Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) In silico model representing the binding of Fab fragment from clones A35795 (A35) and 135c139d6 (135c) compared to the hPD-1/hPD-L1 complex (PDB ID: 4ZQK). ( B ) Binding of A35 and 135c IgG to human PD-1 (left). Competitive binding assay with pembrolizumab and biotin-labeled A35 IgG or 135c IgG (right). Symbols are means of three (left) or two (right) independent experiments. ( C ) Binding of A35 and 135c Fabs to human PD-1 (left). Biolayer interferometry of Fabs–PD-1 interactions (right). Symbols are means of three independent experiments (left). ( D ) Binding of A35 and 135c scFvs-His to human PD-1 (left). Biolayer interferometry of scFvs–PD-1 interactions (right). Symbols are the mean of three independent experiments (left). ( E ) Second-generation CAR design (left). Biotinylated PD-1 binding to anti–PD-1 CARs. Representative flow cytometry and mean of four independent experiments. PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; IgG, immunoglobulin G; Fab, fragment antigen-binding region; K D , equilibrium dissociation constant; scFv, single-chain variable fragment; His, histidine; CAR, chimeric antigen receptor; EGFRt, truncated epidermal growth factor receptor; HD, hinge domain; bPD1-CAR, blocking anti–PD-1 CAR; nbPD1-CAR, nonblocking anti–PD-1 CAR; UT, untransduced cells.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: In Silico, Binding Assay, Clone Assay, Competitive Binding Assay, Labeling, Flow Cytometry, Blocking Assay

Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) Experimental design for engineering human anti–PD-1 CAR-T cells. ( B ) Expansion of primary anti–PD-1 CAR-T cells compared to the anti-CD19 CAR control ( n = 7 donors, seven independent experiments). ( C ) Cumulative data of transduction efficiency for donors reported in (D) and (E). The mean ± SD values eight donors, eight independent experiments, two to three replicates transduced with different MOI is shown. Two-way ANOVA, Tukey’s multiple comparison test. ( D ) Representative flow cytometry plots showing mCherry and PD-1 expression 6 days after transduction (gated on CD3 + living cells). ( E ) Correlation between PD-1 expression and the percentage of transduction efficiency. Cherry-positive and Cherry-negative population are shown. For each experiment the percentage of PD-1 + cells were assessed in Cherry-negative and Cherry-positive cells and normalized to PD-1 expression in untransduced T cells (set as 100%). Eight donors, eight independent experiments each with two to three internal replicates (with different MOI transduction: 1.4 ± 0.6). Simple linear regression tests. ( F ) CD8/CD4 ratio in mCherry ± T cells comparing anti–PD-1 CAR and CD19 CAR-T cells ± PD-1 knockout. Data are presented as mean ± SD values of eight donors, eight independent experiments, two to three internal replicates). Two-way ANOVA, Tukey’s multiple comparisons test. ( G ) Schematic representing the killing assay (left). Cumulative percentage of annexin/Aqua + CD4 + PD-1 + GFP + target cells after 24 hours of coculture (right). Mean ± SD of three donors and three independent experiments is shown. Two-way ANOVA, Tukey’s multiple comparison test. Only statistical differences are reported as follows: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. HD, hinge domain; TMD, transmembrane domain; ICD, intracellular domain; LV, lentivirus; scFv, single-chain variable fragment; CAR, chimeric antigen receptor; bPD1-CAR, blocking anti–PD-1 CAR; nbPD1-CAR, nonblocking anti–PD-1 CAR; FMO, fluorescence minus one; UT, untransduced; KO, knockout.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: Control, Transduction, Comparison, Flow Cytometry, Expressing, Knock-Out, Blocking Assay, Fluorescence

Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) Generation of PD-1-transgenic luciferase + Jurkat cells with increasing PD-1 molecules number. ( B ) Luciferase-based killing assay of blocking and nonblocking CAR-T cells. Mean ± SD of three donors and three independent experiments is shown. ( C ) Phenotype of the K562 cell lines expressing either a PD-1-GFP-F fusion protein (K-PD-1-GFP-F) or PD-1 and GFP proteins with a ribosomal skipping motif in-between (K-PD-1-GFP-noF) used for the trogocytosis assay. Scale bars, 25 μm. ( D ) Representative flow cytometry 1 hour after coculture of K-PD-1-GFP-F or -PD-1-GFP-noF with anti–PD-1 CAR-T cells. ( E ) Cumulative data showing GFP expression in CAR-T cells over time. Mean ± SD values of six donors and four independent experiments is shown. Two-way ANOVA, Tukey’s multiple comparison test. ( F ) Phenotype of the K562 cell lines expressing either the wild-type PD-1 (K-PD-1 WT ) or a truncated PD-1 lacking (K-PD-1 t ) the intracellular domain used for the activation assay. ( G ) Cumulative percentage of CD25 + /CD71 + CAR-T cells after a 48 hours coculture with K-PD-1 WT or K-PD-1 t . Mean ± SD values of four to eight donors and seven independent experiments is shown. Two-way ANOVA, Tukey’s multiple comparison test. ( H ) Luciferase killing assay using PD-1 WT or PD-1 t luciferase + Jurkat cells as targets. Mean ± SD values of three donors, three independent experiments is shown. Two-way ANOVA, Tukey’s multiple comparison test was used to compare the same condition in JPD-1 versus JPD-1 t . Only statistical differences are reported as follows: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. JPD-1, Jurkat-PD-1; CAR, chimeric antigen receptor; bPD1-CAR, blocking anti–PD-1 CAR; nbPD1-CAR, nonblocking anti–PD-1 CAR; E:T, effector:target; K-PD-1-GFP-F, K562 PD-1-GFP-F fusion protein; K-PD-1-GFP-noF, K562 PD-1 + GFP + ; K-PD-1, K562 PD-1 + ; K-PD-1-t, K562 PD-1 truncated; K-PD-1-KO, K562 PD-1 KO; JPD-1-t, Jurkat PD-1 truncated.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: Transgenic Assay, Luciferase, Blocking Assay, Expressing, Trogocytosis Assay, Flow Cytometry, Comparison, Activation Assay

Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) Experimental design. HLA-A2 + human T cells were isolated and activated ± prior editing of PD-1 and/or HLA-A2. ( B ) PD-1 expression in mCherry + and mCherry − cells on days 7 and 10. Mean of three to five donors in three to five independent experiments with one to two replicates transduced at different MOI (1.4 ± 0.6) for each independent experiments. ( C ) HLA-A2 expression in mCherry + and mCherry − cells on day 7 and 10. Mean of two donors in two independent experiments with one to two internal replicates (different MOI transduction: 1.4 ± 0.6). ( D ) Expansion folds of unedited versus edited CAR-T cells on day 10. Mean ± SD values of three to five donors in three to five independent experiments with one to two internal replicates (different MOI transduction: 1.4 ± 0.6). Statistics: Two-way ANOVA, Dunnett’s multiple comparison test was performed comparing each condition with the corresponding HLA-A2 CAR condition. ( E ) Cumulative data showing CD62L/CD45RA expression of mCherry + cells in edited versus unedited condition for PD-1 or HLA-A2. The mean ± SD values of three to five donors in three to five independent experiments with one to two internal replicates (different MOI transduction 1.4 ± 0.6) are shown. Kruskal-Wallis and Dunn’s multiple comparison test was performed comparing each condition with the corresponding HLA-A2 CAR condition. ( F ) Representative flow cytometry showing TIM-3 and LAG-3 expression in the different CAR populations on day 10 (left). Cumulative percentage of LAG-3 + /TIM-3 + double-positive cells (right). Paired data of three to five donors in three to five independent experiments with one to two internal replicates (different MOI transduction: 1.4 ± 0.6) are shown. Two-way ANOVA, Dunnett’s multiple comparison test was performed comparing each condition with the corresponding HLA-A2 CAR condition. Only statistical differences are reported as follows: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: Isolation, Expressing, Transduction, Comparison, Flow Cytometry

Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) Experimental design. Mice received adoptive cell transfer (ACT) of anti–PD-1 CARs versus untransduced (UT) T cells under ART that was interrupted after 2 weeks (three independent experiments). The reporter used for detecting CAR-positive cells was EGFRt. ( B ) Kaplan-Meyer curve showing the percentage of mice maintaining viral control. All mice were included in the analysis. Log-rank (Mantel-Cox) test was performed using the UT control condition as reference. ( C ) Plasma viral load overtime (UT, n = 6; bPD1-CAR, n = 9; nbPD1-CAR, n = 7). ( D ) Percentage of CAR-T cells (EGFRt + gated in CD45 + cells) in the spleen, BM, and blood at the time of euthanasia. Mean ± SD is shown. bPD1-CAR, n = 9, nbPD1-CAR, n = 7. ( E ) Percentage of CD8 + , CD4 + , and CD4 − CAR-T cells detected in the spleen, BM, and blood (bPD1-CAR, n = 3, nbPD1-CAR, n = 4). Mean ± SD is shown. ( F ) Correlation between the percentage of CAR-T cells (defined as CD45 + EGFRt + cells) and the time of viral rebound (bPD1-CAR, n = 9; nbPD1-CAR, n = 7). Simple linear regression was used. ( G ) Correlation between CD4 + PD-1 + cells (gated in huCD45 + EGFRt − cells) and the time of viral rebound (bPD1-CAR, n = 9; nbPD1-CAR, n = 7). Simple linear regression was used. ( H ) Correlation between CD4 + PD-1 + cells (gated in huCD45 + EGFRt − cells) and CAR-T cells detection (bPD1-CAR, n = 9; nbPD1-CAR, n = 7). Simple linear regression was used. ( I ) Correlation between HIV integrated DNA in CD4 + EGFRt- sorted cells from spleen and BM and time of viral rebound (bPD1-CAR, n = 3; nbPD1-CAR, n = 3). Simple linear regression was used. ( J ) Correlation between HIV-integrated DNA in CD4 + EGFRt − sorted cells from spleen and BM and CAR-T cells detection (bPD1-CAR, n = 3; nbPD1-CAR, n = 3). Simple linear regression was used. ART, antiretroviral therapy; ACT, adoptive cell transfer; VC, viral control.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: Control, Clinical Proteomics

Journal: Science Advances

Article Title: Targeting PD-1 + T cells with chimeric antigen receptors to reduce the HIV reservoir

doi: 10.1126/sciadv.aeb7602

Figure Lengend Snippet: ( A ) Immunofluorescence multicolor confocal images of spleens of four control and seven treated hu-mice with detectable CAR-T cells (untransduced treated, n = 4; nbPD1-CAR-T treated, n = 4; bPD1-CAR-T treated, n = 3) 8 to 10 weeks after ACT. The following stainings were performed: DAPI (blue), CD4 (green), CD20 (red), PD-1 (magenta), CD8 (yellow), Grzb (cyan), and CD57 (gray). Scale bars, 100 μm. ( B ) Histo-cytometry analysis showing CD4 + PD-1 + , and CD8 + PD-1 + cell counts/mm 2 in the CD20-enriched areas and CD20 + cell counts/mm 2 in the CD4-enriched zones. Each dot represents a CD20- or CD4-enriched region ( n = 154 and n = 158). Median is shown. Kruskal-Wallis and Dunn’s multiple comparison test. All control mice ( n = 6) and treated mice with detectable CARs ( n = 7) that survived until the end of the experiment were included. ( C ) Mean distance between each B cell and its five nearest neighbors in the CD20-enriched regions in the 13 spleens based on IF staining data. Median is shown. Kruskal-Wallis and Dunn’s multiple comparison test was performed. ( D ) Combined IF and RNAscope for CD8 (green), DAPI (blue), HIV RNA (red), and CAR RNA (magenta) performed on the spleen of one representative control mouse (S2), one HIV suppressed responder mouse (S11), and one nonresponder mouse with viral rebound (S5) 8 weeks after ACT. Scale bars, 50 μm. ( E ) Histo-cytometry analysis showing absolute counts of CAR RNA events (gated on CD8 + cells) and HIV RNA events for each mouse tissue. Two-way ANOVA, Tukey’s multiple comparison test. Only statistical differences are reported as follows: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. S, spleen; bCAR, blocking anti–PD-1 CAR; nbCAR, nonblocking anti–PD-1 CAR; ACT, adoptive cell transfer; UT, untransduced T cells.

Article Snippet: Kinetic analysis of A35/135C IgG, Fab, and scFv binding to PD-1 dimer protein was performed by biolayer interferometry immobilizing PD-1 biotinylated dimer (3 μg/ml) on streptavidin biosensors (Sartorius, Göttingen, Germany, catalog no. 18-5019) dipped into a solution of the Fab/scFv at different concentrations diluted in PBS (ranging from 40 to 240 nM), and the nm shift was recorded on the Octet.

Techniques: Immunofluorescence, Control, Cytometry, Comparison, Staining, RNAscope, Blocking 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: 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: Molecular Therapy. Nucleic Acids

Article Title: Potential effects of endogenous RNA/DNA hybrids on CRISPR-Cas9-mediated homology-directed repair

doi: 10.1016/j.omtn.2026.102880

Figure Lengend Snippet: Modulation of HDR efficiency by R-loop resolution, cell-cycle perturbation, and locus context (A) ddPCR quantification of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or 10,000 vg/cell of scAAVDJ-sg3′ Alb + 10,000 vg/cell ssAAVDJ-donor 3′ Alb. (B) Schematic representation of cell cycle profiles in HEPA1-6 cells and murine hepatocytes. (C) Flow cytometry analysis of HEPA1-6/SpCas9 cells treated for 24 h with rapamycin (1 μM, 5 μM) or palbociclib (PD; 5 μM, 10 μM). DMSO-treated cells served as controls. (D) DRIP-qPCR analysis of HEPA1-6/SpCas9 cells after 24 h treatment with 5 μM rapamycin; DMSO-treated cells served as controls. (E) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with 10,000 vg/cell of scAAVDJ-sgMid Alb + 10,000 vg/cell ssAAVDJ-donor Mid Alb, or scAAVDJ-sg3′ Alb + ssAAVDJ-donor 3′ Alb. (F) IGV visualization of the Alb and Actb loci showing gRNA (sgActb) and donor (donor Actb) design targeting the 5′ region of Actb . (G) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transduced for 72 h with 10,000 vg/cell of scAAVDJ-sgActb + 10,000 vg/cell ssAAVDJ-donor Actb. Scrambled gRNA with donor served as negative control. (H) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells pre-treated with 5 μM rapamycin for 24 h, followed by 72 h transduction with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (I) ddPCR detection of HDR events in HEPA1-6/SpCas9 cells transiently overexpressing human RNase H1 and transduced with scAAVDJ-sgActb + ssAAVDJ-donor Actb. (J) DRIP-qPCR analysis of CD4 + T cells from a healthy donor, pre- and post-activation with 10 μg/mL PHA, 50 IU/mL IL-2, 5 ng/mL IL-7, and 5 ng/mL IL-15. DRIP, immunoprecipitated samples treated with S9.6 antibody which are enriched in R loops; RNAseH + , samples treated with RNAseH1+S9.9 antibody which are depleted of R-loops. (K) Table summarizing the effects of R-loops levels on indels, HDR, and AAV integration. N/A, not affected; ↑ increase; ↓ = decrease. Statistical analysis: (A and E) multiple t test; (C) two-way ANOVA with Dunnett’s post hoc test; (G–I) Student’s t test. p < 0.05, ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001. Error bars represent mean ± SD.

Article Snippet: HEPA1-6/SpCas9 cells were treated with rapamycin (Selleck, S1039) or palbociclib (Selleck, S1116) for 24 h, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry (NovoCyte Penteon, Agilent).

Techniques: Transduction, Flow Cytometry, Negative Control, Activation Assay, Immunoprecipitation

Journal: Genes & Diseases

Article Title: Chloride channel accessory 4 suppresses stem cell-like properties of colorectal cancer and enhances anti-PD-1 immunotherapy

doi: 10.1016/j.gendis.2025.101859

Figure Lengend Snippet: CLCA4 overexpression enhanced the therapeutic effect of anti-PD-1. (A) In vivo tumorigenicity assay in nude mice was performed to detect the therapeutic effect of CLCA4 overexpression combined with anti-PD-1 antibody. (B) Tumor growth curve was monitored from nude mice with different treatment groups. (C) Weights of tumors from nude mice with different treatments were detected. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (D) Analysis of the survival times of mice in each group ( n = 8 per group), and the experiment was terminated 60 days after tumor inoculation. Unpaired two-tailed t -test (mean ± standard deviation). (E) Immunofluorescence or immunohistochemistry staining was performed to detect the infiltration levels of CD8 + T cells, GZMB, Perforin + cells, Ki67 + , Bmi-1 + , and Oct4 + cells in tumors from different treatment groups. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (F) Hematoxylin-eosin staining of liver metastases in each group. (G) Quantitative analysis of the liver weight in each group. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (H) Quantitative analysis of the liver metastasis area/total liver area in each group. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation).

Article Snippet: After 7 days, mice were intraperitoneally treated with either an in vivo blocking antibody against mouse PD-1 (Clone: 29F.1A2, BioXcell, Cat# BP0273) or a rat IgG2a isotype control antibody (Clone: 2A3, BioXcell, Cat# BP0089).

Techniques: Over Expression, In Vivo, Tumorigenicity Assay, Standard Deviation, Two Tailed Test, Immunofluorescence, Immunohistochemistry, Staining