Invariant chain influences the immunological recognition of MHC class II molecules.

Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.     

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.     

Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways

Class I MHC molecules acquire peptides from endogenously synthesized proteins, whereas class II antigens present peptides derived from extracellular compartment molecules. This dichotomy is due to the fact that the invariant chain associates with class II molecules in the endoplasmic reticulum, preventing binding of endogenous peptides. The mutually exclusive binding of peptide and invariant chain to class II molecules suggests that the invariant chain might play a part in autoimmune disease.     

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.     

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.     

Formation of a nine-subunit complex by HLA class II glycoproteins and the invariant chain

HLA class II molecules are heterodimeric transmembrane glycoproteins that bind and present processed antigenic peptides to CD4-positive T lymphocytes. Intracellularly, class II molecules associate with a third subunit termed the invariant (I) chain. Here we describe the physical characteristics of the intracellular class II alpha beta I complex. Chemical crosslinking, size exclusion chromatography and sedimentation velocity studies demonstrate that the alpha beta I complex is a nine-subunit transmembrane protein that contains three alpha beta dimers associated with an I chain trimer. The organization of class II alpha- and beta-subunits in such a multimer may have a role in the documented ability of the I chain to inhibit peptide binding to class II molecules. In addition, the formation of the nine-chain complex may induce the structural changes necessary to overcome the cytoplasmic retention signal responsible for the localization of free I chain in the endoplasmic reticulum, releasing class II-I chain complexes for transport to endosomes.     

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.     

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.     

ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum

The ability of killer T cells carrying the CD8 antigen to detect tumours or intracellular pathogens requires an extensive display of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of potential target cells. These peptides are derived from almost all intracellular proteins and reveal the presence of foreign pathogens and mutations. How cells produce thousands of distinct peptides cleaved to the precise lengths required for binding different MHC class I molecules remains unknown. The peptides are cleaved from endogenously synthesized proteins by the proteasome in the cytoplasm and then trimmed by an unknown aminopeptidase in the endoplasmic reticulum (ER). Here we identify ERAAP, the aminopeptidase associated with antigen processing in the ER. ERAAP has a broad substrate specificity, and its expression is strongly upregulated by interferon-gamma. Reducing the expression of ERAAP through RNA interference prevents the trimming of peptides for MHC class I molecules in the ER and greatly reduces the expression of MHC class I molecules on the cell surface. Thus, ERAAP is the missing link between the products of cytosolic processing and the final peptides presented by MHC class I molecules on the cell surface.     

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.     

MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding

Class II major histocompatibility complex molecules undergo a change in structure upon stable binding of peptide antigen. Analysis of the site and extent of this change among class II molecules of splenic antigen-presenting cells reveals the preference of class II for peptide acquisition outside the endoplasmic reticulum and indicates that the class II presentation system is not saturated with self peptides. There are numerous empty class II molecules on the cell surface and peptide antigen is evidently important in regulating surface class II expression.     

A gene in the human major histocompatibility complex class II region controlling the class I antigen presentation pathway

Major histocompatibility complex (MHC) class I molecules export peptides to the cell surface for surveillance by cytotoxic T lymphocytes. Intracellular peptide binding is critical for the proper assembly and transport of class I molecules. This mechanism is impaired as a result of a non-functional peptide supply factor gene (PSF) in several human mutant cell lines with genomic lesions in the MHC. We have now identified PSF in the MHC class II region by deletion mapping in mutants and chromosome-walking. PSF is homologous to mammalian and bacterial ATP-dependent transport proteins, suggesting that it operates in the intracellular transport of peptides.     

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.     

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.     

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.     

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.     

计算机算法结合实验技术进行T细胞表位筛选

计算机算法与实验技术相结合的方法,已广泛用于各种免疫相关抗原T细胞表位的鉴定,对现在可获得的计算机预测方法,以及计算机算法与实验相结合鉴定T细胞表位的方法进行了综述,主要内容包括表位预测及抗原加工处理的预测。     

Cell-cell adhesion mediated by CD8 and MHC class I molecules

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.     

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.     

Specific binding of antigenic peptides to cell-associated MHC class I molecules

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.