efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Uppsala Biomedical Center, Uppsala University

To understand the interaction between the virus and its host, we used three sources of cDNA microarrays to examine the expression of 12,309 unique genes at 6 h postinfection of HeLa cells with high multiplicities of adenovirus type 2. Seventy-six genes with significantly changed expression ratios were identified, suggesting that adenovirus only modulates expression of a limited set of cellular genes. Quantitative real-time PCR analyses on selected genes were performed to confirm the microarray results. Significantly, a pronounced transcriptional activation by the promiscuous E1A-289R transcriptional activator was not apparent. Instead, promoter sequences in 45% of the upregulated genes harbored a potential E2F binding site, suggesting that the ability of the amino-terminal domain of E1A to regulate E2F-dependent transcription may be a major pathway for regulation of cellular gene expression. CDC25A was the only upregulated gene directly involved in cell cycle control. In contrast, several genes implicated in cell growth arrest were repressed. The transforming growth factor beta superfamily was specifically affected in the expression of both the upstream ligand and an intracellular regulator. In agreement with previous reports, adenovirus also targeted the innate immune response by downregulating several cytokines, including CLL2, CXCL1, and interleukin-6. Finally, stress response genes encoding GADD45B, ATF3, and TP53AP1 were upregulated. Importantly, we also found a novel countermeasure-activation of the apoptosis inhibitor survivin.

Strategic attack on host cell gene expression during adenovirus infection.

The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.

Identification of specific cellular genes up-regulated late in adenovirus type 12 infection.

In cell lines harbouring inducible adenovirus E1A genes, the cytotoxicity of wild type E1A was manifested by poor and subsiding expression of the E1A protein during prolonged induction. In contrast, cells expressing E1A deleted in the C-terminal binding protein (CtBP)-interaction domain (E1ADeltaCID) demonstrated high levels of expression for extended time. Microarray analyses of host cell gene expression demonstrated that approximately 70% of the regulated genes were increased upon E1A induction and that the majority of E1A-regulated genes were similarly regulated by wild type E1A and E1ADeltaCID. However, for 29 genes, regulation by wild type E1A and E1ADeltaCID were different. Consistent with the altered transforming capacity of E1A unable to bind CtBP, genes involved in tumour cell progression and growth suppression were found among the differently regulated genes. Moreover, promoter sequences of genes up regulated by wild type E1A and/or repressed by E1ADeltaCID demonstrated a higher prevalence of potential binding sites for the CtBP-targeted transcription factors Ets, Ikaros and/or partial differentialEF1/ZEB, suggesting that the failure to block CtBP-repression contributed to the "hyper-transforming" phenotype of E1ADeltaCID. Since E1ADeltaCID also specifically activated host cell gene expression, we find it likely that additional, possibly CtBP-independent, mechanisms contribute to the altered phenotype of E1ADeltaCID-expressing cells.

Impact of the interaction between adenovirus E1A and CtBP on host cell gene expression.

Telomerase plays a role in the unlimited replicative capacity of the majority of cancer cells and provides a potential anticancer target. The regulation of telomerase is complex but transcriptional control of its two essential components, hTERC (RNA component) and hTERT (reverse transcriptase component), is of major importance. To investigate this further, we have used the adenoviral protein, E1A, as a tool to probe potential pathways involved in the control of telomerase transcription. The second exon of the adenoviral protein E1A activates both telomerase gene promoters in transient transfections. The corepressor, C terminal binding protein, is one of only two proteins known to bind to this region, and we propose that E1A activates both promoters by sequestering CtBP, thereby relieving repression. Activation by exon 2 of E1A involves the SP1 sites in both promoters, and consistent with this, SP1 and CtBP interact in coimmunoprecipitation studies. Modulation of the chromatin environment has been implicated in the regulation of hTERT transcription and appears to involve the SP1 sites. CtBP can be found within a histone-modifying complex and it is possible that a CtBP complex, associating with the SP1 sites, represses transcription from the telomerase promoters by modifying chromatin structure.

The hTERT and hTERC telomerase gene promoters are activated by the second exon of the adenoviral protein, E1A, identifying the transcriptional corepressor CtBP as a potential repressor of both genes.

Microarray analysis of host cell gene expression during an adenovirus type 2 infection showed that the number of regulated genes, as well as the magnitude of change, was increased as the infection proceeded into the late phase. In contrast to the early phase of infection when the majority of differentially expressed genes were upregulated, expression of most of the regulated genes (82 out of 112) declined during the late phase. In particular, numerous TGF-beta inducible genes and several TGF-beta-independent growth-arrest-inducing genes were targeted. Of the 30 genes upregulated more than 2-fold at 20 h post-infection, nearly two-thirds of encoded proteins are involved in cell metabolism. The data indicate that adenovirus primarily targets cellular genes involved in antiviral defense, cell growth arrest and apoptosis, as well as cell metabolism, to ensure sufficient production of viral progeny.

Modulation of host cell gene expression during onset of the late phase of an adenovirus infection is focused on growth inhibition and cell architecture.

In this report, we have studied gene expression profiles in human primary lung fibroblasts (IMR-90) during the very early phase of an adenovirus infection. Eight out of twelve genes with known functions encoded transcription factors linked to two major cellular processes; inhibition of cell growth (ATF3, ATF4, KLF4, KLF6 and ELK3) and immune response (NR4A1 and CEBPB), indicating that the earliest consequences of an adenovirus infection are growth arrest and induction of an immune response. A time course analysis showed that the induction of these immediate-early response genes was transient and suppressed after the onset of the adenovirus early gene expression.

Adenovirus-induced alterations in host cell gene expression prior to the onset of viral gene expression.

We have previously described a temporal regulation of host cell gene expression during adenovirus type 2 infection (Ad2) of primary human fibroblasts. Among the eleven percent of genes deregulated by Ad2, a large fraction included genes involved in cell cycle, growth control and antiviral defense, consistent with the capacity of Ad2 to efficiently master the infected cell and cause an effectively productive infection. Adenovirus type 12 (Ad12), which belongs to the highly oncogenic subgroup, is characterised by slow progression, less cytopathic effect and lower virus yield compared to the non-oncogenic Ad2. Microarray analysis of host cell gene expression in Ad12 infected human lung fibroblasts (IMR90) demonstrated a quantitatively and qualitatively less impact on host cell gene expression, compared to Ad2. Of the relatively few genes up regulated during the course of Ad12 infection only two (5%) were identified as potential E2F targets, compared to the significant activation of E2F-dependent transcription observed during an Ad2 infection. Although approximately 30% of the genes deregulated by Ad12 were previously identified in Ad2-infected cells, a distinct difference was observed in a group of interferon-stimulated genes (ISGs). G1P2, IFI6, IFI16, IFIT1, IFIT2, IFITM1 and IRF9 were activated during the very late stage of infection, and a consistent induction of IFNbeta gene expression, preceding induction of the ISGs, was demonstrated by quantitative real-time PCR analysis. An activated JAK/STAT signalling pathway was also indicated by the accumulation of all components (STAT1, STAT2 and IRF9) of the ISGF3 transcription factor. Significantly, none of these ISGs was activated in Ad2 infected IMR90 cells. Thus, the inability of Ad12 to evade the interferon response might explain its restricted virulence.

Activation of the interferon-induced STAT pathway during an adenovirus type 12 infection.

Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.

A mouse mammary epithelial cell line permissive for highly efficient human adenovirus growth.

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-β expression was transient, HAdV-12-infected cells maintained high levels of IFN-β expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections.

Catharina Svensson

Department of Medical Biochemistry and Microbiology

Uppsala Biomedical Center, Uppsala University

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Catharina Svensson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medical Biochemistry and Microbiology

Uppsala Biomedical Center, Uppsala University

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Department of Medical Biochemistry and Microbiology