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 human in vitro -activated CD8 + T cells reduces LDLR protein surface expression (A) Schematic representation of the experimental design for in vitro CD8 + T cell activation in the presence of recombinant PCSK9 (10 μg/mL). (B) Representative cell surface geometric mean fluorescence intensity (gMFI) levels of LDLR on CD8 + T cells measured by flow cytometry after 3 days of in vitro activation as described in (A). To visualize cell surface LDLR, CD8 + T cells were stained with anti-LDLR antibody (clone C7) for 30 min at 4 °C. gMFI levels were normalized to the activated (Act.) control. Two-tailed Mann-Whitney test where ∗∗ p < 0.01 compared to activated cells, n = 6 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (C) Normalized LDLR and HMGCR mRNA levels measured by qPCR in CD8 + T cells measured after 24 h of activation with anti-CD3/28 and cytokines with or without PCSK9 (10 μg/mL) supplementation. Two-tailed Mann-Whitney test where ∗∗ p < 0.01, n = 6 healthy donors. Graphs show independent donors normalized to PCSK9-untreated cells. Each dot represents data from a separate donor, and lines depict medians.

Article Snippet: CD8 T cell isolation kit , Miltenyi , 130-096-495.

Techniques: In Vitro, Expressing, Activation Assay, Recombinant, Fluorescence, Flow Cytometry, Staining, Control, Two Tailed Test, MANN-WHITNEY

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 anti-CD3/anti-CD28 activation cultures (A) Normalized ICAM-1 cell surface gMFI levels on CD8 + T cells measured at day 3 of activation with flow cytometry. Two-tailed Mann-Whitney test where ∗∗ p < 0.01 compared to PCSK9 untreated, activated cells, n = 6 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (B) Intracellular granzyme B levels measured with flow cytometry. Two-tailed Mann-Whitney test where ∗∗ p < 0.01 compared to PCSK9 untreated, activated cells, n = 6 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (C) Schematic representation of the experimental design for in vitro CD8 + T cell proliferation in the presence of recombinant PCSK9 (10 μg/mL). (D) CTV proliferation assay of CD8 + T cells; CTV dilution measured with flow cytometry. Kruskal-Wallis test with Dunn’s multiple comparisons test where ∗ p < 0.05, ∗∗∗ p < 0.001 compared to PCSK9 untreated, activated cells, n = 6 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (E) LDLR cell surface gMFI levels on CD8 + T cells measured with flow cytometry. Two-tailed Mann-Whitney test where ∗ p < 0.05, n = 4 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (F) ICAM-1 cell surface gMFI levels on CD8 + T cells measured with flow cytometry. Kruskal-Wallis test with Dunn’s multiple comparisons test where ∗ p < 0.05, n = 4 healthy donors. Each dot represents data from a separate donor, and lines depict medians. (G) Percent of CD8 + T cells positive for intracellular granzyme B. Kruskal-Wallis test with Dunn’s multiple comparisons test where ∗ p < 0.05, n = 4 healthy donors. Graphs show independent donors normalized to PCSK9-untreated cells. Each dot represents data from a separate donor, and lines depict medians.

Article Snippet: CD8 T cell isolation kit , Miltenyi , 130-096-495.

Techniques: Expressing, Activation Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, In Vitro, Recombinant, Proliferation Assay

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

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: 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.

Article Snippet: CD8 T cell isolation kit , Miltenyi , 130-096-495.

Techniques: Cell Function Assay, Flow Cytometry, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: STAR Protocols

Article Title: Protocol for the preparation and analysis of patient-derived organotypic tumor spheroids

doi: 10.1016/j.xpro.2025.104286

Figure Lengend Snippet: PDOTS surface marker staining (A) Scheme showing the steps for surface marker staining of PDOTS “on chip”. (B) Surface markers staining of melanoma PDOTS. The left image is an overlay of the indicated markers. White arrows denote CD38 + CD8 + T cells inside a tumor spheroid.

Article Snippet: CD8 MicroBeads , Miltenyi , Cat#130-045-201.

Techniques: Marker, Staining