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.     

Physical association between MHC class I molecules and immunogenic peptides

Antigenic peptides are presented to T lymphocytes by major histocompatibility complex (MHC) molecules. The binding of peptides to MHC class II molecules has been demonstrated directly, and is found to correlate with the ability of specific class II alleles to restrict the T-cell response to specific peptides. By comparison, a direct demonstration of a physical association between antigenic peptides and MHC class I molecules has proved difficult. A recent report shows that it is possible, however, and the three-dimensional structure of a class I MHC molecule illustrates the site where such binding must occur. Here we describe a simple assay which measures the binding of radiolabelled MHC class I molecules to peptides bound to a solid phase support. We find that class I molecules bind specifically to peptides known to be antigenic for class I-restricted cytotoxic T lymphocytes. Peptides which are recognized by cytotoxic T lymphocytes bind not only to the restricting MHC class I molecule but also to other class I molecules. Our results suggest that quantitative differences in the peptide/MHC class I interaction may influence the-pattern of MHC restriction observed in vivo.     

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.     

New class II-like genes in the murine MHC

Major histocompatibility complex (MHC) class I molecules present endogenous antigens to CD8+ (cytotoxic) T cells. MHC class II molecules present primarily exogenously derived antigens to CD4+ T cells. Three new genes (Ma, Mb1 and Mb2) located between the Pb and Ob genes of the murine MHC have properties indicating that they are members of the MHC class II gene family, but they are the most divergent class II members so far identified and are almost as closely related in sequence to class I genes as they are to the known class II genes.     

MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8.

Interactions between major histocompatibility complex (MHC) molecules and the CD4 or CD8 coreceptors have a major role in intrathymic T-cell selection. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay, and a binding site for CD8 on class I has been identified. Here we demonstrate that a region of the MHC class II beta-chain beta 2 domain, structurally analogous to the CD8-binding loop in the MHC class I alpha 3 domain, is critical for function with both mouse and human CD4.     

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.     

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.     

Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs.

T cells recognize foreign protein antigens in the form of peptide fragments bound tightly to the outer aspect of molecules encoded by the major histocompatibility complex (MHC). Most of the amino-acid differences that distinguish MHC allelic variants line the peptide-binding cleft, and different allelic forms of MHC molecules bind distinct peptides. It has been demonstrated that peptide-binding to MHC class I involves anchor residues in certain positions and that antigenic peptides associated with MHC class I exhibit allele-specific structural motifs. We have previously reported an analysis of MHC class II-associated peptide sequences. Here we extend this analysis and show that certain amino-acid residues occur at particular positions in the sequence of peptides binding to a given MHC class II molecule. These sequence motifs require the amino terminus to be shifted one or two positions to obtain alignment; such shifts occur naturally for a single peptide sequence without qualitatively altering CD4 T-cell recognition.     

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.     

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.     

Class II MHC molecules can use the endogenous pathway of antigen presentation

Models for antigen presentation have divided the world of antigens into two categories, endogenous and exogenous, presented to T cells by class I and class II major histocompatibility complex (MHC) encoded molecules, respectively. Exogenous antigens are though to be taken up into peripheral endosomal compartments where they are processed for binding to class II MHC molecules. Endogenous antigens are either synthesized or efficiently delivered to the cytoplasm before being partially degraded in an as yet undefined way, and complexed with class I MHC molecules. A useful phenotypic distinction between the two pathways has been the sensitivity to weak bases, such as chloroquine, which is a property only of the exogenous pathway. The fungal antibiotic brefeldin A (BFA), which blocks protein transport from the endoplasmic reticulum to the Golgi network, also blocks class I-restricted antigen-presentation, providing us with the corresponding marker of the endogenous pathway. Experiments with influenza virus antigens have supported the view that class II MHC molecules can present exogenous but not endogenous antigen, whereas the observation that class II MHC molecules present measles virus non-membrane antigens by a chloroquine-insensitive pathway suggests that this is not always the case. We show here that influenza A matrix protein can be effectively presented to class II-restricted T cells by two pathways: one of which is chloroquine-sensitive, BFA-insensitive, the other being chloroquine-insensitive and BFA-sensitive. Our results indicate that both class I and class II molecules can complex with antigenic peptides in a pre-Golgi compartment and favour a unified mechanism for MHC-restricted endogenous antigen presentation.     

Tetrameric cell-surface MHC class I molecules.

Purified major histocompatibility complex (MHC) class I molecules have been studied at high resolution by X-ray crystallography; the structure is a complex of a single heavy chain, a beta 2-microglobulin light chain and a tightly bound peptide moiety. We show here that complete MHC class I molecules are post-translationally assembled into tetramers (made up of four heavy chains and four beta 2-microglobulin units) and that this tetrameric species is expressed on the cell surface. The multivalent tetrameric structure of class I molecules can be reconciled with models of T-cell activation that invoke antigen-receptor crosslinking, as opposed to models that depend on an allosteric change.     

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.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

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.     

T-cell recognition of a chimaeric class II/class I MHC molecule and the role of L3T4

In addition to expressing clonally distributed antigen-specific and major histocompatibility complex (MHC)-restricted receptors, T cells also express non-clonally distributed surface molecules that are involved in T-cell function. Among the most intriguing of the latter are L3T4 and Lyt 2, which are expressed on individual T lymphocytes in striking, though not absolute, concordance with their restriction by either class II or class I MHC determinants, and which are thought to contribute to the overall avidity of T-cell interactions by binding to monomorphic determinants on class II and class I MHC molecules, respectively. To examine the ability of T cells to recognize a single class II domain in the absence of the remainder of the Ia molecule, as well as to evaluate the structural basis for the putative interaction of L3T4 with Ia, a recombinant class II/class I murine MHC gene was constructed and introduced into mouse L cells. Here we demonstrate that a subset of class II allospecific cytotoxic T lymphocytes (CTL) can specifically recognize and lyse L-cell transfectants expressing an isolated polymorphic A beta 1 domain, and that anti-L3T4 antibody can block such killing, a result inconsistent with the highly conserved membrane-proximal domains of Ia acting as unique target sites for L3T4 binding.     

Interaction between CD4 and class II MHC molecules mediates cell adhesion

The CD4 glycoprotein is expressed on T-helper and cytotoxic lymphocytes which are restricted to class II major histocompatibility complex (MHC) antigens on target cells. Antibody inhibition studies imply that CD4 acts to increase the avidity of effector-target cell interactions. These observations have led to the speculation that CD4 binds to a monomorphic class II antigen determinant, thereby augmenting low affinity T-cell receptor-antigen interactions. However, no direct evidence has been presented indicating that CD4 and class II molecules interact. To address this issue, we have used a vector derived from simian virus 40 (SV40) to express a complementary DNA (cDNA) encoding the human CD4 glycoprotein. When CV1 cells expressing large amounts of the CD4 protein at the cell surface are incubated with human B cells bearing MHC-encoded class II molecules, they are bound tightly to the infected monolayer, whereas mutant B cells which lack class II molecules fail to bind. Furthermore, the binding reaction is specifically inhibited by anti-class II and anti-CD4 antibodies. Thus, the CD4 protein, even in the absence of T-cell receptor-antigen interactions, can interact directly with class II antigens to function as a cell surface adhesion molecule.     

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.     

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.     

Competition for antigen presentation in living cells involves exchange of peptides bound by class II MHC molecules

T cells recognize foreign proteins as peptides bound to self molecules encoded by the major histocompatibility complex (MHC). The kinetics of interaction between purified class II MHC molecules and peptides is unusual, in that the rate of association is very slow, but once formed, the complexes are extremely stable. This raises the question of how the antigen-presenting cell provides a sufficient number of free MHC binding sites to ensure T cell immunity. We present results suggesting that an exchange of peptide in MHC binding sites may take place under physiological conditions.