rabbit polyclonal anti cd274 pd l1 antibody  (Novus Biologicals)

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: The schematic illustrates the nebulized inhalation of an integrated nanovesicle system (Res-PD-L1@nmEVs) alleviated inflammation, oxidative stress injury, neutrophil activation, and promote mitochondrial integrity to mitigate lung ischemia-reperfusion injury and MRSA-induced bacterial pneumonia.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: In Vitro, Labeling, Incubation, Expressing, Fluorescence, BrdU Incorporation Assay, Staining, Flow Cytometry, Microscopy, Activity Assay, Migration, Wound Healing Assay, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Restores Mitochondrial Homeostasis and Improves Energy Metabolism BEAS-2B cells were pretreated with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs followed by H/R stimulation for subsequent analysis. (A) Representative immunofluorescence images showing the expression and localization of PINK1 (green) and the mitochondrial marker TOMM20 (red), indicating activation of mitophagy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantitative analysis of PINK1 fluorescence intensity. (C) Expression and localization of autophagy-related proteins LC3B and Beclin-1 detected by immunofluorescence. (D-E) Quantitative analysis of LC3B (D) and Beclin-1 (E) fluorescence intensity. (F) Mitochondrial membrane potential assessed by JC-1 staining and flow cytometry. (G) Oxygen consumption rate (OCR) profiles of lung epithelial cells under different treatments. (H-K) Key mitochondrial respiration parameters: basal respiration (H), maximal respiration (I), proton leak (J), and ATP production (K). (L) Representative confocal microscopy images of mitochondria stained with MitoTracker (green) and lysosomes stained with LysoTracker (red), demonstrating mitochondrial-lysosomal colocalization. Scale bar: 5 μm ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Immunofluorescence, Expressing, Marker, Activation Assay, Staining, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation HL60 cells were differentiated into neutrophil-like cells using DMSO and subsequently stimulated with TNF-α to induce activation under conditions simulating IRI. The effects of Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs on neutrophil activation were evaluated. (A) Cell surface PD-1 expression analyzed by flow cytometry. (B) Representative immunofluorescence images of CD206 expression (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Flow cytometric analysis of cell surface CD206 expression. (D) Flow cytometric analysis of cell surface CD95 expression. (E-G) Levels of myeloperoxidase (MPO) (E), neutrophil elastase (NE) (F), and MMP-9 (G) in neutrophil culture supernatants, measured by ELISA. (H-J) BEAS-2B cells were co-cultured with neutrophils in the presence or absence of TNF-α stimulation. Apoptosis levels (I) and migration capacity (J) of BEAS-2B cells were assessed under different treatment conditions. ∗ vs. Control; # vs. TNF-a; & vs. TNF-a+PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Expressing, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Nebulized Res-PD-L1@nmEVs Target and Attenuate Lung Ischemia-Reperfusion Injury (A) Experimental timeline: rats undergoing lung IRI received nebulized treatments (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion, with sample collection 2 h post-reperfusion. (B) Ex vivo organ fluorescence imaging 24 h after intravenous or bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs. (C) In vivo lung distribution of nebulized DiL-labeled PD-L1@mEVs and PD-L1@nmEVs evaluated using a small animal dynamic imaging system. Blue: CD31 (vascular marker), Red: DiL. (D-E) Quantitative fluorescence intensity in ex vivo organs (heart, liver, spleen, lungs, kidneys) at 0–24 h after bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs in Sham and IRI groups. (F-G) Representative H&E-stained lung sections (F) and corresponding lung injury scores (G). (H) Lung wet/dry weight ratio. (I-K) Levels of inflammatory cytokines in lung tissue. (L-N) Pulmonary oxidative stress markers: T-SOD2 activity (L), GSH/GSSG ratio (M), and MDA content (N). (O) Representative fluorescence images of ROS in lung tissue. Scale bar: 50 μm. (P-R) Immunofluorescence staining and co-localization of tight junction proteins Occludin-1 (green) and ZO-1 (red) in lung tissues (DAPI: blue). Scale bar: 50 μm. Quantitative analysis of ZO-1 (Q) and Occludin-1 (R) fluorescence intensity. ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Ex Vivo, Fluorescence, Imaging, Labeling, In Vivo, Marker, Staining, Activity Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation and Preserves Mitochondrial Integrity via PD-L1 Delivery (A-B) Rats subjected to lung IRI received nebulized administration of different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion. Lung tissues were collected 2 h post-reperfusion. (A) Representative immunofluorescence images showing the expression and localization of CD11b (green), MPO (red), and PD-1 (yellow) in lung sections across treatment groups. (B) Enlarged view of the IRI group from (A). (C-D) mRNA levels of CD95 (C) and CD206 (D) in lung tissues. (E-F) Levels of myeloperoxidase (MPO) (E) and matrix metalloproteinase-9 (MMP-9) (F) in bronchoalveolar lavage fluid (BALF). (G-I) (G) Representative transmission electron microscopy (TEM) images of lung tissues (scale bar: 2 μm). (H) Proportion of damaged mitochondria. (I) Average number of mitophagic events per cell. (J) Immunofluorescence co-localization of mitochondrial marker TOMM20 (red) and EpCAM (green) in lung tissues (nuclei stained with DAPI, scale bar: 50 μm). (K-L) Protein expression levels of Beclin-1 (K) and LC3 (L) in lung tissues, with insets showing immunofluorescence co-localization of Beclin-1 (green) and LC3 (red) across treatment groups (nuclei stained with DAPI, scale bar: 50 μm). ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Marker, Staining

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Transcriptomic Analysis Reveals the Mechanism of Res-PD-L1@nmEVs Against IRI-Induced Lung Injury (A-B) Transcriptome sequencing of lung tissues from the Res-PD-L1@nmEVs-treated IRI group (N = 3) and the IRI-only group (N = 3). (A) Volcano plot and (B) heatmap display differentially expressed genes (DEGs) between the IRI + Res-PD-L1@nmEVs and IRI groups. (C-D) GO term and KEGG pathway enrichment analyses of upregulated DEGs after Res-PD-L1@nmEVs treatment. (E-F) GO term and KEGG pathway enrichment analyses of downregulated DEGs following Res-PD-L1@nmEVs treatment. (G-J) Gene Set Enrichment Analysis (GSEA) revealed enrichment in energy metabolism pathways (G) (TCA cycle and oxidative phosphorylation), biosynthetic pathways (H) (ribosome, amino acid biosynthesis, DNA replication), immune pathways (I) (allograft rejection, PD-L1 expression and PD-1 checkpoint pathway), and inflammatory responses (J) (chemokine signaling pathway, ECM-receptor interaction, cytokine-cytokine receptor interaction).

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Sequencing, Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Effectively Attenuates MRSA-Induced Pneumonia (A-B) Rats with MRSA-induced pneumonia received three bronchial nebulization treatments over one week with different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs). (A) Representative H&E-stained lung sections and (B) corresponding lung injury scores are shown (n = 5). (C) TUNEL staining of lung tissues to assess apoptosis. (D) Representative micro-CT images of anesthetized rats. (E-G) Flow cytometric analysis of immune cell proportions in lung single-cell suspensions: CD8 + T cells (E), neutrophils (F), and classical monocytes (G). (H-J) Plasma levels of inflammatory cytokines IL-6 (H), IL-1β (I), and TNF-α (J) (n = 5). (K) Immunofluorescence staining of tight junction proteins Occludin (green) and ZO-1 (red) in lung tissues (nuclei stained with DAPI). Scale bar: 50 μm. (L-N) Pulmonary function parameters: lung compliance (L), airway resistance (M), and oxygenation index (N) (n = 4). ∗ vs. Sham; # vs. MRSA; & vs. MRSA + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Staining, TUNEL Assay, Micro-CT, Single Cell, Clinical Proteomics, Immunofluorescence

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Bar charts showing PD-L1 expression via flow cytometry. (B) Confocal microscopy images of MOC1, MOC2, and MOC2( PD-L1 ) cells merged from 3 channels (Supplemental Fig. 1A). Scale = 20 µm. MFI = median fluorescence intensity.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Fluorescence

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Saturation binding assay of 89 Zr-DFO-PD-L1 mAb on MOC2( PD-L1 ) cells. Curves for total bound (TB), specifically bound (SB), and nonspecifically bound (NSB) fractions fitted using given equation. (B) Specificity of binding assay of 89 Zr-DFO-PD-L1 mAb (2 nM; 1 h) on cancer cell lines pretreated with IFN-γ (20 ng/mL; 24 h) or PBS. Blocking with 100-fold molar excess of unlabeled PD-L1 mAb . Data were normalized to nontreated MOC2( PD-L1 ) cell–associated radioactivity. Significance determined using 2-way ANOVA with Bonferroni adjustment. C bound = bound ligand concentration; C ligand = ligand concentration; B max = maximum specific binding; K d = dissociation constant; ns = not significant.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Saturation Assay, Binding Assay, Blocking Assay, Radioactivity, Concentration Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Coregistered PET/CT scans of MOC1, MOC2, and MOC2( PD-L1 ) tumor–bearing mice 48 h after 89 Zr-DFO-PD-L1 mAb injection (PET, coronal slice; CT, 3-dimensional maximum intensity projection). (B) 89 Zr-DFO-PD-L1 mAb biodistribution profile of MOC1, MOC2, and MOC2( PD-L1 ) tumor–bearing mice 48 h after injection (110 μg; adjusted specific activity = 0.018 MBq/μg). Data pooled from 3 studies. Inset graph shows correlation of PET quantification and biodistribution (BioD). Linear regression fit with 95% CI (dashed lines). (C) PD-L1 immunohistochemistry of tumor sections. BAT = brown adipose tissue; p.i. = postinjection.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Activity Assay, Immunohistochemistry

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: Experimental timeline of 89 Zr-DFO-PD-L1 mAb immuno-PET studies to monitor PD-L1 expression after single intratumoral dose of RP1 or PBS in MOC1 tumor–bearing mice. Figure created with BioRender.com. i.v. = intravenous; s.c. = subcutaneous.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Expressing

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Coregistered PET/CT scans of RP1 and PBS-treated mice on days 3 and 7 after treatment (PET, coronal slice; CT, 3-dimensional maximum intensity projection). Spleens of RP1-treated mice are highlighted (arrowheads). (B) Bar charts of 89 Zr-DFO-PD-L1 mAb uptake on day 3 vs. day 7 after RP1 or PBS. Significance determined using 2-way ANOVA with Bonferroni adjustment on log-transformed data. (C) Radiomics heatmaps of tumor and spleen VOIs from day 3 scans. x -axis shows feature numbers from Supplemental Tables 3 and 4. Feature values normalized to z score. Decision trees shown to left of each heatmap. TD Ln = tumor-draining lymph node.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Positron Emission Tomography-Computed Tomography, Transformation Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) PD-L1 immunohistochemistry of MOC1 tumor and spleen sections from immuno-PET studies on day 3 and 7 after single intratumoral RP1 or PBS dose. (B) Concentrations of GM-CSF, IFN-α, IFN-β and IFN-γ in tumors on day 3 after RP1 or PBS. Significance determined using multiple unpaired t tests on log-transformed data with Benjamini-Hochberg FDR correction (FDR < 1%).

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Immunohistochemistry, Transformation Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Representative fluorescent images of MOC1 and MOC2 cells after 48 h of incubation with increasing titers of GFP-expressing RP1-15. GFP shown in gray scale. (B) MOC1 and MOC2 cell viability after 48 h with increasing RP1 titers in vitro, assessed via CellTiter-Glo assay. Significance determined using 1-way ANOVA with Dunnett test. RLU = relative light units.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Incubation, Expressing, In Vitro, Glo Assay

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Elevated ABHD16A triggered by ACh was associated with poor prognosis in patients with gastric cancer. A, Representative IHC images of ABHD16A expression in human normal gastric tissues and gastric cancer tissues from the TMA with magnifications of 40×, 100×, and 200×. Scale bar for 200×, 100 μm. B–D, Relationship of ABHD16A IHC score to clinical stage ( B ), pT stage ( C ), and lymph node metastasis ( n = 80; D ). E, Cumulative survival curves of patients with gastric cancer with high or low ABHD16A expression based on the TMA. F, IHC images and score of ABHD16A in S100 + or S100 − gastric cancer tissues. Scale bar, 100 μm. G, Procedure for coculture of DRG neurons and gastric cancer cells. H, Western blotting was used to analyze ABHD16A levels in MFC cells cocultured with DRG neurons or DRG neuron–derived CM. GAPDH served as the loading control. I, Expression of HIF1A and ABHD16A in neurotransmitter [HA, dopamine (DA), 5-HT, norepinephrine (NE), ACh]-treated MFC cells and ACh-treated MGC-803 cells. J, ACh concentration was detected in DRG CM by ELISA. K, Tumor volume of orthotopic gastric cancer tumors with or without vagotomy. L, Representative mIF staining images of FOXP3, CD163, CD11b, CD8, PD-L1, and Pan-CK in gastric cancer tissues with high or low expression of ABHD16A. Scale bar, 100 μm. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Expressing, Western Blot, Derivative Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: ABHD16A promotes ILC3 aggregation and IL22 release in the immune microenvironment. A, A total of 5 × 10 6 MFC-luc or Abhd16a -knockdown MFC-luc cells were injected into the epidermis of the greater curvature of the stomach in the 615 mice. Tumor growth was monitored weekly through bioluminescence imaging from the first day of MFC cell inoculation. Scale bar, 2.000e+4 – 5.000e + 5 p/s/cm 2 /sr. B and C, Representative images of the excised stomachs ( B ) and the orthotopic tumor volume ( C ) on day 28 after gastric cancer cells were implanted orthotopically ( n = 9 per group). D, H&E images of the stomach, peritoneum, liver, intestine, and lung on day 42 after MFC or Abhd16a -knockdown MFC cells were implanted into 615 mice. Scale bar, 400 μm. E–G, Representative images ( E ), volume ( F ), and weight ( G ) of control and Abhd16a -knockdown subcutaneous tumors ( n = 5 per group). H, IHC images of PD-L1 in control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues. Scale bar, 500 μm. I, Detection of mouse cytokines in the TIF of orthotopic gastric cancer tissues by proteome profiler mouse XL cytokine array. J, Flow cytometry gating strategy and frequencies of ILC3s and Th17/Th22 cells out of CD45 + cells in control and Abhd16a -knockdown gastric cancer tissues in 615 mice. K, Flow cytometry gating strategy and frequencies of ILC3s out of ILCs in control and Abhd16a -knockdown gastric cancer tissues in Rag1 −/− mice. ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Knockdown, Injection, Imaging, Control, Flow Cytometry

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: High expression of ABHD16A mediates the cumulative release of LysoPS into the TME. A, mIF images of the orthotopic gastric cancer (GC) tissues stained for PD-L1, RORC, and CD3. Yellow, PD-L1 + tumor cells; red, RORC + ILC3s; green, CD3 + cells. Scale bars, 100 μm (left) and 25 μm (right). B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). D and E, Flow cytometry gating strategy and frequencies of ILC3s ( D ) and proportions of ILC3s in total CD45 + cells ( E ) derived from the peripheral blood of healthy controls (HC) and patients with gastric cancer ( n = 8 per group). F and G, LC-MS/MS analysis of 18:0 and 18:1 LysoPS in the control and Abhd16a -knockdown orthotopic ( F ) and subcutaneous ( G ) gastric cancer tissues ( n = 3 per group). H–J, ELISA was used to measure levels of LysoPS in gastric cancer cell supernatant ( H ), TIF ( I ), and in vitro tumor culture supernatant ( J ) of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tumors ( n = 3 per group). K, LysoPS levels in the serum of healthy controls and patients with gastric cancer detected by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Control, Knockdown, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: IL22 upregulates PD-L1 expression in gastric cancer cells through the UPR IRE1α–XBP1 axis. A, mIF images show the alterations of PD-L1 + tumor cells (purple), CD4 + (green), and CD8 + (red) T cells in orthotopic gastric cancer tumors from control and Abhd16a -knockdown mice following IL22 treatment. Scale bar, 50 μm. B, KEGG pathway enrichment analysis of RNA-seq data of gastric cancer tissues with or without IL22 treatment. C, RT-PCR was used to assess the mRNA expression of key downstream molecules of the UPR branches ( XBP1 , ATF4 , ATF6 ) in control and IL22RA1 -knockdown gastric cancer cells. D, Western blotting analysis of PD-L1 and XBP1s levels in control and IL22RA1 -knockdown MGC-803 cells treated with IL22 (100 μg/L). E, Western blotting detection of PD-L1 and XBP1s levels in XBP1- knockdown MGC-803 cells treated with IL22 and MGC-803 cells treated with IL22 or XBP1s inhibitor (STF083010, 30 μmol/L) in combination with IL22. F, The binding sequence of XBP1 on the CD274 promoter. G and H, ChIP ( G ) and luciferase reporter assay ( H ) showing the transcriptional regulation of CD274 by XBP1s under IL22 stimulation. I, Orthotopic gastric cancer mouse models ( n = 5 per group) were injected with anti-IL22 (200 μg per mouse), anti-CD90.2 antibody (150 μg per mouse), anti-CD90.2 antibody in combination with IL22 (500 ng per mouse), or anti-CD90.2 antibody in combination with XBP1s inhibitors (STF083010, 30 mg/kg) and IL22 for 2 weeks. IHC analysis was used to show IL22, XBP1s, and PD-L1 levels in gastric cancer tissues. Scale bar, 200 μm. J, Tumor volume of orthotopic gastric cancer models under treatments the same as in I . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Expressing, Control, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Injection

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Combination therapy enhances the anti–PD-L1 immunotherapeutic effect in gastric cancer. A and B, After tumor formation, the orthotopic gastric cancer mice ( n = 5 per group) were treated with anti–PD-L1 (100 μg per mouse), GPR34 inhibitor (20 mg/kg), or XBP1s inhibitor (30 mg/kg) every 3 days or ACh inhibitor (2.5 mg/kg) daily. Combinations of anti–PD-L1 with each inhibitor followed the every 3-day dosing schedule for a total duration of 2 weeks via i.p. injection. Living images were used to monitor tumor progression at 5-day intervals from the time of drug administration ( A ); IHC and mIF were performed to detect PD-L1 and XBP1s levels and proportions of CD4 + (green) and CD8 + (red) T cells in gastric cancer tissues at the end of treatments ( B ). Scale bars, 1.000e+5 –∼ 5.000e + 5 p/s/cm 2 /sr for living images; 200 μm for IHC; 50 μm for immunofluorescence. C and D, Representative images ( C ) and tumor volume ( D ) of subcutaneous tumors. The administration protocol for the mice was consistent with the description provided in A and B . **, P < 0.01; ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Injection, Immunofluorescence

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Elevated ABHD16A triggered by ACh was associated with poor prognosis in patients with gastric cancer. A, Representative IHC images of ABHD16A expression in human normal gastric tissues and gastric cancer tissues from the TMA with magnifications of 40×, 100×, and 200×. Scale bar for 200×, 100 μm. B–D, Relationship of ABHD16A IHC score to clinical stage ( B ), pT stage ( C ), and lymph node metastasis ( n = 80; D ). E, Cumulative survival curves of patients with gastric cancer with high or low ABHD16A expression based on the TMA. F, IHC images and score of ABHD16A in S100 + or S100 − gastric cancer tissues. Scale bar, 100 μm. G, Procedure for coculture of DRG neurons and gastric cancer cells. H, Western blotting was used to analyze ABHD16A levels in MFC cells cocultured with DRG neurons or DRG neuron–derived CM. GAPDH served as the loading control. I, Expression of HIF1A and ABHD16A in neurotransmitter [HA, dopamine (DA), 5-HT, norepinephrine (NE), ACh]-treated MFC cells and ACh-treated MGC-803 cells. J, ACh concentration was detected in DRG CM by ELISA. K, Tumor volume of orthotopic gastric cancer tumors with or without vagotomy. L, Representative mIF staining images of FOXP3, CD163, CD11b, CD8, PD-L1, and Pan-CK in gastric cancer tissues with high or low expression of ABHD16A. Scale bar, 100 μm. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Western Blot, Derivative Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: ABHD16A promotes ILC3 aggregation and IL22 release in the immune microenvironment. A, A total of 5 × 10 6 MFC-luc or Abhd16a -knockdown MFC-luc cells were injected into the epidermis of the greater curvature of the stomach in the 615 mice. Tumor growth was monitored weekly through bioluminescence imaging from the first day of MFC cell inoculation. Scale bar, 2.000e+4 – 5.000e + 5 p/s/cm 2 /sr. B and C, Representative images of the excised stomachs ( B ) and the orthotopic tumor volume ( C ) on day 28 after gastric cancer cells were implanted orthotopically ( n = 9 per group). D, H&E images of the stomach, peritoneum, liver, intestine, and lung on day 42 after MFC or Abhd16a -knockdown MFC cells were implanted into 615 mice. Scale bar, 400 μm. E–G, Representative images ( E ), volume ( F ), and weight ( G ) of control and Abhd16a -knockdown subcutaneous tumors ( n = 5 per group). H, IHC images of PD-L1 in control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues. Scale bar, 500 μm. I, Detection of mouse cytokines in the TIF of orthotopic gastric cancer tissues by proteome profiler mouse XL cytokine array. J, Flow cytometry gating strategy and frequencies of ILC3s and Th17/Th22 cells out of CD45 + cells in control and Abhd16a -knockdown gastric cancer tissues in 615 mice. K, Flow cytometry gating strategy and frequencies of ILC3s out of ILCs in control and Abhd16a -knockdown gastric cancer tissues in Rag1 −/− mice. ***, P < 0.001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Knockdown, Injection, Imaging, Control, Flow Cytometry

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: High expression of ABHD16A mediates the cumulative release of LysoPS into the TME. A, mIF images of the orthotopic gastric cancer (GC) tissues stained for PD-L1, RORC, and CD3. Yellow, PD-L1 + tumor cells; red, RORC + ILC3s; green, CD3 + cells. Scale bars, 100 μm (left) and 25 μm (right). B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). D and E, Flow cytometry gating strategy and frequencies of ILC3s ( D ) and proportions of ILC3s in total CD45 + cells ( E ) derived from the peripheral blood of healthy controls (HC) and patients with gastric cancer ( n = 8 per group). F and G, LC-MS/MS analysis of 18:0 and 18:1 LysoPS in the control and Abhd16a -knockdown orthotopic ( F ) and subcutaneous ( G ) gastric cancer tissues ( n = 3 per group). H–J, ELISA was used to measure levels of LysoPS in gastric cancer cell supernatant ( H ), TIF ( I ), and in vitro tumor culture supernatant ( J ) of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tumors ( n = 3 per group). K, LysoPS levels in the serum of healthy controls and patients with gastric cancer detected by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Control, Knockdown, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: IL22 upregulates PD-L1 expression in gastric cancer cells through the UPR IRE1α–XBP1 axis. A, mIF images show the alterations of PD-L1 + tumor cells (purple), CD4 + (green), and CD8 + (red) T cells in orthotopic gastric cancer tumors from control and Abhd16a -knockdown mice following IL22 treatment. Scale bar, 50 μm. B, KEGG pathway enrichment analysis of RNA-seq data of gastric cancer tissues with or without IL22 treatment. C, RT-PCR was used to assess the mRNA expression of key downstream molecules of the UPR branches ( XBP1 , ATF4 , ATF6 ) in control and IL22RA1 -knockdown gastric cancer cells. D, Western blotting analysis of PD-L1 and XBP1s levels in control and IL22RA1 -knockdown MGC-803 cells treated with IL22 (100 μg/L). E, Western blotting detection of PD-L1 and XBP1s levels in XBP1- knockdown MGC-803 cells treated with IL22 and MGC-803 cells treated with IL22 or XBP1s inhibitor (STF083010, 30 μmol/L) in combination with IL22. F, The binding sequence of XBP1 on the CD274 promoter. G and H, ChIP ( G ) and luciferase reporter assay ( H ) showing the transcriptional regulation of CD274 by XBP1s under IL22 stimulation. I, Orthotopic gastric cancer mouse models ( n = 5 per group) were injected with anti-IL22 (200 μg per mouse), anti-CD90.2 antibody (150 μg per mouse), anti-CD90.2 antibody in combination with IL22 (500 ng per mouse), or anti-CD90.2 antibody in combination with XBP1s inhibitors (STF083010, 30 mg/kg) and IL22 for 2 weeks. IHC analysis was used to show IL22, XBP1s, and PD-L1 levels in gastric cancer tissues. Scale bar, 200 μm. J, Tumor volume of orthotopic gastric cancer models under treatments the same as in I . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Control, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Injection

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Combination therapy enhances the anti–PD-L1 immunotherapeutic effect in gastric cancer. A and B, After tumor formation, the orthotopic gastric cancer mice ( n = 5 per group) were treated with anti–PD-L1 (100 μg per mouse), GPR34 inhibitor (20 mg/kg), or XBP1s inhibitor (30 mg/kg) every 3 days or ACh inhibitor (2.5 mg/kg) daily. Combinations of anti–PD-L1 with each inhibitor followed the every 3-day dosing schedule for a total duration of 2 weeks via i.p. injection. Living images were used to monitor tumor progression at 5-day intervals from the time of drug administration ( A ); IHC and mIF were performed to detect PD-L1 and XBP1s levels and proportions of CD4 + (green) and CD8 + (red) T cells in gastric cancer tissues at the end of treatments ( B ). Scale bars, 1.000e+5 –∼ 5.000e + 5 p/s/cm 2 /sr for living images; 200 μm for IHC; 50 μm for immunofluorescence. C and D, Representative images ( C ) and tumor volume ( D ) of subcutaneous tumors. The administration protocol for the mice was consistent with the description provided in A and B . **, P < 0.01; ***, P < 0.001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Injection, Immunofluorescence