Journal: iScience
Article Title: PCSK9-mediated degradation of cell-surface LDL receptors impairs human CD8+ T cell effector functions
doi: 10.1016/j.isci.2026.114859
Figure Lengend Snippet: PCSK9 exposure of activated CD8 + T cells induces a decrease in ICAM-1 expression and granzyme B production in an antigen-driven activation model (A) Schematic representation of the experimental design, where CD8 + T cells specific to NLVPMVATV/HLA-A2 complexes were co-cultured with T2 cells loaded with NLVPMVATV peptide or the irrelevant MART-1-derived ELAGIGILTV peptide. NLVPMVATV peptide is derived from the CMV protein pp65. In the figure, “(−) ctrl” represents the co-culture in presence of the irrelevant MART-1-derived ELAGIGILTV peptide, while in all other conditions, CMV-derived NLVPMVATV peptide was added. Where indicated, recombinant PCSK9 (10 μg/mL) and alirocumab (2 μM) were supplemented to the co-culture. (B) Normalized LDLR cell surface gMFI levels on the CMV-specific CD8 + T cells measured with flow cytometry. Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗ p < 0.05, n = 4 independent replicates. Each dot represents an independent replicate, and lines depict medians. (C) Normalized ICAM-1 cell surface expression on the CMV-specific CD8 + T cells measured with flow cytometry. 3 h before measuring, cells were treated with GolgiStop (1,500x, BD Biosciences). Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗∗ p < 0.01, n = 6 independent replicates. Each dot represents an independent replicate, and lines depict medians. (D) Normalized intracellular granzyme B levels in the CMV-specific CD8 + T cells measured with flow cytometry. 3 h before measuring, cells were treated with GolgiStop (1,500x, BD Biosciences). Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗∗∗ p < 0.001, n = 6 independent replicates. Each dot represents an independent replicate and lines depict medians. (E) Normalized concentrations of secreted granzyme B by CMV-specific CD8 + T cells. Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗∗ p < 0.01, n = 5 independent replicates. Each dot represents an independent replicate, and lines depict medians. (F) Normalized LDLR cell surface expression on the CMV-specific CD8 + T cells measured with flow cytometry. Kruskal-Wallis test with Dunn’s multiple comparisons test, n = 3 independent replicates, ∗ p < 0.05. Each dot represents an independent replicate, and lines depict medians. (G) Normalized intracellular granzyme B levels in the CMV-specific CD8 + T cells measured with flow cytometry. 3 h before measuring, cells were treated with GolgiStop (1,500x, BD Biosciences). Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗ p < 0.05, n = 4 independent replicates. Each dot represents an independent replicate, and lines depict medians. (H) Normalized concentrations of secreted granzyme B by CMV-specific CD8 + T cells. Kruskal-Wallis test with Dunn’s multiple comparisons test, ∗ p < 0.05, n = 5 independent replicates. Each dot represents an independent replicate, and lines depict medians.
Article Snippet: CD8 T cell isolation kit , Miltenyi , 130-096-495.
Techniques: Expressing, Activation Assay, Cell Culture, Derivative Assay, Co-Culture Assay, Recombinant, Flow Cytometry
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![CD8 + T cells from individuals with hoFH validate a role of LDLR signaling for CD8 + T cell function (A) A schematic overview of the LDLR protein domains and LDLR-adaptor protein 1 (LDLRAP1), highlighting the hoFH mutations included in the experiments with blue reversed triangles. The empty reverse triangle shapes represent hoFH patients with mutations in the LDLR, while the filled reverse triangle shape represents hoFH patients with mutations in the LDLRAP1. (B) Cell surface LDLR gMFI levels on CD8 + T cells from hoFH patients ( n = 5) and healthy controls (HCs) ( n = 5). Cells were activated for the indicated duration of time with anti-CD3/CD28 Dynabeads and a cytokine mix (interleukin [IL]-2, IL-7, and IL-15). (C) Graphical illustration of the LDL-pHrodo uptake experiment. (D) Uptake of LDL-pHrodo measured by flow cytometry, where CD8 + T cells were activated for 24 h, followed by a 2-h culturing in lipoprotein-deprived medium and 2-h incubation with the LDL-pHrodo complex (20 μg/mL). Where indicated, anti-LDLR (5 μg/mL) was added when cells were cultured in lipoprotein-deprived medium. One-way ANOVA with Šídák’s multiple comparisons test, where ∗∗∗∗ p < 0.0001 ( n = 5 HCs and n = 5 hoFH patients). Each dot represents data from a separate donor, and lines depict medians. (E–H) Cells were activated for 72 h with anti-CD3/CD28 Dynabeads and cytokine mix (IL-2, IL-7, and IL-15). (E) Intracellular Ki67 levels measured with flow cytometry. Two-tailed Mann-Whitney test, where ∗ p < 0.05 ( n = 5 HCs and n = 5 hoFH patients). Each dot represents data from a separate donor, and lines depict medians. (F) Cell surface ICAM-1 levels measured with flow cytometry. Two-tailed Mann-Whitney test, where ∗ p < 0.05 ( n = 5 HCs and n = 5 hoFH patients). Each dot represents data from a separate donor, and lines depict medians. (G) Intracellular granzyme B levels measured with flow cytometry. Two-tailed Mann-Whitney test ( n = 5 HCs and n = 5 hoFH patients). (H) Secreted granzyme B levels measured with ELISA. Two-tailed Mann-Whitney test ( n = 5 HCs and n = 5 hoFH patients). Each dot represents data from a separate donor, and lines depict medians.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6826/pmc12936826/pmc12936826__gr5.jpg)