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Peter Cresswell - One of the best experts on this subject based on the ideXlab platform.
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three Tapasin docking sites in tap cooperate to facilitate transporter stabilization and heterodimerization
Journal of Immunology, 2014Co-Authors: Ralf M Leonhardt, Peter Cresswell, Parwiz Abrahimi, Susan M MitchellAbstract:The TAP translocates peptide Ags into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex, an oligomeric complex that the chaperone Tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three Tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains of TAP1 and TAP2. The third binding site is present in the core transmembrane (TM) domain of TAP1 and is used only by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular, the role of Tapasin binding to the core TM domain of TAP1 single chains is mysterious because this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic TM helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants that lack Tapasin binding to either N-terminal domain, we conclude that all three Tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization.
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identification of an alternate splice form of Tapasin in human melanoma
Human Immunology, 2010Co-Authors: Alan Belichavillanueva, Peter Cresswell, Michelle Golding, Sarah Mcevoy, Nilofar Sarvaiya, Sandra O Gollnick, Naveen BangiaAbstract:Assembly of major histocompatibility complex (MHC) class I molecules with peptide in the endoplasmic reticulum requires the assistance of Tapasin. Alternative splicing, which is known to regulate many genes, has been reported for Tapasin only in the context of mutations. Here, we report on an alternate splice form of Tapasin (tpsnΔEx3) derived from a human melanoma cell line that does not appear to be caused by mutations. Excision of exon 3 results in deletion of amino acids 70 to 156 within the beta barrel region, but the membrane proximal Ig domain, the transmembrane domain, and cytoplasmic tail of Tapasin are intact. Introduction of tpsnΔEx3 into a Tapasin-deficient cell line does not restore MHC class I expression at the cell surface. Similar to a previously described Tapasin mutant (tpsnΔN50), tpsnΔEx3 interacts with TAP. Therefore, we used these altered forms of Tapasin to test the importance of MHC class I interaction with TAP. In the presence of wild-type Tapasin, transfection of tpsnΔN50, but not tpsnΔEx3, reduced MHC class I expression at the cell surface likely due its ability to compete MHC class I molecules from TAP. Together these findings suggest that tumor cells may contain alternate splice forms of Tapasin which may regulate MHC class I antigen presentation.
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functional significance of Tapasin membrane association and disulfide linkage to erp57 in mhc class i presentation
European Journal of Immunology, 2009Co-Authors: Nathalie Vigneron, David R. Peaper, Ralf M Leonhardt, Peter CresswellAbstract:Tapasin is disulfide linked to ERp57 within the peptide loading complex. In cell-free assays, a soluble variant of the Tapasin/ERp57 dimer recruits MHC class I molecules and promotes peptide binding to them, whereas soluble Tapasin alone does not. Here we show that within cells, Tapasin conjugation with ERp57 is as critical as its integration into the membrane for efficient MHC class I assembly, surface expression, and Ag presentation to CD8(+) T cells. Elimination of both of these properties severely compromises Tapasin function, in keeping with predictions from in vitro studies.
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insights into mhc class i peptide loading from the structure of the Tapasin erp57 thiol oxidoreductase heterodimer
Immunity, 2009Co-Authors: Gang Dong, Peter Cresswell, David R. Peaper, Pamela A Wearsch, Karin M ReinischAbstract:Summary Tapasin is a glycoprotein critical for loading major histocompatibility complex (MHC) class I molecules with high-affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here, we present the 2.6 A resolution structure of the Tapasin-ERp57 core of the PLC. The structure revealed that Tapasin interacts with both ERp57 catalytic domains, accounting for the stability of the heterodimer, and provided an example of a protein disulfide isomerase family member interacting with substrate. Mutational analysis identified a conserved surface on Tapasin that interacted with MHC class I molecules and was critical for peptide loading and editing functions of the Tapasin-ERp57 heterodimer. By combining the Tapasin-ERp57 structure with those of other defined PLC components, we present a molecular model that illuminates the processes involved in MHC class I peptide loading.
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selective loading of high affinity peptides onto major histocompatibility complex class i molecules by the Tapasin erp57 heterodimer
Nature Immunology, 2007Co-Authors: Pamela A Wearsch, Peter CresswellAbstract:Major histocompatibility complex (MHC) class I glycoproteins bind peptides in the endoplasmic reticulum after incorporation into the peptide-loading complex, whose core is the transporter associated with antigen processing. Other components are the chaperone calreticulin, the thiol oxidoreductase ERp57, and Tapasin. Tapasin and ERp57 have been shown to exist in the peptide-loading complex as a disulfide-linked heterodimer. Here, using a cell-free system, we demonstrate that although recombinant Tapasin was ineffective in recruiting MHC class I molecules and facilitating peptide binding, recombinant Tapasin-ERp57 conjugates accomplished both of those functions and also 'edited' the repertoire of bound peptides to maximize their affinity. Thus, the Tapasin-ERp57 conjugate is the functional unit of the peptide-loading complex that generates MHC class I molecules with stably associated peptides.
Kajsa Paulsson - One of the best experts on this subject based on the ideXlab platform.
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hla class i is most tightly linked to levels of Tapasin compared with other antigen processing proteins in glioblastoma
British Journal of Cancer, 2015Co-Authors: Camilla Thuring, Linda Geironson, Mikkel Harndahl, Soren Buus, Elna Follin, Eva Freyhult, Victoria Junghans, Kajsa PaulssonAbstract:Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of Tapasin has been linked to higher malignancy in several different tumours.
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Tapasin and human leukocyte antigen class i dysregulation correlates with survival in glioblastoma multiforme
Anti-cancer Agents in Medicinal Chemistry, 2014Co-Authors: Camilla Thuring, Linda Geironson, Kajsa PaulssonAbstract:Human leukocyte antigen class I (HLA-I) molecules present antigenic peptides to cytotoxic CD8(+) T cells. Downregulation of peptide:HLA-I complexes is common in tumors and results in tumor immune escape variants. Also molecules involved in the maturation of HLA-I have been demonstrated to be dysregulated in malignant neoplasms. We here set out to investigate the antigen presentation capabilities of a set of 12 glioblastoma multiforme (GBM) tumors based on the expression of HLA-I. Moreover, we analyzed the expression of Tapasin, a protein dedicated and essential to HLA-I maturation, as well as the infiltration of CD8+ cells using immunohistochemistry on paraffin-embedded sections. Comparison of different GBMs showed a variation in expression of both HLA-I heavy chain (HC) and Tapasin. Interestingly, the expression of Tapasin and HLA-I HC correlated significantly (p=0.0002) suggesting Tapasin to be a key factor for efficient HLA-I antigen presentation in GBMs. Although no statistically significant correlation between CD8(+) cells and survival was found, probably due to a very low number of infiltrating CD8(+) cells at the time of surgical resection, both Tapasin and HLA-I HC levels significantly correlated with survival. We suggest that analysis of expression of Tapasin and/or HLA-I may be of value as prognostic tool for GBM patients, especially when considering immunotherapy.
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Tapasin facilitation of natural hla a and b allomorphs is strongly influenced by peptide length depends on stability and separates closely related allomorphs
Journal of Immunology, 2013Co-Authors: Linda Geironson, Camilla Thuring, Mikkel Harndahl, Michael R Rasmussen, Soren Buus, Gustav Roder, Kajsa PaulssonAbstract:Despite an abundance of peptides inside a cell, only a small fraction is ultimately presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and are restricted in length. So far, detailed length studies have been limited to few allomorphs. Peptide-HLA-I (pHLA-I) complexes of different allomorphs are qualitatively and quantitatively influenced by Tapasin to different degrees, but again, its effect has only been investigated for a small number of HLA-I allomorphs. Although both peptide length and Tapasin dependence are known to be important for HLA-I peptide presentation, the relationship between them has never been studied. In this study, we used random peptide libraries from 7- to 13-mers and studied binding in the presence and absence of a recombinant truncated form of Tapasin. The data show that HLA-I allomorphs are differentially affected by Tapasin, different lengths of peptides generated different amounts of pHLA-I complexes, and HLA-A allomorphs are generally less restricted than HLA-B allomorphs to peptides of the classical length of 8-10 aa. We also demonstrate that Tapasin facilitation varies for different peptide lengths, and that the correlation between high degree of Tapasin facilitation and low stability is valid for different random peptide mixes of specific lengths. In conclusion, these data show that Tapasin has specificity for the combination of peptide length and HLA-I allomorph, and suggest that Tapasin promotes formation of pHLA-I complexes with high on and off rates, an important intermediary step in the HLA-I maturation process.
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stability of peptide hla i complexes and Tapasin folding facilitation tools to define immunogenic peptides
FEBS Letters, 2012Co-Authors: Linda Geironson, Kajsa Paulsson, Gustav RoderAbstract:Only a small fraction of the peptides generated inside the cell end up being presented by HLA-I on the cell surface. High stability of peptide-HLA-I complexes and a low HLA-I Tapasin-facilitation have been proposed to predict immunogenicity. We here set out to investigate if these parameters correlated and defined immunogenic peptides. Both peptide-HLA-B*08:01 and peptide-HLA-A*02:01 complexes showed small differences in Tapasin-facilitation and larger differences in stability. This suggests that the stability of immunogenic peptide-HLA-I complexes vary above an HLA-I allomorph dependent lower limit (e. g. > 2 h for HLA-A*02:01), immunogenicity predicted by Tapasin-facilitation may be defined by an equally allomorph unique upper value (e. g. Tapasin-facilitation <1.5 for HLA-A*02:01), and variation above the stability-threshold does not directly reflect a variation in Tapasin-facilitation. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved. (Less)
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Tapasin discriminates peptide human leukocyte antigen a 02 01 complexes formed with natural ligands
Journal of Biological Chemistry, 2011Co-Authors: Gustav Roder, Kajsa Paulsson, Linda Geironson, Mikkel Harndahl, Michael R Rasmussen, Soren BuusAbstract:A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of Tapasin, Tpn1–87, assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established Tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn1–87 could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of Tapasin, are major features of Tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.
James Mccluskey - One of the best experts on this subject based on the ideXlab platform.
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optimization of the mhc class i peptide cargo is dependent on Tapasin
Immunity, 2002Co-Authors: Anthony P Williams, Anthony W Purcell, James Mccluskey, Tim ElliottAbstract:The loading of MHC class I molecules with their peptide cargo is undertaken by a multimolecular peptide loading complex within the endoplasmic reticulum. We show that MHC class I molecules can optimize their peptide repertoire over time and that this process is dependent on Tapasin. Optimization of the peptide repertoire is both quantitatively and qualitatively improved by Tapasin. The extent of optimization is maximal when MHC class I molecules are allowed to load within the fully assembled peptide loading complex. Finally, we identify a single natural polymorphism (116D>Y) in HLA-B*4402 that permits Tapasin-independent loading of HLA-B*4405 (116Y). In the presence of Tapasin, the Tapasin-independent allele B*4405 (116Y) acquires a repertoire of peptides that is less optimal than the Tapasin-dependent allele B*4402 (116D).
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Tapasin mediated retention and optimization of peptide ligands during the assembly of class i molecules
Journal of Immunology, 2000Co-Authors: Megan J Barnden, Anthony W Purcell, Jeffrey J Gorman, James MccluskeyAbstract:The murine class I H-2Kb molecule achieves high level surface expression in Tapasin-deficient 721.220 human cells. Compared with their behavior in wild-type cells, Kb molecules expressed on 721.220 cells are more receptive to exogenous peptide, undergo more rapid surface decay, and fail to form macromolecular peptide loading complexes. As a result, they are rapidly transported to the cell surface, reflecting a failure of endoplasmic reticulum retention mechanisms in the absence of loading complex formation. Despite the failure of Kb molecules to colocalize to the TAP and their rapid egress to the cell surface, Kb is still capable of presenting TAP-dependent peptides in the absence of Tapasin. Furthermore, pool sequencing of peptides eluted from these molecules revealed strict conservation of their canonical H-2Kb-binding motif. There was a reduction in the total recovery of peptides associated with Kb molecules purified from the surface of Tapasin-deficient cells. Comparison of the peptides bound to Kb in the presence and absence of Tapasin revealed considerable overlap in peptide repertoire. These results indicate that in the absence of an interaction with Tapasin, Kb molecules fail to assemble with calreticulin and TAP, yet they are still capable of acquiring a diverse array of peptides. However, a significant proportion of these peptides appear to be suboptimal, resulting in reduced cell surface stability of Kb complexes. Taken together, the findings indicate that Tapasin plays an essential role in the formation of the class I loading complex, which retains class I heterodimers in the endoplasmic reticulum until optimal ligand selection is completed.
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distinct functions of Tapasin revealed by polymorphism in mhc class i peptide loading
Journal of Immunology, 2000Co-Authors: Chen Au Peh, Scott R Burrows, Nihay Laham, Yong Zhu, James MccluskeyAbstract:Peptide assembly with class I molecules is orchestrated by multiple chaperones including Tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine Tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine Tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine Tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the Tapasin-deficient human cell line 721.220. These findings indicate distinct roles for Tapasin in class I peptide loading. First, Tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, Tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of Tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.
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hla b27 restricted antigen presentation in the absence of Tapasin reveals polymorphism in mechanisms of hla class i peptide loading
Immunity, 1998Co-Authors: Scott R Burrows, Peter Cresswell, James Mccluskey, Megan J Barnden, Rajiv Khanna, Denis J MossAbstract:Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on Tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of Tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human Tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires Tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or Tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.
Guoqing Zang - One of the best experts on this subject based on the ideXlab platform.
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correlation between low Tapasin expression and impaired cd8 t cell function in patients with chronic hepatitis b
Molecular Medicine Reports, 2016Co-Authors: Yuyan Tang, Jieling Wang, Yi Zhang, Meng Zhuo, Linlin Song, Zhenghao Tang, Guoqing Zang, Xiaohua ChenAbstract:Abstract Recent studies have demonstrated that chronic hepatitis B virus (HBV) infection is associated with reduced antigen‑presenting capacity and insufficient cytotoxic T lymphocyte (CTL) production. The molecular chaperone Tapasin mediates binding of the transporter associated with antigen processing (TAP), and has an important role in endogenous antigen processing and presentation, and the induction of specific CTL responses. The present study aimed to determine whether Tapasin is associated with chronic HBV (CHB) infection. The mRNA expression levels of Tapasin were detected in peripheral blood mononuclear cells from 27 patients with CHB, 20 patients with acute HBV (AHB) and 26 healthy controls by reverse transcription‑quantitative polymerase chain reaction. In addition, CD8+ T immune responses were evaluated in all groups, and the correlation between Tapasin expression and CD8+ responses was analyzed. The results demonstrated that the mRNA expression levels of Tapasin were significantly downregulated in patients with CHB compared with in healthy controls and patients with AHB. Furthermore, the apoptotic rate of CD8+ T cells was increased in patients with CHB compared with in the other two groups. The percentage of interferon (IFN)‑γ+CD8+ T cells was reduced in patients with CHB compared with in patients with AHB and healthy controls, and serum cytokine levels (IFN‑γ, interleukin‑2 and tumor necrosis factor‑α) were generally low in patients with CHB. Furthermore, the mRNA expression levels of Tapasin were positively correlated with IFN‑γ production by CD8+ T cells, and were inversely correlated with the apoptotic ratio of CD8+ T cells. These results indicate that decreased expression of Tapasin may be closely associated with CHB, and suggest an important role for Tapasin in the pathogenesis of CHB.
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Tapasin modification on the intracellular epitope hbcag18 27 enhances hbv specific ctl immune response and inhibits hepatitis b virus replication in vivo
Laboratory Investigation, 2014Co-Authors: Xiaohua Chen, Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Guoqing ZangAbstract:HBV-specific cytotoxic T-lymphocyte (CTL) activity has a very important role in hepatitis B virus clearance. Present studies suggest that Tapasin, a endoplasmic reticulum (ER) chaperone, stabilizes the peptide-receptive MHC I conformation, allowing peptide exchange and increasing more peptides to be translocated into the ER. We have previously testified that cytoplasmic transduction peptide (CTP)-HBcAg18–27-Tapasin fusion protein could enter cytoplasm of dendritic cells, and enhance T cells’ response to generate specific CTLs efficiently in vitro. In the present study, we evaluated specific immune responses of CTP-HBcAg18–27-Tapasin fusion protein in HLA-A2 transgenic mice (H-2Kb) and anti-viral ability in HBV transgenic mice, and explored the mechanisms probably involved in. The studies showed that CTP-HBcAg18–27-Tapasin not only increased production of cytokine IFN-γ and interleukin-2 (IL-2), compared with CTP-HBcAg18–27, HBcAg18–27-Tapasin, and PBS, but also significantly induced the higher percentages of IFN-γ+CD8+ T cells and specific CTL responses in HLA-A2 transgenic mice. Moreover, enhancement of specific CTL activity induced by the fusion protein reduced HBV DNA and hepatitis B surface antigen (HBsAg) levels and decreased the expression of HBsAg and hepatitis B core antigen (HBcAg) in liver tissue of HBV transgenic mice. In addition, CTP-HBcAg18–27-Tapasin could upregulate the expression of JAK2, Tyk2, STAT1, and STAT4 in T lymphocytes in HLA-A2 transgenic mice splenocytes. However, there was no significant difference on the expressions of JAK1, JAK3, and STAT6 between each group. In conclusion, CTP-HBcAg18–27-Tapasin fusion protein could enhance not only the percentages of CTLs but also induce robust specific CTL activity and inhibits hepatitis B virus replication in vivo, which was associated with activation of the JAK/STAT signaling pathway.
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fusion protein of Tapasin and hepatitis b core antigen 18 27 enhances t helper cell type 1 2 cytokine ratio and antiviral immunity by inhibiting suppressors of cytokine signaling family members 1 3 in hepatitis b virus transgenic mice
Molecular Medicine Reports, 2014Co-Authors: Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Xiaohua Chen, Peng Wang, Guoqing ZangAbstract:Persistent hepatitis B virus (HBV) infection is characterized by a weak adaptive immune response, which is considered to be due to an imbalance of T helper cell types 1 and 2 (Th1/Th2). Suppressors of cytokine signaling (SOCS) family members, particularly SOCS1 and SOCS3, have been demonstrated to be important in the regulation of T cell differentiation. Previous studies by our group showed that the expressed and purified fusion protein of cytoplasmic transduction peptide (CTP) and HBV core antigen 18‑27 (HBcAg18‑27)‑Tapasin was able to enter the cytoplasm of bone marrow‑derived dendritic cells (BMDCs), promoting the maturation of BMDCs and efficiently enhancing T cell immune responses in vitro. In the present study, HBcAg‑specific immune responses induced by CTP‑HBcAg18‑27‑Tapasin in HBV were assessed in transgenic mice, and SOCS1 and SOCS3 were identified as negative regulators of this response. The Th1/Th2 cytokine ratio was analyzed by ELISA. The expression of T cell‑specific T‑box transcription factor (T‑bet) and GATA‑binding protein 3 (GATA‑3), SOCS1 and SOCS3 were detected by real‑time quantitative polymerase chain reaction and western blot analysis. The results demonstrated that CTP‑HBcAg18‑27‑Tapasin significantly increased the Th1/Th2 cytokine ratio in HBV transgenic mice. CTP‑HBcAg18‑27‑Tapasin immunization more efficiently suppressed the expression of serum hepatitis B surface antigen (HBsAg), HBV DNA as well as liver HBsAg and HBcAg in HBV transgenic mice. Furthermore, CTP‑HBcAg18‑27‑Tapasin promotes T‑bet but reduces GATA‑3 expression. In addition, the expression of SOCS1 and SOCS3 was significantly downregulated in the CTP‑HBcAg18‑27‑Tapasin group compared with the control groups. In conclusion, the present study demonstrated that CTP‑HBcAg18‑27‑Tapasin enhanced the Th1/Th2 cytokine ratio and antiviral immunity by suppressing SOCS1/3 in HBV transgenic mice.
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the fusion protein of ctp hbcag18 27 Tapasin mediates the apoptosis of cd8 t cells and cd8 t cell response in hla a2 transgenic mice
Hepatitis Monthly, 2014Co-Authors: Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Guoqing Zang, Xiaohua ChenAbstract:Background: HBV-specific cytotoxic T lymphocyte (CTL) activity is believed to play a critical role in controlling HBV infection. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway manipulates cell fate decisions in many different cell types by regulating the activity of downstream effectors. We have previously testified that the fusion protein of CTP-HBcAg18-27--Tapasin could enter the cytoplasm of dendritic cells and efficiently induce robust specific CTL response in vitro. Objectives: In the present study, we evaluated specific CTL response and the level of apoptosis of CD8+ T cells induced by CTP-HBcAg18-27- Tapasin in HLA-A2 transgenic mice (H-2Kb). Meanwhile, we preliminary investigated PI3K, phosphorylation level of Akt, and mammalian target of rapamycin (mTOR) as positive regulator of the magnitude and effector function of the hepatitis B virus-specific cytotoxic T lymphocytes in HLA-A2 transgenic mice. Materials and Methods: HLA-A2 transgenic mice were immunized by intramuscular injection in the hind legs three times at one-week intervals with PBS, CTP-HBcAg18-27-Tapasin (50 μg), CTP-HBcAg18-27 (50 μg), HBcAg18-27-Tapasin (50 μg), and HBcAg18-27 (50 μg). One week after the last immunization, mice were sacrificed and splenocytes were harvested in strile condition. The specific CTL response was analyzed by flow cytometry and enzyme linked immunosorbent assay (ELISA); the expression of (PI3K)/Akt signaling was detected by RT- PCR and western blot. Results: The results showed that CTP-HBcAg18-27-Tapasin significantly increased the percentages of IFN-γ + CD8α + T cells, the numbers of these polyfunctional triple-cytokine-producing (IFN-γ, TNF-α, and IL-2) CD8 + T cells, the secretion of cytokine IFN-γ, IL-2, and TNF-α, while in comparison to control group, it significantly decreased the percentage of apoptotic CD8 + T cells in HLA-A2 transgenic mice. Moreover, the expression of PI3K, P-Akt, and P-mTOR was significantly upregulated in CTP-HBcAg18-27-Tapasin group compared with control groups. Conclusions: In conclusion, CTP-HBcAg18-27-Tapasin could reduce apoptosis of CD8 + T cells, increase the percentages of IFN-γ + CD8α + T cells, and elicit cell-mediated immunity in HLA-A2 transgenic mice; these processes were associated with activation of the PI3K/Akt signaling pathway.
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the modification of Tapasin enhances cytotoxic t lymphocyte activity of intracellularly delivered ctl epitopes via cytoplasmic transduction peptide
Acta Biochimica et Biophysica Sinica, 2013Co-Authors: Xiaohua Chen, Zhenghao Tang, Honghong Liu, Guoqing ZangAbstract:Previous studies have demonstrated that the therapeutic vaccine based on the enhancement of hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) activity may lead to viral clearance in HBV-infected individuals. The endoplasmic reticulum (ER) chaperone Tapasin plays an important role in major histocompatibility complex (MHC) class I assembly and enhances specific MHC class Irestricted CTL activity by allowing more peptides to be translocated into the ER. Combining the specificity of hepatitis B core antigen (HBcAg) CTL epitope, the cell-penetrating property of cytoplasmic transduction peptide (CTP), and chaperone Tapasin may elicit robust specific HBV immune responses. In the present study, we confirmed the cytoplasmic localization preference of CTP-HBcAg 18 –2 7-Tapasin fusion protein in vitro and evaluated the effects on promoting bone marrow-derived dendritic cells (BMDCs) maturation and enhancing T cells response to generate specific CTLs. Our results showed that CTP-HBcAg18 – 27-Tapasin fusion protein could not only penetrate into the cytoplasm exactly and effectively to elevate Tapasin expression, but also increase the expression of surface molecules (CD80, CD83, CD86, and MHC-I) and secretion of cytokine (IL-12p70) of DCs. Moreover, DCs treated with the above fusion proteins increased significantly the cytokine secretion of proliferated Tc ellsin vitro, the percentages of IFN-g 1 CD8 1 T cells and specific CTL responses compared with control groups. In conclusion, the modification of Tapasin can enhance the presentation of targeting antigens via intracellular delivery to DCs and elicit specific CTL immune responses efficiently.
Xiaohua Chen - One of the best experts on this subject based on the ideXlab platform.
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correlation between low Tapasin expression and impaired cd8 t cell function in patients with chronic hepatitis b
Molecular Medicine Reports, 2016Co-Authors: Yuyan Tang, Jieling Wang, Yi Zhang, Meng Zhuo, Linlin Song, Zhenghao Tang, Guoqing Zang, Xiaohua ChenAbstract:Abstract Recent studies have demonstrated that chronic hepatitis B virus (HBV) infection is associated with reduced antigen‑presenting capacity and insufficient cytotoxic T lymphocyte (CTL) production. The molecular chaperone Tapasin mediates binding of the transporter associated with antigen processing (TAP), and has an important role in endogenous antigen processing and presentation, and the induction of specific CTL responses. The present study aimed to determine whether Tapasin is associated with chronic HBV (CHB) infection. The mRNA expression levels of Tapasin were detected in peripheral blood mononuclear cells from 27 patients with CHB, 20 patients with acute HBV (AHB) and 26 healthy controls by reverse transcription‑quantitative polymerase chain reaction. In addition, CD8+ T immune responses were evaluated in all groups, and the correlation between Tapasin expression and CD8+ responses was analyzed. The results demonstrated that the mRNA expression levels of Tapasin were significantly downregulated in patients with CHB compared with in healthy controls and patients with AHB. Furthermore, the apoptotic rate of CD8+ T cells was increased in patients with CHB compared with in the other two groups. The percentage of interferon (IFN)‑γ+CD8+ T cells was reduced in patients with CHB compared with in patients with AHB and healthy controls, and serum cytokine levels (IFN‑γ, interleukin‑2 and tumor necrosis factor‑α) were generally low in patients with CHB. Furthermore, the mRNA expression levels of Tapasin were positively correlated with IFN‑γ production by CD8+ T cells, and were inversely correlated with the apoptotic ratio of CD8+ T cells. These results indicate that decreased expression of Tapasin may be closely associated with CHB, and suggest an important role for Tapasin in the pathogenesis of CHB.
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Tapasin modification on the intracellular epitope hbcag18 27 enhances hbv specific ctl immune response and inhibits hepatitis b virus replication in vivo
Laboratory Investigation, 2014Co-Authors: Xiaohua Chen, Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Guoqing ZangAbstract:HBV-specific cytotoxic T-lymphocyte (CTL) activity has a very important role in hepatitis B virus clearance. Present studies suggest that Tapasin, a endoplasmic reticulum (ER) chaperone, stabilizes the peptide-receptive MHC I conformation, allowing peptide exchange and increasing more peptides to be translocated into the ER. We have previously testified that cytoplasmic transduction peptide (CTP)-HBcAg18–27-Tapasin fusion protein could enter cytoplasm of dendritic cells, and enhance T cells’ response to generate specific CTLs efficiently in vitro. In the present study, we evaluated specific immune responses of CTP-HBcAg18–27-Tapasin fusion protein in HLA-A2 transgenic mice (H-2Kb) and anti-viral ability in HBV transgenic mice, and explored the mechanisms probably involved in. The studies showed that CTP-HBcAg18–27-Tapasin not only increased production of cytokine IFN-γ and interleukin-2 (IL-2), compared with CTP-HBcAg18–27, HBcAg18–27-Tapasin, and PBS, but also significantly induced the higher percentages of IFN-γ+CD8+ T cells and specific CTL responses in HLA-A2 transgenic mice. Moreover, enhancement of specific CTL activity induced by the fusion protein reduced HBV DNA and hepatitis B surface antigen (HBsAg) levels and decreased the expression of HBsAg and hepatitis B core antigen (HBcAg) in liver tissue of HBV transgenic mice. In addition, CTP-HBcAg18–27-Tapasin could upregulate the expression of JAK2, Tyk2, STAT1, and STAT4 in T lymphocytes in HLA-A2 transgenic mice splenocytes. However, there was no significant difference on the expressions of JAK1, JAK3, and STAT6 between each group. In conclusion, CTP-HBcAg18–27-Tapasin fusion protein could enhance not only the percentages of CTLs but also induce robust specific CTL activity and inhibits hepatitis B virus replication in vivo, which was associated with activation of the JAK/STAT signaling pathway.
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fusion protein of Tapasin and hepatitis b core antigen 18 27 enhances t helper cell type 1 2 cytokine ratio and antiviral immunity by inhibiting suppressors of cytokine signaling family members 1 3 in hepatitis b virus transgenic mice
Molecular Medicine Reports, 2014Co-Authors: Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Xiaohua Chen, Peng Wang, Guoqing ZangAbstract:Persistent hepatitis B virus (HBV) infection is characterized by a weak adaptive immune response, which is considered to be due to an imbalance of T helper cell types 1 and 2 (Th1/Th2). Suppressors of cytokine signaling (SOCS) family members, particularly SOCS1 and SOCS3, have been demonstrated to be important in the regulation of T cell differentiation. Previous studies by our group showed that the expressed and purified fusion protein of cytoplasmic transduction peptide (CTP) and HBV core antigen 18‑27 (HBcAg18‑27)‑Tapasin was able to enter the cytoplasm of bone marrow‑derived dendritic cells (BMDCs), promoting the maturation of BMDCs and efficiently enhancing T cell immune responses in vitro. In the present study, HBcAg‑specific immune responses induced by CTP‑HBcAg18‑27‑Tapasin in HBV were assessed in transgenic mice, and SOCS1 and SOCS3 were identified as negative regulators of this response. The Th1/Th2 cytokine ratio was analyzed by ELISA. The expression of T cell‑specific T‑box transcription factor (T‑bet) and GATA‑binding protein 3 (GATA‑3), SOCS1 and SOCS3 were detected by real‑time quantitative polymerase chain reaction and western blot analysis. The results demonstrated that CTP‑HBcAg18‑27‑Tapasin significantly increased the Th1/Th2 cytokine ratio in HBV transgenic mice. CTP‑HBcAg18‑27‑Tapasin immunization more efficiently suppressed the expression of serum hepatitis B surface antigen (HBsAg), HBV DNA as well as liver HBsAg and HBcAg in HBV transgenic mice. Furthermore, CTP‑HBcAg18‑27‑Tapasin promotes T‑bet but reduces GATA‑3 expression. In addition, the expression of SOCS1 and SOCS3 was significantly downregulated in the CTP‑HBcAg18‑27‑Tapasin group compared with the control groups. In conclusion, the present study demonstrated that CTP‑HBcAg18‑27‑Tapasin enhanced the Th1/Th2 cytokine ratio and antiviral immunity by suppressing SOCS1/3 in HBV transgenic mice.
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the fusion protein of ctp hbcag18 27 Tapasin mediates the apoptosis of cd8 t cells and cd8 t cell response in hla a2 transgenic mice
Hepatitis Monthly, 2014Co-Authors: Yuyan Tang, Yi Zhang, Meng Zhuo, Zhenghao Tang, Guoqing Zang, Xiaohua ChenAbstract:Background: HBV-specific cytotoxic T lymphocyte (CTL) activity is believed to play a critical role in controlling HBV infection. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway manipulates cell fate decisions in many different cell types by regulating the activity of downstream effectors. We have previously testified that the fusion protein of CTP-HBcAg18-27--Tapasin could enter the cytoplasm of dendritic cells and efficiently induce robust specific CTL response in vitro. Objectives: In the present study, we evaluated specific CTL response and the level of apoptosis of CD8+ T cells induced by CTP-HBcAg18-27- Tapasin in HLA-A2 transgenic mice (H-2Kb). Meanwhile, we preliminary investigated PI3K, phosphorylation level of Akt, and mammalian target of rapamycin (mTOR) as positive regulator of the magnitude and effector function of the hepatitis B virus-specific cytotoxic T lymphocytes in HLA-A2 transgenic mice. Materials and Methods: HLA-A2 transgenic mice were immunized by intramuscular injection in the hind legs three times at one-week intervals with PBS, CTP-HBcAg18-27-Tapasin (50 μg), CTP-HBcAg18-27 (50 μg), HBcAg18-27-Tapasin (50 μg), and HBcAg18-27 (50 μg). One week after the last immunization, mice were sacrificed and splenocytes were harvested in strile condition. The specific CTL response was analyzed by flow cytometry and enzyme linked immunosorbent assay (ELISA); the expression of (PI3K)/Akt signaling was detected by RT- PCR and western blot. Results: The results showed that CTP-HBcAg18-27-Tapasin significantly increased the percentages of IFN-γ + CD8α + T cells, the numbers of these polyfunctional triple-cytokine-producing (IFN-γ, TNF-α, and IL-2) CD8 + T cells, the secretion of cytokine IFN-γ, IL-2, and TNF-α, while in comparison to control group, it significantly decreased the percentage of apoptotic CD8 + T cells in HLA-A2 transgenic mice. Moreover, the expression of PI3K, P-Akt, and P-mTOR was significantly upregulated in CTP-HBcAg18-27-Tapasin group compared with control groups. Conclusions: In conclusion, CTP-HBcAg18-27-Tapasin could reduce apoptosis of CD8 + T cells, increase the percentages of IFN-γ + CD8α + T cells, and elicit cell-mediated immunity in HLA-A2 transgenic mice; these processes were associated with activation of the PI3K/Akt signaling pathway.
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the modification of Tapasin enhances cytotoxic t lymphocyte activity of intracellularly delivered ctl epitopes via cytoplasmic transduction peptide
Acta Biochimica et Biophysica Sinica, 2013Co-Authors: Xiaohua Chen, Zhenghao Tang, Honghong Liu, Guoqing ZangAbstract:Previous studies have demonstrated that the therapeutic vaccine based on the enhancement of hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) activity may lead to viral clearance in HBV-infected individuals. The endoplasmic reticulum (ER) chaperone Tapasin plays an important role in major histocompatibility complex (MHC) class I assembly and enhances specific MHC class Irestricted CTL activity by allowing more peptides to be translocated into the ER. Combining the specificity of hepatitis B core antigen (HBcAg) CTL epitope, the cell-penetrating property of cytoplasmic transduction peptide (CTP), and chaperone Tapasin may elicit robust specific HBV immune responses. In the present study, we confirmed the cytoplasmic localization preference of CTP-HBcAg 18 –2 7-Tapasin fusion protein in vitro and evaluated the effects on promoting bone marrow-derived dendritic cells (BMDCs) maturation and enhancing T cells response to generate specific CTLs. Our results showed that CTP-HBcAg18 – 27-Tapasin fusion protein could not only penetrate into the cytoplasm exactly and effectively to elevate Tapasin expression, but also increase the expression of surface molecules (CD80, CD83, CD86, and MHC-I) and secretion of cytokine (IL-12p70) of DCs. Moreover, DCs treated with the above fusion proteins increased significantly the cytokine secretion of proliferated Tc ellsin vitro, the percentages of IFN-g 1 CD8 1 T cells and specific CTL responses compared with control groups. In conclusion, the modification of Tapasin can enhance the presentation of targeting antigens via intracellular delivery to DCs and elicit specific CTL immune responses efficiently.
