Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

An assay for peptide binding to HLA-Cw*0102

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) molecules is based on the ability of peptides to stabilize MHC class I molecules synthesized by transporter associated with antigen processing (TAP)-deficient cell. The TAP-deficient cell line T2 has previously been used in the assembly assay to analyze peptide binding to HLA-A*0201 and -B*5101. In this study, we have extended this technique to assay for peptides binding to endogenous HLA-Cw*0102 molecules. We have analyzed the peptide binding of 20 peptides with primary anchor motifs for HLA-Cw*0102. One-third of the peptides analyzed bound with high affinity, half of the peptides examined did not bind, whereas the remaining peptides displayed intermediate binding activity. Interest in HLA-C molecules has increased significantly in recent years, since it has been shown that HLA-C molecules both can present peptides to cytotoxic T lymphocytes (CTL) and in addition are able to inhibit natural killer (NK)-mediated lysis.     

Functional regulation of immunoproteasomes and transporter associated with antigen processing

The central event in the cellular immune response to invading pathogens is the presentation of non-self antigenic peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). As peptide binding and transport proteins, MHC class I molecules have evolved distinct biochemical and cellular strategies for acquiring antigenic peptides, providing CTLs an extracellular representation of the intracellular antigen content. Whereas efficient generation of MHC class I binding peptides depends on the intracellular, immunoproteasome-mediated proteolysis machinery, translocation of peptides into the lumen of the endoplasmic reticulum requires the endoplasmic reticulum-resident, adenosine 5'-triphosphate (ATP) binding cassette transporter associated with antigen processing (TAP). Here we show, for the first time, that immunoproteasomes, TAP complexes, and MHC class I molecules are physically associated, providing an effective means of transporting MHC class I binding peptides from their sites of generation into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. In this review, we assess the current understanding of the functional regulation of immunoproteasomes and transporter associated with antigen processing.     

The ABC-transporter signature motif is required for peptide translocation but not peptide binding by TAP

The transporter associated with antigen processing (TAP) translocates peptides from their site of generation in the cytosol to the lumen of the endoplasmic reticulum for binding to MHC class I molecules. TAP is a member of the ATP-binding cassette (ABC) transporter family whose members utilize energy from ATP hydrolysis to translocate substrates across membranes. The highly conserved nucleotide-binding domains of ABC transporters couple ATP hydrolysis to substrate translocation by the membrane domains. The conserved 'signature motif' can be identified in the nucleotide-binding domains of all ABC transporters, and may play a role in ATP hydrolysis. Here we show that introduction of mutations into the signature motifs of either TAP1 or TAP2 inhibits the translocation of peptide without affecting binding of either peptide or ATP by TAP. We therefore conclude that the signature motifs in both TAP1 and TAP2 are required after peptide binding to facilitate peptide translocation by TAP.     

Peptide selection for presentation by HLA class I: A role for the human transporter associated with antigen processing?

Peptide epitopes presented by HLA class I are supplied by an elaborate system of antigen processing which subjects candidate epitopes to a process of selection according to littleunderstood rules. Transport of peptides from the cytosol into the endoplasmic reticulum by the TAP (transporter associated with antigen processing) complex is likely to play an important role in peptide selection for presentation. The recent development of an assay measuring substrate binding to the TAP complex has led to the identification of the major structural properties of peptides selected by the human transporter. Human TAP favors transport of peptides with structural features common to HLA class I ligands. However, TAP dislikes peptide ligands preferred by some HLA class I alleles, which may therefore frequently rely on alternative sources of peptide ligands.     

Induction of herpes simplex virus gB-specific cytotoxic T lymphocytes in TAP1-deficient mice by genetic immunization but not HSV infection

Loading of most endogenous peptides on major histocompatibility complex class I molecules is conditional on their transport into the endoplasmic reticulum (ER) by the peptide transporter TAP. We describe an HSV-2/1 cross-reactive cytotoxic T-cell (CTL) epitope that is processed in a TAP1-independent manner in vivo following immunization of TAP1-/- mice with naked DNA or a recombinant vaccinia virus. These data indicated that TAP1-independent processing of endogenous proteins is sufficient to prime CTLs in vivo. TAP1-independent processing of this epitope was not due to ER targeting by signal sequences and exogenous loading of MHC-I molecules and was not influenced by the amino acids flanking this epitope. In contrast, TAP1-/- mice infected with HSV-2 or HSV-2 mutants did not mount a CTL response against this epitope.     

抗原提呈相关转运体基因多态性与慢性HBV感染研究进展

抗原提呈相关转运体（TAP）在内源性抗原提呈过程中有重要作用,负责内源性抗原从胞浆到内质网腔的转运。TAP是由TAP1基因和TAP2基因编码的TAP1和TAP2组成的异二聚体。TAP基因具有多态性。乙型肝炎病毒（HBV）作为细胞内抗原,主要是经过内源性抗原加工和提呈途径而启动免疫应答的,TAP1／TAP2基因多态性的改变造成了分子结构异常,则会出现抗原呈递的功能障碍,抗原提呈不能有效进行,HBV抗原肽不能被细胞毒细胞识别而清除,则形成HBV的慢性持续感染。     

Exogenous peptides enter the endoplasmic reticulum of TAP-deficient cells and induce the maturation of nascent MHC class I molecules

MHC class I molecules assemble within the endoplasmic reticulum (ER) in complexes that include beta2-microglobulin (beta(2)m), the transporter associated with antigen processing (TAP)and several additional chaperones. Release of class I complexes from the ER is thought to require the binding of an appropriate endogenous peptide, predominantly delivered from the cytosol to the ER by TAP. It was recently demonstrated that exogenous synthetic peptide could 'directly' enter the ER of intact cells, independently of TAP function, and bind to the class I molecule H-2K(b).In TAP-deficient cells, we show that nascent K(b) or K(b)-L(d) chimeric molecules have a high trafficking background; 50-80% of these class I molecules are released from the ER independently of TAP function or the addition of exogenous peptide. The addition of exogenous K(b) cognate peptides enhanced the release of these class I molecules only slightly over the high background. The chimeric class I-b molecule, M3-L(d), differs from K(b)-L(d) only in its peptide binding domains, and M3-L(d) preferentially binds N-formylated peptides, which are rare in eukaryotic cells. Release of M3-L(d) from the ER in the absence of exogenous peptide was negligible. Addition of exogenous formylated peptides induced significant trafficking and surface expression of M3-L(d). These observations suggest that peptide binding is necessary for class I release from the ER even in TAP-deficient cells. These results demonstrate that exogenous peptide not only enters the ER of intact cells independently of TAP but also functionally induces class I antigen presentation.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Analysis of the fine specificity of rat,mouse and human TAP peptide transporters

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.     

A point mutation in the human transporter associated with antigen processing (TAP2) alters the peptide transport specificity

The heterodimeric transporter associated with antigen processing (TAP1/TAP2) translocates peptides from the cytosol into the endoplasmic reticulum where loading of major histocompatibility complex class I molecules takes place. TAP transporters from different species are known to exhibit distinct transport specificities with regard to the C-terminal amino acid (aa) of peptides. Thus, human TAP (hTAP), and rat TAP (rTAP) containing the rTAP2^a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2^u allele are restrictive and select against peptides with C-terminal small polar/hydrophobic or positively charged aa. The structural basis for this selectivity is not clear. To assess the relative contribution of the TAP1 and TAP2 subunits to transport specificity, we have constructed and analyzed interspecies TAP hybrids and point mutants of hTAP2 expressed in Sf9 insect cells and in TAP-deficient T2 cells. Transport assays with 20 C-terminal variants of the peptide RYWA-NATRSX showed that: first, transport specificity with regard to C-terminal aa is mainly influenced by TAP2, but TAP1 can also contribute. Second, the selective transport of peptides with C-terminal positively charged aa is critically controlled by the amino-terminal region (1–361) on the TAP2 chain, while transport of peptides with C-terminal small polar/hydrophobic aa is determined by residues located within as well as outside the region 1–361. Third, a single point mutation in hTAP2 (374A → D) resulted in a drastic alteration of the transport pattern. These results indicate that both TAP1 and TAP2 contribute to efficient peptide transport and that single point mutations in hTAP2 are able to alter the peptide transport specificity. This opens the possibility that naturally occurring mutations in one of the hTAP subunits may alter epitope selection in vivo.     

Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules

The cyclin-D1 protein, which was found to be overexpressed in various human tumors, promotes cell cycle progression from the G1 into the S phase. It is normally expressed at low levels in several tissues and is likely to induce immunological tolerance. We have recently shown in a murine system that T cell tolerance to a widely expressed protein was circumvented by raising cytotoxic T lymphocytes (CTL) from major histocompatability complex mismatched donors. In this study, we tested whether it is possible to raise human allo-restricted CTL against the cyclin-D1 protein. The human cell line T2 is deficient in the genes encoding the transporter associated with antigen processing (TAP), resulting in inefficient loading of HLA-A2 class I molecules with endogenous peptides. Thus, a large number of A2 molecules can bind exogenously supplied synthetic peptides. Peripheral blood mononu clear cells from HLA-A2-negative donors were stimulated with T2 cells presenting cyclin-D1-derived synthetic peptides. Cloning of bulk cultures revealed that a large proportion of CTL clones were peptide specific. One peptide induced CTL which lysed cyclin-D1-expressing breast cancer cells, but not control Epstein-Barr virus-transformed B lymphoid cells. The results show that HLA-A2-negative donors can be used to isolate tumor-reactive CTL spe cific for cyclin-D1 peptides presented by HLA-A2 class I molecules.     

Synthetic peptides based on Chlamydia trachomatis antigens identify cytotoxic T lymphocyte responses in subjects from a trachoma-endemic population

CD8⁺ cytotoxic T lymphocytes (CTL) recognize peptide antigens in the context of class I MHC antigen molecules. To identify peptides capable of eliciting anti-Chlamydia trachomatis CTL responses, 13 synthetic peptides conforming to human leucocyte antigen (HLA)-B8- or -B35-predicted binding motifs were synthesized using sequences based on C. trachomatis major outer membrane protein (MOMP) and heat shock protein 60 (hsp60). Two of 11 HLA-B35-predicted binding peptides were able to stabilize HLA-B35 in an in vitro binding assay. All peptides were tested in CTL assays using peripheral blood mononuclear cells (PBMC) isolated from 26 HLA-B8 or -B35 individuals resident in a trachoma-endemic community. Responses to MOMP and hsp60 peptides were identified in a minority of both HLA-B8 and -B35 individuals. Two of 12 HLA-B8 subjects responded to MOMP and 1/13 to hsp60 peptides. Responses in HLA-B35 subjects were similar, 1/13 subjects responding to MOMP and 2/13 to hsp60 peptides. CTL responses were observed only in children resolving current infection and in adults without scarring of the conjunctiva. These results suggest that anti-chlamydial CTL occur at low levels in peripheral blood, but may be important in the resolution of naturally acquired human ocular chlamydial infection.     

Identification of sequences in the human peptide transporter subunit TAP1 required for transporter associated with antigen processing (TAP) function

The heterodimeric peptide transporter associated with antigen processing (TAP) consisting of the subunits TAP1 and TAP2 mediates the transport of cytosolic peptides into the lumen of the endoplasmic reticulum (ER). In order to accurately define domains required for peptide transporter function, a molecular approach based on the construction of a panel of human TAP1 mutants and their expression in TAP1(-/-) cells was employed. The characteristics and biological activity of the various TAP1 mutants were determined, and compared to that of wild-type TAP1 and TAP1(-/-) control cells. All mutant TAP1 proteins were localized in the ER and were capable of forming complexes with the TAP2 subunit. However, the TAP1 mutants analyzed transported peptides with different efficiencies and displayed a heterogeneous MHC class I surface expression pattern which was directly associated with their susceptibility to cytotoxic T lymphocyte-mediated lysis. Based on this study, the TAP1 mutants can be divided into three categories: those expressing a similar phenotype compared to TAP1(-/-) or wild-type TAP1 cells respectively, and those representing an intermediate phenotype in terms of peptide transport rate, MHC class I surface expression and immune recognition. Thus, the results provide evidence that specific regions in the TAP1 subunit are crucial for the proper processing and presentation of cytosolic antigens to MHC class I-restricted T cells, whereas others may play a minor role in this process.     

TAP polymorphism does not influence transport of peptide variants in mice and humans

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) delivers cytosolic peptides to the lumen of the endoplasmic reticulum (ER) for presentation by MHC class I molecules. For the rat, it has been demonstrated that TAP polymorphism results in the selection of different sets of peptides, the nature of the C terminus being of particular importance. Here, we investigated whether TAP polymorphism in mice and humans has functional consequences for transport of peptide sets variable at the C-terminal residues. Using cell lines of H-2^d, H-2^k, and H-2^dxk haplotype and a panel of human lymphoblastoid cell lines expressing eight different TAP alleles, we detected species-specific transport patterns, but no significant influence of TAP polymorphism on peptide selection. In addition, peptides with different core sequences were translocated to the same extent by different TAP. These results suggest that a major contribution of human TAP polymorphism to disease progression and autoimmunity is not very likely.     

TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

Redox-regulated peptide transfer from the transporter associated with antigen processing to major histocompatibility complex class I molecules by protein disulfide isomerase

Most antigenic peptides are generated by proteasomes in the cytosol and are transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind with nascent major histocompatibilitiy complex class I molecule (MHC-I). Although the overall process of peptide-MHC-I complex assembly is well studied, the mechanism by which free peptides are delivered from TAP to MHC-I is unknown. In this study, we investigated the possible role of protein disulfide isomerase (PDI) as a peptide carrier between TAP and MHC-I. Analysis of PDI-peptide complexes reconstituted in vitro showed that PDI exhibits some degree of specificity for peptides corresponding to antigenic ligands of various human leukocyte antigen (HLA) alleles. Mutations of either anchor residues of the peptide ligand or the peptide-binding site of PDI inhibited the PDI-peptide interaction. The PDI-peptide interaction increased under reducing conditions, whereas binding of the peptide to PDI decreased under oxidizing conditions. TAP-associated PDI was predominantly present in the reduced form, whereas the MHC-I-associated PDI was present in the oxidized form. Further, upon binding of optimal peptides, PDI was released from TAP and sequentially associated with HLA-A2.1. Our data revealed a redox-regulated chaperone function of PDI in delivering antigenic peptides from TAP to MHC-I.     

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.     

Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways

Major histocompatibility complex (MHC) class I molecules usually present endogenous peptides at the cell surface. This is the result of a cascade of events involving various dedicated proteins like the peptide transporter associated with antigen processing (TAP) and the ER chaperone tapasin. However, alternative ways for class I peptide loading exist which may be highly relevant in a process called cross-priming. Both pathways are described here in detail. One major difference between these pathways is that the proteases involved in the generation of peptides are different. How proteases and peptidases influence peptide generation and degradation will be discussed. These processes determine the amount of peptides available for TAP translocation and class I binding and ultimately the immune response.     

A major role for tapasin as a stabilizer of the TAP peptide transporter and consequences for MHC class I expression

Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin-deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAP. The low amount of TAP moleculesin Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides.