Journal: mBio

Article Title: Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity

doi: 10.1128/mbio.01191-24

Figure Lengend Snippet: PD-L1 regulation by UL23 leads to decreased IFN-γ secretion from T cells. ( A ) HFFs were infected with Towne or Towne-ΔUL23 at an MOI of 3. Venn diagram and heatmap illustrating representative genes involved in the regulation of T cell activity in the Towne group compared to the ΔUL23 group. ( B ) HFFs were infected with Towne or Towne-ΔUL23 and treated with different indicated neutralizing antibodies or a control antibody, followed by incubation with activated Jurkat cells. Prior to co-culture, purified anti-human CD66 (CEACAM) neutralizing antibody (CEACAM1 NAb), as well as anti-human IL-10R beta neutralizing antibody (IL-10RB NAb), were individually added to the medium at a final concentration of 10 µg/mL for 30 min. qPCR analysis was performed to measure IFN-γ mRNA expression. ( C and D ) HFFs were transfected with NC or siR-PD-L1 for 24 h and subsequently infected with HCMV (Towne or ΔUL23). PD-L1 mRNA levels ( C ) and protein levels ( D ) were analyzed using qPCR and Western blot. Representative Western blot analysis of PD-L1 protein levels (down) and the densitometry values normalized to GAPDH (up) were presented. ( E and F ) siR-PD-L1-transfected HFFs were infected with HCMV ( E ), while HCMV-infected HFFs were treated with PD-L1 neutralizing antibodies ( F ). The resulting cells were then incubated with activated Jurkat cells, followed by ELISA analysis to measure IFN-γ secretion. ( G ) HFF-UL23 cells or HFF-C cells were infected with Towne (labeled as “T”) or Towne-ΔUL23 (labeled as “Δ”) and subsequently co-cultured with Jurkat cells in the presence of either Fc control (Fc) or PD-L1 Fc. The secretion of IFN-γ was quantified. ( H–M ) In the co-culture system, IL-2 secretion ( H and I ), T cell proliferation ( J and K ), and T cell apoptosis ( L and M ) were assessed by transfecting siR-PD-L1 or adding PD-L1 antibodies along with their respective controls. Each assay was performed three times, yielding similar results. Data are presented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student’s t -test ( * P < 0.05, ** P < 0.01, and *** P < 0.001). Three independent experiments were conducted.

Article Snippet: HEK 293T cells were co-transfected with 0.5 µg of pGL3-Promoter-PD-L1, 0.2 µg of the pRL-TK Renilla plasmid (Promega, Madison, WI, USA; catalog number E2241), and specific expression plasmids using the Lipo293 transfection reagent (Beyotime, Shanghai, China; catalog number C0521), following the manufacturer’s instructions.

Techniques: Infection, Activity Assay, Control, Incubation, Co-Culture Assay, Purification, Concentration Assay, Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture, Two Tailed Test

Journal: mBio

Article Title: Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity

doi: 10.1128/mbio.01191-24

Figure Lengend Snippet: UL23 promotes the expression of PD-L1. ( A ) 293T cells were co-transfected with UL23 plasmids of varying quality and the PD-L1-Luc reporter for 24 h. The luciferase activity of PD-L1 was measured (up), while the expression of UL23 was detected by immunoblot analysis (down). (B–D) The expression of PD-L1 in HFF-UL23 cells, HFF-C cells, or HFF-EGFP cells was assessed using qPCR analysis ( B ), Western blot analysis ( C ), and flow cytometry analysis ( D ). (E–H) HFFs were infected with Towne or Towne-ΔUL23 at the indicated time point, and the expression of PD-L1 was measured by qPCR analysis ( E ), Western blot analysis ( F ), flow cytometry analysis ( G ), and ELISA analysis ( H ). Representative Western blot analysis of PD-L1 protein levels (left) and the densitometry values normalized to GAPDH (right) were presented. (I–L) HFF-UL23 cells or HFF-C cells were infected with Towne or Towne-ΔUL23, and the expression of PD-L1 was measured by qPCR analysis ( I ), Western blot analysis ( J ), flow cytometry analysis ( K ), and ELISA analysis ( L ). Representative Western blot analysis of PD-L1 protein levels (left) and the densitometry values normalized to GAPDH (right) were presented. Each bar represents the mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 (unpaired, two-tailed Student’s t -test).

Article Snippet: HEK 293T cells were co-transfected with 0.5 µg of pGL3-Promoter-PD-L1, 0.2 µg of the pRL-TK Renilla plasmid (Promega, Madison, WI, USA; catalog number E2241), and specific expression plasmids using the Lipo293 transfection reagent (Beyotime, Shanghai, China; catalog number C0521), following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Western Blot, Flow Cytometry, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: mBio

Article Title: Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity

doi: 10.1128/mbio.01191-24

Figure Lengend Snippet: UL23-upregulated PD-L1 protects HCMV from T cell cytotoxicity. ( A ) HFFs were transfected with siR-NC or siR-PD-L1, followed by infection with HCMV (Towne or ΔUL23). The infected cells were then co-cultured with activated Jurkat cells, and viral titers were determined. ( B ) HFFs were infected with Towne or Towne-ΔUL23 and co-cultured with activated Jurkat cells in the presence or absence of a PD-L1 neutralizing antibody. Viral titers were subsequently determined. ( C and D ) HFFs were transfected with siR-NC or siR-PD-L1, followed by infection with HCMV (Towne or ΔUL23). The infected cells were then co-cultured with activated Jurkat cells. qPCR ( C ) and Western blot analysis ( D ) were used to analyze IE1 mRNA and protein levels. Representative Western blot analysis of IE1 protein levels (left) and the densitometry values normalized to GAPDH (right) were presented. ( E and F ) HFFs were infected with Towne or Towne-ΔUL23 and co-cultured with activated Jurkat cells in the presence or absence of a PD-L1 neutralizing antibody. qPCR ( E ) and Western blot analysis ( F ) were used to analyze IE1 mRNA and protein levels. Representative Western blot analysis of IE1 protein levels (left) and the densitometry values normalized to GAPDH (right) were presented. ( G ) HFF-UL23 cells or HFF-C cells were transfected with siR-NC or siR-PD-L1, followed by infection with HCMV (Towne or ΔUL23). The infected cells were then co-cultured with activated Jurkat cells, and qPCR analysis was performed to measure IE1 mRNA expression. ( H ) HFF-UL23 cells or HFF-C cells were infected with Towne or Towne-ΔUL23, followed by co-culture with activated Jurkat cells in the presence or absence of a PD-L1 neutralizing antibody. Viral titers were subsequently determined. Each bar represents the mean ± SD from three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t -test ( *** P < 0.001).

Article Snippet: HEK 293T cells were co-transfected with 0.5 µg of pGL3-Promoter-PD-L1, 0.2 µg of the pRL-TK Renilla plasmid (Promega, Madison, WI, USA; catalog number E2241), and specific expression plasmids using the Lipo293 transfection reagent (Beyotime, Shanghai, China; catalog number C0521), following the manufacturer’s instructions.

Techniques: Transfection, Infection, Cell Culture, Western Blot, Expressing, Co-Culture Assay, Two Tailed Test

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: EGFR mutation-specific durable clinical benefit proposes intrinsic determinants of response to immune checkpoint inhibitors (ICIs) and transcriptome analyses identify EGFR as the major nonimmunological driver of PD-L1 (A) TMB was calculated for the indicated alleles of EGFR from 5 datasets and the p value was calculated using the Kruskal-Wallis test. All of the values were mean centered and normalized. The number of patients per group and durable clinical benefit (DCB) from anti-PD-1 or anti-PD-L1 monotherapies are indicated. Statistics were calculated using the log likelihood ratio test (p < 0.01). (B) Regression analysis was performed on the following RNA sequencing (RNA-seq) datasets: left panel (lung cancer): The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) (n = 516) and TCGA lung squamous cell carcinoma (LUSC) (n = 501). Right panel (breast cancer): TCGA-breast cancer gene (BRCA) datasets of 1,093 patients with breast cancer. Following integration of the datasets, we transformed the expression values to log TPM and normalized. p value, 1.14e−14. (C) MCF10A cells were stimulated with EGF, TGF-β, or IFN-γ (each at 30 ng/mL), harvested at the indicated time points and extracts were probed. (D) Band intensities from (C) were quantified and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (E) The indicated NSCLC cell lines, along with the non-cancerous NL20 cells (3 × 10 6 ), were incubated with EGF (30 ng/mL) and extracted at the indicated time points.

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Mutagenesis, RNA Sequencing, Transformation Assay, Expressing, Incubation

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: EGFR signaling increases PD-L1 mRNA stability (A) The indicated cell lines were treated with EGF and qPCR was performed to determine levels of the mRNA of PD-L1. Signals were normalized to actin transcripts. (B and C) PC9ER cells were transfected with the indicated reporters corresponding to human PD-L1 . Plasmids containing different length inserts of genomic DNA lying upstream to the initiator codon of the PD-L1 gene were used. Cells were treated for 8 (B) or 24 h (C) with medium only, EGF (30 ng/mL), or IFN-γ (10 ng/mL). The numbers correspond to the region selected from the human reference genome assembly GRCh38. The normalized data (averages ± SEs of triplicates) are representatives of 3 independent experiments. (D) PC9ER cells were co-treated with EGF (or vehicle), actinomycin D, or the respective combination, and qPCR analysis was used to determine the transcript levels of PD-L1. (E) PC9ER and H1975 cells were transfected with siRNAs targeting AU-rich element binding proteins, or with siScrambled (siSc). Cells were analyzed 48 h later for PDL1 transcript (in triplicates). (F) PC9ER cells were transfected with siRNAs as in (E). Extracts were analyzed 48 h later using immunoblotting (IB). (G) PC9ER cells were starved overnight and later transfected with luciferase reporter plasmids containing wild-type (ATTTA) 6 or mutant (ATGTA) 6 versions of the PD-L1 3′ UTR. Luciferase signals were determined 24 h after transfection. Data depict the normalized means ± SEs.

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Transfection, Binding Assay, Western Blot, Luciferase, Mutagenesis

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: High expression of PD-L1 instigates an invasive phenotype and enhances tumorigenesis (A) PC9ER cells and cells pretransfected with a plasmid encoding a PD-L1-GFP fusion protein (or GFP alone; NT ) were extracted 48 h after transfection. Extracts were immunoblotted. (B and C) PD-L1-GFP-overexpressing PC9ER cells (4 × 10 6 ) were assayed in Transwell (B) or Matrigel-coated chambers (C). Paraformaldehyde was used to fix cells that reached the lower side of the intervening filter. Cell images and average signals are shown. (D) HeLa cells were transfected with a vector encoding PD-L1 fused to GFP or GFP only. Shown are frames from live cell videos captured 36 h later. The framed area is enlarged in the upper right corner. (E) PC9ER cells pretransfected with either a pGIPZ-EGFP plasmid or with the pGIPZ-PD-L1-EGFP plasmid were analyzed. 3 H-thymidine (1 μCi) was added after 16 h, and radioactivity incorporated into DNA was measured 48 h later. Shown are averages ± SDs of quadruplicates. (F and G) Control PC9ER cells or cells transiently overexpressing PD-L1-GFP were analyzed using either qPCR (F) or IB (G). (H and I) PD-L1-overexpressing H1975 cells (1 × 10 6 ) or cells transfected with an empty vector (EV) were implanted in nude mice (n = 6). Tumor volumes were estimated once per week. At the end of the experiment, tumors were excised and their weights were determined (right panel; averages ± SEMs).

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Expressing, Plasmid Preparation, Transfection, Radioactivity, Control

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: Loss of PD-L1 enhances adhesion and inhibits proliferation and migration, as well as tumorigenesis and metastasis (A) Extracts from parental and PD-L1 KO clones were analyzed using IB. The lower panel presents cytometry results obtained using antibodies recognizing PD-L1. (B) Parental PC9ER cells and the G4 clone (KO) were subjected to wound closure assays. Migration fronts are shown at time 0 and 10 h later, and velocities are presented. (C) Derivatives of PC9ER cells were plated onto fibronectin-coated plates. Thirty minutes later, non-adherent cells were removed and adherent cells were stained (means ± SEMs of triplicates). (D) Parental PC9ER and PD-L1-KO cells (1 × 10 4 cells/well) were subjected to 3 H-thymidine incorporation assays (averages ± SDs of quadruplicates). (E) The indicated derivatives of PC9ER cells were incubated for 60 min with bromodeoxyuridine (BrdU), fixed, and subjected to staining for PI and BrdU. Cell-cycle distributions are shown. (F) Whole extracts of the indicated cells were immunoblotted using the indicated antibodies. (G and H) Control or KO PC9ER cells were grown in medium containing fetal bovine serum (FBS; 1%) and analyzed for migration as in Figure 3 B in the presence of the indicated growth factors. (I and J) WT and KO derivatives of PC9ER cells (1 × 10 6 ) were implanted in NSG (I) or in nu/nu (J) mice (n = 10). Shown are averages ± SDs of tumor volumes. (K and L) Luciferase-labeled PC9ER (WT) and PD-L1 KO cells (1 × 10 6 ) were injected into the tail vein of NSG mice. Ten days later, we determined luminescence signals (1-way ANOVA).

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Migration, Clone Assay, Cytometry, Staining, Incubation, Control, Luciferase, Labeling, Injection

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: By altering GTP loading onto small GTPases and recruiting phospholipase C-γ1, PD-L1 instigates transcriptional programs and chemotaxis (A) PC9ER and KO cells were mixed with collagen and then cultured for 48 h. The left parts of chemotaxis chambers were filled with control media and the right sides were filled with EGF-containing media (30 ng/mL). Live imaging was performed for 16 h. The respective rose plots were processed using dedicated software. (B) PC9ER cells were pretreated with durvalumab (0.05 mg/mL) or osimertinib (40 nM) for 24 h. The cells were later assayed as in (A). (C and D) Activities of RhoA, RAC1, and CDC42 were determined using an ELISA-based kit. Both PD-L1-overexpressing PC9ER cells and KO clones were used, along with a positive control and an EV (2-way ANOVA). (E) Extracts of PC9ER cells were subjected to a co-immunoprecipitation (coIP) assay using a control antibody or an antibody to PD-L1. Shown are input blots, along with immunoblots probed for PLC-γ1, FLNA, and PD-L1. (F) PC9ER cells transiently overexpressing PD-L1 and PLC-γ1 were fixed and probed with the indicated antibodies. Thereafter, cells were processed for PLA that used TRITC (red). Counterstaining used DAPI (blue) and phalloidin-fluorescein isothiocyanate (FITC) (green). The squared areas are magnified. Single antibody controls are shown. Scale bar, 10 μm. (G) PC9ER cells transiently overexpressing PD-L1-GFP were seeded on coverslips (0.5 × 10 6 ). Thereafter, cells were fixed and incubated with PLC-γ1- or FLNA-specific antibodies, followed by a TRITC-conjugated antibody. The arrows mark the co-stained areas. Bars, 10 μm. (H) RNA was isolated from PC9ER cells transiently overexpressing PD-L1 fused to GFP, or from cells that were pretreated for 48 h with siPD-L1. RNA-seq libraries were sequenced at 10 million reads per sample. Up- and downregulated genes were analyzed using GeneAnalytics ( https://geneanalytics.genecards.org/ ).

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Chemotaxis Assay, Cell Culture, Control, Imaging, Software, Enzyme-linked Immunosorbent Assay, Clone Assay, Positive Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Incubation, Staining, Isolation, RNA Sequencing

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: PD-L1 facilitates EGF-induced activation of PLC-γ1, which is recognized by a cytoplasmic motif of PD-L1 (A) Schematic diagrams of full-length PD-L1 and the C-terminally truncated mutants. Full-length PD-L1 comprises a signal peptide (SP), an extracellular domain (ECD), a transmembrane segment (TM) and an intracellular domain (ICD). The numbers of amino acids (aa) in each domain are indicated. (B) HEK293 cells were pretransfected with plasmids encoding PD-L1 (full length) or the truncation mutants PD-L1-6aaDel or PD-L1-16aaDel. Proteins were extracted 48 h later and resolved using IB. (C) HEK293 cells were transfected as in (A) and subjected to IP and IB assays. Shown are blots probed for PLC-γ1, PD-L1, and GAPDH. (D) A549 cells were transfected with increasing amounts of a PD-L1-GFP plasmid (0.5–4 μg). Extracts were resolved using IB. (E) A549 cells were transfected as in (D). After 48 h, cells were stimulated for 30 min with EGF and analyzed as in (D). (F) Chinese hamster ovary (CHO) cells were transfected with plasmids encoding EGFR (WT), F5-EGFR (lacking all 5 auto-phosphorylation sites), and PD-L1-GFP (0.5 or 1 μg). Posttransfection, cells were stimulated with EGF for 30 min, and later they were extracted for IB.

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Phospho-proteomics

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet: PD-L1 abundance regulates PLC-γ1 phosphorylation, PKC-α activation, and calcium fluxes (A and B) PC9ER cells were pretransfected with either pGIPZ- EGFP or pGIPZ-PD-L1-EGFP (A). Alternatively, we used PD-L1 KO cells and parental cells (B). Cells were incubated for 30 min with Calcium Orange (2 μM) and stimulated with EGF 2 min after initial recording. The quantification of time-lapse fluorescence was performed over 17 min. (C) PC9ER, KO, and cells transiently expressing hemagglutinin (HA)-tagged PD-L1 were preloaded with Indo-1. EGF was added 1 min after the beginning of the experiment, and cells were subjected to flow cytometry. Calcimycin (A23187) was added after 180 s (arrows). Ca 2+ influx was followed by measuring changes in the mean fluorescence ratio of Indo-1 at violet (405 nm) to blue laser (510 nm). (D) PC9ER cells were transfected with the pGIPZ-EGFP (control) or PD-L1-GFP plasmids. Alternatively, we used PD-L1 KO cells (1.4 × 10 7 ). Steady-state DAG levels were measured (4 biological repeats). (E) Control PC9ER cells and KO cells were serum starved, treated with EGF, and then fixed. Thereafter, cells were incubated with a PKC-α-specific antibody, followed by a secondary, FITC-conjugated antibody. Images were quantified in 2 experiments. Arrows mark PKC-α recruited to the plasma membrane. (F) The endogenous activity of RhoA was determined. PC9ER cells (0.5 × 10 6 ) were transfected with a PD-L1 plasmid (3 μg). After 48 h, cells were serum starved overnight and later preincubated for 60 min with either U73122 (15 μM), a PLC inhibitor, or PP2 (50 μM), a SRC inhibitor. This was followed by stimulation for 30 min at 37°C with either EGF (30 ng/mL) or PMA. Cleared cell extracts were assayed for RhoA GTPase levels. Statistical analyses were performed using 1-way ANOVA. (G) The model depicts the herein reported interactions between a short cytoplasmic segment of PD-L1 and PLC-γ1. Formation of this complex enhances the activation of PLC-γ1 by ligand- or mutation-activated EGFRs. Once stimulated by means of phosphorylation, PLC-γ1 degrades phosphatidylinositol 4,5-bisphosphate (PIP2) to generate both diacylglycerol (which activates PKC) and inositol triphosphate (which elevates cytoplasmic Ca 2+ ). The direct interaction between PD-L1 and PLC-γ1 may explain the herein reported involvement of PD-L1 in chemotaxis and metastasis.

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Phospho-proteomics, Activation Assay, Incubation, Fluorescence, Expressing, Flow Cytometry, Transfection, Control, Clinical Proteomics, Membrane, Activity Assay, Plasmid Preparation, Mutagenesis, Chemotaxis Assay

Journal: Cell Reports

Article Title: PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR

doi: 10.1016/j.celrep.2021.109181

Figure Lengend Snippet:

Article Snippet: pGL3 3′UTR reporter MUT 1.3 kb CD274 Hs 3′UTR , Addgene , Cat# 107010; RRID:Addgene_107010.

Techniques: Control, Injection, Recombinant, Isolation, SYBR Green Assay, Reverse Transcription, Reporter Assay, Enzyme-linked Immunosorbent Assay, In Situ, Activation Assay, Luciferase, RNA Sequencing, shRNA, Plasmid Preparation, Software

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A ) Schematic of the deubiquitinase CRISPR knockout screening workflow. ( B ) Guides enriched in PD-L1–low and PD-L1–high populations with their fold enrichment. The guide code of each gene for further validation is indicated. ( C ) Guide hits described were validated by flow using individual guide knockouts. ( D ) Western blotting validation of ATXN3 knockout and PD-L1 expression with specific sgRNAs in LLC1 cells. WT, cells transfected with empty vector; KO, ATXN3-knockout stable cell strains. ( E and F ) Representative flow cytometry plots and quantification by MFI of cell-surface PD-L1 in LLC1 cells. ( G ) Western blotting analysis of ATXN3 and PD-L1 expression in A549 cells with knockout of ATXN3 . ( H and I ) Representative flow cytometry plots and quantification of cell-surface PD-L1 in A549 cells with knockout of ATXN3 . ( J and K ) Cd274 and Atxn3 mRNA levels were analyzed by reverse transcription quantitative PCR (RT-qPCR) in LLC1 cells. ( L and M ) Cd274 and Atxn3 mRNA levels were analyzed by RT-qPCR in B16 cells. ( N ) Correlation of CD274 mRNA levels with ATXN3 mRNA levels in lung cancer patients ( n = 22). ( O ) Correlation of CD274 with ATXN3 expression in multiple tumors based on TCGA data ( n = 40). C , F , and I – M : 2-tailed unpaired t test; N : Pearson’s correlation analysis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Western Blot, Expressing, Transfection, Plasmid Preparation, Stable Transfection, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A and B ) WT and ATXN3-KO cells were treated with IFN-γ (10 ng/mL) for 24 hours, and surface PD-L1 levels were analyzed. ( C ) ATXN3 interacts with IRF1 in transiently transfected HEK293T cells. ( D ) Interaction of endogenous ATXN3 and IRF1 in A549 cells. ( E ) HA-ubiquitin and FLAG-IRF1 expression plasmids were cotransfected with Myc-ATXN3 into HEK293T cells. IRF1 ubiquitination was determined by immunoprecipitation of IRF1 and immunoblotting with HA antibody. ( F and G ) FLAG-IRF1 was cotransfected with or without Myc-ATXN3 plasmids into HEK293T cells. The transfected cells were treated with cycloheximide (CHX) for different times. The protein levels of FLAG-IRF1 (top panel) and Myc-ATXN3 (middle panel) with β-actin control (bottom panel) were analyzed by Western blotting. Representative images ( F ) and quantification data from 3 independent experiments are shown ( G ). ( H and I ) Immunoblot analysis of IRF protein stability in WT and ATXN3-KO A549 cells as in F and G . ( J ) Interaction between ATXN3 and STAT3 in transfected HEK293T cells. ( K ) Endogenous interaction between ATXN3 and STAT3 in A549 cells. ( L ) The effect of ATXN3 on STAT3 ubiquitination was determined as in E . ( M and N ) The effects of ATXN3 on STAT3 protein stability were analyzed as in F and G . ( O and P ) Immunoblot analysis of STAT3 protein stability in WT and ATXN3-KO A549 cells as in H and I . ( Q ) The interaction between ATXN3 and STAT1 was tested in A549 cells. ( R ) ATXN3 enhances tumoral PD-L1 expression through protecting IRF1 and STAT3 from ubiquitination-induced protein degradation. B : Ordinary 1-way ANOVA; G , I , N , and P : 2-tailed unpaired t test; * P < 0.05, ** P < 0.01, *** P < 0.001. WCL, whole-cell lysate.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A and B ) A549 cells were cultured under normoxia and hypoxia (hyp) (1% pO 2 ) for 48 hours, and surface PD-L1 levels were analyzed by flow cytometry and quantification. ( C ) ATXN3 specifically interacts with HIF-2α. HA–HIF-2α expression plasmid was cotransfected with or without FLAG-ATXN3 into HEK293T cells. Their interactions were examined by co-IP with anti-FLAG antibodies and by Western blotting with anti-HA antibodies. ( D ) The interaction between ATXN3 and HIF-1α was tested in transfected HEK293T cells. ( E ) Endogenous interaction between ATXN3 and HIF-2α in A549 cells. ( F ) HA-ubiquitin, FLAG–HIF-2α, and Myc-ATXN3 plasmids were cotransfected into HEK293T cells. HIF-2α ubiquitination was determined by immunoprecipitation of HIF-2α with anti-FLAG antibodies and immunoblotting with anti-HA antibody. ( G and H ) HIF-2α was cotransfected with or without ATXN3 plasmids into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels of HIF-2α (top panel) and ATXN3 (middle panel) were analyzed by Western blotting. β-Actin was used as a loading control (bottom panel). ( I and J ) Immunoblot analysis of HIF-2α protein stability in WT and ATXN3-KO A549 cells. ( K ) ATXN3 enhances hypoxia-induced PD-L1 expression through protecting HIF-2α from ubiquitination-induced protein degradation. B : Ordinary 1-way ANOVA; H and J : 2-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Cell Culture, Flow Cytometry, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A – C ) WT or ATXN3-KO LLC1 cells were injected subcutaneously into C57BL/6 mice ( n = 10). Tumor growth curve ( A ), photograph ( B ), and weight ( C ) are shown. ( D ) MFI of surface PD-L1 on LLC1 tumors ( n = 5). ( E – G ) Quantification of CD4 + ( E ) and CD8 + T cell ( F ) and Treg ( G ) percentages ( n = 5–10). ( H and I ) Quantification of cell-surface PD-1 ( H ) and PD-L1 ( I ) MFI on CD8 + T cells ( n = 5). ( J and K ) Quantification of cell-surface CTLA-4 MFI ( J ) and Tim3 percentage ( K ) in CD8 + T cells ( n = 5). ( L ) MFI of cell-surface LAG3 and percentage in CD8 + T cells ( n = 5). ( M – O ) Intracellular staining of Blimp1 + CD8 + T cell ( M ), EOMES + CD8 + T cell ( N ), and T-bet + CD8 + T cell ( O ) percentage in LLC1 tumors ( n = 5-7). ( P ) Quantification of cell-surface CD44 + CD8 + T cell percentage from LLC1 tumors ( n = 5–10). ( Q ) Apoptotic CD8 + T cells in the tumors were analyzed ( n = 5–7). ( R and S ) Representative flow staining and quantification of intracellular cytokine staining of granzyme B + CD8 + and IFN-γ + CD8 + in CD45 + T cell populations from LLC1 tumors ( n = 5). ( T ) Tumor growth curve and tumor photograph of C57BL/6 mice injected subcutaneously with WT and ATXN3-KO LLC1 cells with or without treatment of anti-CD8 depleting antibodies ( n = 5). ( U ) Left: Tumor cell-surface PD-L1 expression. Right: Tumor growth of WT or ATXN3-KO LLC1 cells stably expressing PD-L1 (as shown in the left plot) in C57BL/6 mice ( n = 5). A and C – S : 2-tailed unpaired t test; T and U : ordinary 1-way ANOVA. * P < 0.05, ** P < 0.01,*** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Injection, Staining, Expressing, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A ) Representative images from immunohistochemical staining of PD-L1, ATXN3, IRF1, and HIF-2α in human lung adenocarcinoma (LUAD) and melanoma patients. Scale bar: 50 μm. ( B ) PD-L1, ATXN3, IRF1, and HIF-2α protein levels in tumor tissues compared with normal tissues in LUAD patients ( n = 61); “PD-L1%” means the percentage of PD-L1–positive area versus all tissue area. ( C ) PD-L1, ATXN3, IRF1, and HIF-2α protein levels in tumor tissues compared with normal tissues in patients with melanoma ( n = 48). ( D ) Correlation analysis of ATXN3 expression with PD-L1, IRF1, and HIF-2α expression in LUAD patients ( n = 61). ( E ) Correlation analysis of ATXN3 expression with PD-L1, IRF1, and HIF-2α expression in melanoma patients ( n = 48). ( F ) ATXN3 is a positive regulator for PD-L1 transcription through stabilizing multiple transcription factors including HIF-2α, IFR1, STAT3, and JunB and enhances tumor evasion. B and C : 2-tailed unpaired t test; D and E : Pearson’s correlation analysis. ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 3 Expression of immunosuppressive markers correlates to BRAF mutation. (A) The tumor tissues of 19 PTC cases were stained with CD274 antibody, and the expression intensity was scored by a COH pathologist. Grade 1 represents the lowest expression, and grade 3+ represents the highest. (B) The tumor tissue was processed to detect CD73, and the expression intensity was scored where grade 1 represents the lowest expression and grade 3+ represents the highest. (C) The images represent the cells positive for cancer cells (TTF-brown), T cells CD8 (blue) and CD4 (pink) in the two representative tumor tissue of BRAF-mutated and WT samples. (D) The plot illustrates the percentage of lymphocyte infiltration within the tumor area, as assessed by the pathologist. The lymphocytic infiltration ratio is notably higher for mutant patients (red dots) compared with wild-type patients (blue). Additionally, the percentages of CD4 and CD8 lymphocytes were analyzed. The findings suggest a higher contribution of CD4 lymphocytes compared with CD8, reinforcing the cell type infiltration data. PTC, papillary thyroid cancer; WT, wild-type. * indicates a p value of <0.05

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Expressing, Mutagenesis, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 4 BRAF knockdown induced CD274 expression. (A) The RNA extracted from four PTC cell lines was used to determine the expression of BRAF, CD274, CD73, CD200, CD276, ENTPD1, and CD200. The expression in the mutant was compared with the WT cell type. (B) The immunoblot represents the expression of proteins in the BRAF-mutated and WT cell lines. (C) The bar graph represents the changes in the mRNA expression of CD274 and other immunosuppressive markers on knocking down BRAF. (D) Immunoblot represents the knockdown of BRAF and upregulation of CD274 in the BRAF-mutated cell lines T32 and T85 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance; PTC, papillary thyroid cancer; WT, wild-type.

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Knockdown, Expressing, Mutagenesis, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 5 Cell cycle arrest correlates with CD274 expression. (A, B) The T85 cell line was treated with increasing concentration of BRAF inhibitors, and changes in the proliferation were measured using the live cell imaging system. (C, D) Data representing the proliferation changes in the T68 cell lines with respect to BRAF inhibitor treatment. (E) FACS data representing the changes in the cell cycle stages of T68 with respect to BRAF inhibitors where dabrafenib induced stronger cell cycle arrest. (F) The qPCR data represents the changes in the expression of BRAF, CD274 and CD73 in the T68 and T85 cells with respect to the BRAF inhibitor treatment. (G) The immunoblot represents the changes in the CD274 and CD73 expression on combining dabrafenib with either binimetinib, temsirolimus, or abemaciclib (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Expressing, Concentration Assay, Live Cell Imaging, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 6 BRAF inhibitor-induced transcriptional changes upstream of CD274. (A) Cartoon showing the design of CD274- promoter-luciferase constructs used for determining the changes in transcriptional activity postdrug treatment. (B) The box plot representing the luciferase activity determined in the cells post 48 hours of transfection, where 2 Kb promoter sequence correlated with maximum activity. (C) Luciferase activity measured in the cells post 48 hours of BRAF or KRAS knockdown. (D) The bar graph represents the difference in the promoter activity between the T85 and T32 cell lines. (E, F) Luciferase (Promoter) activity correlated with increased BRAF inhibitor concentration in the T68 and T85 cells. (****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Luciferase, Construct, Activity Assay, Transfection, Sequencing, Knockdown, Concentration Assay

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 7 The drug combination effect on CD274 expression. (A, B) The bar graph represents the percentage change in the growth of T68 or T85 cells on binimetinib, abemaciclib or temsirolimus exposure. The immunoblot shows the change in the expression of CD274 and CD73 on drug treatment. (C) The bar graph represents the effect of drug combinations on the cell viability of T85 cells as measured by CCK8 assay. (D–F) The immunoblot represents the increase in the expression of CD274 and CD73 expression on combining dabrafenib with either binimetinib or temsirolimus, or abemaciclib (*****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Expressing, Western Blot, CCK-8 Assay

Journal: Journal for immunotherapy of cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies.

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Figure 8 The synergistic effect of BRAF and checkpoint inhibitors in immune cell-mediated killing. (A) The line graph represents the growth kinetics of T85 cells when cultured in combination with activated CD8 cells. The bar graph represents the comparison in growth between monoculture and cocultured T85 cells at 0 and 72 hours time points. (B) The growth kinetics of T32 cells when cultured in combination with activated CD8 cells. (C) The images represent the effect of dabrafenib, CD8, atezolizumab, and IL-2 on the T85 (GFP) cell growth. (D) The growth kinetics of T85 cells when cultured in combination with activated CD8 cells, IL-2, and atezolizumab. (E) The growth kinetics of T85 cells, when cultured in combination with dabrafenib, activated CD8 cells, IL-2 and atezolizumab. (F) The images represent the effect of CD8, atezolizumab, and IL-2 on the T85 (GFP) derived spheroids. Loss of GFP represents a loss of spheroid viability. (G) The bar graph represents the changes in the spheroid viability when cocultured with CD8, IL-2, and atezolizumab. (H) The changes in the spheroid viability when cocultured with dabrafenib, CD8, IL-2, and atezolizumab. (I) The immunoblot represents the changes in the CD274 expression in T85 cells on exposure to IL-2 and dabrafenib n monoculture or coculture. **** indicates P value < 0.001.

Article Snippet: The pGL3 plasmids harboring the 1 kb, 2 kb or 3 kb region of the CD274 promoter were purchased from Addgene (# 107002, #107 003 and #107004).

Techniques: Cell Culture, Comparison, Derivative Assay, Western Blot, Expressing