MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens.     

Restoration by IL-15 of MHC class I antigen-processing machinery in human dendritic cells inhibited by tumor-derived gangliosides

We have recently reported that MHC class I Ag-processing machinery (APM) component expression in dendritic cells (DC) might be down-regulated by tumor cells. However, the tumor-derived factors responsible for inhibition of the APM component expression in DC generated in the tumor microenvironment as well as potential protective mechanism have not yet been investigated. In this article, we demonstrate that expression of several MHC class I APM components, including MB1 (beta5), LMP2, LMP7, LMP10, and ERp57, is significantly down-regulated in human DC generated in the presence of primary oral squamous cell carcinoma cell lines or coincubated with purified gangliosides. Suppression of MHC class I APM component expression in DC generated in the presence of tumor cells was significantly attenuated by the inhibition of glucosyl transferase in tumor cells, suggesting that tumor-induced MHC class I APM component down-regulation in DC was mediated in part by oral squamous cell carcinoma-derived gangliosides. Furthermore, rIL-15 restored both tumor cell-induced and ganglioside-induced MHC class I APM component expression in DC, as well as their ability to present Ags to autologous Ag-specific T cells. These results demonstrate that IL-15 restores MHC class I APM component expression in DC down-regulated by tumor-derived gangliosides.     

Association of HLA class I antigen abnormalities with disease progression and early recurrence in prostate cancer

Defects in HLA class I antigen processing machinery (APM) component expression often have a negative impact on the clinical course of tumors and on the response to T cell-based immunotherapy. Since only scant information is available about the frequency and clinical significance of HLA class I APM component abnormalities in prostate cancer, the APM component expression pattern was analyzed in 59 primary prostate carcinoma, adjacent normal tissues, as well as in prostate carcinoma cell lines. The IFN-γ inducible proteasome subunits LMP2 and LMP7, TAP1, TAP2, calnexin, calreticulin, ERp57, and tapasin are strongly expressed in the cytoplasm of normal prostate cells, whereas HLA class I heavy chain (HC) and β₂-microglobulin are expressed on the cell surface. Most of the APM components were downregulated in a substantial number of prostate cancers. With the exception of HLA class I HC, TAP2 and ERp57 not detectable in about 0.5% of tumor lesions, all other APM components were not detected in at least 21% of lesions analyzed. These APM component defects were associated with a higher Gleason grade of tumors and an early disease recurrence. Prostate carcinoma cell lines also exhibit a heterogeneous, but reduced constitutive APM component expression pattern associated with lack or reduced HLA class I surface antigens, which could be upregulated by IFN-γ. Our results suggest that HLA class I APM component abnormalities are mainly due to regulatory mechanisms, play a role in the clinical course of prostate cancer and on the outcome of T cell-based immunotherapies.     

The low rate of HLA class I molecules on the human embryonic stem cell line HS293 is associated with the APM components' expression level

Human embryonic stem cells (hESCs) represent a promise for future strategies of tissue replacement. However, there are different issues that should be resolved before these cells can be used in cellular therapies; among others, the rejection of transplantable hESCs as a result of HLA incompatibility between donor cells and recipients. The hESCs exhibit a weak HLA class I expression on the cell surface, but today the responsible mechanisms are unknown. We have analyzed the level expression of HLA class I heavy chain, beta2-microglobulin (beta2-m), and antigen-processing machinery (APM) components (TAP1, TAP2, LMP2, LMP7, and Tapasin) using the HS293 hESC line by real-time quantitative RT-PCR. This analysis has revealed a low expression of beta2-m, HLA-B, and Tapasin, and an absence of expression of: TAP1, TAP2, LMP2, and LMP7 genes in the HS293 hESC line respect to the embryoid bodies (EBs) and the induced stem cells with IFNgamma (with significant differences, p<0.05). The lack or loss of HLA class I molecules due to the down-regulation of the APM components has been frequently found in tumors of different histology as specific mechanisms of immune-evasion. We described for the first time in this report that the hESCs shared similar mechanisms with respect to tumor cells responsible for the weak HLA class I expression on the cell surface.     

Interferon regulatory factor 8 mediates tumor-induced inhibition of antigen processing and presentation by dendritic cells

Introduction  Suppression of dendritic cells (DCs) is a crucial mechanism by which tumor cells escape immune recognition and elimination. We have recently reported that MHC class I antigen processing machinery (APM) component expression in human DCs is down-regulated by tumor-derived gangliosides. However, the molecular mechanisms underlying this abnormality were not identified. Thus, the aim of this work was to analyze the role of interferon regulatory factor 8 (IRF-8) in APM protein expression and the antigen presenting capacity of DCs developed in the tumor microenvironment. Results  We demonstrate that the expression of several MHC class I APM components, including delta, MB-1, LMP-10, ERp57, and tapasin, is significantly decreased in murine DCs generated in the presence of prostate cancer cells. APM component down-regulation was associated with decreased ability of DCs to present model antigen to antigen-specific T cells. Notable, impaired antigen-presenting activity of DCs co-cultured with tumor cells was accompanied by decreased levels of IRF-8. Transduction of DCs with the silencing RNA for the IRF-8 gene also led to reduced expression of APM components in DCs and decreased antigen presenting function. Conclusion  Together, our data suggest that tumor-induced inhibition of antigen processing and presenting function of DCs is mediated by IRF-8, a member of the interferon regulatory factor family. These results provide a new molecular target for optimizing the generation of efficient DC vaccines for cancer therapy.     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

The escape of cancer from T lymphocytes: immunoselection of MHC class I loss variants harboring structural-irreversible “hard” lesions

The discovery of tumor antigens recognized by T lymphocytes has stimulated the development of a variety of cancer treatment protocols aimed at enhancing antitumor-specific T cell responses and tumor rejection. However, immunotherapy-mediated regression of established tumors and clearly positive clinical response to such treatment has not been achieved yet despite the induction of T cells directed against tumor antigens. The failure of the modern immunotherapy protocols can be explained by different tumor escape mechanisms that have been defined in various types of malignancy. The loss or downregulation of MHC class I antigens in tumor cells is one of the best analyzed mechanisms. In this review, we show experimental evidence obtained in our laboratory on human tumors and in a mouse cancer model suggesting that the molecular mechanism responsible for the MHC class I alteration in tumor cells might have a crucial impact on tumor recovery of normal H-2/HLA expression during the natural history of tumor development or after immunotherapy. When the preexisting molecular lesion underlying tumor MHC class I alteration is reversible (regulatory or soft), class I expression can be recovered leading to regression of tumor lesion. In contrast, if the HLA class I alteration is irreversible in nature (structural or hard), the lesion will progress killing the host. This is a new vision of the role of MHC class I alteration in tumors that can explain the failure of immunotherapy in a variety of different clinical protocols.     

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy. Supported partially by The Heidi Leukemia Research Fund, Buffalo, NY, and by PHS grants CA 67108 and CA 16056 awarded by the National Cancer Institute, DHHS.     

Complex interplay of activating and inhibitory signals received by Vgamma9Vdelta2 T cells revealed by target cell beta2-microglobulin knockdown

Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vgamma9Vdelta2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vgamma9Vdelta2 T cell activation, we used stable beta(2)-microglobulin knockdown to generate tumor cells with a approximately 10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vgamma9Vdelta2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vgamma9Vdelta2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vgamma9Vdelta2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.     

RelB nuclear translocation mediated by C-terminal activator regions of Epstein-Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells.

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappa B activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative I kappa B alpha S32/36A, an NF-kappa B inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappa B subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.     

Expression of class I histocompatibility antigens in neuroectodermal tumors is independent of the expression of a transfected neuroblastoma myc gene

We have evaluated the relationship between the neuronal myc gene (NMYC) and class I major histocompatibility complex (MHC) expression in human neuroblastoma (NB) tumor cell lines. Class I MHC surface Ag expression in NB cell lines varied from nearly undetectable to levels nearly as high as in a lymphoblastoid cell line. Class I MHC mRNA levels in NMYC-amplified NB cell lines were lower than levels observed in single copy NMYC NB cell lines. However, considerable variation in class I MHC surface Ag and mRNA expression was evident in NMYC-amplified cell lines. To determine directly whether NMYC might modulate class I MHC expression in NB, we transfected a plasmid containing a recombinant NMYC gene into two tumor cell lines derived from a NB and a related neuroepithelioma tumor. Constitutive overexpression of the recombinant NMYC gene produced no consistent change in class I MHC surface Ag or mRNA levels. To determine whether class I MHC expression might be developmentally regulated in adrenal medullary cells, the precursor cells of adrenal NB tumors, beta 2-microglobulin expression was measured in fetal and adult adrenal glands. beta 2-Microglobulin expression was not evident in the neuroblasts of a 24-wk-old fetal adrenal gland, whereas beta 2-microglobulin expression was present in the adult adrenal medulla. These data suggest that variation in class I MHC expression among NB cells may reflect the developmental stage at which neuroblasts were arrested during tumorigenesis.     

Using the TAP component of the antigen-processing machinery as a molecular adjuvant

We hypothesize that over-expression of transporters associated with antigen processing (TAP1 and TAP2), components of the major histocompatibility complex (MHC) class I antigen-processing pathway, enhances antigen-specific cytotoxic activity in response to viral infection. An expression system using recombinant vaccinia virus (VV) was used to over-express human TAP1 and TAP2 (VV-hTAP1,2) in normal mice. Mice coinfected with either vesicular stomatitis virus plus VV-hTAP1,2 or Sendai virus plus VV-hTAP1,2 increased cytotoxic lymphocyte (CTL) activity by at least 4-fold when compared to coinfections with a control vector, VV encoding the plasmid PJS-5. Coinfections with VV-hTAP1,2 increased virus-specific CTL precursors compared to control infections without VV-hTAP1,2. In an animal model of lethal viral challenge after vaccination, VV-hTAP1,2 provided protection against a lethal challenge of VV at doses 100-fold lower than control vector alone. Mechanistically, the total MHC class I antigen surface expression and the cross-presentation mechanism in spleen-derived dendritic cells was augmented by over-expression of TAP. Furthermore, VV-hTAP1,2 increases splenic TAP transport activity and endogenous antigen processing, thus rendering infected targets more susceptible to CTL recognition and subsequent killing. This is the first demonstration that over-expression of a component of the antigen-processing machinery increases endogenous antigen presentation and dendritic cell cross-presentation of exogenous antigens and may provide a novel and general approach for increasing immune responses against pathogens at low doses of vaccine inocula.     

Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune responses efficiently, have developed a unique membrane transport pathway linking the lumen of endocytic compartments and the cytosol. Endosome-to-cytosol transport is restricted to dendritic cells, specific to internalized antigens and selective for the size of the transported molecules. Thus, in dendritic cells, internalized antigens gain access to the cytosolic antigen-processing machinery and to the conventional MHC class I antigen-presentation pathway.     

Regulation of MHC protein expression in pancreatic beta-cells by interferon-gamma and tumor necrosis factor-alpha

Isolated human and mouse pancreatic islet cells and the rat insulinoma cell line RIN-m5F were used to examine the ability of recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to regulate the expression of the class I and class II major histocompatibility (MHC) surface proteins and mRNA in beta-cells. Each cytokine increased significantly the expression of class I MHC proteins as determined by double indirect immunofluorescence microscopy and flow cytofluorimetric analysis. In the RIN-m5F cells, this increase in surface expressed class I MHC proteins was mirrored by an increase in the level of class I MHC mRNA. The order of potency of the cytokines on class I MHC expression was TNF-alpha plus IFN-gamma greater than or equal to IFN-gamma greater than or equal to TNF-alpha. While IFN-gamma or TNF-alpha alone were without effect, in combination they were found to induce class II MHC proteins on 30-40% of human or murine beta-cells. In contrast, IFN-gamma plus TNF-alpha did not induce detectable class II MHC proteins or mRNA in the RIN-m5F cells. These findings indicate that 1) TNF-alpha, in addition to IFN-gamma, upregulates the expression of beta-cell class I MHC proteins and mRNA, and 2) more than one signal is required for the induction of class II MHC proteins on beta-cells. The ability of IFN-gamma plus TNF-alpha to induce class II MHC proteins on only a fraction of the normal beta-cell population and not on RIN-m5F cells suggests that this response is related to the differentiation state of the beta-cell.     

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.