A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.     

Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.     

Cytolytic T-lymphocyte response to isolated class I H-2 proteins and influenza peptides

T cells recognize antigenic peptides in the context of major histocompatibility complex (MHC) proteins. Peptide binding to class II MHC proteins, and T-cell recognition of these complexes at the functional level has been demonstrated. Although considerable evidence suggests that class I-restricted cytotoxic T lymphocytes (CTL) recognize class I-peptide complexes, this has not yet been directly demonstrated. Chen and Parham have recently detected a low level of direct binding of radiolabelled influenza peptides to class I HLA proteins, but the relevance of this binding to T-cell recognition remains uncertain. We report here that purified class I proteins pulsed with influenza peptides can trigger antigen-specific, TCR-mediated degranulation by CTL. Effective pulsing depends on both peptide concentration and time, and can occur within 60 minutes. These results provide strong support for the formation of an antigenic complex that is recognized by CTL in which peptide antigens are bound to isolated class I proteins.     

Peptide binding to empty HLA-B27 molecules of viable human cells

Intracellular binding of antigenic peptides by polymorphic class I major histocompatibility complex molecules creates the ligands recognized by receptors of CD8+ T cells. Previously described in vitro assays of peptide binding to class I molecules have been limited by either the low proportion of accessible binding sites or the lack of allelic specificity. Here we describe a system in which the human class I molecule HLA-B27 binds considerable amounts of an influenza peptide with precise allelic discrimination. Binding requires viable cells, is stimulated by gamma-interferon and is inhibited by brefeldin A. Our results are consistent with the presence of fairly stable 'empty' HLA-B27 molecules at the cell surface. By contrast, analysis of the binding of a second influenza peptide indicates that empty HLA-Aw68 molecules are relatively short-lived. We speculate that HLA-B27 might bind extracellular peptides in vivo and that this property could underlie its association with autoimmune disease.     

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.     

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.     

Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with beta 2-microglobulin, in vitro

Cytotoxic T lymphocytes kill virally infected cells when they detect antigenic fragments presented by class I major histocompatibility complex (MHC) antigens (HLA in humans). The crystal structures of HLA-A2 and HLA-Aw68 reveal that peptide-antigen forms an integral part of the HLA structure, being retained in a prominent groove even after purification and crystallization. Here we report that the heavy chain and beta 2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, beta 2-microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. Reconstitution is not stimulated by either of two peptides not restricted to HLA-A2. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, Np (85-94), crystallizes under conditions previously used to crystallize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence.     

Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the alpha 2 domain of HLA-A2

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.     

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.     

Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA

The extreme polymorphism in the human leukocyte antigen (HLA) class I region of the human genome is suggested to provide an advantage in pathogen defence mediated by CD8+ T cells. HLA class I molecules present pathogen-derived peptides on the surface of infected cells for recognition by CD8+ T cells. However, the relative contributions of HLA-A and -B alleles have not been evaluated. We performed a comprehensive analysis of the class I restricted CD8+ T-cell responses against human immunodeficiency virus (HIV-1), immune control of which is dependent upon virus-specific CD8+ T-cell activity. In 375 HIV-1-infected study subjects from southern Africa, a significantly greater number of CD8+ T-cell responses are HLA-B-restricted, compared to HLA-A (2.5-fold; P = 0.0033). Here we show that variation in viral set-point, in absolute CD4 count and, by inference, in rate of disease progression in the cohort, is strongly associated with particular HLA-B but not HLA-A allele expression (P < 0.0001 and P = 0.91, respectively). Moreover, substantially greater selection pressure is imposed on HIV-1 by HLA-B alleles than by HLA-A (4.4-fold, P = 0.0003). These data indicate that the principal focus of HIV-specific activity is at the HLA-B locus. Furthermore, HLA-B gene frequencies in the population are those likely to be most influenced by HIV disease, consistent with the observation that B alleles evolve more rapidly than A alleles. The dominant involvement of HLA-B in influencing HIV disease outcome is of specific relevance to the direction of HIV research and to vaccine design.     

Class I cross-restricted T cells reveal low responder allele due to processing of viral antigen

Cytotoxic T lymphocytes (CTL) recognize protein antigens which have been processed by the target cell and then presented in association with the relevant class I molecule of the major histocompatibility complex (MHC). Short synthetic peptides, which are able to associate directly with target cells, may substitute for these processed fragments in stimulating antigen-specific CTL responses. Using this approach, a dominant HLA-A2-restricted epitope has previously been mapped to residues 58-68 of influenza A virus matrix protein. Here we report HLA-A2-restricted CTL which are also able to recognize this short synthetic peptide in association with HLA-Aw69, but which fail to recognize HLA-Aw69 expressing cells infected with influenza A virus. Furthermore, individuals possessing HLA-Aw69 who respond to influenza A virus, do not respond to M58-68. These results imply that the low response to this epitope on infection of HLA-Aw69 individuals with influenza A is due to failure of the naturally processed product of matrix protein to associate with Aw69.     

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.     

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.     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis

Antigenic peptides are presented to CD8+T lymphocytes by class I major histocompatibility complex (MHC) molecules. Peptides specifically bind to purified class I molecules in vitro, and to class I molecules on cells at nonphysiological temperatures. We report here the kinetic and equilibrium parameters for the binding of radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter homologues) to the murine H-2Db molecule on intact, viable cells at 37 degrees C. In contrast to earlier reports, we show that peptide binding is rapid and reversible, with dissociation constants ranging from nanomolar to micromolar, suggestive of typical ligand-receptor interactions. Only 10% of cell-surface Db molecules can bind these peptides. To address the relationship between peptide binding and T-cell recognition of the antigen-MHC complex, we determined the minimum number of complexes required to sensitize a target cell for lysis by class I-restricted cytotoxic T-lymphocytes. Our data indicate that EL4 thymoma cells (H-2b) can be sensitized for lysis by cytotoxic T-lymphocytes when as few as 200 class I-peptide complexes (less than 0.08% of surface Db molecules) are present per cell.     

HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides.

The mutant human cell line T2 is defective in antigen presentation in the context of class I major histocompatibility complex (MHC) molecules, and also in that transfected T2 cells show poor surface expression of exogenous human class I (HLA) alleles. Both defects are thought to lie in the transport of antigenic peptides derived from cytosolic proteins into the endoplasmic reticulum (ER), as peptide-deficient class I molecules might be expected to be either unstable or retained in the ER. The products of several mouse class I (H-2) genes, and the endogenous gene HLA-A2 do, however, reach the surface of T2 cells at reasonable levels although they are non-functional. We report here that, as expected, poorly surface-expressed HLA molecules do not significantly bind endogenous peptides. Surprisingly, H-2 molecules expressed in T2 also lack associated peptides, arguing that 'empty' complexes of mouse class I glycoproteins with human beta 2-microglobulin are neither retained in the ER nor unstable. HLA-A2 molecules, however, do bind high levels of a limited set of endogenous peptides. We have sequenced three of these peptides and find that two, a 9-mer and an 11-mer, are derived from a putative signal sequence (of IP-30, an interferon-gamma-inducible protein), whereas a third, a 13-mer, is of unknown origin. The unusual length of two of the peptides argues that the 9-mers normally associated with HLA-A2 molecules may be generated before their transport from the cytosol rather than in a pre-Golgi compartment. To our knowledge, this is the first report of the isolation of a fragment of a eukaryotic signal peptide generated in vivo.     

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.     

Specificity of peptide presentation by a set of hybrid mouse class I MHC molecules

The class I molecules of the major histocompatibility complex (H-2 in mouse, HLA in man) are membrane proteins composed of a polymorphic heavy chain associated with beta-2-microglobulin. Recent studies suggest that class I molecules present peptides derived from processed antigens to the receptor of cytolytic T cells. In particular, in the H-2d haplotype, synthetic HLA peptides can be recognized on Kd-bearing target cells by Kd-restricted cytolytic T cells specific for HLA. Here we analyse the specificity of presentation of two HLA peptides by a set of chimaeric Kd/Dd molecules to four different cytolytic T-cell clones. We identify two distinct regions within the second external (alpha 2) domain of Kd that contribute to its specificity as a restriction element. Our results indicate that the binding of an immunogenic peptide by a class I molecule is not always sufficient for its recognition by the T-cell antigen receptor. This suggests that the major histocompatibility complex restriction element either interacts with the T-cell antigen receptor or induces the recognized conformation of the peptide.     

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.