Role of the cellular transcription factor YY1 in the latent-lytic switch of Kaposi's sarcoma-associated herpesvirus.

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) is initiated by expression of the ORF50 gene. Here we show that YY1 protein specifically binds to the ORF50 promoter (ORF50p) region in vitro and in vivo. After treatment with chemical inducers, including sodium butyrate (SB) and TPA, the levels of YY1 protein are inversely correlated with the lytic induction of KSHV in cells. Overexpression of YY1 completely blocks the ORF50p activation in transient reporter assays, while mutation at the YY1 site in the ORF50p or knockdown of YY1 protein confers an enhancement of the ORF50p activation induced by SB and TPA. YY1 overexpression in a stable cell clone HH-B2(Dox-YY1) also inhibits expression of the ORF50 and its downstream lytic genes. On the other hand, a chimeric YY1 construct that links to its coactivator E1A can disrupt viral latency. These results imply that YY1 is involved in the regulation of KSHV reactivation.

Transcriptional regulation of the ORF61 and ORF60 genes of Kaposi's sarcoma-associated herpesvirus.

The ORF61 and ORF60 genes of Kaposi's sarcoma-associated herpesvirus (KSHV) encode the ribonucleotide reductase large and small subunits, respectively. Here we show that ORF50 protein, a latent-lytic switch transactivator, activates the transcription of these two early-lytic genes through different mechanisms. Activation of the ORF61 promoter by ORF50 protein is dependent on an intact RBP-Jkappa-binding site within the identified responsive element and the expression of RBP-Jkappa protein in cells. The critical element in the ORF60 promoter in response to ORF50 was mapped to a 40-bp region. Binding of YY1, Sp1/Sp3 or unknown proteins to this element may contribute to repression or activation of the ORF60 promoter. Although ORF50 protein does not directly bind to the ORF61 and ORF60 promoters in vitro, we show the association of ORF50 protein with these two promoters in vivo. Our results provide further insights into the regulatory network of the viral lytic genes in KSHV reactivation.

Construction and characterization of a novel fusion protein consisting of anti-CD3 antibody fused to recombinant interleukin-2.

T cells can be activated in vitro by monoclonal antibodies to CD3 (anti-CD3) to become non-MHC restricted killer cells (CD3-AK). Anti-CD3 activation upregulates the expression of the interleukin (IL)-2 receptors on T cells whose expansion is facilitated by IL-2. The therapeutic effect of in vivo administration of anti-CD3 and IL-2 has been investigated in many types of human cancers. To circumvent the toxicities posed by systemic administration of high-dose IL-2, there is interest in forming a strategy for targeting and concentrating IL-2 at the site where it is needed. This study investigates the feasibility of constructing a novel fusion protein consisting of IL-2 fused to the constant region of anti-CD3 antibody. Our results indicate that the specific IL-2 receptor-binding capability and bioactivity of the IL-2 portion as well as the CD3-binding and biological functions of anti-CD3 portion remain intact in this anti-CD3/IL-2 fusion protein. Thus, cytokines fused to anti-CD3 antibody by genetic engineering is feasible and may provide a new class of immunotherapeutics for cancer.

Clonal restriction of the expansion of antigen-specific CD8+ memory T cells by transforming growth factor-{beta}.

Recent evidence showed that transforming growth factor-beta (TGF-beta) regulates the global expansion of CD8+ T cells, which are CD44hi, a marker for memory cells. However, it is not clear whether this regulatory mechanism also applies to the antigen-specific CD8+ memory cells. By using a murine mixed lymphocyte culture (MLC) model, we examined the effect of TGF-beta on antigen-specific CD8+ memory cells [cytotoxic T lymphocyte (CTL)]. We found that the secondary CTL response in CD8+ memory cells from untreated MLC was not affected by TGF-beta but augmented by interleukin (IL)-2, whereas the CD8+ memory cells from TGF-beta-pretreated MLC (MLC-TGF-beta) failed to mount a significant, secondary CTL response, even when IL-2 was added. In exploring this dichotomy, in combination with flow cytometry analysis, we found that prolonged exposure to TGF-beta reduces the CTL activity in CD8+ memory cells. The increase by IL-2 and the reduction by TGF-beta of the CTL responses were clonal-specific. TGF-beta did not affect the CTL response to a third-party antigen or polyclonal T cell activation. Experiments performed with transgenic 2C cells gave similar results. Cell-cycle study performed with adoptive transfer of the cell tracker-labeled MLC cells revealed that the in vivo expansion of CD8+ memory cells from MLC-TGF-beta was restricted severely, and the restriction was clonal-specific, thus offering direct evidence to show that TGF-beta induces clonal restriction of CD8+ memory cell expansion.

Inducing long-term survival with lasting anti-tumor immunity in treating B cell lymphoma by a combined dendritic cell-based and hydrodynamic plasmid-encoding IL-12 gene therapy.

In a previous study we showed that immunization with dendritic cells (DC) pulsed with idiotype (Id) fused with CD40 ligand (CD40L) could break the tolerance to Id which is expressed on B lymphoma cells and restored the responsiveness of T(h) cells, and, subsequently, induced IgG antibody response. However, this treatment had no therapeutic effect. In the present study, we found that using a hydrodynamic transfection-based technique, a high level of IL-12 production was noticed as early as 7 h after administering plasmid encoding IL-12 (pIL-12) and persisted at a detectable level for at least 9 days. In evaluating the efficacy of DC-based and/or IL-12 gene-based therapy in the treatment of 38C13 B cell lymphoma, it was found that either treatment alone was ineffective. However, a combined treatment induced 100% long-term survival. Furthermore, a long-lasting anti-tumor immunity was induced in these mice which resisted further tumor challenge at 58 days after initial inoculation. The surviving mice showed a strong IFN-gamma-producing T(h) cell response and humoral antibody response, but there were no detectable cytotoxic T lymphocytes. The antibody from the immune sera mediated a complement-dependent lysis of tumor cells that was tumor specific. Furthermore, immunization of mice with DC-based vaccine and pIL-12 treatment elicited higher levels of anti-Id IgG titer and an enhanced IgG2a response which increased the efficacy in mediating 38C13 tumor lysis. On examining the mechanism for this isotype change, we found that IFN-gamma production by CD4(+) T cells is not the only determining factor for achieving a successful therapy. DC-based treatment alone could induce the increase of IFN-gamma production, but lacked any therapeutic effect. The deciding factor appears to be the abrogation of IL-4 production that was achieved by combing with IL-12 gene therapy. Our study provides a basis for exploring the combined use of cytokines or cytokine genes in DC-based treatment for achieving effective cancer immunotherapy.