Binding of immunogenic peptides to Ia histocompatibility molecules

Most cellular interactions essential for the development of an immune response involve the membrane glycoproteins encoded in the major histocompatibility gene complex. The products of the I region, the class II histocompatibility molecules (Ia molecules), are essential for accessory cells such as macrophages to present polypeptide antigens to helper T cells. This interaction, antigen presentation, is needed for T-cell recognition of the antigen and its consequent activation. How the Ia molecules regulate the immune response during antigen presentation is not known, although it is commonly thought to result from their association with the presented antigen. Recent studies, including the elucidation of the structure of the T-cell receptor, favour recognition of a single structure, an antigen-Ia complex. Here we report attempts to determine whether purified Ia glycoproteins have an affinity for polypeptide antigens presented by intact cells in an Ia-restricted manner. We first identified the epitope of a peptide antigen involved in presentation. Several laboratories have shown that globular proteins are altered (processed) in intracellular vesicles of the antigen-presenting cell before antigen presentation. A major component of the T-cell response is directed toward determinants found in the unfolded or denatured molecule, and our laboratory has shown that the determinant of the hen-egg lysozyme protein (HEL), presented in H-2k mice to T cells, is a sequence of only 10 amino acids. This portion resides in an area of the native molecule partially buried inside the molecule, in a beta-sheet conformation. To be presented, intact or native HEL must first be processed in acidic intracellular vesicles. Having isolated the peptide responsible for T-cell recognition of HEL, we sought a physical association of this peptide with purified, detergent-solubilized I-Ak molecules from B-hybridoma cells. We have found such an association, which may explain the role of the Ia glycoproteins in cellular interactions.     

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.     

Variable expression of Ia antigens on the vascular endothelium of mouse skin allografts

Ia antigens are membrane-bound glycoproteins that play a part in antigen recognition and subsequent cell-cell interactions in the immune response. In the mouse they are coded for by the I region of the major histocompatibility complex H-2 and have been demonstrated on B lymphocytes, monocytes, activated T cells, macrophages and dendritic cells, including Langerhans cells. Ia-like antigens have also been detected on the vascular endothelium in man and on epidermal keratinocytes in rats but expression on the latter cells was induced by a graft-versus-host reaction or by contact hypersensitivity. In the mouse, previous studies have suggested that Ia antigens in skin are restricted to epidermal Langerhans cells and it was thought that these were the targets for Ia-dependent rejection of skin allografts. The results presented here show that Ia antigens in mouse allografts are also present on the vascular endothelium but their expression is variable and dependent on the immunological status of the recipient. These findings suggest that vascular endothelial cells can act as targets in Ia-incompatible skin allograft rejection.     

Immune recognition. A new receptor for beta-glucans.

The carbohydrate polymers known as beta-1,3-d-glucans exert potent effects on the immune system - stimulating antitumour and antimicrobial activity, for example - by binding to receptors on macrophages and other white blood cells and activating them. Although beta-glucans are known to bind to receptors, such as complement receptor 3 (ref. 1), there is evidence that another beta-glucan receptor is present on macrophages. Here we identify this unknown receptor as dectin-1 (ref. 2), a finding that provides new insights into the innate immune recognition of beta-glucans.     

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.     

Role of gamma-interferon in antibody-producing responses

Interferon preparations, especially those containing gamma-interferon (IFN-gamma), have long been known to modulate immune responses. However, because many studies used only partially purified interferons, it has been difficult to separate the immunoregulatory effects of the interferons from those of other biologically active molecules contaminating the preparations. Recently, with the cloning of the interferon genes in mouse and man, it has become possible to use these cloned interferons directly to test their effects in assays other than those involving the protection of cells from viruses. For example, cloned IFN-gamma has been shown to be a potent inducer of Ia antigen expression on macrophages. Similarly, cloned IFN-gamma has been reported to act as a macrophage activation factors, as judged by the ability of activated macrophages to kill tumour cells in vitro. We demonstrate here that cloned murine IFN-gamma can also substitute for a late-acting helper factor which acts synergistically with other helper factors in the stimulation of B-cell antibody responses in vitro.     

Mouse Ia antigens are receptors for lactate dehydrogenase virus

Infection of mice with lactate dehydrogenase virus (LDV) leads to elevation of plasma lactate dehydrogenase, lifelong viraemia and perturbations of cell-mediated and humoral immune responses. The virus replicates exclusively in a restricted set of macrophages, but the basis for restricted cell susceptibility is unknown. By immunofluorescence techniques we have found that the per cent infected was the same as the per cent expressing antigens encoded by the I region of the major histocompatibility complex (Ia). Infection of CBA strain I-A+ peritoneal macrophages was blocked when cells were treated simultaneously with monoclonal antibody to I-A and I-E, but not with either antibody separately. LDV infectivity was inactivated when virus was treated with purified rat glycoprotein homologous to mouse I-A and I-E antigens. These results indicate that the receptors for LDV are I-A and I-E antigens. Selective infection of Ia-positive macrophages may have an important effect on the immunological capability of infected mice.     

Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.     

T-cell antigen receptor genes and T-cell recognition

The four distinct T-cell antigen receptor polypeptides (alpha, beta, gamma, delta) form two different heterodimers (alpha:beta and gamma:delta) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.     

Thymic cortical epithelial cells lack full capacity for antigen presentation

Several recent studies have suggested that interactions between thymocytes and thymic stromal cells are essential for the development and elimination of antigen-reactive T lymphocytes. It is important, therefore, to characterize the stromal cells involved in presentation of antigen in the thymus. In a previous report, we demonstrated, using T-cell hybridomas, that three distinct types of antigen presenting cells in the thymus (cortical epithelial cells, macrophages, and dendritic cells) constitutively expressed self haemoglobin/Ia complexes. Here we report that one of these cell types, the cortical epithelial cell, does not induce stimulation of T-lymphocyte clones even though the antigen/Ia complex required for antigen-specific recognition is present. This lack of response occurs with both TH1 and TH2 clones. Responsiveness of the TH2 clone can be restored by adding the murine lymphokine interleukin-1 beta to the culture system.     

SB-restricted presentation of influenza and herpes simplex virus antigens to human T-lymphocyte clones

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.     

Recognition of bacterial glycosphingolipids by natural killer T cells

Natural killer T (NKT) cells constitute a highly conserved T lymphocyte subpopulation that has the potential to regulate many types of immune responses through the rapid secretion of cytokines. NKT cells recognize glycolipids presented by CD1d, a class I-like antigen-presenting molecule. They have an invariant T-cell antigen receptor (TCR) alpha-chain, but whether this invariant TCR recognizes microbial antigens is still controversial. Here we show that most mouse and human NKT cells recognize glycosphingolipids from Sphingomonas, Gram-negative bacteria that do not contain lipopolysaccharide. NKT cells are activated in vivo after exposure to these bacterial antigens or bacteria, and mice that lack NKT cells have a marked defect in the clearance of Sphingomonas from the liver. These data suggest that NKT cells are T lymphocytes that provide an innate-type immune response to certain microorganisms through recognition by their antigen receptor, and that they might be useful in providing protection from bacteria that cannot be detected by pattern recognition receptors such as Toll-like receptor 4.     

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.     

Presentation of antigen, foreign major histocompatibility complex proteins and self by thymus cortical epithelium

In mouse and man most peripheral T cells bear clonally variable receptors made up of alpha- and beta-chains which bind ligands on target cells consisting of peptide fragments of foreign antigens, complexed with cell surface proteins encoded by the major histocompatibility complex (MHC) of the individual. In the thymus, developing T cells are selected to mature only if their receptors will be able to participate in self-MHC plus antigen recognition in the periphery. This positive selection occurs in the presence of self-MHC, but in the apparent absence of antigen, leading to the paradoxical conclusion that developing thymocytes must be positively selected by engagement of their receptors and self-MHC alone, although thymocytes that react too well with self-MHC are eliminated. To account for this, it has been suggested that MHC molecules in the thymus are not identical to those found elsewhere. To test this and other hypotheses, we have examined the ability of the presumed selecting cells, those of the thymus cortical epithelium, to present various MHC complexes to T cells. Our results indicate that MHC molecules on thymus epithelium are not always the same as those found elsewhere.     

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.     

Recognition and plasma clearance of endotoxin by scavenger receptors.

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.     

Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1

Cell surface molecules have been implicated in cell interactions which underlie formation of the nervous system. The analysis of the functional properties of such molecules has profited from the combined use of antibodies and cell culture systems. It has been suggested that the interplay between these molecules modulates cell-to-cell interaction at critical developmental stages. In the mouse, N-CAM and L1 antigen have been shown to mediate Ca2+-independent adhesion among neural cells. N-CAM plays a role in fasciculation of neurites and formation of neuromuscular junction. L1 is apparently not involved in synaptogenesis, but in migration of granule cell neurones in the developing mouse cerebellar cortex. The two antigens are distinct molecular and functional entities which act synergistically in aggregation of neuroblastoma and early postnatal cerebellar cells. In view of a certain similarity in function between the two groups of molecules, it was not surprising to find that structural similarities are detectable by the monoclonal antibody L2. We show here that a carbohydrate moiety recognized by L2 and HNK-1 monoclonal antibodies, is present in mouse N-CAM and L1. The L2 epitope appears on all major neural cell types but not all N-CAM molecules express it. This heterogeneity points to a previously undetected molecular diversity which may have functional implications for modulating cell adhesion during development.     

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.     

Lectin-induced expression of DR antigen on human cultured follicular thyroid cells

HLA-DR antigens, the human equivalent of mouse I region-associated or Ia products, are polymorphic cell surface sialoglycoproteins involved in initiation of the immune response. Their expression is normally restricted to B lymphocytes, macrophages, dendritic and other antigen-presenting cells and vascular endothelium and possibly some cells of the mucosa lining body cavities. HLA-DR expression can be modified during cell differentiation; B lymphocytes become negative on maturing to plasma cells and human T lymphocytes acquire these antigens when activated in vitro or in vivo. We report here that human thyroid follicular cells which are normally negative for HLA-DR molecules, can be induced to express these antigens when cultured with phytohaemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM). These lectins exert their action directly on the thyroid cells with no concomitant mitogenic effect.     

Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope

The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.