Journal: Clinical & Translational Immunology

Article Title: Parallel processing of T cells and natural killer cells to enhance in vivo CAR T cell activity against blood and solid cancers

doi: 10.1002/cti2.70120

Figure Lengend Snippet: Phenotypic characterisation of expanded natural killer (NK) cells. NK cells were characterised on Day 14 of expansion. Uniform Manifold Approximation and Projection (UMAP) plots show the 10 distinct meta clusters of NK cells generated via FLOWSOM (a) , with a contour UMAP indicating proportions of NK cells belonging to each cluster (b) . A clustered heatmap visualisation displays the MFI of each indicated marker belonging to NK cells within the corresponding clusters (c) . To model ADCC, PD‐L1 was over‐expressed on MCF‐7 cells (d) prior to cytotoxicity assay in the presence/absence of 10 μg/mL avelumab (e) . NK Cell phenotyping, n = 3. Cytotoxicity displayed as individual donors ( n = 4). ** P ≤ 0.005, by multiple paired t ‐tests.

Article Snippet: The human PD‐L1 (NCBI 29126) gene with endogenous signal sequence was synthesised by Twist Biosciences and cloned into SfiI sites of pSBbiP—a modified pSBbiGP encoding puromycin resistance but with the original GFP gene removed.

Techniques: Generated, Marker, Cytotoxicity Assay

Journal: Clinical & Translational Immunology

Article Title: Parallel processing of T cells and natural killer cells to enhance in vivo CAR T cell activity against blood and solid cancers

doi: 10.1002/cti2.70120

Figure Lengend Snippet: Recycled natural killer (NK) cells enhance CAR T cell therapy of MCF‐7. NSG mice were engrafted with Her2 h i PDL1 hi MCF‐7 cells on Day 0 prior to sequential administration of NK cells (d10) and CAR T cells (d12) (a) . Quality control of both cell types ensured high NK cell purity and transduction efficiency of T cells prior to administration (b, c) . Mice were imaged weekly (d, e) , and callipers were used every other day to monitor tumor burden (f) . Mice were euthanised when radiance and/or tumor volume reached endpoint thresholds (g) . Data from one mouse experiment. n = 4–5 mice per group. Tumor burdens displayed as mean ± SEM. * P < 0.05 by two‐way ANOVA with Bonferroni correction. Survival analysed with Kaplan–Meier, log‐rank mantel‐cox test. ** P ≤ 0.005. White out‐lined box conceals mouse removed from study because of failed tumor engraftment.

Article Snippet: The human PD‐L1 (NCBI 29126) gene with endogenous signal sequence was synthesised by Twist Biosciences and cloned into SfiI sites of pSBbiP—a modified pSBbiGP encoding puromycin resistance but with the original GFP gene removed.

Techniques: Control, Transduction

Journal: The Journal of Gene Medicine

Article Title: PCAT6 Regulates IGF2BP1/PD‐L1 to Promote Immune Escape in Breast Cancer

doi: 10.1002/jgm.70105

Figure Lengend Snippet: Knockdown of PCAT6 suppresses PD‐L1 expression and enhances CD8 + T cell‐mediated cytotoxicity. (A) Correlation analysis between PCAT6 and PD‐L1 expression in tumor tissues from TNBC patients ( n = 68). (B) RT‐qPCR analysis of PD‐L1 expression in PCAT6‐knockdown TNBC cells ( n = 3). (C) Cytotoxic activity of CD8 + T cells assessed by LDH release assay after co‐culture with TNBC cells. (D, E) Levels of IFN‐γ (D) and granzyme B (E) in co‐culture supernatants measured by ELISA ( n = 3). (F) FCA of IFN‐γ expression in CD8 + T cells after co‐culture ( n = 3). Comparisons among three or more groups were conducted using two‐way analysis of variance, followed by Tukey's post hoc test. * p < 0.05.

Article Snippet: The membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by overnight incubation at 4°C with primary antibodies against PD‐L1 (1:3000, A1645, ABclonal) or GAPDH (1:10,000, AC001, ABclonal) as a loading control.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Activity Assay, Lactate Dehydrogenase Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Gene Medicine

Article Title: PCAT6 Regulates IGF2BP1/PD‐L1 to Promote Immune Escape in Breast Cancer

doi: 10.1002/jgm.70105

Figure Lengend Snippet: PCAT6 interacts with IGF2BP1. (A) Subcellular localization of PCAT6 in TNBC cells detected by FISH ( n = 3). (B) Effect of PCAT6 knockdown on the half‐life of PD‐L1 mRNA ( n = 3). (C) Venn diagram showing the overlap between PCAT6‐interacting RBPs predicted by starBase and prognosis‐associated genes from UALCAN. (D) Expression of IGF2BP1 in normal and BC tissues based on UALCAN. (E) Correlation between IGF2BP1 expression and overall survival in BC patients (UALCAN). (F) RIP assay demonstrating the binding between PCAT6 and IGF2BP1 ( n = 3). (G) Schematic diagram of full‐length (FL) PCAT6 and its different truncation fragments (T1, T2, and T3). (H) RNA pull‐down assay detecting the binding of different PCAT6 segments to IGF2BP1 in MDA‐MB‐468 cells. Comparisons among three or more groups were conducted using two‐way analysis of variance, followed by Tukey's post hoc test. * p < 0.05.

Article Snippet: The membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by overnight incubation at 4°C with primary antibodies against PD‐L1 (1:3000, A1645, ABclonal) or GAPDH (1:10,000, AC001, ABclonal) as a loading control.

Techniques: Knockdown, Expressing, Binding Assay, Pull Down Assay

Journal: The Journal of Gene Medicine

Article Title: PCAT6 Regulates IGF2BP1/PD‐L1 to Promote Immune Escape in Breast Cancer

doi: 10.1002/jgm.70105

Figure Lengend Snippet: PCAT6 promotes the binding and stabilization of PD‐L1 mRNA by IGF2BP1, leading to upregulation of PD‐L1 expression. (A) Correlation analysis between IGF2BP1 and PD‐L1 expression in tumor tissues from TNBC patients ( n = 68). (B) RT‐qPCR analysis of IGF2BP1 and PD‐L1 expression in IGF2BP1‐knockdown TNBC cells ( n = 3). (C) RIP assay evaluating the binding between IGF2BP1 and PD‐L1 mRNA ( n = 3). (D) RNA pull‐down assay assessing the interaction of IGF2BP1 with PD‐L1 mRNA ( n = 3). (E) RIP assay measuring the enrichment of PD‐L1 mRNA in anti‐IGF2BP1 immunoprecipitates following PCAT6 overexpression ( n = 3). (F) RT‐qPCR analysis of IGF2BP1 and PD‐L1 expression in PCAT6‐knockdown TNBC cells with or without IGF2BP1 overexpression ( n = 3). (G) Effect of PCAT6 knockdown and IGF2BP1 overexpression on PD‐L1 mRNA half‐life ( n = 3). Comparisons among three or more groups were conducted using two‐way analysis of variance, followed by Tukey's post hoc test. * p < 0.05.

Article Snippet: The membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by overnight incubation at 4°C with primary antibodies against PD‐L1 (1:3000, A1645, ABclonal) or GAPDH (1:10,000, AC001, ABclonal) as a loading control.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Knockdown, Pull Down Assay, Over Expression

Journal: ACS Omega

Article Title: Fast-Food Component-Induced Epigenetic Modifications Modulate Immunotherapy in Metastatic Breast Cancer

doi: 10.1021/acsomega.5c10558

Figure Lengend Snippet: Primer Sequences Used for qRT-PCR Analysis

Article Snippet: The primary antibody against PD-L1 (Cat. No. A1645) was purchased from ABclonal.

Techniques:

Journal: ACS Omega

Article Title: Fast-Food Component-Induced Epigenetic Modifications Modulate Immunotherapy in Metastatic Breast Cancer

doi: 10.1021/acsomega.5c10558

Figure Lengend Snippet: Relative mRNA expression levels of (a–b) CD47 and (c–d) PD-L1 in MDA-MB-231 cells following 7 days of treatment with IC 25 and IC 50 concentrations of MSG, AA, NaNO 2 , and SS. Relative protein expression of (e) CD47 and (f) PD-L1 was determined by indirect ELISA experiment. (g) Percentage of apoptotic Jurkat cells (human T-cell leukemia cell line) following 24 h of co-culture with fast-food component-treated MDA-MB-231 cells. Data are presented as the mean ± SEM ( n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, whereas ns indicates no significant difference compared with the vehicle control. PBS-treated MDA-MB-231 cells served as the vehicle control.

Article Snippet: The primary antibody against PD-L1 (Cat. No. A1645) was purchased from ABclonal.

Techniques: Expressing, Indirect ELISA, Co-Culture Assay, Comparison, Control