Journal: Journal for immunotherapy of cancer

Article Title: Inhibition of PNCK inflames tumor microenvironment and sensitizes head and neck squamous cell carcinoma to immune checkpoint inhibitors.

doi: 10.1136/jitc-2024-009893

Figure Lengend Snippet: Figure 5 PNCK knockdown enhances the efficacy of immune checkpoint inhibition therapy. (a) The experimental schedule for the SCC7 cell challenge in the C3H mice tumor model is as follows. Intraperitoneal injections of ICIs were administered at a dose of 200 µg per mouse on the 5th, 8th, and 11th days after tumor implantation. The experiment was terminated on the 23rd day, and the tumors were removed for subsequent analysis. If the tumor volume reached 2000 mm³ during the experiment, euthanasia was performed immediately. (b) Overall tumor growth curves in different groups of mice. Mice were intraperitoneally treated with 200 µg anti-PD-L1 or 200 µg anti-CTLA4 on days 5, 8 and 11 after tumor inoculation. A rat IgG isotype antibody was applied as a control. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean values±SEM. The statistical comparisons among these groups were listed as follows: WT+IgG vs KD+IgG, p=0.0258; WT+CTLA4 vs KD+CTLA4, p=0.0994; WT+PD-L1 vs KD+PD-L1, p=0.0016; WT+CTLA4 + PD-L1 vs KD+CTLA4 + PD-L1, p=0.0412; KD+IgG vs KD+CTLA4, p=0.0016; KD+IgG vs KD+PD-L1, p=0.0177; KD+CTLA4 vs KD+CTLA4 + PD-L1, p=0.0066; KD+PD-L1 vs KD+CTLA4 + PD- L1, p<0.0001. (c) Individual tumor growth curves and percentages in different groups of mice. (d) Tumor growth curve in mice with cured tumors after subcutaneous reimplantation of HNSCC. Two-way ANOVA with Tukey’s multiple comparison test was performed. Data are presented as mean values±SD. (e) Expression levels of immune markers and cytokines in tumors of mice in different groups. Kruskal-Wallis with Dunn’s multiple comparison test are reported. ANOVA, analysis of variance; ICIs, immune checkpoint inhibitors.

Article Snippet: For ICI therapy, mice with WT or Pnck- KD SCC7 cells received intraperitoneal injections of anti- mouse CTLA4 mAbs (200 μg per mouse; BioXCell, Cat No. BE0032, clone: UC10- 4F10- 11, RRID: AB_1107598), anti- PD- L1 mAbs (200 μg per mouse; MCE, Cat No. HY- P99145, clone: 10F.9G2), or corresponding isotype control mAbs on days 5, 8, and 11.

Techniques: Knockdown, Inhibition, Tumor Implantation, Control, Comparison, Expressing

Journal: Neurosurgical Review

Article Title: Dual biomarker role of PD-L1 and LC3B in glioblastoma: prognostic and therapeutic potential

doi: 10.1007/s10143-025-04050-7

Figure Lengend Snippet: (A) Diffuse/fibrillary strong PD-L1 expression in glioblastoma (IHC, x400). (B) Diffuse/fibrillary PD-L1 moderate expression in glioblastoma (IHC, x200). (C) Diffuse/fibrillary and cytoplasmic weak PD-L1 expression in glioblastoma (IHC, x400). (D) High LC3B cytoplasmic expression in glioblastoma (IHC, x400). (E) Low LC3B expression in glioblastoma (IHC, x200)

Article Snippet: The sections placed in an endogenous peroxide block for 15 min and For purpose of antigen retrieval for both PDL-1 & LC3B, sections were treated in microwave of (600 W) by immersion of the slides in citrate puffer solution (PH 7) for 20 min subsequently applied PD-L1 (mouse monoclonal antibodies, CUSABIO, Houston, USA, clone no.14D8C2, product code: CSB-MA878942A1m, 1: 200 [ ]) and LC3B (mouse monoclonal antibody, CUSABIO, Houston, USA, Clone No. 18F2, Product Code CSB-MA171423, a dilution of 1: 100 for 60 min, and then immunocomplexes were visualized with diaminobenzidine for 10 min and covered by a coverslip.

Techniques: Expressing

Journal: Neurosurgical Review

Article Title: Dual biomarker role of PD-L1 and LC3B in glioblastoma: prognostic and therapeutic potential

doi: 10.1007/s10143-025-04050-7

Figure Lengend Snippet: OS according to expression of each PD-L1& LC3B and PFS according to LC3B; ( A ) OS in patients with high PD-L1 expression= 8.07 ± 0.62(Mean±SE) with 95% C.I (6.85-9.281), moderate expression OS= 9.65 ± 1.00 with 95% C.I (7.68-11.62) and weak PD-L1 expression OS=23.14 ±1.26with 95%C.I (20.67-25.60). ( B ) OS in cases with high LC3B expression=9.88 ± 1.19 (Mean±SE) with 95%C.I (7.55-12.21), and in those with low expression =20.77 ± 1.42 with 95%C.I (17.99-23.56). ( C ) PFS in cases with High LC3B expression = 2.93 ± 0.33(Mean±S.E )with 95%C.I=2.27-3.58. PFS in cases with Low expression =8.47 ± 0.36 with 95%C.I =7.75-9.18

Article Snippet: The sections placed in an endogenous peroxide block for 15 min and For purpose of antigen retrieval for both PDL-1 & LC3B, sections were treated in microwave of (600 W) by immersion of the slides in citrate puffer solution (PH 7) for 20 min subsequently applied PD-L1 (mouse monoclonal antibodies, CUSABIO, Houston, USA, clone no.14D8C2, product code: CSB-MA878942A1m, 1: 200 [ ]) and LC3B (mouse monoclonal antibody, CUSABIO, Houston, USA, Clone No. 18F2, Product Code CSB-MA171423, a dilution of 1: 100 for 60 min, and then immunocomplexes were visualized with diaminobenzidine for 10 min and covered by a coverslip.

Techniques: Expressing

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet: Canine CD20-BB-ζ CAR-T cells express cPD-1 after CAR engagement (A) Schematic representation of cCD20-BB-ζ CAR construct. ICD, intracellular domain; TM, transmembrane domain. (B) Surface expression of cCD20-BB-ζ CAR on CD4 + and CD8 + T cells evaluated by flow cytometry 4 days post transduction. Plots are gated on lymphocytes>single>live>CD5 + >CD4 + or CD8 + cells. (C) Surface expression of cCD20 and cPD-L1 on engineered K562 cells. Plots are gated on live single cells. (D) cPD-1 expression on CAR-T cells after co-culture with indicated target cells at different E:T ratios. CAR-T cells were generated as described and 6 days after transduction (8 days post activation) cells were co-cultured with either K562-WT or K562-cCD20 cells with or without cPD-L1, and cPD-1 expression was determined by flow cytometry after 72 h. Plots are gated on lymphocyte>single>live>CD5 + >CAR + cells.

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Construct, Expressing, Flow Cytometry, Transduction, Co-Culture Assay, Generated, Activation Assay, Cell Culture

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet: cPD-L1 expressing targets suppress canine CAR-T cell effector functions (A) Proliferation index of canine CD4 + and CD8 + CD20-BB-ζ CAR T cells from 4 healthy donors, 72 h after stimulation with the indicated K562 target cells at an E:T ratio of 1:1. (B) cCD20-BB-ζ CAR-T cells were co-cultured with the indicated K562 target cells at different E:T ratios and cytotoxicity was measured after 24 h. Data are mean ± SD ( n = 4 biological replicates). (C) IFN-γ, IL-2, and TNF-α production by cCD20-BB-ζ CAR T cells cultured with K562 (wild-type) or K562-cCD20 cells with or without PD-L1 at an E:T ratio of 1:1. Data are mean ± SD ( n = 4 biological replicates). (D and E) Effects of target cPD-L1 expression on CAR-T cell phenotype. cCD20-BB-ζ CAR T cells were stimulated three times with K562-cCD20 or K562-cCD20-cPD-L1 cells and immunophenotyped 6 days after the third stimulation. (D) Representative two-dimensional dot plot of CD45RA and CD62L expression on CD8 + CAR T cells. (E) Data for each of the four dogs are shown. ∗ p < 0.05; ∗∗ p ≤ 0.01; ns: not significant. Naive-like (CD45RA + CD62L + ), TCM-like (CD45RA − CD62L + ), TEM-like (CD45RA − CD62L − ), TEMRA-like (CD45RA + CD62L − ).

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Expressing, Cell Culture

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet: An active PD-1/CD28 CSR augments cCD20-BB-ζ CAR T cell function against cPD-L1 expressing target cells (A) Design of CSR WT and CSR MUT CAR constructs. ECD, extracellular domain; TM, transmembrane; ICD, intracellular domain. (B) Cartoon showing the CD28 mutations that prevent Grb2 and Lck binding in the CSR MUT construct. (C) Canine CD4 + and CD8 + T cells successfully co-express the cCD20-BB-ζ CAR and the CSR WT and CSR MUT constructs. Representative data from one of four CAR-T cell products generated from 4 healthy donor dogs 6 days post-transduction. Plots are gated on lymphocyte>single>live>CD5 + >CD4 + or CD8 + cells. (D) CAR expression on CD4 + and CD8 + T cells from 4 healthy donor dogs transduced with CAR alone, CAR+CSR WT or CAR+CSR MUT . Data are mean ± SD ( n = 4 biological replicates). ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ns: not significant.

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Cell Function Assay, Expressing, Construct, Binding Assay, Generated, Transduction

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet: An active PD-1/CD28 CSR augments CAR-T function against PD-L1 engineered target cells (A) IFN-γ, TNF-α, and IL-2 production by CAR+CSR WT or CAR+CSR MUT after 24 h co-culture with K562-cCD20 or K562-cCD20-cPD-L1 target cells at an E:T ratio of 1:1. Data are mean ± SD ( n = 4 biological replicates). (B) Cytotoxicity of CAR, CAR+CSR WT , or CAR+CSR MUT against K562-cCD20 and K562-cCD20-cPD-L1 target cells at E:T ratio of 8:1. Data are mean ± SD ( n = 4 biological replicates). (C) Memory phenotype of CAR, CAR+CSR WT , or CAR+CSR MUT T cells determined 6 days after the third stimulation with K562-cCD20 and K562-cCD20-cPD-L1 target cells at an E:T ratio of 1:1. Data for each of the four dogs are shown. ∗ p < 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001, ns: not significant.

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Co-Culture Assay

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet: An active PD-1/CD28 CSR augments CAR-T function against canine B cell lymphoma (A) Schematic of experimental timeline of repeat stimulation of CAR-T cells with WT or CLBL-1ΔcPD-L1 target cells. (B) Confirmation of lack of cPD-L1 expression by CLBL-1ΔcPD-L1 cells by flow cytometry. Plots are gated on 7AAD − cells. (C) Cell surface expression of CAR on CD4 + and CD8 + CAR-T and CAR+CSR WT T cells from healthy donor dogs following cell sorting as determined by flow cytometry. Data from three individual dogs are shown. (D) IFN-γ and TNF-α production by CAR-T cells stimulated with CLBL-1 cells or CLBL-1ΔcPD-L1 cells after 5 rounds of stimulation at an E:T of 1:1. IFN-γ and TNF-αproduction was assessed in supernatants 24 h after the fifth stimulation. Data are mean ± SD of 3 technical replicates for each dog. (E) CAR-T and CSR WT CAR-T cells from healthy dogs were co-cultured with CLBL-1 target cells at an E:T ratio of 1:1. Supernatants were harvested at 24 h after the 5 th stimulation and assayed for IFN-γ and TNF-α production. (F and G) CAR-T and CSR WT CAR-T cells from 2 healthy dogs were stimulated 3 times every 3 days with K562-cCD20-cPD-L1 target cells and then co-cultured with (F) CLBL-1 cells or (G) CLBL-1 cells pre-treated with IFN-γ for 48 h, at increasing E:T ratios. Cytotoxicity was determined by luciferase assay at 24 h. Data are mean ± SD of 3 technical replicates for each dog are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Expressing, Flow Cytometry, FACS, Cell Culture, Luciferase

Journal: iScience

Article Title: Validation of a PD-1/CD28 chimeric switch receptor to augment CAR-T function in dogs with spontaneous B cell lymphoma

doi: 10.1016/j.isci.2024.110863

Figure Lengend Snippet:

Article Snippet: Seventy-two hours after electroporation, edited CLBL-1 cells were labeled with mouse anti-canine PD-L1 antibody (QED Bioscience), and editing efficiency was determined by flow cytometry.

Techniques: Purification, Recombinant, Conjugation Assay, Modification, Enzyme-linked Immunosorbent Assay, Luciferase, Software