Expression of transporter associated with antigen processing 1 and 2 (TAP1/2) in malignant melanoma cell lines

TAP1 and TAP2 molecules are involved in the transport of peptides prior to their association with class I molecules and are mandatory for efficient antigen presentation. To investigate whether loss of expression of TAP1 or TAP2 is a likely mechanism of immune escape in malignant melanoma, TAP1 and TAP2 mRNA was analyzed by RT-PCR in 39 melanoma cell lines expressing at least 2 of the known melanoma-associated antigens, tyrosinase, Melan-A/MART-1, gp100, MAGE-1 and MAGE-3. All 39 cell lines expressed both TAP1 and TAP2 at the mRNA level. To investigate other factors potentially involved in immune escape, the expression of LMP2, LMP7, HLA class I molecules, β₂-microglobulin (β₂m) and specific HLA-A alleles was evaluated by RT-PCR and FACS analyses. All 39 cell lines expressed LMP2, LMP7 and β₂m. A single cell line (FM37) had lost the expression of class I molecules, and this same cell line showed loss of expression of the HLA-A2 heavy chain. No cell lines showed loss of expression of the HLA-A1 heavy chain. Based on our studies of in vitro established cell lines, loss of TAP1/2 or LMP2/7 expression does not appear to be a common mechanism of immune escape in malignant melanoma.Int. J. Cancer 70:582–586. © 1997 Wiley-Liss Inc     

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.     

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.     

Defects in the human leukocyte antigen class I antigen processing machinery in head and neck squamous cell carcinoma: association with clinical outcome.

PURPOSE: Human leukocyte antigen (HLA) class I antigen defects, which are frequently present in head and neck squamous cell carcinoma (HNSCC) cells may provide the tumor with an escape mechanism from immune surveillance. Scanty information is available about mechanisms underlying HLA class I antigen defects in both lesions and cell lines from HNSCC. In this study, we investigate the role of antigen processing machinery (APM) component abnormalities in the generation of deficient HLA class I surface expression of HNSCC cells. EXPERIMENTAL DESIGN: Using immunohistochemistry, Western blot, and RT-PCR analyses we correlated the expression of the IFN-gamma inducible proteasome subunits and of the peptide transporter TAP with that of HLA class I antigens in biopsies and cell lines from primary, recurrent, and metastatic HNSCC. Furthermore, APM component and HLA class I antigen expression in surgically removed lesions were correlated with the course of the disease in order to assess the clinical significance of deficient expression of these molecules. RESULTS: A high frequency of LMP2, LMP7, and TAP1 down-regulation or loss was found in tumor lesions and cell lines obtained from HNSCC cancer patients. These defects could be corrected by incubating cells with IFN-gamma. Furthermore, LMP2, LMP7, TAP1, TAP2, and HLA class I antigen expression rates in primary HNSCC lesions were found to predict overall survival. Lastly, the level of LMP7 expression was significantly associated with disease recurrence at 2 years. CONCLUSIONS: Our results suggest that the analysis of APM component expression in HNSCC lesions can provide useful prognostic information in patients with HNSCC.     

Multiple defects of the antigen-processing machinery components in human neuroblastoma: immunotherapeutic implications

Low expression of human leukocyte antigen (HLA) class I in human tumors may be related to defects of the antigen-processing machinery (APM) components. Neuroblastoma cells are virtually HLA class I negative, but (i) the underlying mechanisms are unknown, and (ii) expression of the APM components has never been investigated. Here we have used a panel of novel monoclonal antibodies to proteasomal and immunoproteasomal components, chaperons and transporter associated with antigen processing (TAP) to characterize 24 stroma-poor neuroblastoma tumors and six neuroblastoma cell lines. Primary tumors showed defects in the expression of zeta, tapasin, TAP1 or TAP2, HLA class I heavy chain and beta2 microglobulin, LMP2 and LMP7, as compared to normal adrenal medulla. Neuroblastoma cell lines displayed roughly similar patterns of APM expression in comparison to primary tumors. Incubation of neuroblastoma cell lines with interferon-gamma caused upregulation of HLA class I molecules and reduced lysis by killer inhibitory receptor HLA ligand-matched NK cells. Defects in APM components explain reduced peptide loading on HLA class I molecules, their instability and failure to be expressed on the cell surface. HLA class I upregulation by interferon-gamma, although enhancing neuroblastoma cell recognition by cytotoxic T cells, dampens their susceptibility to NK cells.     

Transcriptional regulation of the major histocompatibility complex (MHC) class I heavy chain, TAP1 and LMP2 genes by the human papillomavirus (HPV) type 6b, 16 and 18 E7 oncoproteins

We have examined the possibility that the E7 proteins of the high-risk human papillomavirus (HPV) type 16 and 18 and the oncogenic adenovirus (Ad) type 12 E1A protein share the ability to down-regulate the expression of components of the antigen processing and presentation pathway, as a common strategy in the evasion of immune surveillance during the induction of cell transformation. Expression of the HPV 18 E7 oncoprotein, like Ad 12 E1A, resulted in repression of the major histocompatibility complex (MHC) class I heavy chain promoter, as well as repression of a bidirectional promoter that regulates expression of the genes encoding the transporter associated with antigen processing subunit 1 (TAP1) and a proteasome subunit, low molecular weight protein 2 (LMP2). HPV 16 E7 also caused a reduction in class I heavy chain promoter activity, however it did not have any significant effect on the activity of the bidirectional promoter. Interestingly, expression of the low-risk HPV 6b E7 protein resulted in an increase in MHC class I heavy chain promoter activity, while repressing the TAP1/LMP2 promoter. Interference with the class I pathway could also explain the ability of low-risk HPVs in inducing benign lesions.     

Molecular basis for lack of expression of HLA class I antigens in human small-cell lung carcinoma cell lines

HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and thus play an important role in immune surveillance of cells infected with virus or altered by malignant transformation. Immunochemical studies have demonstrated a marked deficiency or lack of expression of class I molecules on the surface of many different types of tumor cells. It is likely that this allows these cells to escape immune surveillance. In the present study, we examined the molecular basis for lack of expression of class I antigens in small-cell lung carcinoma cell lines. Our results demonstrate that these cell lines also lacked products of MHC-encoded proteasome subunit LMP2 and the putative peptide transporter TAP1. In contrast, LMP7 and TAP2 genes were expressed in these cell lines. Pulse-chase experiments showed that class I molecules were unstable and thus not transported to the cell surface from endoplasmic reticulum. Our results suggest that antigenic peptides were not available for binding to class I &agr: chains due to genes showed that the tumor cells lacked trans-regulatory nuclear protein(s), which binds to the interferon-γ (IFN-γ) response element (ISRE) in the TAP I, LMP2 bidirectional intergenic promoter. Treatment of tumor cells with IFN-γ induced ISRE-binding nuclear protein(s) and resulted in expression of TAP 1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. Our data provide credence for a role of TAP and LMP genes in immune response. © 1996 Wiley-Liss, Inc.     

MHC class I-deficient metastatic tumor variants immunoselected by T lymphocytes originate from the coordinated downregulation of APM components

Previous reports from our group indicated that the MHC class I phenotype of metastatic lung colonies produced by a mouse fibrosarcoma tumor clone (B9) were, depending on the immune status of the host, MHC class I negative in immunocompetent mice and MHC class I positive in immunodeficient athymic nude/nude mice. Now we report the identification of the molecular alterations responsible for the changes of MHC class I molecules in both situations. Metastatic nodes were analyzed for the mRNA level of H-2 class I and beta2-microglobulin genes, and several gene components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM). These included the genes coding for the low-molecular-weight proteins LMP2, LMP7, LMP10, the transporter associated with antigen processing (TAP-1, TAP-2), and calnexin, calreticulin, tapasin, PA-28-alpha, PA-28-beta, ERP-59 and ER-60. Analyses with RT-PCR showed that TAP-1, TAP2, LMP-2, LMP7, LMP10, tapasin and calnexin mRNA specific for these genes was absent in metastases produced in immunocompetent mice. In contrast, similar techniques with mRNA preparations obtained from metastatic nodes from immunodeficient mice showed that the mRNA expression level of these genes was highly positive. Interestingly, the MHC class I-positive or negative phenotypes of the metastatic colonies correlated with in vivo immunogenicity. H-2 positive metastasis grew more slowly than the H-2 negative ones when injected intrafootpat in syngeneic immunocompetent animals and were finally rejected. These results provide evidence of the role of T cells in immune surveillance against tumors and identify a mechanism targeted by antitumor T lymphocytes to generate MHC class I-negative tumor escape variants.     

Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products

Upon primary infection, EBV establishes a latent infection in B cells, characterized by maintenance of the viral genome in the absence of viral replication. The Epstein-Barr Nuclear Antigen 1 (EBNA1) plays a crucial role in maintenance of the viral DNA episome and is consistently expressed in all EBV-associated malignancies. Compared to other EBV latent gene products, EBNA1 is poorly recognized by CD8(+) T lymphocytes. Recent studies are discussed that shed new light on the mechanisms that underlie this unusual lack of CD8(+) T cell activation. Whereas the latent phase is characterized by the expression of a limited subset of viral gene products, the full repertoire of over 80 EBV lytic gene products is expressed during the replicative phase. Despite this abundance of potential T cell antigens, which indeed give rise to a strong response of CD4(+) and CD8(+) T lymphocytes, the virus can replicate successfully. Evidence is accumulating that this paradoxical situation is the result of actions of multiple viral gene products, inhibiting discrete stages of the MHC class I and class II antigen presentation pathways. Immediately after initiation of the lytic cycle, BNLF2a prevents peptide-loading of MHC class I molecules through inhibition of the Transporter associated with Antigen Processing, TAP. This will reduce presentation of viral antigens by the large ER-resident pool of MHC class I molecules. Synthesis of new MHC class I molecules is blocked by BGLF5. Viral-IL10 causes a reduction in mRNA levels of TAP1 and bli/LMP2, a subunit of the immunoproteasome. MHC class I molecules present at the cell surface are downregulated by BILF1. Also the antigen presenting capacity of MHC class II molecules is severely compromised by multiple EBV lytic gene products, including gp42/gH/gL, BGLF5, and vIL-10. In this review, we discuss how concerted actions of these EBV lytic proteins result in highly effective interference with CD8(+) and CD4(+) T cell surveillance, thereby providing the virus with a window for undisturbed generation of viral progeny.     

MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma

Colorectal tumorigenesis has been associated with the progressive acquisition of a variety of genetic alterations. These include mutations of the Ki-ras proto-oncogene in codons 12 and 13, which account for 85% of genetic changes in colorectal cancer. In murine in vitro models of oncogenic transformation, an association between ras-mediated transformation and downregulation of different components of the MHC class I antigen processing machinery (APM) has been described. In order to investigate whether this association also exists in human tumors, 10 cases of high-grade intraepithelial neoplasia (HIN), as well as primary tumors and autologous lymph node metastases from 42 patients with colorectal carcinoma, were monitored by allele-specific restriction analysis for Ki-ras mutations. In parallel, APM component expression and tumor cell proliferation were analyzed by immunohistochemistry. In comparison to autologous colorectal mucosa, TAP1, LMP2 and tapasin loss was found in 68%, 67% and 80% of HIN, respectively. In contrast, impaired TAP1, LMP2 and tapasin expression was found in 42%, 42% and 63% of primary adenocarcinomas of stage III disease and in 63%, 47% and 79% of the matched lymph node metastases, respectively. More than 60% of colorectal tumor lesions with TAP1, LMP2 and/or tapasin defects displayed Ki-ras mutations. The frequency of TAP1, LMP2 and tapasin loss varied between 33% of primary adenocarcinomas, 40% of HIN to approximately 67% of metastases. These data suggest that i) APM component deficiencies occur more frequently in Ki-ras-mutated colorectal carcinoma lesions and ii) APM abnormalities in conjunction with Ki-ras mutations appear to be associated with disease stage. These findings support the hypothesis that Ki-ras mutations may contribute to immune escape mechanisms of tumors by downregulating the MHC class I APM component expression.     

Systemic deficits in transporter for antigen presentation (TAP)-1 or proteasome subunit LMP2 have little or no effect on tumor incidence

Some tumor cells have deficits in class I MHC antigen processing, suggesting that T cells exert selective pressure on tumor cells. Previous studies have not revealed increased tumor incidence in mice with deficits in T-cell immunity, including mice lacking TAP1 (a subunit of the transporter for antigen presentation) or LMP2 (a regulated subunit of the 20S proteasome). The incidence of spontaneous tumors in these mice, however, is too low to assess differences in host resistance to tumors. To increase tumor incidence and better assess the role of systemic expression of TAP1 and LMP2 in responses to tumors, TAP1-/- and LMP2-/- mice were bred with p53-/- mice to create TAP1-/-p53-/- and LMP2-/-p53-/- double knockout mice. Lymphomas and sarcomas (malignant fibrous histiocytoma and angiosarcoma) occurred with high incidence in all p53-deficient populations. Tumor incidence and death rate were similar in TAP1-/-p53-/- mice and closely matched control TAP1+/+p53-/- mice. Tumor incidence and death rate were slightly accelerated in LMP2-/-p53-/- mice relative to control LMP2+/+p53-/- mice, but the biological significance of this difference was unclear. The relative incidence of lymphomas vs. sarcomas was not significantly altered by variation in TAP1 or LMP2. In conclusion, systemic absence of TAP1 did not alter tumor incidence, while absence of LMP2 was associated with only a slight acceleration of tumor incidence of uncertain significance. These observations are consistent with other evidence that normal T-cell responses do not effectively limit tumorigenesis. Even though T cells can attack some tumor cells, the ability of tumors to alter their immunogenicity and evade T-cell surveillance may render the native immune system ineffective at providing a rate-limiting barrier to tumorigenesis and preventing cancer.     

The mutation in the ATP-binding region of JAK1, identified in human uterine leiomyosarcomas, results in defective interferon-gamma inducibility of TAP1 and LMP2

The presentation of human leukocyte antigens (HLA) class I requires the coordinated expression of numerous components involved in antigen presentation. Tumor cells may alter the antigen presentation by HLA class I, allowing them to evade antitumor immunity. In many cases, the lack of antigen presentation can be attributed to the downregulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP) 1, and the proteasomal component, low molecular weight proteins (LMP) 2. The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an interferon (IFN)-gamma-response factor element; thus, the IFN-gamma-signal strongly induces both TAP1 and LMP2 expression. Low molecular weight proteins2-deficient mice exhibited the development of uterine leiomyosarcomas. Here, the differential responsiveness to IFN-gamma of the SKN human uterine leiomyosarcomas cell line was investigated. We now identify the G871E mutation in the ATP-binding region of Janus kinases 1, suggesting that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma-signal pathway, resulting in the inability of SKN cells to upregulate the antigen-processing pathway. Understanding the mechanisms by which these tumors circumvent cytokine signalling, thereby evading antitumor-specific immunity, would greatly aid the efficacy of immunotherapy for treating uterine leiomyosarcomas.     

Resistance to lysis by cytotoxic T cells: a dominant effect in metastatic mouse prostate cancer cells

Better understanding of the immunology of prostate cancer is needed for the development of new therapeutic approaches that can be used in conjunction with current treatment methods. The present study was designed to compare the immunological properties of a genetically matched pair of primary tumor- and metastasis-derived prostate cancer cell lines generated from the mouse prostate reconstitution (MPR) model. Only the primary prostate cancer cells were immunogenic in that prior immunization with irradiated primary but not the metastatic prostate cancer cells delayed the growth of subsequently injected live cancer cells. The lack of immunogenicity of the metastatic cells was not attributable to their inability to induce antitumor cytotoxic T cells. Both primary and metastatic cells induced antitumor CTLs in syngeneic hosts, but unlike the primary cells, the metastatic cells were resistant to CTL lysis. Differential resistance to cytolysis in metastatic versus primary prostate cancer cells was not attributable to the differential expression of molecules such as transporter associated with antigen processing (TAP)-1, TAP-2, low molecular weight protein of the proteasome complex (LMP)-2, and LMP-7 that contribute to antigen presentation by class I MHC. IFN-gamma induced surface class I MHC expression, as well as gene expression of TAP-1, TAP-2, LMP-2, and LMP-7 in the metastatic cells, yet the cells remained resistant to cell lysis induced by CTLs. Interestingly, although in comparison to the primary cells the metastatic cells were resistant to cytolysis, both cell types were susceptible to DNA fragmentation induced by CTLs. Cell fusion between primary and metastatic cancer cells resulted in hybrids that also resisted the cytolytic activity of CTLs. Therefore, there is a dominant factor(s) in the metastatic prostate cancer cells that confers specific protection against CTL cytolysis in this model system.     

T cell recognition of HLA‐A2 restricted tumor antigens is impaired by the oncogene HER2

The HER2 oncogene is frequently over‐expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over‐expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor‐antigen specific cytotoxic T lymphocytes (CTLs) in HLA‐A2.1⁺ melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA‐A2. A significant reduction of HLA‐A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling‐competent HER2 molecule was crucial for the observed HLA‐A2 down‐regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA‐A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen‐specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN‐γ mediated HLA‐A2 up‐regulation and improved recognition by HLA‐A2‐restricted CTLs in treated cells. Moreover, key APM molecules were down‐regulated by HER2. These findings implicate that HER2 over‐expressing tumors may be more prone to escape from HLA‐A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.     

Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma

To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.