Activation of natural killer cells by hepatitis C virus particles in vitro.

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection.

Corking the bottleneck: the transporter associated with antigen processing as a target for immune subversion by viruses.

In this chapter, mechanisms are reviewed that viruses use to inhibit the function of the peptide transporter associated with antigen processing (TAP), which translocates cytosolic peptides into the endoplasmic reticulum (ER) for binding to MHC class I molecules. Although some DNA viruses, such as adenovirus or EBV, downmodulate TAP expression on the transcriptional level, members of the alpha and beta subfamily of herpesviruses, such as herpes simplex virus (HSV) and human cytomegalovirus (HCMV), express proteins that bind to TAP and interfere with peptide translocation. The modes of action of the HSV-encoded cytosolic TAP inhibitor ICP47 and the HCMV-encoded ER-resident TAP inhibitor gpUS6 are discussed in detail. Viral interference with antigen presentation through TAP inhibition is not only relevant for the immunobiology of persistent viral infections but also contributes to the understanding of the translocation mechanism utilized by the ATP-binding cassette transporter TAP.

Tapasin-the keystone of the loading complex optimizing peptide binding by MHC class I molecules in the endoplasmic reticulum.

MHC class I molecules are loaded with peptides that mostly originate from the degradation of cytosolic protein antigens and that are translocated across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). The ER-resident molecule tapasin (Tpn) is uniquely dedicated to tether class I molecules jointly with the chaperone calreticulin (Crt) and the oxidoreductase ERp57 to TAP. As learned from the study of a Tpn-deficient cell line and from mice harboring a disrupted Tpn gene, the transient association of class I molecules with Tpn and TAP is critically important for the stabilization of class I molecules and the optimization of the peptide cargo presented to cytotoxic T cells. The different functions of molecular domains of Tpn and the highly coordinated formation of the TAP-associated peptide loading complex will also be discussed in this review.

Deficient expression of components of the MHC class I antigen processing machinery in human cervical carcinoma.

In cervical carcinomas abnormalities in the MHC class I surface expression are a frequent event, which are often associated with the deficient expression of the peptide transporter subunit TAP1 thereby resulting in impaired T cell response. In order to understand the role of other components of the MHC class I antigen processing machinery (APM) in the immune escape, 16 surgically removed primary cervical carcinoma lesions were analyzed for their mRNA expression of the heterodimeric peptide transporter TAP, the constitutive and interferon (IFN)-gamma inducible proteasome subunits and their activators PA28alpha/beta, various chaperones as well as MHC class I antigens. High expression levels of all APM components were detected in normal cervical tissue, whereas 15/16 of cervical carcinoma lesions exhibited an impaired expression of at least one APM component, including the proteasome subunits, their activators PA28alpha/beta, the peptide transporter subunits TAP1 and TAP2, different chaperones, HLA class I heavy chains and beta2-microglobulin (beta2-m). In particular, calnexin expression was strongly downregulated in 69% of cervical cancer lesions analyzed. Such abnormalities were neither associated with a specific human papilloma virus (HPV) or HLA class I phenotype nor with tumor grading and staging. Analysis of five cervical carcinoma cell lines demonstrated a reduced MHC class I surface expression due to deficient expression and function of TAP, LMP subunits or specific HLA-alleles which could be mostly corrected by IFN-gamma treatment. The high frequency of abnormalities of APM component expression together with their potential negative influence on T cell-mediated immune recognition emphasize the need to evaluate the antigen processing pathway in cervical carcinoma patients, particularly in those selected for T-cell-based immunotherapies.

Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection.

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.

A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides.

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.

Characterization of the major histocompatibility complex class I deficiencies in B16 melanoma cells.

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.

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.

Coordinate downregulation of multiple MHC class I antigen processing genes in chemical-induced murine tumor cell lines of distinct origin.

In murine tumor cell lines, downregulation of MHC class I surface expression has been frequently detected, but the underlying molecular mechanisms of such deficiencies have not been defined. In this study, murine tumor cell lines of different histology derived from spontaneous or from chemical-induced tumors were analyzed for the expression of multiple components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM), including the peptide transporter TAP, the interferon (IFN)-gamma inducible proteasome subunits and several chaperones. The tumor cell lines analyzed demonstrated a heterogeneous expression pattern of various APM components. In comparison to control cells an impaired coordinated expression of at least three APM components was detected. In particular, extensive APM deficiencies were found in cell lines derived from chemical-induced tumors. A strong coordinated downregulation of expression and/or function of TAP, the low molecular weight proteins (LMP) subunits, the proteasome activator PA28 and/or tapasin was found in 5 of 10 tumor cells, which was associated with impaired MHC class I surface expression. In contrast, the expression of beta2-microglobulin (beta2-m), PA28beta, the constitutive proteasome subunits X, Y, Z and of the chaperones calnexin, calreticulin, ER60 and phospho disulfide isomerase (PDI) was unaltered or only weakly decreased. The deficient expression of APM components could be corrected by IFN-gamma treatment, which also reconstituted MHC class I surface expression. However, impaired expression of APM molecules appears not to be the only cause of abnormal MHC class I expression, since it could neither be corrected by the addition of exogeneous MHC class I binding peptides nor by incubation at low temperature. These results suggest that one major mechanism of murine tumor cells, in particular chemical-induced tumors, to evade the immune system is the combined dysregulation of various APM components and other factors, which still have to be identified.

Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance.

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.

Functional deficiencies of components of the MHC class I antigen pathway in human tumors of epithelial origin.

An association between oncogenic transformation and repression of different components of the MHC class I antigen processing machinery (APM) have been described in murine model systems. In order to discover whether a similar correlation exists, human tumor cell lines of distinct histology with altered ras protein were analyzed for the expression of APM components utilizing RT-PCR and Western blot analyses. A heterogeneous expression pattern of MHC class I antigens, TAP peptide transporter, proteasome subunits, proteasome activator PA28 and the chaperones calnexin, calreticulin as well as tapasin was displayed by these tumor cell lines. Single or combined deficiencies in the expression and/or function of TAP, LMP2, LMP10 and tapasin were demonstrated in 11 of 12 cell lines studied, whereas the expression of calnexin, calreticulin, beta2-microglobulin, LMP7 and PA28alpha was unaltered or only weakly decreased. The impaired expression of TAP, LMP subunits and tapasin was not associated with altered ras, but resulted in reduced MHC class I surface expression. In particular, a significant allele- and locus-specific downregulation of the HLA-A and HLA-B haplotypes was found. IFN-gamma treatment corrected the TAP, LMP and tapasin deficiencies and enhanced the constitutive PA28alpha, LMP7, calnexin and calreticulin expression which was accompanied with increased levels of MHC class I antigens. Thus, dysregulation rather than structural alterations of different APM components might be one mechanism of colon carcinoma, small cell lung carcinoma and pancreatic carcinoma cell lines to evade immune recognition.

Export of antigenic peptides from the endoplasmic reticulum intersects with retrograde protein translocation through the Sec61p channel.

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.

Impaired immune responses and altered peptide repertoire in tapasin-deficient mice.

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.

Membrane topology and dimerization of the two subunits of the transporter associated with antigen processing reveal a three-domain structure.

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.

Antigen presentation in syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules.

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.

Cytotoxic T lymphocytes define multiple peptide isoforms derived from the melanoma-associated antigen MART-1/Melan-A.

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.

Molecular analysis of the HLA-A2 antigen loss by melanoma cells SK-MEL-29.1.22 and SK-MEL-29.1.29.

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.

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts.

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.