Apolipoprotein-mediated pathways of lipid antigen presentation

Peptide antigens are presented to T cells by major histocompatibility complex (MHC) molecules, with endogenous peptides presented by MHC class I and exogenous peptides presented by MHC class II. In contrast to the MHC system, CD1 molecules bind lipid antigens that are presented at the antigen-presenting cell (APC) surface to lipid antigen-reactive T cells. Because CD1 molecules survey endocytic compartments, it is self-evident that they encounter antigens from extracellular sources. However, the mechanisms of exogenous lipid antigen delivery to CD1-antigen-loading compartments are not known. Serum apolipoproteins are mediators of extracellular lipid transport for metabolic needs. Here we define the pathways mediating markedly efficient exogenous lipid antigen delivery by apolipoproteins to achieve T-cell activation. Apolipoprotein E binds lipid antigens and delivers them by receptor-mediated uptake into endosomal compartments containing CD1 in APCs. Apolipoprotein E mediates the presentation of serum-borne lipid antigens and can be secreted by APCs as a mechanism to survey the local environment to capture antigens or to transfer microbial lipids from infected cells to bystander APCs. Thus, the immune system has co-opted a component of lipid metabolism to develop immunological responses to lipid antigens.     

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.     

Striking similarities between antigen receptor J pieces and sequence in the second chain of the murine CD8 antigen

The CD8 antigen is a marker for T-lymphocyte subsets that is absent from helper T cells but expressed on cytotoxic T cells which recognize foreign determinants in association with class I major histocompatibility complex (MHC) antigens. It has been suggested that CD8 plays some part in recognition by CD8+ cytotoxic T cells since anti-CD8 antibodies can block their functions and the human CD8 antigen contains a domain with clear similarities to immunoglobulin and T-cell receptor (TCR) variable-region (V) domains. Human CD8 antigen is thought to be a homodimer but in the mouse and rat the equivalent antigens (alternatively called Lyt2,3 and OX8) are heterodimeric. Rat CD8 contains two chains of relative molecular mass 32,000 (32K) and 37K: the 32K chain is the rat homologue of human CD8 and mouse Lyt2. We describe here the molecular cloning of the rat 37K chain using an oligonucleotide probe predicted from peptide sequence. The full protein sequence is derived from the complementary DNA and matches limited peptide sequence for mouse Lyt3. The new sequence is more like immunoglobulin and T-cell receptor V domains than other T-cell antigens and includes a patch that is almost identical to some joining (J) piece sequences. This suggests that the CD8 and receptor heterodimers may have evolved directly from a common ancestor.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.     

Human gamma delta+ T cells respond to mycobacterial heat-shock protein

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.     

Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction

CD8+ T cells have a crucial role in resistance to pathogens and can kill malignant cells; however, some critical functions of these lymphocytes depend on helper activity provided by a distinct population of CD4+ T cells. Cooperation between these lymphocyte subsets involves recognition of antigens co-presented by the same dendritic cell, but the frequencies of such antigen-bearing cells early in an infection and of the relevant naive T cells are both low. This suggests that an active mechanism facilitates the necessary cell-cell associations. Here we demonstrate that after immunization but before antigen recognition, naive CD8+ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell-CD4+ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1alpha and MIP-1beta) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4+ T cells to promote memory CD8+ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell-cell interactions within lymph nodes, optimizing CD8+ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire.     

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.     

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.     

Deletion of self-reactive T cells before entry into the thymus medulla

The thymus is important in the differentiation of bone marrow-derived precursor cells into functional T cells; humoral factors, as well as physical interactions with nurse cells, dendritic cells and epithelial cells, are thought to be instrumental in this process. Thymic lymphocytes mature during their migration from the cortical to the medullary region of the thymus, when they undergo phenotypic changes that include the acquisitions of T-cell antigen receptors, hormone receptors and differentiation antigens. Cortical T cells are thus mostly CD4+CD8+, whereas medullary T cells are either CD4+CD8- or CD4-CD8+. During this period T cells are subjected to two types of repertoire selection: all T cells recognizing self-MHC with low affinity may be preferentially amplified (positive selection), and in a second step T cells with high-affinity receptors for self-MHC determinants plus self antigens are eliminated (negative selection). We have described two monoclonal antibodies specific for the V beta 6 gene segment of the alpha/beta heterodimeric T-cell antigen receptor and have shown that most CD4+/V beta 6+ T cell recognize the Mlsa antigenic determinant but not Mlsb; similar results have been reported for V beta 8.1 and Mlsa. In both situations, tolerance to Mlsa correlated in an MHC-dependent fashion with absence of V beta 6 or V beta 8.1 T-cell antigen receptor expressing T cells in the periphery. We show here by immunostaining of thymus cryosections and cytofluorometric analysis that V beta 6-expressing cortical T cells are present at high density in both Mlsa and Mlsb mice, but do not enter the medullary region of Mlsa animals.     

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.     

B cells acquire antigen from target cells after synapse formation

Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.     

Activated CD8 binding to class I protein mediated by the T-cell receptor results in signalling

The CD8 glycoprotein of T cells bind nonpolymorphic regions of class I major histocompatibility complex proteins on target cells and these interactions promote antigen recognition and signalling by the T-cell receptor. Studies using artificial membranes indicated that effective CD8/class I interaction is critical for response by alloantigen-specific cytotoxic T lymphocytes when class I protein is the only ligand on the antigen-bearing surface. But significant CD8-mediated binding of cytotoxic T lymphocytes to non-antigenic class I protein could not be detected in the absence of the alloantigen. These apparently contradictory findings indicate that CD8 binding to class I protein might be activated through the T-cell receptor and the results reported here demonstrate that this is the case. Treatment of cytotoxic T lymphocytes with soluble anti-T-cell receptor antibody activates adhesion of the cytotoxic T lymphocytes to class I, but not class II proteins. The specificity of this binding implies that it is mediated by CD8 and blocking by anti-CD8 antibodies confirmed this. Furthermore, binding of CD8 to class I protein resulted in generation of an additional signal(s) necessary to initiate response at low T-cell receptor occupancy levels.     

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.     

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.     

Evidence from in vitro studies that tolerance to self antigens is MHC-restricted

Mature T cells respond to foreign antigens in the context of self major histocompatibility complex (MHC)-encoded products: T helper cells recognize antigen in the context of class II molecules, while cytotoxic T cells (CTL) recognize antigen plus class I molecules. Recent evidence suggests that the MHC-restricted T cell is unable to recognize either the foreign antigen or the self-MHC product alone, but only a complex of the two. Unresponsiveness to self antigens--self tolerance--implies the deletion or suppression of clones of T cells having reactivity to self antigens. Here we demonstrate the presence in normal mice of T cells which recognize self antigens together with allogeneic MHC products. This finding suggests the MHC restriction of T-cell recognition during the entire process of T-cell ontogeny, that is, MHC restriction of self tolerance.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Induction of regulatory T-lymphocyte responses by liposomes carrying major histocompatibility complex molecules and foreign antigen

Regulatory (helper and suppressor) T lymphocytes become activated only when foreign antigen is presented to them on the surface of antigen-presenting cells (APC), together with class II major histocompatibility complex (MHC) molecules (heterodimers of polypeptides of 28,000 and 35,000 relative molecular mass). Once activated by a certain foreign antigen--MHC combination, T cells react to the same antigen only in combination with the same MHC molecule, a phenomenon termed MHC restriction of T-cell recognition (reviewed in refs 1,5). Studies of the mechanisms involved in antigen presentation and MHC restriction have been hampered mainly by the virtual impossibility of inducing T-cell responses in the absence of APC. We describe here the production of synthetic lipid vesicles with inserted class II MHC molecules and a protein antigen coupled covalently to the lipid. These liposomes are shown to stimulate cloned helper T cells and T-cell hybridomas in an antigen-specific, MHC-restricted manner in the absence of APC. Thus, the recognition of foreign antigen together with class II MHC molecules seems to be the only signal required for the activation of antigen-primed regulatory T cells. Furthermore, 'processing' of antigen by APC is not essential for its recognition by T cells.     

Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen

Effector T cells are restricted to recognizing antigens associated with major histocompatibility complex (MHC) molecules. Specific recognition is mediated by the alpha beta heterodimer of the T-cell receptor (TCR)/CD3 complex, although other membrane components are involved in T-cell antigen recognition and functions. There has been much controversy in this regard over the part played by the CD4 glycoprotein. It is known that expression of CD4 correlates closely with the cell's ability to recognize antigens bound to class II MHC molecules and that CD4 can bind to class II molecules. Also monoclonal antibodies to CD4 can modify signals generated through the TCR/CD3 complex. It has therefore been proposed that CD4 binds to class II molecules, coaggregates with the TCR-CD3 complex and aids the activation of T cells. But given that TCR can itself impart restriction on the cell, it remains unclear whether the contribution of CD4-derived signals to those generated through the TCR alpha beta-CD3 complex is central to this activation. Here we report that when preceded by ligation of CD4, signalling through TCR alpha beta results in T cell unresponsiveness due to the induction of activation dependent cell death by apoptosis. These results imply that CD4 is critically involved in determining the outcome of signals generated through TCR, and could explain why the induction of effector T cells needs to be MHC-restricted.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.