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Supplementary Data from Tumor-Localized Ligation of CD3 and CD28 with Systemic Regulatory T-Cell Depletion Induces Potent Innate and Adaptive Antitumor Responses

Purpose: Tumor-localized activation of immune cells by membrane-tethered anti-CD3 antibodies (CD3L) is under investigation to treat poorly immunogenic tumors. Here we sought to elucidate the mechanism of antitumor immunity elicited by CD3L.Experimental Design: CD3L and CD86 were expressed on poorly immunogenic B16 melanoma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4+ and CD8+ T cells, natural killer T (NKT) cells, and regulatory T cells, on antitumor activity was investigated.Results: B16/3L86 cells activated naïve T cells; suppressed tumor growth in subcutaneous, peritoneal, and metastasis models; and protected mice from rechallenge with B16 melanoma cells. However, in vivo antitumor activity against primary B16/3L86 tumors unexpectedly depended on NKT cells rather than CD4+ or CD8+ T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatory T cells unmasked latent T-cell antitumor activity; the number of activated CD8+ T cells in tumors increased and B16/3L86 tumors were completely rejected in a CD8+ and CD4+ T-cell–dependent fashion. Furthermore, fibroblasts expressing CD3L and CD86 suppressed the growth of neighboring B16 cancer cells in vivo, and direct intratumoral injection of adenoviral vectors expressing CD3L and CD86 or CD3L and a membrane-tethered anti-CD28 antibody significantly suppressed the growth of subcutaneous tumors.Conclusions: Tumor-located ligation of CD3 and CD28 can activate both innate (NKT cells) and adaptive (CD4+ and CD8+ T cells) responses to create a tumor-destructive environment to control tumor growth, but modulation of regulatory T cells is necessary to unmask local adaptive antitumor responses.

Purpose: Tumor-localized activation of immune cells by membrane-tethered anti-CD3 antibodies (CD3L) is under investigation to treat poorly immunogenic tumors. Here we sought to elucidate the mechanism of antitumor immunity elicited by CD3L.Experimental Design: CD3L and CD86 were expressed on poorly immunogenic B16 melanoma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4+ and CD8+ T cells, natural killer T (NKT) cells, and regulatory T cells, on antitumor activity was investigated.Results: B16/3L86 cells activated naïve T cells; suppressed tumor growth in subcutaneous, peritoneal, and metastasis models; and protected mice from rechallenge with B16 melanoma cells. However, in vivo antitumor activity against primary B16/3L86 tumors unexpectedly depended on NKT cells rather than CD4+ or CD8+ T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatory T cells unmasked latent T-cell antitumor activity; the number of activated CD8+ T cells in tumors increased and B16/3L86 tumors were completely rejected in a CD8+ and CD4+ T-cell–dependent fashion. Furthermore, fibroblasts expressing CD3L and CD86 suppressed the growth of neighboring B16 cancer cells in vivo, and direct intratumoral injection of adenoviral vectors expressing CD3L and CD86 or CD3L and a membrane-tethered anti-CD28 antibody significantly suppressed the growth of subcutaneous tumors.Conclusions: Tumor-located ligation of CD3 and CD28 can activate both innate (NKT cells) and adaptive (CD4+ and CD8+ T cells) responses to create a tumor-destructive environment to control tumor growth, but modulation of regulatory T cells is necessary to unmask local adaptive antitumor responses.

The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV G2335A expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro . In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes. IMPORTANCE Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to the TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.

Background The covalently closed-circular DNA (cccDNA) of hepatitis B virus (HBV) is associated with viral persistence in HBV-infected hepatocytes. However, the regulation of cccDNA and its transcription in the host cells at different growth stages is not well understood. Methods We took advantages of a stably HBV-producing cell line, 1.3ES2, and examine the dynamic changes of HBV cccDNA, viral transcripts, and viral replication intermediates in different cellular growth stages. Results In this study, we showed that cccDNA increased suddenly in the initial proliferation phase of cell growth, probably attributable to its nuclear replenishment by intracellular nucleocapsids. The amount of cccDNA then decreased dramatically in the cells during their exponential proliferation similar to the loss of extrachromosomal plasmid DNA during cell division, after which it accumulated gradually while the host cells grew to confluency. We found that cccDNA was reduced in dividing cells and could be removed when proliferating cells were subjected to long term of lamivudine (3TC) treatment. The amounts of viral replicative intermediates were rapidly reduced in these proliferating cells and were significantly increased after cells reaching confluency. The expression levels of viral transcripts were increased in parallel with the elevated expression of hepatic transcription factors (HNF4α, CEBPα, PPARα, etc.) during cell growth confluency. The HBV transcripts were transcribed from both integrated viral genome and cccDNA, however the transcriptional abilities of cccDNA was less efficient then that from integrated viral genome in all cell growth stages. We also noted increases in the accumulation of intracellular viral particles and the secretion of mature virions as the cells reached confluency and ceased to grow. Conclusions Based on the dynamics of HBV replication, we propose that HBV replication is modulated differently in the different stages of cell growth, and can be divided into three phases (initial proliferation phase, exponential proliferation phase and growth confluency phase) according to the cell growth curve. The regulation of cccDNA in different cell growth phase and its importance regarding HBV replication are discussed.

Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples. (Cancer Epidemiol Biomarkers Prev 2008;17(3):535–42)

Transforming growth factor–beta1 (TGF-β1) is a pleiotropic cytokine with pivotal roles in the regulation of cellular functions and immune responses. In this study, we found that TGF-β1 was able to effectively suppress hepatitis B virus (HBV) replication. In the presence of TGF-β1, the level of viral replicative intermediates was dramatically decreased, both in actively dividing cells and in confluent cells. At the same time, the levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF-β1 treatment. Interestingly, the inhibitory activity of TGF-β1 was associated with preferential reduction of the level of pregenomic RNA compared with pre-C mRNA. Further analysis indicated that TGF-β1 might exert its antiviral effect primarily through reducing expression of the HBV core protein by transcriptional regulation instead of posttranscriptional modification. Conclusion: TGF-β1 may play a dual role in HBV infection, in the suppression of immune responses against viral infection and in the direct inhibition of viral replication, resulting in minimization of liver damage in patients with chronic hepatitis. (HEPATOLOGY 2007.)