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Major Histocompatibility Class I (MHC-I) molecules are present at the cell surface either as fully conformed trimolecular complexes composed of heavy chain, beta-2-microglobulin (β2m) and antigenic peptide or as various open forms, devoid of the peptide and/or β2m. While the role of fully conformed MHC-I is well studied, the physiological role of open conformers is neglected. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I from the early endosomal/juxtanuclear recycling route. As a result, open MHC-I conformers are internalized with a higher rate than fully conformed counterparts. Although the majority of internalized open MHC-I is directed into the acidic late endosomal (LE) compartments, only a fraction of them is degraded. Namely, a significant fraction of open MHC-I is present in a subset of LEs with the capacity of recycling and/or exocytosis. Therefore, it should be examined whether exogenous peptide loading may occur during traveling of MHC-I proteins through LE compartments, especially in a subset of less acidic LEs that detach from the core of perinuclear acidic LEs and migrate toward the cell periphery. Given that the acidic LE environment is not favorable for peptide loading, an endosomal compartment with the recycling capacity and less acidic environment that allows stabilization of newly formed trimolecular complexes is proper site for exogenous peptide loading. We propose that a LE compartment which collect and retain open MHC-I conformers should be taken into consideration as a site of exogenous peptide loading., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8(+) T-cell responses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA., Experimental Design: Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells., Results: Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced., Conclusions: This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.

Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection.

Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8(+) T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and β(2)-microglobulin (β(2)m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8(+) T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMV-infected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS(Ist2)) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS(Ist2) targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P(2) at the PM created ER-PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca(2+) entry (SOCE). The CSS(Ist2) and the C-terminus of the ER-located Ca(2+) sensor STIM2 were sufficient to bind PI(4,5)P(2) and PI(3,4,5)P(3) at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P(2), suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN-gamma ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu-specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN-alpha and reduced transforming growth factor (TGF)beta1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFbeta1, reduced IFN-alpha, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis.

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3zeta lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.

Heat shock proteins (HSPs) induce cross-presentation of antigens by dendritic cells (DC) as well as DC maturation. These properties make HSP antigen complexes good candidates to prime CD8 T cell responses against tumor-associated antigens. In this study, we analyzed four different members of the HSP70 family fused to a fragment of ovalbumin (OVA) as a model tumor antigen. E. coli-derived recombinant HSP70-OVA fusion proteins efficiently primed antigen-specific cytotoxic T cells in short-term in vivo immunization assays. Because of concerns that the adjuvant effect of HSPs may be due to endotoxin contamination, we studied this issue in detail. Induction of OVA-specific cytotoxicity was significantly decreased in mice deficient for the LPS receptor, TLR4. After careful removal of endotoxins, immunization with HSP70-OVA failed to prime cytotoxic T cell responses. However, we obtained strong in vivo kill responses when endotoxin-depleted HSP70-OVA was used in combination with the TLR9 ligand CpG oligodeoxynucleotide 1668. Importantly, prophylactic and therapeutic treatment with endotoxin-depleted HSP70-OVA together with CpG significantly delayed the outgrowth of OVA-expressing B16 melanoma cells. However, we were unable to detect significant differences in the magnitudes of immune responses against endotoxin-depleted recombinant OVA vs. endotoxin-depleted HSP70-OVA fusion protein. Thus, immunization with recombinant HSP70-antigen fusion protein does not provide an advantage over recombinant antigen alone when combined with a suitable adjuvant. Altogether, our data suggest that the adjuvant effect of the HSP70 part of the fusion protein is completely lost after endotoxin removal.

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini., Methodology/principal Findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains., Conclusions/significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

Background: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer., Methodology/principal Findings: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase., Conclusion/significance: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.

The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.

MHC class I molecules present peptides derived from the ectodomains of endogenous transmembrane proteins; however, the processing of these Ags is incompletely understood. As model transmembrane Ags we investigated the processing of MHC-I-derived fusion proteins containing the N-terminally extended K(b)-restricted OVA epitope SIINFEKL in the extracytoplasmic domain. In TAP-deficient, nonprofessional APCs, the epitope was cleaved out of various sequence contexts and presented to T cells. Ag presentation was inhibited by acidophilic amines and inhibitors of the vacuolar proton pump, indicating processing in endosomes. Endosomal aspartic-type cathepsins, and to some extent also the trans-Golgi network protease furin, were involved in processing. Clathrin-dependent and independent internalization from the cell surface targeted MHC-I fusion proteins to early and late endosomes, where SIINFEKL/K(b) complexes were detected by immunofluorescence microscopy. Targeting of MHC-I fusion proteins to processing compartments was independent of sequence motifs in the cytoplasmic tail. Not only TAP-deficient cells, but also TAP-competent APCs used the vacuolar pathway for processing of MHC-I fusion proteins. Thus, endosomal processing of internalized endogenous transmembrane proteins represents a novel alternate pathway for the generation of MHC-I-binding peptides.

Expression of the neural cell adhesion molecule (NCAM) on malignant cells of neuroendocrine, epithelial and hematopoeitic origin has been reported, but its role for tumor cell recognition by the immune system remained uncertain so far. We have studied the cytotoxicity of the natural killer (NK) cell line NK92 and polyclonal NK cells from different donors, against NCAM-deficient and NCAM-transfected tumors. While the pancreatic carcinoma PANC-1 and the glioblastoma T98G showed no enhanced susceptibility to NK lysis after NCAM transfection, de novo NCAM expression in HeLa cervical carcinoma, SHEP neuroblastoma and the multiple myeloma lines RPMI-8226 and LP-1 was associated with significantly decreased lysis by NK cells. Binding of an NCAM-specific monoclonal antibody to NCAM-positive target cells was able to reverse the reduced lysis susceptibility. Conjugate formation of NCAM-expressing tumor cells with NK cells was blocked and could be restored by anti-NCAM. NK cell-expressed NCAM molecules which might engage in homotypic cis- or trans-interactions had no apparent inhibitory function. The known cis-ligands of NCAM, heparan sulfate proteoglycan and L1-CAM, were also not directly involved in NK inhibition. ICAM-1 mRNA and cell surface expression was downmodulated in NCAM-transfected HeLa cells. ICAM-1 is involved in killer cell immune synapse formation. Its downmodulation may therefore contribute to the reduced lysis of NCAM-expressing target cells. We conclude that aberrant expression of NCAM on tumor cells of different histogenetic origin can lead to inhibition of target cell recognition and lysis by NK cells.

The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading of major histocompatibility complex class I (MHC-I) molecules with an optimal spectrum of antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide transporter. In a detailed mutational analysis, the transmembrane domain (TMD) and ER-luminal connecting peptide (CP) of mouse Tpn were analyzed for their capacity to stabilize the TAP2 subunit. Replacement of the TMD of Tpn by TMDs from calnexin or the Tpn-related protein, respectively, completely abolished TAP2 stabilization after transfection of Tpn-deficient cells, whereas TMDs derived from distantly related Tpn molecules (chicken and fish) were functional. A detailed mutational analysis of the TMD and adjacent residues in the ER-luminal CP of mouse Tpn was performed to elucidate amino acids that control the stabilization of TAP2. Single amino acid substitutions, including a conserved Lys residue in the center of the putative TMD, did not affect TAP2 expression levels. Mutation of this Lys plus four additional residues, predicted to be neighbors in an assumed alpha-helical TMD arrangement, abrogated the TAP2-stabilizing capacity of Tpn. In the presence of a wild-type TMD, also the substitution of a highly conserved Glu residue in the CP of Tpn strongly affected TAP2 stabilization. Defective TAP2 stabilization resulted in impaired cell surface expression of MHC-I molecules. This study thus defines a novel, spatially arranged motif in the TMD of Tpn essential for stable expression of the TAP2 protein and a novel protein interaction mode involving an ER-luminal Glu residue close to the membrane.

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.

The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing (TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8+ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6.

The thiol-oxidoreductase ERp57 is an integral component of the peptide-loading complex of the major histocompatibility complex (MHC) class I pathway, but its function is unknown. To investigate its function in antigen presentation, we generated ERp57-deficient mice. Death in utero caused by ubiquitous ERp57 deletion was prevented by specific deletion in the B cell compartment. We demonstrate that ERp57 was central for recruitment of MHC class I molecules into the loading complex. In ERp57-deficient cells, we found short-lived interaction of MHC class I molecules with the loading complex. Thus, in the steady state, very few MHC class I molecules were present in the loading complex. Surface H-2K(b)-peptide expression and stability were reduced, and presentation of a model antigen was decreased. Our results indicate that ERp57 does not influence the redox state of MHC class I molecules but is an essential structural component required for stable assembly of the peptide-loading complex.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an IFN-gamma-induced aminopeptidase in the endoplasmic reticulum that trims longer precursors to the antigenic peptides presented on MHC class I molecules. We recently reported that purified ERAP1 trimmed N-extended precursors but spared peptides of 8-9 residues, the length required for binding to MHC class I molecules. Here, we show another remarkable property of ERAP1: that it strongly prefers substrates 9-16 residues long, the lengths of peptides transported efficiently into the ER by the transporter associated with antigen processing (TAP) transporter. This aminopeptidase rapidly degraded a model 13-mer to a 9-mer and then stopped, even though the substrate and the product had identical N- and C-terminal sequences. No other aminopeptidase, including the closely related ER-aminopeptidase ERAP2, showed a similar length preference. Unlike other aminopeptidases, the activity of ERAP1 depended on the C-terminal residue of the substrate. ERAP1, like most MHC class I molecules, prefers peptides with hydrophobic C termini and shows low affinity for peptides with charged C termini. Thus, ERAP1 is specialized to process precursors transported by TAP to peptides that can serve as MHC class I epitopes. Its "molecular ruler" mechanism involves binding the hydrophobic C terminus of the substrate 9-16 residues away from the active site.

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2-/- knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2K(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

Assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum is a highly coordinated process that results in abundant class I/peptide complexes at the cell surface for recognition by CD8(+) T cells and natural killer cells. During the assembly process, a number of chaperones and accessory molecules, such as transporter associated with antigen processing, tapasin, ER60, and calreticulin, assist newly synthesized class I molecules to facilitate loading of antigenic peptides and to optimize the repertoire of surface class I/peptide complexes. This review focuses on the relative importance of these accessory molecules for CD8(+) T-cell responses in vivo and discusses reasons that may help explain why some CD8(+) T-cell responses develop normally in mice deficient in components of class I assembly, despite impaired antigen presentation.

MHC class I heavy chains (HC) that fail to acquire a mature conformation in the endoplasmic reticulum (ER) as a result of defective folding or assembly with beta2-microglobulin, or lack of appropriate peptide cargo are retrotranslocated through the Sec61 channel to the cytosol for degradation by proteasomes. The mechanisms involved in ER retrotranslocation of HC are as yet incompletely understood. Using a microsomal system, we characterized the molecular requirements for the release of HC into the soluble fraction. Extraction of ubiquitinated HC was facilitated by cytosol, or by addition of proteins that stabilized the membrane association of the cytoplasmic ATPase p97. Functional proteasomes were not needed for HC mobilization. ATP supply to the ER lumen was found to be an essential factor since an inhibitor of the ATP importing pump in the ER membrane blocked HC release. Also non-hydrolyzable ATP analogs delivered to the ER lumen facilitated HC export suggesting that ATP binding by ER chaperones rather than ATP hydrolysis is involved.

Human cytomegalovirus has evolved multiple strategies to interfere with immune recognition by the host. A variety of mechanisms affect antigen presentation by major histocompatibility complex class I molecules resulting in a reduced class I cell-surface expression. This downregulation is expected to trigger natural killer (NK) cytotoxicity, requiring counteraction by the virus to establish long-term infection. Here we describe that the human cytomegalovirus gpUS6 protein, which has been demonstrated to downregulate the expression of human leukocyte antigen (HLA) class I and the presentation of cytotoxic T lymphocyte epitopes by blocking transporter associated with antigen presentation (TAP function), does not affect the ability of HLA-E to inhibit NK cell mediated lysis of K-562 cells by interaction with CD94/NKG2A expressed on NK cells. Cell surface expression and function of HLA-E is not altered although gpUS6 inhibits TAP-dependent peptide transport by 95%. Moreover, HLA-E molecules presenting HLA class I signal sequence-derived peptides are functionally detectable on transfected TAP-deficient RMA-S cells.

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.

The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/beta(2)-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410-->F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient.220.B*4402 cells. Consistent with suboptimal peptide loading, the t(1/2) of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.

In both the MHC class II and class I pathways of antigen presentation, accessory molecules influence formation of MHC-peptide complexes. In the MHC class II pathway, DM functions in the loading and editing of peptides; recent work demonstrated that it is acting not only in late endosomal compartments but also in recycling compartments and on the surface of B cells and immature dendritic cells. DM activity is modulated by another accessory molecule, DO, but this modulation is mainly operative in B cells, where it may lead to preferential activation of B cells producing high-affinity antibodies. In the MHC class I pathway of antigen presentation, recent in vivo experiments with knockout mice confirmed the role of tapasin in antigen presentation and indicate that it acts as a peptide editor and as a chaperone for TAP and the MHC class I heavy chain. In the class I loading complex, calreticulin and the thiol-dependent oxidoreductase ER60/ERp57 appear to support the function of tapasin in an as-yet-unknown fashion. The picture emerges that DM and tapasin have analogous functions in shaping the peptide repertoire presented by the respective MHC class II and class I molecules.

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8-11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K(b)-restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODC-LEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K(b) and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides.

In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207-214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.

The murine B16 melanoma system represents an important in vivo model for the evaluation of T cell-based immunization and vaccination strategies, although deficient MHC class I surface expression has been identified in these cells. We postulate here that the MHC class I-deficient phenotype of B16 melanoma cells is attributable to down-regulation or the loss of the expression and function of multiple components of the MHC class I antigen-processing pathway, including the peptide transporter associated with antigen processing, the proteasome subunits LMP2, LMP7, and LMP10, PA28alpha and -beta, and the chaperone tapasin. In contrast, calnexin, calreticulin, ER60, and protein disulfide isomerase expression are unaltered or only marginally suppressed in these cells. The level of down-regulation of the components of the antigen-processing pathway is either transcriptionally or posttranscriptionally controlled and could be corrected in all cases by IFN-y treatment, which also reconstituted MHC class I surface expression. Thus, B16 melanoma cells can be used as a model for the characterization of the mechanisms underlying the coordinated dysregulation of the antigen-processing components, which should provide new insights into the development of tumors and the factors controlling this process.

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.

Tapasin is a component of the major histocompatibility complex (MHC) class I antigen-loading complex. Here we show that mice with a disrupted tapasin gene display reduced MHC class I expression. Cytotoxic T cell (CTL) responses to viruses are impaired, and dendritic cells of tapasin-deficient mice do not cross-present protein antigen via the MHC class I pathway, indicating a defect in antigen processing. Natural killer (NK) cells from tapasin-deficient mice have an altered repertoire and are self-tolerant. In addition, the repertoire of class I-bound peptides is altered towards less stably binding ones. Thus tapasin plays a role in CTL and NK immune responses and in optimal peptide selection.

Antigenic peptides are translocated by the TAP peptide transporter from the cytosol into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. Peptides that fail to bind need to be removed from the ER. Here we provide evidence that peptide export utilizes the Sec61p translocon as demonstrated by blocking this channel with bacterial exotoxin. Peptide export interferes with the retrotranslocation of beta2-microglobulin from the ER to the cytosol, suggesting similar pathways for the disposal of proteins and oligopeptides. Peptide export requires ATP supply to the ER lumen but is independent of ATP hydrolysis.

Presentation of peptides derived from cytosolic and nuclear proteins by MHC class I molecules requires their translocation across the membrane of the endoplasmic reticulum (ER) by a specialized ABC (ATP-binding cassette) transporter, TAP. To investigate the topology of the heterodimeric TAP complex, we constructed a set of C-terminal deletions for the TAP1 and TAP2 subunits. We identified eight and seven transmembrane (TM) segments for TAP1 and TAP2, respectively. TAP1 has both its N and C terminus in the cytoplasm, whereas TAP2 has its N terminus in the lumen of the ER. A putative TM pore consists of TM1-6 of TAP1 and, by analogy, TM1-5 of TAP2. Multiple ER-retention signals are present within this region, of which we positively identified TM1 of both TAP subunits. The N-terminal domain containing TM1-6 of TAP1 is sufficient for dimerization with TAP2. A second, independent dimerization domain, located between the putative pore and the nucleotide-binding cassette, lies within the cytoplasmic peptide-binding domains, which are anchored to the membrane via TM doublets 7/8 and 6/7 of TAP1 and TAP2, respectively. We present a model in which TAP is composed of three subdomains: a TM pore, a cytoplasmic peptide-binding pocket, and a nucleotide-binding domain.

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.

Peptides derived from the melanoma-associated MART-1/Melan-A antigen are currently implemented in immunotherapy for inducing or augmenting T-cell responses directed against peptides expressed by autologous tumor cells in HLA-A2+ patients with melanoma. Here, we describe the specificity of the T-cell clone SK29-FFM1.1, which secretes GM-CSF in response to a panel of synthetic MART-1/Melan-A-derived peptides, including the naturally presented ILTVILGVL(32-40), but exhibits cytotoxicity and IFN-gamma secretion exclusively to the MART-1/Melan-A derived peptide AAGIGILTV(27-35). In addition, cytotoxic T-lymphocyte (CTL) clone SK29-FFM1.1 recognizes 3 different naturally processed and presented peptides on HLA-A2+ MART-1/Melan-A+ melanoma cells, as defined by cytotoxicity and IFN-gamma and GM-CSF secretion. Processing and presentation of MART-1/Melan-A peptides appears to be different in cells of non-melanocytic origin, as shown by the characterization of naturally presented peptides displayed by HLA-A2+ colorectal cancer cells transduced with a MART-1/Melan-A gene-containing retrovirus. Our data suggest that multiple epitopes, including ILTVILGVL and different isoforms of AAGIGILTV derived from MART-1/Melan-A may be naturally presented by melanoma cells to the immune system.

Due to the potential clinical relevance of HLA class I antigen losses in melanoma cells and the scanty information about the molecular mechanisms underlying these defects, we have characterized the cause of the HLA-A2 antigen loss by autologous melanoma cell lines SK-MEL-29.1.22 and SK-MEL-29.1.29. Both cell lines have structural defects of HLA-A2 genes, which cause lack of their transcription. In SK-MEL-29.1.22 cells the 5'-flanking region, exon 1, intron 1, and a region at the 5' end of exon 2 of the HLA-A2 gene are deleted. The breakpoint of the HLA-A2 gene, which is recombined with a DNA fragment of unknown origin, was localized between two GTTCG sequence repeats at position 101 of exon 2. These repeats may provide the sequence basis for misalignment in the process of DNA deletion. In SK-MEL-29.1.29 cells, loss of HLA-A2 antigens, as well as of HLA-B44 and HLA-Cw5 alleles, is caused by the loss of one copy of chromosome 6. Down-regulation of the expressed HLA class I alleles in the two HLA-A2 loss variants and in the parental cells was found to be associated with a low TAP1 expression and a reduced function of peptide transporters. Therefore, multiple defects result in loss or down-regulation of HLA class I alleles in SK-MEL-29.1.22 and SK-MEL-29.1.29 melanoma cells.

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 degrees C) or by loading these cells with either exogenous human beta2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-gamma treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

Human cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, beta2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face.

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 approximately 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.

The endoplasmic reticulum (ER)-resident stress protein gp96 induces a major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response against antigens present in the cells from which it has been prepared. In this study, photoreactive peptides were translocated into the ER by the transporter associated with antigen processing (TAP). These peptides can be cross-linked specifically to gp96. Thus, we provide the first evidence that gp96 binds TAP-translocated peptides which have been implicated in the induction of specific CTL responses after immunization with gp96 (Srivastava, P. K. et al., Immunogenetics 1994. 39: 93).

MHC class I molecules present antigenic peptides that are mostly derived from endogenous cytosolic proteins. Recent studies addressing the function of the proteasome and its activator complexes have advanced our understanding of the cytosolic processing of peptides. Transporters associated with antigen processing (TAPs) translocate these peptides to the endoplasmic reticulum where MHC class I molecules, which are retained in transient complexes with chaperones and TAPs, await them for binding. The sequence specificity and the peptide length preference of TAPs roughly meet the requirements of class I molecules in a range of different species, suggesting evolutionary shaping of the specificity of TAPs.

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 rTAP2a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2a 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 RYWANATRSX 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.

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.