MHC-E-Restricted CD8+ T Cells Target Hepatitis B Virus-Infected Human Hepatocytes.
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ABSTRACT:
SUBMITTER: Burwitz BJ
PROVIDER: S-EPMC8109620 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:AimTo find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray.MethodsPrimary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-kappaB luciferase reporter assay.ResultsFrom the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-alpha, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-kappaB activation.ConclusionDuring the initial state of HBV infection, hepatocytes facilitate the activation of NF-kappaB through up regulation of LT-alpha, TRAF2, and NIK.
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Project description:Hepatitis delta virus (HDV) requires hepatitis B virus (HBV) to complete its infection cycle and causes severe hepatitis, with limited therapeutic options. To determine the prospect of T cell therapy in HBV/HDV co-infection, we study the impact of HDV on viral antigen processing and presentation. Using in vitro models of HBV/HDV co-infection, we demonstrate that HDV boosts HBV epitope presentation, both in HBV/HDV co-infected and neighboring mono-HBV-infected cells through the upregulation of the antigen processing pathway mediated by IFN-β/λ. Liver biopsies of HBV/HDV patients confirm this upregulation. We then validate in vitro and in a HBV/HDV preclinical mouse model that HDV infection increases the anti-HBV efficacy of T cells with engineered T cell receptors. Thus, by unveiling the effect of HDV on HBV antigen presentation, we provide a framework to better understand HBV/HDV immune pathology, and advocate the utilization of engineered HBV-specific T cells as a potential treatment for HBV/HDV co-infection.
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Project description:HIV-1-specific CD4+ T cells (TCD4+s) play a critical role in controlling HIV-1 infection. Canonically, TCD4+s are activated by peptides derived from extracellular ("exogenous") Ags displayed in complex with MHC class II (MHC II) molecules on the surfaces of "professional" APCs such as dendritic cells (DCs). In contrast, activated human TCD4+s, which express MHC II, are not typically considered for their APC potential because of their low endocytic capacity and the exogenous Ag systems historically used for assessment. Using primary TCD4+s and monocyte-derived DCs from healthy donors, we show that activated human TCD4+s are highly effective at MHC II-restricted presentation of an immunodominant HIV-1-derived epitope postinfection and subsequent noncanonical processing and presentation of endogenously produced Ag. Our results indicate that, in addition to marshalling HIV-1-specific immune responses during infection, TCD4+s also act as APCs, leading to the activation of HIV-1-specific TCD4+s.
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Project description:Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.
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Project description:BackgroundHepatitis B virus (HBV) is a major cause of liver infection in human. Because of the lack of an appropriate cell culture system for supporting HBV infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. Duck heptatitis B virus (DHBV) can naturally infect primary duck hepatocytes (PDHs) that provide valuable model systems for studying hepadnavirus infection in vitro. In this report, we explored global changes in cellular protein expression in DHBV infected PDHs by two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF/TOF tandem mass spectrometry (MS/MS).ResultsThe effects of hepadnavirus infection on hepatocytes were investigated in DHBV infected PDHs by the 2-DE analysis. Proteomic profile of PDHs infected with DHBV were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected PDHs, and 75 differentially expressed protein spots were revealed by 2-DE analysis. Among the selected protein spots, 51 spots were identified corresponding to 42 proteins by MS/MS analysis; most of them were matched to orthologous proteins of Gallus gallus, Anas platyrhynchos or other avian species, including alpha-enolase, lamin A, aconitase 2, cofilin-2 and annexin A2, etc. The down-regulated expression of beta-actin and annexin A2 was confirmed by Western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed.ConclusionsDifferentially expressed proteins of DHBV infected PDHs revealed by 2-DE, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis.
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Project description:Background & aimsHepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle.MethodsWe knocked down or overexpressed TM6SF2 in hepatic cells and examined HCV infection, measuring viral RNA and protein levels and infectious LVP titers. The density of secreted LVPs was evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells.ResultsTM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCV to increase the expression of TM6SF2 in hepatic cells.ConclusionsIn studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.
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Project description:In this study, we generated human MHC Class I-restricted CD4+ T cells specific for Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two herpesviridae associated with lymphoma, nasopharyngeal carcinoma and medulloblastoma, respectively. Retroviral transfer of virus-specific, HLA-A2-restricted TCR-coding genes generated CD4+ T cells that recognized HLA-A2/peptide multimers and produced cytokines when stimulated with MHC Class II-deficient cells presenting the relevant viral peptides in the context of HLA-A2. Peptide titration revealed that CD4+ T cells had a 10-fold lower avidity than CD8+ T cells expressing the same TCR. The impaired avidity of CD4+ T cells was corrected by simultaneously transferring TCR- and CD8-coding genes. The CD8 co-receptor did not alter the cytokine signature of CD4+ T cells, which remained distinct from that of CD8+ T cells. Using the xenogeneic NOD/SCID mouse model, we demonstrated that human CD4+ T cells expressing a specific TCR and CD8 can confer efficient protection against the growth of tumors expressing the EBV or CMV antigens recognized by the TCR. In summary, we describe a robust approach for generating therapeutic CD4+ T cells capable of providing MHC Class I-restricted immunity against MHC Class II-negative tumors in vivo.
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