Journal: Nature Biomedical Engineering

Article Title: Engineered outer membrane vesicles enhance solid tumour CAR-T cell therapy

doi: 10.1038/s41551-025-01575-6

Figure Lengend Snippet: a , BROAD-CAR was prepared by extracting OMVs from genetically engineered E. coli expressing aPD-L1 scFv (C5H9V2), followed by electroporation with plasmids encoding the ECD of HER2. b , CAR-T targeting and antitumour activity driven by BROAD-CAR are achieved through two mechanisms: (1) remodelling the TME by activating innate immune responses via OMVs and blocking the PD-1/PD-L1 axis, thereby enhancing CAR-T cell infiltration and effector function and (2) delivering HER2-encoding plasmids into antigen-negative tumour cells, resulting in membrane HER2 expression and subsequent recognition and killing by anti-HER2 CAR-T cells. MDSC, myeloid-derived suppressor cell. c , Representative FCM histogram of POMVs. A total of 20 μg of POMVs were incubated with 5 μl of 4 μm aldehyde/sulfate latex beads for 15 min at room temperature, followed by incubation with PE-conjugated anti-Myc antibody for 30 min at 4 °C. d , Representative transmission electron microscope image of POMVs. Scale bar, 50 nm. e , Confocal microscopy of 4T1 cells cocultured with 10 μg ml −1 DiI-labelled OMVs or POMVs at 37 °C for 4 h; the cell membranes are stained with DiO and the nuclei with Hoechst. Scale bar, 10 μm. f , PD-L1 expression on 4T1 cells was detected by PE-conjugated anti-mouse PD-L1 antibody after 4 h cocultured with 10 μg ml −1 OMVs or POMVs; representative FCM histogram and frequency of PD-L1 expression on 4T1. g , Molecular structure design of HER2-targeting CAR. h , Differential interference contrast (DIC) image (left) and fluorescence microscopy image (right) of T cells transfected with pCDH-CAR. Scale bar, 50 μm. i , Representative FCM histograms showing GFP (left) and CAR (right) expression on transfected T cells. For CAR detection, CAR-T cells were collected, incubated with 1 mg ml −1 biotinylated protein L for 45 min at 4 °C and stained with PE-conjugated streptavidin for 30 min before analysis. Untransfected T cells and T cells transfected with mock plasmids (the multiple cloning site without insertion of the CAR gene) served as negative controls for GFP and CAR detection, respectively. j , The measurement of the mean fluorescence intensity (MFI) of PD-L1 on 4T1-HER2 cells following 24 h coculture with mock or anti-HER2 CAR-T cells at an E:T ratio of 1:2. k , The measurement of the MFI of PD-L1 on 4T1-HER2 cells after 24 h coculture with 10 μg ml −1 OMVs or POMVs and CAR-T cells at an E:T ratio of 1:2. l , The cytotoxicity of CAR-T cells against 4T1-HER2 cells at the indicated E:T ratios after 48 h coculture with 10 μg ml −1 OMVs, OMVs + 20 μg ml −1 PD-L1 antibodies, POMVs or equal volume of PBS, evaluated by an LDH assay. m , The dose-dependent cytotoxicity of CAR-T cells (E:T of 1:2) against 4T1-HER2 cells treated with POMVs (0–32 μg ml −1 ) for 48 h. n = 6 independent biological replicates per group for the data in f and j – m . The data and error bars represent the mean ± s.d. A statistical analysis was performed using one-way ANOVA for f and m or two-way ANOVA for l , followed by a Tukey’s multiple comparisons test and unpaired two-tailed Student’s t -tests for j and k . CTL, cytotoxic T lymphocyte; ISO, isotype control; M1, M1 macrophages; M2, M2 macrophages; scRNA-seq, single-cell RNA sequencing;T EM , T effector memory cell.

Article Snippet: PD-L1 expression was induced with 1:1,000 1 M isopropyl β- D -1-thiogalactopyranoside (CAS no. HY-15921, MedChemExpress) diluted 1:1,000 in lysogeny broth broth.

Techniques: Expressing, Electroporation, Activity Assay, Blocking Assay, Membrane, Derivative Assay, Incubation, Transmission Assay, Microscopy, Confocal Microscopy, Staining, Fluorescence, Transfection, Cloning, Lactate Dehydrogenase Assay, Two Tailed Test, Control, Single Cell, RNA Sequencing

Journal: Biochemistry and Biophysics Reports

Article Title: NOHA as a key biomarker to link NOS2 activity to immune checkpoint and cytokine signaling in ER-negative breast cancer

doi: 10.1016/j.bbrep.2026.102468

Figure Lengend Snippet: Breast cancer 3D spheroid cell line 7-week measurements of ER–/ER + AA and CA origins. [A] medium NOHA, [B] cellular NOHA, [C] cellular PD-L1, [D] cellular IL8, [E] cellular NOS expression, and [F] cellular nitric oxide as total nitrite. n = 6, ∗, from ER+ and/or control groups, p < 0.01. #, between ER– groups, p < 0.01.

Article Snippet: Commercial assay kits were used for the following: NOS2 expression (Antibodies-online, Atlanta, GA); NOS2 enzymatic activity via total nitrite detection by fluorometric assay (Cayman Chemical, Ann Arbor, MI); total PD-L1 expression (Cell Signaling Technology, Danvers, MA); and IL-8 expression (Abbexa, Sugar Land, TX).

Techniques: Expressing, Control

Journal: Biochemistry and Biophysics Reports

Article Title: NOHA as a key biomarker to link NOS2 activity to immune checkpoint and cytokine signaling in ER-negative breast cancer

doi: 10.1016/j.bbrep.2026.102468

Figure Lengend Snippet: Longitudinal NOHA to PD-L1 ratio comparison in ER– AA and CA groups over 7-weeks [A] cellular PD-L1 expression to medium NOHA levels, and [B] cellular PD-L1 expression to cellular NOHA levels. n = 6 ∗, ratio significance between ER– groups, p < 0.01.

Article Snippet: Commercial assay kits were used for the following: NOS2 expression (Antibodies-online, Atlanta, GA); NOS2 enzymatic activity via total nitrite detection by fluorometric assay (Cayman Chemical, Ann Arbor, MI); total PD-L1 expression (Cell Signaling Technology, Danvers, MA); and IL-8 expression (Abbexa, Sugar Land, TX).

Techniques: Comparison, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: NOHA as a key biomarker to link NOS2 activity to immune checkpoint and cytokine signaling in ER-negative breast cancer

doi: 10.1016/j.bbrep.2026.102468

Figure Lengend Snippet: NOHA inversely correlate with PD-L1 and IL-8 in ER– breast cancer spheroids. [A] PD-L1 vs. medium NOHA in ER– CA (i.e., MDB-436), [B] PD-L1 vs. medium NOHA in ER– AA (i.e., MDA-MB157); [C] IL-8 vs. medium NOHA in ER– CA (i.e., MDB-436), and [D] IL-8 vs. NOHA in ER– AA (i.e., MDA-MB157). Across all panels [A-D], increasing NOHA is associated with decreased PD-L1 or IL-8, demonstrating consistent inverse relationships in both ethnic groups.

Article Snippet: Commercial assay kits were used for the following: NOS2 expression (Antibodies-online, Atlanta, GA); NOS2 enzymatic activity via total nitrite detection by fluorometric assay (Cayman Chemical, Ann Arbor, MI); total PD-L1 expression (Cell Signaling Technology, Danvers, MA); and IL-8 expression (Abbexa, Sugar Land, TX).

Techniques:

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: Sepsis and M1 polarization induce the upregulation of PD-L1 expression in monocytes and macrophages. ( A ) Analysis of three GEO datasets ( GSE57065 , GSE28750 , and GSE95233 ) revealed significantly higher PD-L1 expression in sepsis patients than in healthy controls. ( B ) Venn intersection analysis of commonly upregulated genes across sepsis datasets revealed CD274 as a key hub gene (ranked 74th among 100 hub genes) via protein‒protein interaction network analysis. ( C ) The mRNA expression levels of PD-L1 in septic monocytes were measured by qRT‒PCR ( n =3 individuals per group). ( D ) THP-1-derived macrophages (THP-1-M) were treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) or an equivalent volume of PBS for 6 h, followed by RNA sequencing analysis. ( E ) THP-1-M cells were stimulated with LPS + IFN-γ for 6, 12, and 24 h. Upregulated PD-L1 expression in stimulated cells was validated by qRT‒PCR (left) and Western blotting (middle). The relative expression levels of PD-L1 were normalized and quantified using ImageJ (right) ( n =3 independent biological replicates). ( F ) THP-1-M cells were treated with LPS + IFN-γ for 24 h and analyzed via laser scanning confocal microscopy (scale bar = 20 μm). ( G ) Single-gene GSEA was conducted using three sepsis-related datasets ( GSE57065 , GSE65682 , and GSE95233 ) retrieved from the GEO database to investigate the relationship between PD-L1 expression and inflammatory pathways. The data are presented as the mean ± SEM (exclude A). Statistical analysis for A was performed by Wilcoxon test. Statistical analysis for C was performed by Student’s t test, and that for E was determined by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: Expressing, Derivative Assay, RNA Sequencing, Western Blot, Confocal Microscopy

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 mediates the regulation of chemokine biosynthesis in M1-polarized macrophages, as determined by RNA-seq. ( A ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h and subjected to RNA sequencing analysis. The KEGG database was used to identify enriched signaling pathways whose activation significantly differed (up). Single-gene GSEA was conducted using the GSE57065 , GSE65682 , and GSE95233 datasets to identify the relationship between PD-L1 and chemokine signaling pathways (down). ( B ) Intersection analysis of upregulated genes between the PD-L1 HI /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + LPS + IFN-γ group and between the PD-L1 NC /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + PBS group via a Venn diagram, followed by further intersection with chemokine datasets, revealed three highly expressed PD-L1-mediated chemokines (CCL8, CXCL9, and CXCL10) under LPS + IFN-γ stimulation. ( C ) RNA sequencing analysis revealed the expression of CCL8, CXCL9 and CXCL10 upon PD-L1 overexpression. ( D ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( E ) Human monocytes were induced into macrophages, transfected with scramble siRNA and PD-L1 siRNA, and then treated with LPS + IFN-γ for 12 h. CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( F ) PD-L1 NC /THP-1-M and PD-L1 KO /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for D, E and F was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: RNA Sequencing, Protein-Protein interactions, Activation Assay, Expressing, Over Expression, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 may regulate chemokine expression via the Toll-like receptor signaling pathway, as determined by single-gene GSEA. ( A ) Venn diagram of PD-L1-activated signaling pathways in sepsis-related GEO datasets ( GSE57065 , GSE65682 , and GSE95233 ) and RNA-seq data from LPS (100 ng/mL) + IFN-γ (20 ng/mL)-treated PD-L1 HI /THP-1-M cells versus PD-L1 NC /THP-1-M cells (left). Top 5 pathways according to the RNA-seq data (right). ( B ) Mechanistic summary of NOD-like receptor pathway- and Toll-like receptor pathway-mediated chemokine regulation. ( C ) AIM2 expression in RNA-seq data from LPS + IFN-γ-treated PD-L1 HI /THP-1-M cells versus PD-L1 NC /THP-1-M cells (left). AIM2 mRNA levels were determined by qRT‒PCR (right) ( n = 3 independent biological replicates). ( D ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were stimulated with LPS + IFN-γ for 12 h. Upregulated AIM2 expression in stimulated cells was validated by Western blotting (left). The relative expression levels of PD-L1 were normalized and quantified using ImageJ (right) ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C and D was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: Expressing, Protein-Protein interactions, RNA Sequencing, Western Blot

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: TLR4 acts as a critical upstream regulator of PD-L1 in modulating CCL8 and CXCL9 expression. ( A ) GSEA of RNA-seq data to investigate the relationship between PD-L1 expression and Toll-like receptor signaling pathway activation. ( B ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TLR4 siRNA, respectively, followed by 12 h of stimulation with LPS (100 ng/mL) + IFN-γ (20 ng/mL). The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: Expressing, RNA Sequencing, Activation Assay, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 binds to the TLR4 downstream molecule TRAF6 to regulate CCL8 and CXCL9 expression. ( A ) Venn diagram analysis of proteins coimmunoprecipitated with PD-L1 in THP-1-M cells stimulated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h via IP‒MS. After excluding proteins bound to the IgG control group, 289 PD-L1-specific interacting proteins were identified (up). Representative MS/MS spectrum showing peptide fragments of the TRAF6 protein identified in the immunoprecipitation complex (down). ( B ) THP-1-M (up) and human monocyte-derived (down) macrophages were treated with or without LPS+IFN-γ for 6 h and then harvested for Co-IP. TRAF6 protein levels were measured via Western blotting. ( C ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TRAF6 siRNA, respectively, followed by 12 hours of stimulation with LPS+IFN-γ. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). ( D ) Structural models of full-length PD-L1 and TRAF6 predicted by AlphaFold (PAE plots were obtained from AlphaFold). ( E ) Molecular docking generic matching information. ( F ) A protein‒protein interaction cartoon model generated by PyMOL (left). Predicted binding sites between PD-L1 and TRAF6 (right). ( G ) PD-L1 NC /THP-1-M, PD-L1 HI /THP-1-M and PD-L1 Δ62-139 /THP-1-M cells were treated with LPS+IFN-γ for 12 h. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C and G was performed by one-way ANOVA with LSD test for post hoc analyses. * p <0.05.

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: Expressing, Control, Tandem Mass Spectroscopy, Immunoprecipitation, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Generated, Binding Assay

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 knockout attenuates septic mouse mortality and suppresses CCL8 and CXCL9 expression. ( A – C ) PD-L1 WT /mice and PD-L1 KO /mice were intraperitoneally injected with LPS (15 mg/kg) and IFN-γ (10 µg/kg) to induce endotoxemia-induced sepsis. ( B ) The survival rate was determined over a 60-hour observation period and is presented as a Kaplan‒Meier survival curve ( n = 15 mice per group). The results are presented as Kaplan–Meier survival curves. The results of the statistical analysis were analyzed with the log-rank test. * p < 0.05. ( C ) Peripheral blood was collected 12 h posttreatment for serum isolation and PBMC separation. CCL8 and CXCL9 protein levels in serum and their corresponding mRNA levels in PBMCs were subsequently analyzed ( n = 3 independent biological replicates). ( D ) Peritoneal macrophages (PMs) were extracted from PD-L1 wild-type and knockout mice and then treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). (E) Peripheral blood monocytes were extracted from PD-L1 wild-type and knockout mice and then treated with LPS + IFN-γ for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C was performed by Student’s t test, and that for D and E was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: Genechem (Shanghai, China) established and cultured the engineered THP-1 variants: a negative control cell line with baseline PD-L1 expression (PD-L1 NC /THP-1), PD-L1-overexpressing (PD-L1 HI /THP-1) and PD-L1-knockout (PD-L1 KO /THP-1) cell lines.

Techniques: Knock-Out, Expressing, Injection, Isolation