Additional file 1 of HBV DNA polymerase upregulates the transcription of PD-L1 and suppresses T cell activity in hepatocellular carcinoma
Creators: name: Jia, Yan
name: Zhao, Jianing
name: Wang, Chunqing
name: Meng, Jing
name: Zhao, Liqing
name: Yang, Hongwei
name: Zhao, Xiaoqing
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Space Science
subject: Cell Biology
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. HBV-DNA-Pol+ Huh7 cells inhibited the function of Jurkat cells. A. Validation of stable transfer cell lines. Protein or RNA extracts from the HBV-DNA-Pol+ Huh7 cells or control cells were harvested and HBV-P expression was analyzed by Western blotting (upper) or qRT-PCR (lower) assays. GAPDH protein or mRNAs were used as a control. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student’s t-test). ***, P &lt; 0.001. B. HBV DNA polymerase inhibits the proliferation of Jurkat cells after direct co-culture. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated and cell viability was detected by MTT assays at 24 h and 48 h after Con A stimulation, respectively. C. HBV DNA polymerase inhibits the activation of Jurkat cells after direct co-culture. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated and RNA extracts were harvested after 24 h of Con A stimulation and CD69 levels were analyzed by qRT-PCR. D and E. HBV DNA polymerase inhibits the cytokine secretion of Jurkat cells after direct co-culture. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated to measure the production of IFN-γ (D) and TNF-a (E) by ELSA 24 h after Con A stimulation. F. HBV DNA polymerase does not impair the proliferation of Jurkat cells after indirect co-culture. Jurkat cells were indirectly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated and cell viability was detected by MTT assays at 24 h and 48 h after Con A stimulation, respectively. G. HBV DNA polymerase do not impair the activation of Jurkat cells after indirect co-culture. Jurkat cells were indirectly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated and RNA extracts were harvested after 24 h of Con A stimulation and CD69 levels were analyzed by qRT-PCR. H and I. HBV DNA polymerase do not impair the cytokine secretion of Jurkat cells after indirect co-culture. Jurkat cells were indirectly co-cultured with HBV-DNA-Pol+ Huh7 cells or control cells for 24 h, then isolated to measure the production of IFN-γ (H) and TNF-a (I) by ELSA 24 h after Con A stimulation. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student’s t-test). ***, P &lt; 0.001, ns, no significant. Figure S2. HBV-DNA-Pol upregulates PD-L1 expression. A. PD-L1 protein levels in different hepatocellular carcinoma cell lines. Extracts from different hepatocellular carcinoma cell lines were collected and subjected to Western blotting with anti-PD-L1 and anti-GAPDH antibodies. B. HBV-DNA-Pol increases PD-L1 protein levels. HepG2.2.15 cells were transfected with the indicated plasmids. Extracts were collected at 48 h post-transfection and PD-L1 expression was analyzed by Western blotting. C. HBV-DNA-Pol increases PD-L1 protein levels in a somewhat dose-dependent manner. Huh7 cells were transfected with the indicated plasmids. Extracts were collected at 48 h post-transfection and PD-L1 expression was analyzed by Western blotting. Figure S3. HBV-DNA-Pol upregulates PD-L1 expression via PARP1 and STAT3. A. HBV-DNA-Pol upregulates PD-L1 expression via PARP1. pLVX-HA-HBV-P cells were transfected with the indicated plasmids. Extracts were collected at 48 h post-transfection and PD-L1 expression was analyzed by Western blotting and qRT-PCR assays. B. HBV-DNA-Pol upregulates PD-L1 expression via STAT3. pLVX-HA-HBV-P cells were transfected with the indicated plasmids. Extracts were collected at 48 h post-transfection and PD-L1 expression was analyzed by Western blotting and qRT-PCR assays. Figure S4. PD-L1 blockade reverses the inhibition of HBV DNA polymerase on the function of Jurkat cells. A. PD-L1 blockade reverses the inhibition of HBV DNA polymerase on the proliferation of Jurkat cells. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells for 24 h in the presence of anti-PD-L1 (5 μg/ml) or isotype control, then isolated and stained with CFSE, proliferation rates were measured by flow cytometry after 48 h of Con A stimulation. B. PD-L1 blockade reverses the inhibition of HBV DNA polymerase on the activation of Jurkat cells. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells for 24 h in the presence of anti-PD-L1 (5 μg/ml) or isotype control, then isolated and CD69 expression was determined by flow cytometry after 24 h of Con A stimulation. C and D. PD-L1 blockade reverses the inhibition of HBV DNA polymerase on the cytokine secretion of Jurkat cells. Jurkat cells were directly co-cultured with HBV-DNA-Pol+ Huh7 cells for 24 h in the presence of anti-PD-L1 (5 μg/ml) or isotype control, then isolated to measure the production of IFN-γ (C) and TNF-a (D) by flow cytometry 24 h after Con A stimulation. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student’s t-test). **, P &lt; 0.01, ***, P &lt; 0.001, ns, no significant. Figure S5. Inhibition of immune cell function by HBV-DNA-Pol is dependent on its interaction with PARP1. A and B. RT domain deletion reverses the inhibition of HBV DNA polymerase on the proliferation of Jurkat cells. (A) Huh7 cells were transfected with the indicated plasmids and directly co-cultured with Jurkat cells for 24 h, then isolated and stained with CFSE, proliferation rates were measured by flow cytometry after 48 h of Con A stimulation. (B) The results were plotted after quantitation. C and D. RT domain deletion reverses the inhibition of HBV DNA polymerase on the activation of Jurkat cells. (C) Huh7 cells were transfected with the indicated plasmids and directly co-cultured with Jurkat cells for 24 h, then isolated and CD69 expression was determined by flow cytometry after 24 h of Con A stimulation. (D) The quantified results were plotted. E and F. RT domain deletion reverses the inhibition of HBV DNA polymerase on the cytokine secretion of Jurkat cells. (E) Huh7 cells were transfected with the indicated plasmids and directly co-cultured with Jurkat cells for 24 h, then isolated to measure the production of IFN-γ and TNF-a by flow cytometry 24 h after Con A stimulation. (F) IFN-γ and TNF-a production was summarized. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student’s t-test). **, P &lt; 0.01, ***, P &lt; 0.001, ns, no significant.