenous protein antigen to helper (CD4+)T-lymphocytes by antigen presenting cells (APC) generally requires that the APCs degrade the native protein antigen into an immunogenic peptide, a process termed 'antigen processing', and that this peptide bind to a major histocompatibility complex (MHC) class II molecule. The complex of peptide and MHC molecule on the APC surface provides the stimulatory ligand for the alpha beta T cell receptor. The intracellular pathways and molecular mechanisms involved in the generation of the peptide-MHC complex are not well understood. Here, we describe several mutant APCs which are altered in their ability to present native exogenous protein antigens but effectively present immunogenic peptides derived from these proteins. The lesions in these mutants are not in the class II structural genes, but they affect the conformation of mature class II dimers.     

T cells can present antigens such as HIV gp120 targeted to their own surface molecules

To trigger class II-restricted T cells, antigen presenting cells have to capture antigens, process them and display their fragments in association with class II molecules. In most species, activated T cells express class II molecules; however, no evidence has been found that these cells can present soluble antigens. This failure may be due to the inefficient capture, processing or display of antigens in a stimulatory form by T-cells. The capture of a soluble antigen, which is achieved by nonspecific mechanisms in macrophages and dendritic cells, can be up to 10(3) times more efficient in the presence of surface receptors, such as surface immunoglobulin on B cells that specifically bind antigen with high affinity. We asked whether T cells would be able to present soluble antigens that bind to their own surface molecules. Here we show that such antigens can be effectively processed and presented by both CD4+- and CD8+-bearing human T cells. This indicates that T cells are fully capable of processing and displaying antigens and are mainly limited in antigen presentation by their inefficiency at antigen capture.     

Empty MHC class I molecules come out in the cold

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.     

Functionally distinct subsites on a class II major histocompatibility complex molecule

Mature T lymphocytes are activated by recognition of the combination of foreign protein antigen and membrane products of the major histocompatibility complex (MHC). Studies of peptide antigen binding to detergent-solubilized class II MHC molecules (Ia) have established that peptide-Ia interaction occurs in the absence of the T-cell receptor and varies according to allele-specific features of Ia molecules. The residues of immunogenic peptides thus contribute to two largely independent functions--the control of association with Ia molecules and the determination of the specificity of T-cell receptor binding. Two analogous and potentially independent functional sites have been postulated for Ia molecules--a region that controls binding to peptides and a region that interacts with T-cell receptors. Here we present evidence from functional analysis of recombinant class II molecules that these two postulated functional regions of Ia molecules do exist and can be independently manipulated, consistent with our recent demonstration of the segmental nature of Ia molecule structure-function relationships.