In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.     

Identification of classical minor histocompatibility antigen as cell-derived peptide.

Histocompatibility antigens expressed on tissue grafted between individuals are recognized by host T cells, which reject the graft. The major histocompatibility complex (MHC) antigens have been identified on the molecular level, whereas the molecules representing the remaining ones, the minor histocompatibility antigens, are unknown, apart from some exceptions. The cytotoxic T lymphocyte (CTL) response against minor histocompatibility antigens shares many aspects with that against virus-infected cells. Virus-specific CTL recognize peptides derived from viral proteins produced in the infected cell. These peptides are presented by MHC class I molecules, as indicated by functional and crystallographic data. By analogy, minor histocompatibility antigens have been postulated to be peptides derived from normal cellular proteins presented by MHC class I molecules. Here we report that peptides derived from normal cellular proteins can indeed be recognized by CTL raised in the classical minor histoincompatible mouse strain combination, C57BL/6 against BALB.B. Thus, we have proven the above postulate, and isolated one of the minor histocompatibility molecules elusive for several decades.     

Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection

After an infection, cytotoxic T lymphocyte precursors proliferate and become effector cells by recognizing foreign peptides in the groove of major histocompatibility complex (MHC) class I molecules expressed by antigen-presenting cells (APCs). Professional APCs specialized for T-cell activation acquire viral antigen either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells, followed by transfer of antigen to the cytosol, processing and MHC class I loading in a process referred to as cross-presentation. An alternative way, referred to as 'cross-dressing', by which an uninfected APC could present antigen was postulated to be by the transfer of preformed peptide-MHC complexes from the surface of an infected cell to the APC without the need of further processing. Here we show that this mechanism exists and boosts the antiviral response of mouse memory CD8(+) T cells. A number of publications have demonstrated sharing of peptide-loaded MHC molecules in vitro. Our in vitro experiments demonstrate that cross-dressing APCs do not acquire peptide-MHC complexes in the form of exosomes released by donor cells. Rather, the APCs and donor cells have to contact each other for the transfer to occur. After a viral infection, we could isolate cross-dressed APCs able to present viral antigen in vitro. Furthermore, using the diphtheria toxin system to selectively eliminate APCs that could only acquire viral peptide-MHC complexes by cross-dressing, we show that such presentation can promote the expansion of resting memory T cells. Notably, naive T cells were excluded from taking part in the response. Cross-dressing is a mechanism of antigen presentation used by dendritic cells that may have a significant role in activating previously primed CD8(+) T cells.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cellular peptide composition governed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present peptides derived from cellular proteins to cytotoxic T lymphocytes (CTLs), which check these peptides for abnormal features. How such peptides arise in the cell is not known. Here we show that the MHC molecules themselves are substantially involved in determining which peptides occur intracellularly: normal mouse spleen cells identical at all genes but MHC class I express different patterns of peptides derived from cellular non-MHC proteins. We suggest several models to explain this influence of MHC class I molecules on cellular peptide composition.     

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.     

Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.

In mammalian cells, short peptides derived from intracellular proteins are displayed on the cell membrane associated with class I molecules of the major histocompatibility complex (MHC). The surface presentation of class I-peptide complexes presumably alerts the immune system to intracellular viral protein synthesis. Peptides derived from the cytosol must reach the cisternae of the endoplasmic reticulum where they are required for the assembly of stable class I molecules, and it has been proposed that the products of the two MHC-encoded ATP-binding cassette (ABC) transporter genes function to deliver the peptides across the membrane of the endoplasmic reticulum. This idea is supported by experiments in which transfection of a human cell line defective in class I expression with a complementary DNA of one of these genes restored cell surface expression levels. Here we show that the complete phenotype of the mouse mutant cell line RMA-S, in which lack of surface expression of stable class I molecules correlates with an inability to present viral peptides originating in the cytosol, is repaired by the cDNA of the other transporter gene. These results are consistent with the possibility that the two transporter polypeptides form a heterodimer.     

Transient aggregation of ubiquitinated proteins during dendritic cell maturation

Dendritic cells (DCs) are antigen-presenting cells with the unique capacity to initiate primary immune responses. Dendritic cells have a remarkable pattern of differentiation (maturation) that exhibits highly specific mechanisms to control antigen presentation restricted by major histocompatibility complex (MHC). MHC class I molecules present to CD8(+) cytotoxic T cells peptides that are derived mostly from cytosolic proteins, which are ubiquitinated and then degraded by the proteasome. Here we show that on inflammatory stimulation, DCs accumulate newly synthesized ubiquitinated proteins in large cytosolic structures. These structures are similar to, but distinct from, aggresomes and inclusion bodies observed in many amyloid diseases. Notably, these dendritic cell aggresome-like induced structures (DALIS) are transient, require continuous protein synthesis and do not affect the ubiquitin-proteasome pathway. Our observations suggest the existence of an organized prioritization of protein degradation in stimulated DCs, which is probably important for regulating MHC class I presentation during maturation.     

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.     

Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments

Class II major histocompatibility complex (MHC) glycoproteins associate with peptides derived from material endocytosed by antigen-presenting cells and processed along the endocytotic pathway. No consensus exists as to what extent class II molecules themselves are endocytosed and it is not known whether endocytosed MHC class II molecules can be recycled again to the cell surface--an itinerary which might allow a single cell-surface MHC molecule to associate with different peptides during its lifetime. We now show by using new cleavable labelling reagents that class II and class I MHC on B lymphoblastoid cells are continually endocytosed and recycled to the cell surface. The intracellular pool size is normally kept small by efficient recycling, but in the presence of primaquine the rate of recycling is slowed, thereby increasing the size of this pool substantially. On removal of the amine, the intracellular population recycles rapidly to restore the original distribution. These results reveal a cycle that might explain the rapid binding and turnover of some peptide/class II MHC complexes and the exchange of pre-existing for new peptides observed in living cells.     

Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments.

Traffic of MHC molecules dictates the source of peptides that are presented to T cells. The intracellular distribution of MHC class I and class II molecules reflects the dichotomy in presentation of antigen from endogenous and exogenous origin, respectively. In human B lymphoblastoid cells, class I molecules are present in compartments constituting the biosynthetic pathway, whereas class II molecules enter structures related to lysosomes during their biosynthesis.     

Structural and serological similarity of MHC-linked LMP and proteasome (multicatalytic proteinase) complexes

Major histocompatibility complex (MHC) class I molecules associate with peptides derived from endogenously synthesized antigens. Cytotoxic T-lymphocytes can thus scan class I molecules and bound peptide on the surface of cells for foreign antigenic determinants. Recent evidence demonstrates that the products of trans-acting, non-class I genes in the class II region of the MHC are required in the class I antigen-processing pathway. There are genes (called HAM1 and HAM2 in the mouse) in this region that encode proteins postulated to be involved in the transport of peptide fragments into the endoplasmic reticulum for association with newly synthesized class I molecules. But, the mechanism by which such peptide fragments are produced remains a mystery. At least two genes encoding subunits of the low-molecular mass polypeptide (LMP) complex are tightly linked to the HAM1 and HAM2 genes. We show that the LMP complex is closely related to the proteasome (multicatalytic proteinase complex), an intracellular protein complex that has multiple proteolytic activities. We speculate that the LMP complex may have a role in MHC class I antigen processing, and therefore that the MHC contains a cluster of genes required for distinct functions in the antigen processing pathway.     

Presentation of viral antigen by MHC class I molecules is dependent on a putative peptide transporter heterodimer.

Major histocompatibility complex (MHC) class I molecules present peptides derived from the endogenous protein pool to cytotoxic T lymphocytes, which can thus recognize intracellular antigen. This pathway may depend on a transporter (PSF1) to mediate entry of the cytosolic peptides into a pre-Golgi compartment where they bind to class I heavy chains and promote their stable assembly with beta 2-microglobulin. There is, however, only indirect support for this function of PSF1. Here we show that PSF1 is necessary for the efficient assembly of class I molecules and enables them to present a peptide epitope derived from endogenously synthesized viral antigen. Immunochemical and genetic data demonstrate that the PSF1 polypeptide is associated with a complementary transporter chain, which is polymorphic and is encoded by the PSF2 gene, which is closely linked to PSF1.     

Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters

Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.