anti human pd 1 antibody  (Bio X Cell)

Journal: Signal Transduction and Targeted Therapy

Article Title: Exogenous Epstein–Barr virus nuclear antigen 1 induces ADAR1-driven tumor resistance against immunotherapy

doi: 10.1038/s41392-026-02574-y

Figure Lengend Snippet: Interaction between EBNA1 and the m 6 A reader IGF2BP3. a Proteins that interact with EBNA1 were identified in HEK293FT cells through Co-IP and mass spectrometry, and Venn analysis was used to identify the proteins related to m 6 A modification. b The EBNA1-FLAG plasmid was transfected into HEK293FT cells, and the EBNA1-FLAG and cell lysate complexes were immunoprecipitated with an anti-FLAG antibody. The presence of IGF2BP1, IGF2BP2, and IGF2BP3 was then detected. NC: empty control plasmid, EBNA1-FLAG: EBNA1 overexpression plasmid. c Relative mRNA levels of IGF2BP1, IGF2BP2, and IGF2BP3 in normal (n = 10) and NPC (n = 31) tissue samples from the GSE12452 dataset. d Relative mRNA levels of IGF2BP1, IGF2BP2, and IGF2BP3 in normal (n = 26) and NPC (n = 26) tissue samples from the GSE51575 dataset. e The EBNA1-FLAG plasmid and IGF2BP3-HA plasmid were transfected into HEK293FT or HK1 cells. The EBNA1-FLAG and IGF2BP3-HA complexes were immunoprecipitated with anti-FLAG and anti-HA antibodies, and HA (IGF2BP3) and FLAG (EBNA1) were detected. The data are representative of two independent experiments. f Immunofluorescence assays using anti-FLAG and anti-IGF2BP3 antibodies were conducted to detect the colocalization of exogenous FLAG (representing EBNA1) and endogenous IGF2BP3 proteins in OE EBNA1 HK1 cells. Cellular nuclei were stained with DAPI and appeared blue. IGF2BP3 and FLAG are visualized in red and green, respectively. The merged image displays overlapping signals of DAPI, IGF2BP3, and FLAG, with colocalization indicated in yellow. Scatter analysis revealed signals in channels 647 (IGF2BP3) and 561 (FLAG). The Pearson correlation coefficient for colocalization is shown. Magnification: 100×. Scale bar = 10 µm. The data are representative of two independent experiments. g In C666-1 cells, the EBNA1–IGF2BP3 complex was immunoprecipitated via anti-EBNA1 and anti-IGF2BP3 antibodies, and IGF2BP3 and EBNA1 were detected. The data are representative of three independent experiments. h In C666-1 cells, immunofluorescence staining with anti-EBNA1 and anti-IGF2BP3 antibodies was performed to examine the colocalization of the EBNA1 and IGF2BP3 proteins. The cell nuclei were stained with DAPI and appeared blue. IGF2BP3 and EBNA1 are visualized in red and green, respectively. In the merged images, overlapping signals of DAPI, IGF2BP3, and EBNA1 appeared yellow, indicating colocalization. Scatter plot analysis revealed signals in channels 647 (IGF2BP3) and 561 (EBNA1). The Pearson correlation coefficient for colocalization is shown. Magnification: 100×. Scale bar = 10 µm. The data are representative of three independent experiments. i Diagram of the different domains in both the wild-type (WT) and mutant IGF2BP3 constructs. R represents the RRM. j , k The IGF2BP3-HA mutants and EBNA1-FLAG plasmids were cotransfected into HEK293FT cells. The EBNA1-FLAG ( h ) and IGF2BP3-HA ( i ) complexes were immunoprecipitated with anti-FLAG and anti-HA antibodies, respectively. The IGF2BP3-HA mutant and EBNA1-FLAG complexes were detected via anti-HA and anti-FLAG antibodies. The data are representative of two independent experiments. l Wild-type (WT) or GxxG mutant (GxxGΔ) IGF2BP3-HA plasmids were cotransfected with EBNA1-FLAG plasmids into HEK293FT cells. RNA pull-down assays were performed to study the in vitro binding of IGF2BP3 to single-stranded m 6 A-RNA probes. The data are representative of three independent experiments. c , d Data are shown as the mean ± s.e.m. Two-tailed unpaired t-tests were used for statistical analysis. *P < 0.05; **P < 0.01; ns, not significant

Article Snippet: In PBMC-humanized NCG mice, 1.0 × 10 7 C666-1 cells were inoculated subcutaneously, and 200 μg of InvivoMAb anti-human PD-1 antibody (Bio X Cell, clone: J116) was administered intraperitoneally on days 6, 9, 12, and 15.

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Modification, Plasmid Preparation, Transfection, Immunoprecipitation, Control, Over Expression, Immunofluorescence, Staining, Mutagenesis, Construct, In Vitro, Binding Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Exogenous Epstein–Barr virus nuclear antigen 1 induces ADAR1-driven tumor resistance against immunotherapy

doi: 10.1038/s41392-026-02574-y

Figure Lengend Snippet: EBNA1 promotes ADAR1 translation but does not affect its mRNA stability. a Integrative Genomics Viewer (IGV) tracks displaying the distribution of m 6 A peaks and EBNA1 binding peaks in the ADAR1 transcript. b MeRIP experiments were performed using an anti-m 6 A antibody, followed by qPCR analyses in HK1 or HONE1 cells. Rabbit IgG served as a control. The enrichment of the indicated genes was normalized to the input level. c MeRIP experiments were performed in C666-1 cells using an anti-m 6 A antibody, followed by qPCR analysis. Rabbit IgG was used as a control. The enrichment of the indicated genes was normalized to the input level. d FLAG(EBNA1)-RIP-qPCR and IGF2BP3-RIP-qPCR were performed in HONE1 and HK1 cells (OE EBNA1 group and NC group) to analyze the effect on ADAR1 mRNA enrichment. e In C666-1 cells, EBNA1 was knocked down, and EBNA1-RIP-qPCR and IGF2BP3-RIP-qPCR were performed to analyze their effects on ADAR1 mRNA enrichment. f After EBNA1 overexpression, Western blotting was used to examine ADAR1 protein levels in NPC cells (HK1, HONE1). g After IGF2BP3 was knocked down in NPC cells (HK1, HONE1), ADAR1 protein levels were detected. h In EBNA1-overexpressing HEK293FT cells, IGF2BP3 was knocked down followed by reintroduction of IGF2BP3-MUT1 or IGF2BP3-WT, and changes in ADAR1 protein levels were examined. i An RNA pull-down assay was used to confirm the enrichment of the IGF2BP3 protein on ADAR1 mRNA. j Co-IP and RNA pull-down assays were performed, followed by mass spectrometry, to identify proteins interacting with EBNA1, IGF2BP3, and ADAR1 mRNA, respectively. Venn analysis was conducted to analyze the three groups of proteins. k An EBNA1-FLAG plasmid was transfected into HEK293FT cells, and Co-IP was performed using anti-FLAG and anti-EIF4G1 antibodies to isolate the EBNA1-FLAG and EIF4G1 complexes, respectively. Western blotting was used to detect the presence of FLAG (EBNA1) and EIF4G1. l IF staining was performed using anti-IGF2BP3, anti-FLAG, and anti-EIF4G1 antibodies to detect the colocalization of exogenous FLAG (EBNA1), endogenous IGF2BP3, and EIF4G1 proteins in EBNA1-overexpressing HK1 cells. Cellular nuclei were stained with DAPI (blue). EIF4G1, FLAG (EBNA1), and IGF2BP3 were visualized in red, pink, and green, respectively. The signals of the two proteins and three proteins were merged, respectively. Scale bar: 50 µm. m Sucrose gradient-based polysome profiling analyses were conducted on NC and OE EBNA1 HK1 cells. n ADAR1 mRNA in each polysome fraction was quantified via qRT-PCR and plotted as a percentage of the total amount. o Sucrose gradient-based polysome profiling analyses were performed on control cells and EBNA1-knockdown cells in C666-1. p qRT‒PCR was used to quantify ADAR1 mRNA in each polysome fraction of shNC and shEBNA1 C666-1 cells, and the results were plotted as a percentage of the total amount. q Knockdown of EIF4G1 in OE EBNA1 HK1 and NC HK1 cells, followed by Western blot analysis of ADAR1 protein levels. b – q The results are representative of three independent experiments. b – e The data are shown as the mean ± s.e.m. Two-way ANOVA with Tukey’s test was used for multiple comparisons. **P < 0.01; ***P < 0.001

Article Snippet: In PBMC-humanized NCG mice, 1.0 × 10 7 C666-1 cells were inoculated subcutaneously, and 200 μg of InvivoMAb anti-human PD-1 antibody (Bio X Cell, clone: J116) was administered intraperitoneally on days 6, 9, 12, and 15.

Techniques: Binding Assay, Control, Over Expression, Western Blot, Pull Down Assay, Co-Immunoprecipitation Assay, Mass Spectrometry, Plasmid Preparation, Transfection, Staining, Quantitative RT-PCR, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Exogenous Epstein–Barr virus nuclear antigen 1 induces ADAR1-driven tumor resistance against immunotherapy

doi: 10.1038/s41392-026-02574-y

Figure Lengend Snippet: The presence of EBNA1 reduces tumor responsiveness to immunotherapy. a Control cells (HK1-NC) and EBNA1-overexpressing cells (HK1-EBNA1) were cocultured with CD8 + T cells, respectively. The immunotherapy effect on both groups of tumor cells was evaluated. b The secretion levels of IFNγ and IFNβ were measured in the cell supernatants shown in ( a ). c At effector-to-target (E:T) ratios of 0:1, 5:1, 10:1, and 20:1, EBNA1-overexpressing HK1 cells and control cells were cocultured with CD8 + T lymphocytes. The secretion levels of IFNβ and IFNγ were measured. d After stimulation with IFNγ, IFNβ, or a combination of IFNγ + IFNβ, the growth and viability of OE EBNA1 HK1 cells and NC HK1 cells were assessed. e ELISA was used to measure the secretion levels of IFNβ and IFNγ in the culture supernatants of NPC cells (EBNA1 HK1 and NC HK1) under the following conditions: Unstimulated, IFNβ-stimulated, IFNγ-stimulated, and combined IFNβ + IFNγ-stimulated. f EBNA1-overexpressing or IGF2BP3 knockdown HK1 cells were stimulated with IFNβ, and western blotting was used to detect the protein expression levels of ADAR1 and downstream RNA sensor molecules. g EBNA1-overexpressing or ADAR1 knockdown HK1 cells were stimulated with IFNβ, and western blotting was used to detect changes in downstream RNA sensor molecules. h Poly I:C was used to treat EBNA1-overexpressing HK1 cells at different time points, and western blotting was performed to detect changes in the following proteins: MDA5, PKR, RIG-I, pPKR (phosphorylated PKR), and MAVS. i C666-1 cells with EBNA1 knockdown were reintroduced with ADAR1 and cocultured with CD8⁺ T cells (effector-to-target ratio = 20:1). The immunotherapy effects on four groups of tumor cells (shNC, shEBNA1, shEBNA1+NC, and shEBNA1 + ADAR1) were evaluated using the CCK8 assay. j In C666-1 cells, after EBNA1 knockdown followed by ADAR1 reintroduction, Western blot analysis was performed to examine the changes in the expression of RNA sensors (MDA5, MAVS, pPKR, and RIG-I) after interferon stimulation. k Differentially expressed genes in EBNA1-overexpressing cells after IFNβ stimulation and differentially expressed genes in control tumor cells after IFNβ stimulation were subjected to Venn analysis, yielding 96 interferon-related genes. Among these genes, 12 interferon-related genes (UBE2L6, SP100, IFIH1, EIF2AK2, LGALS9, CMPK2, IDO1, DHX58, HERC6, HSH2D, IFIT3, and OAS3) of interest were selected for A-to-I analysis (n = 3 for each condition). l In whole-transcriptome sequencing, A-to-I editing changes were detected in the SINE region of HK1 cells under conditions with or without IFN stimulation and with or without EBNA1 expression. These changes were then mapped to specific transcript locations (red lines). a – e , i The results are shown as the means ± s.e.m.s. Two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0. 00; ns, not significant. The results are representative of three independent experiments

Article Snippet: In PBMC-humanized NCG mice, 1.0 × 10 7 C666-1 cells were inoculated subcutaneously, and 200 μg of InvivoMAb anti-human PD-1 antibody (Bio X Cell, clone: J116) was administered intraperitoneally on days 6, 9, 12, and 15.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Expressing, CCK-8 Assay, Sequencing, Two Tailed Test