TAP1 Expression Research Articles

Pseudomonas aeruginosa reduces the immune response to respiratory viruses (99.33)

Abstract P. aeruginosa chronically colonizes the respiratory tract of patients with chronic obstructive pulmonary disease, bronchiectasis and Cystic Fibrosis. These patients experience debilitating and life-threatening viral exacerbations of unknown etiology. Cif, a bacterial toxin secreted by P. aeruginosa, promotes the degradation of several ABC transporters involved in innate immunity, including CFTR and Pgp. The current study tests the hypothesis that Cif enhances the degradation of the transporter associated with antigen processing (TAP), resulting in reduced adaptive T-cell responses to viral pathogens. Cif inhibited the deubiquitinating enzyme, ubiquitin specific protease 10 (USP10), resulting in a time-dependent increase in the polyubiquitination and proteasomal degradation of TAP1. Cif inhibited USP10 activity by stabilizing an inhibitory protein-protein interaction between USP10 and G3BP1. The Cif-mediated decrease in TAP1 expression resulted in decreased MHC class I viral antigen presentation, CD8+ cytotoxic T lymphocyte (CTL) recognition of influenza virus-infected cells and dramatically reduced MHC class I cell surface levels on CD11c+ cells in the BAL of mice treated with P. aeruginosa. We propose that P. aeruginosa, by secreting the Cif toxin, inhibits the ability of CTL to recognize and eliminate viral infections, severely compromising the immune defenses of the lung. This is the first identified bacterial toxin that inhibits the viral immune response of the host.

136 REDUCED EXPRESSION OF ALPHA-TOCOPHEROL-ASSOCIATED PROTEIN IS ASSOCIATED WITH TUMOR CELL PROLIFERATION AND THE INCREASED RISK OF PROSTATE CANCER RECURRENCE

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011136 REDUCED EXPRESSION OF ALPHA-TOCOPHEROL-ASSOCIATED PROTEIN IS ASSOCIATED WITH TUMOR CELL PROLIFERATION AND THE INCREASED RISK OF PROSTATE CANCER RECURRENCE Xingqiao Wen and Xiaojuan Li Xingqiao WenXingqiao Wen Guangzhou, China, People's Republic of More articles by this author and Xiaojuan LiXiaojuan Li Guangzhou, China, People's Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.203AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To examine the impact and prognostic significance of alpha-tocopherol associated protein (TAP) expression in a series of prostate cancer patients. METHODS Tissues from 87 patients underwent radical prostatectomy were examined for TAP expression by immunohistochemistry. The relationships of the staining results, the clinic pathological characteristics and the recurrence times were analyzed. RESULTS Compared with the adjacent areas of normal and benign glands, immunoreactivity of TAP was reduced in areas of prostate cancer. A lower TAP-positive cell number per mm(2) of the largest cancer area (defined as TAP-PN) was associated with higher clinical stage (r = −0.248, P = 0.0322). Inverse associations were found among the TAP-PN and positive lymph nodes (r = −0.231, P = 0.0325), preoperative prostate-specific antigen (PSA) levels (r = −0.423, P = 0.0043), tumor size (r= −0.315, P= 0.0210) and elevated tumor cell proliferation, which was indicated by the staining of Ki-67 (r = −0.308, P = 0.0026). TAP-PN was a significant predictor of recurrence univariately (P = 0.0006), as well as multivariately, adjusted for known markers including preoperative PSA, clinical stage, Gleason score, surgical margin, extra-prostatic extension, seminal vesicle invasion and lymph node metastasis (P = 0.0012). CONCLUSIONS Reduced expression of TAP was associated with the cell proliferation status of prostate cancer, adverse pathological parameters and the increased risk of recurrence. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e57 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xingqiao Wen Guangzhou, China, People's Republic of More articles by this author Xiaojuan Li Guangzhou, China, People's Republic of More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Disparate Antiviral Responses in Molluscum contagiosum Virus–Induced Skin Lesions

Molluscum contagiosum virus (MCV) is a poxvirus that causes tumor-like skin lesions. New evidence indicates that plasmacytoid dendritic cells, type I interferon production, and interferon-induced dendritic cells have prominent roles in anti-MCV responses, and these features characterize the inflammatory response in lesions that will likely undergo spontaneous regression.

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

We have recently reported that chemotherapeutic agents in ultra low noncytotoxic concentrations may block the ability of tumor cells to suppress functional activation of dendritic cells (DCs). HCT-116 human colon cancer cells were treated with 0.5 nM paclitaxel (PAC) or 2 nM doxorubicin (DOX) with the aim of defining the immunogenic changes induced by ultra low noncytotoxic concentrations of antineoplastic chemotherapeutic agents. Genetic alterations were screened by DNA microarray that revealed increased expression of genes involved in antigen processing and presentation, including the heat-shock protein, calmodulin, and proteasome 26 genes. As the proteins encoded by these genes are involved in the cytosolic route of antigen processing machinery, we next evaluated whether PAC and DOX in noncytotoxic concentrations changed expression of MHC class I antigen processing machinery (APM) components in three different colon cancer cell lines. Our results showed that PAC and DOX increased the intracellular expression of APM proteins, including calmodulin, LMP2, LMP7, TAP1 and tapasin. The biological significance of modulation of antigen processing and presentation proteins in tumor cells by ultra low nontoxic concentrations of chemotherapeutic drugs was revealed when non-treated and treated tumor cells were used as a source of tumor antigens for the generation of tumor-specific cytotoxic T cells (CTLs) in vitro. We demonstrated that (i) DCs that engulf tumor cells pretreated with noncytotoxic concentrations of chemotherapeutic agents induced CTLs with a higher cytotoxic potential than DCs loaded with nontreated tumor cells, and (ii) CTLs induced by tumor lysate-pulsed DCs killed live tumor cells more efficiently if these tumor cells were pretreated with noncytotoxic concentrations of chemotherapeutic drugs. These results demonstrate that chemomodulation of human tumor cells with noncytotoxic concentrations of chemotherapeutic agents increases tumor immunogenicity and results in the generation of more efficient DC vaccines and CTLs, which can be used for cell-based anticancer immunotherapies.

Human Cytomegalovirus Disrupts the Major Histocompatibility Complex Class I Peptide-Loading Complex and Inhibits Tapasin Gene Transcription

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.

Molecular characterization of subcellular localization and nucleocytoplasmic shuttling of PRV UL54

Pseudorabies virus (PRV) UL54 protein localized almost exclusively to the nucleolus. By constructing a series of mutants, the putative nuclear localization signal (NLS), nucleolar localization signal (NoLS) and nuclear export signal (NES) of UL54 were for the first time mapped to amino acids 45RRRRGGRGGRAAR57, 61RQRRR65 and 240LQNLRLKLGPFL251, respectively. In addition, nuclear localization of UL54 was important for its transcriptional regulation of glycoprotein C promoter. The nuclear import of UL54 was abrogated by dominant negative RanGTP and importin β1, respectively, indicating that UL54 targeted to the nucleus by means of a classic Ran- and importin β-dependent nuclear import mechanism. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1). However, ectopic expression of the mRNA export receptor TAP(NXF1) promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP(NXF1), but not CRM1, dependent nuclear export pathway. The present study demonstrated that UL54 is a nucleolar protein, adding UL54 to the growing list of transactivators which localize to the nucleolus and shuttle between the nucleus and the cytoplasm.

RNA Interference of Human Papillomavirus Type 16 E7 Increases HLA Class I Antigen Expression in HaCaT-E7 Cells

High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Effect of α-tocopherol associated protein in the regulation of the apoptosis induced by oxidative stress in prostate cancer cells

Objective To investigate the effect of α-tocopherol associated protein (TAP) gene in the regulation of the apoptosis induced by oxidative stress.Methods The plasmid pEGFP-N3-TAP expressing human TAP gene was transfected into PC-3 cells by the transfection reagent DMRIEC,and the expression of TAP was detected by Western blotting.Reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting were used to detect the change of TAP gene expression in the oxidative stress cell model established by using hydrogen peroxide ( H2O2 ).The apoptosis rate under oxidative stress in PC-3 cells was evaluated by the flow cytometry.Results After transfection,the expression level of TAP gene was significantly higher than the wild type PC-3 cell line.The TAP mRNA expression level in the groups of hydrogen peroxide alone (2.457 ± 0.113 ) or hydrogen peroxide combined with 250 μmol/L BSO (3.643 ± 0.162) was significantly higher than in control group (0.721 ± 0.048 ) ( P< 0.01 ).The flow cytometry suggested that in control group,the apoptosis rate in PC-3 cells,PC-3-TAP cells,PC-3-vector cells was 2.42%,3.69% and 15.37% respectively; In the hydrogen peroxide-stimulated groups,the apoptosis rate was 7.56%,16.71% and 43.93%,respectively; In the group of BSO combined with hydrogen peroxide,the apoptosis rate was 13.73%,26.36% and 49.19%,respectively.Conclusion TAP gene was involved in the response of oxidative stress in prostate cancer cells,and could promote the apoptosis induced by oxidative stress. Key words: Prostate carcinoma; Oxidative stress; Tocopherol associated protein; Apoptosis

A novel approach to rescue immune escape in oral squamous cell carcinoma: Combined use of interferon-γ and LY294002

Major histocompatibility complex (MHC) class I molecules have been found to be downmodulated in many tumors. The antigen-processing machinery (APM) genes, especially transporters associated with antigen processing (TAP)-1 and tapasin play important roles in the processing of class I antigens. In this study, we investigated the expression of TAP-1 and tapasin in oral squamous cell carcinoma (OSCC); the result indicated significant down-regulation in the expression of these genes. Interferon (IFN)-γ treatment was applied. After the addition of IFN-γ, unexpectedly, the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway was activated, which induced the proliferation of tumor cells. With the combined application of LY294002 (specific inhibitor of AKT signaling) and IFN-γ, tumor cell apoptosis was induced and the expression of TAP-1 and tapasin was still up-regulated. Hence, our method is a novel and efficient approach to use IFN-γ for rescuing the cells from immunosurveillance.

Paclitaxel blocks immunologic escape through up-regulating TAP-1, TAP-2 and eliminatiing Treg cells in 3LL-bearing mice

背景与目的化疗是否可以逆转肿瘤的免疫逃逸尚未得到肯定的回答。本研究旨在探讨紫杉醇类抗肿瘤药物紫杉醇参与逆转肺部肿瘤细胞免疫逃逸的机制。方法体外实验应用流式单抗TAP-1、TAP-2检测分析3LL细胞株（Lewis肺腺癌细胞）和经紫杉醇5×10-8 mol/L处理的3LL细胞株抗原肽转运体（transporters associated with antigen processing, TAP）的表达；体内实验中，3LL细胞经尾静脉注射成瘤，8 d后化疗组小鼠腹腔内注射紫杉醇：0.012 5 mg/只；对照组小鼠腹腔内注射生理盐水；设立空白对照组；21 d后取各组小鼠的肺部组织，制备单细胞悬液，流式三染法检测调节性T细胞（Regulatory T cells, Treg）。结果经紫杉醇处理的3LL细胞高表达TAP-1（5.68%±0.65%）和TAP-2（89.54%±4.8%），明显高于3LL细胞组TAP-1（1.93%±0.25%）和TAP-2（67.78%±5.08%）的表达（P分别为0.006和0.036）；体内实验中：荷瘤小鼠肺部肿瘤中Treg细胞的表达（25.46%±2.23%）明显高于正常对照组的小鼠（12.46%±1.21%），差异具有统计学意义（P < 0.001）；紫杉醇化疗组明显抑制小鼠肺部肿瘤组织中Treg细胞的表达（17.53%±1.24%），与荷瘤小鼠肺部组织的Treg细胞相比，差异具有统计学意义（P=0.004）。结论化疗药物紫杉醇通过上调肿瘤细胞表面抗原转运肽的表达以及消除肿瘤局部调节性T细胞的产生，可能部分参与了逆转肺癌免疫逃逸的功能。

M13.5 Homeobox genes show altered expression levels in pre-eclamptic placentae

Cell surface expression of HLA class I (including non-classical HLA-G) in JEG3 (choriocarcinoma cell line) was blocked by stable transfection with the sequence encoding the Herpes simplex virus protein, infected cell peptide 47 (ICP47) inserted into a vector pCEP4. Intracellular expression of ICP47 protein in ICP47-transfected cells was demonstrated. The lack of HLA cell surface expression was likely to be due to blockage of peptide transport from the cytoplasm into the endoplasmic reticulum by ICP47. ICP47 is known to block the heterodimeric transporter associated with antigen processing (formed from TAP1 and TAP2). Western blotting with a polyclonal antibody to the C-terminus of TAP1 showed high expression of TAP1 in BeWo and JEG3, but not JAR cells, expression that was strongly upregulated by γ-interferon. γ-Interferon also upregulated the cell surface expression of HLA class I. TAP1 was strongly expressed in MC2 and MC3 extravillous cytotrophoblast cell lines immortalised with the SV40 large T antigen. The results suggest a role for non-classical HLA in the presentation of antigenic peptides to the immune system.

Down-regulation of the antigen processing machinery is linked to a loss of inflammatory response in colorectal cancer

Antitumor inflammatory response is known to inhibit tumor growth in colorectal carcinoma. The density and functionality of tumor-infiltrating lymphocytes (TIL) is regulated by the antigen processing machinery through regulator proteins such as transporters associated with antigen processing (TAP) and major histocompatibility complex (MHC) class I antigen. We aimed to investigate the in vivo association of those factors and their impact on prognosis in colorectal cancer. TAP1, TAP2 and MHC class I antigen expression, inflammatory infiltrate and TIL (CD4(+), CD8(+), and CD20(+)) were assessed by immunohistochemistry in 336 sporadic colorectal carcinomas. The factors were correlated with each other and with clinic-pathological parameters and patient outcome. We found TAP1 and TAP2 expression to be significantly associated with MHC class I antigen expression (TAP1: r = 0.363, P < .001; TAP2: r = 0.393, P < .001). Increased density of CD8(+) TIL was predominantly found in TAP1, TAP2 and MHC class I antigen-positive cases. Increased density of CD4(+) TIL was linked with TAP1 and TAP2, but not with MHC class I antigen. High CD4(+) and CD8(+) cell count but not TAP1, TAP2 and MHC class I antigen expression had favorable prognostic impact in colorectal cancer (P = .003 and P = .003, respectively). In conclusion, our data show that the expression of key components of the antigen processing machinery is tightly linked to the density of TIL, which are positive prognostic factors in colorectal cancer in vivo. This implies that modulation of these factors may help to enhance antitumor inflammatory response which in turn may improve patient prognosis.

The effect of monotherapy and combined therapy with NSC-631570 (Ukrain) on growth of low- and high-metastasizing B16 melanoma in mice

NSC-631570 (ukrain) is a semisynthetic derivative of the Chelidonium majus alcaloids and the alkylans thiotepa. It exerts a selective cytotoxic effect on tumor cells in vitro and in vivo and shows the ability to modulate immunocyte functions. Purpose. The aim of our work was to carry out a comparative investigation of the effects of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice. NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. The effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the organism.

HLA‐F is a surface marker on activated lymphocytes

Of the three nonclassical class I antigens expressed in humans, HLA-F has been least characterized with regard to expression or function. In this study, we examined HLA-F expression focusing on lymphoid cells, where our previous work with homologous cell lines had demonstrated surface HLA-F expression. HLA-F protein expression was observed by Western blot analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes, all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA-F intracellularly showed an induction of surface HLA-F protein. An examination of peripheral blood from individuals genetically deficient for TAP and tapasin expression demonstrated the same activation expression profiles for HLA-F,but with altered kinetics post-activation. Further analysis of CD41+CD25+1 Treg showed that HLA-F was not upregulated on the major fraction of these cells when they were activated,whereas CD41+CD25- T cells showed strong expression of surface HLA-F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA-F and its potential clinical use as a marker of an activated immune response.

Effects of fumonisin B1 on HLA class I antigen presentation and processing pathway in GES-1 cells in vitro

Fumonisin B1 (FB1) is a food-borne mycotoxin produced by genus Fusarium and was classified as possible carcinogen to humans by the International Agency for Research on Cancer (IARC). Human leukocyte antigen (HLA) class I antigen presentation pathway plays an important role in immunosurveillance. Defects in HLA class I antigen presentation pathway can down-regulate the expression of HLA class I antigen on the surface of nucleated cells that will confer a survival advantage to randomly mutant cells and may lead to malignant transformation. In the present study, we analyzed the effects of FB1 on the expression of HLA class I heavy chain (classical HLA-A, -B and -C genes included), beta2-microglobulin (β2m), LMP2 and TAP1 genes in human gastric epithelial immortalized GES-1 cells in vitro using semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blot and immunocytochemical methods in dose- and time-effect studies. Our results revealed that FB1 have an effect on HLA class I antigen presentation pathway via the decreased expression of HLA class I heavy chain and/or defects of LMP2 and TAP1 expression. However, the importance of this effect in carcinogenesis needs further investigation.

Aflatoxin G 1 reduces the molecular expression of HLA-I, TAP-1 and LMP-2 of adult esophageal epithelial cells in vitro

Aflatoxins (AF) are the mycotoxin with the carcinogenic effect produced by the fungi like Aspergillus flavus, including AFB 1, B 2, G 1, G 2, M 1, and so on. The detection rate of Aflatoxin G 1 (AFG 1) is very high in the diet of residents in the high incidence area of the gastric carcinoma and esophageal carcinoma in China, which is the main polluting mycotoxin of the local grain. However, there were no available data regarding the gastric toxicity of AFG 1 up to now. In the present study, The toxicity of AFG 1 in vitro-cultured human primary adult esophageal epithelial cells was explored by employing the methods of Western blot, semi-quantitative RT-PCR and observing the effect of AFG 1 on the expression of the HLA-I molecules of the human esophageal epithelial cells and the antigen processing relative genes TAP-1 and LMP-2. The results showed that AFG 1 treatment could decrease both the protein expression of HLA-ABC of the cell surface and the mRNA expression of HLA-A and HLA-B. Moreover, In the AFG 1 treatment groups of different densities, both the TAP-1 mRNA and protein expression level of the human esophageal epithelial cells decreased. Furthermore, the LMP-2 protein expression level of the human esophageal epithelial cells decreased, while the mRNA expression level was not obviously changed by AFG 1 treatment. It is indicated that the decrease of the HLA-I expression of the cell surface was probably dependent on the decrease of the TAP-1 and LMP-2 expression to a great extent. The decrease of LMP-2 expression resulted in the decrease of HLA-I expression and stability in the cell membrane and finally led to the defect in the antigen presentation, which hindered its identification by the T lymphocytes, made it escape the immune surveillance, and then caused the tumor genesis.

Impaired Antigen Processing and Presentation Machinery is Associated with Immunotolerant State in Chronic Hepatitis B Virus Infection

The mechanism of hepatitis B virus (HBV)-specific T cell hyporesponsiveness in hepatitis Be antigen (HBeAg)-positive subjects is not well understood. Inefficient antigen processing and transport to major histocompatibility complex class I molecules, namely due to low molecular weight protein (LMP) 2 and 7 and transporter associated with antigen processing (TAP) 1 and 2 genes could be playing a role. Forty patients with chronic hepatitis B (CHB) infection, hepatitis B surface antigen, and HBeAg positive; 26 with raised (Gr. I) and 14 with persistently normal ALT levels (Gr. II) and 11 healthy controls (Gr. III) were studied. Total RNA was isolated from peripheral blood mononuclear cells and mRNA expression of TAP1, TAP2, LMP2, and LMP7 genes was analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction method. Gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels were quantified by enzyme-linked immunosorbent assay (ELISA) using log-log and linear graphs, respectively. Group II CHB patients had significantly lower mRNA expression for TAP1 (p = 0.003) and LMP2 (p = 0.002) genes as compared to Gr. I patients. The mRNA expression of TAP2 and LMP7 genes was comparable between the groups. However, expression of TAP1 (p = 0.02), TAP2 (p = 0.035), and LMP2 (p = 0.041) was found to be significantly higher in Gr. III subjects compared to Gr. I and Gr. II patients. In Gr. I and II, the IFN-gamma {s54.2{9.4-165} pg/ml), (59.5{28.5-110} pg/ml)}, and TNF-alpha {12.0 (8.0-23.2)},{10.8(6.2-20.8)} pg/ml levels were comparable but were significantly (p = 0.00,0.004, respectively) higher than Gr. III subjects. Low expression of TAP1 and LMP2 suggests an important role of these genes in defective viral antigen processing in immune tolerant state of CHB patients. Higher IFN-gamma and TNF-alpha production in CHB are probably enough to potentiate liver injury but not enough to clear the chronic HBV infection. These novel observations could pave way for new therapeutic strategies for immune restoration in CHB infected patients.

Down-Regulation of HLA Class I Antigen in Human Papillomavirus Type 16 E7 Expressing HaCaT Cells

High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P < 0.001). Moreover, the expression of HPV-16 E7 in HaCaT cells resulted in decreased expression of TAP-1 that was essential for HLA class I expression at the cell surface, a statistical significant decrease of approximately 40% compared with that with the empty vector control (P < 0.001). Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.

Up-regulate HLA Class I Expression Following Hepatitis B Virus Transfection in a Hepatocellular Carcinoma Cell Line BEL7405

Chronic hepatitis B virus infection is associated with a high risk of developing into hepatocellular carcinoma, while tumor recognition is important during the immune surveillance process that prevents cancer development in humans. The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis B virus (HBV) are still unclear. In the present study, 1 copy or 1.2 copies of HBV genome was transfected into a hepatocellular carcinoma cell line BEL7405. RT-PCR, Western blot and flow cytometry analysis were used to evaluate the expression of HLA class I molecules and transporter associated with antigen processing 1 (TAP1). Finally, the cytotoxic activity of natural killer (NK) cells against HBV transfected liver cells was detected by MTT colorimetry method. Following transfection of 1 copy or 1.2 copies of HBV genome, HLA class I expression was up-regulated in BEL7405 cell line in a dose-dependent manner. Furthermore, increased the surface HLA class I expression were caused by enhanced expression of TAP1 at mRNA and protein levels in those transfected cells. Consequently, a significantly down-regulated cytotoxic activity of NK cells against HBV transfected liver cells was observed. These results may demonstrate a way by which HBV avoids recognition by NK cells that might be associated with the establishment of chronic infection and tumor formation.

Calreticulin maintains the low threshold of peptide required for efficient antigen presentation

Calreticulin (CRT) plays a critical role in MHC class I antigen processing and elicits peptide-specific CD8(+) T cell responses against tumours when administered with peptides. However, how CRT contributes to class I antigen processing and the mechanism of its adjuvant effect in anti-tumour responses, remain to be elucidated. Here we show that reduced class I expression in CRT deficient cells can be restored by the direct delivery of peptides into the ER or by incubation at low temperature. CRT deficient cells exhibited a TAP-deficient phenotype in terms of class I assembly, without loss of TAP expression or functionality. Furthermore, a higher concentration of antigen in the cytosol is required for specific T cell stimulation, suggesting that CRT has a functional role in the maintenance of the low peptide concentration threshold required in the ER for efficient antigen presentation. In the absence of CRT, ERp57 is up-regulated, which indicates that they collaborate with each other in class I antigen processing.