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: Molecular Biomedicine

Article Title: TBC1 domain family member 23 is essential for STING-mediated anti-melanoma effect

doi: 10.1186/s43556-026-00418-3

Figure Lengend Snippet: High TBC1D23 expression is correlated with greater immune cell infiltration in melanoma. a Expression level of TBC1D23 in melanoma (n = 558) and adjacent normal (n = 461) tissues from TCGA database was analyzed using the GEPIA 2 on 12 October, 2024 ( http://gepia2.cancer-pku.cn/#analysis ). Bars indicate the median expression level in each group. Statistical analysis was performed using t test. b Prognostic value of TBC1D23 in melanoma was analyzed using the Kaplan–Meier Plotter database ( http://kmplot.com/analysis/ ), which integrates multiple GEO datasets. Overall survival, event free survival and post progression survival were analyzed in all patients with melanoma. Log-rank tests were performed. HR below 1 implies greater survival probabilities for TBC1D23 high group compared with TBC1D23 low group. Detailed information on tumor samples can be found in the individual GEO dataset in the NCBI GEO website. c The estimated enrichment of 7 immune cells in the TBC1D23 high and TBC1D23 low groups was calculated by CIBERSORT. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. d Kaplan–Meier plots for CD8 + T cells infiltrates and TBC1D23 mRNA expression to visualize survival differences in melanoma. e Representative images of mIHC staining of the expression pattern of TBC1D23 and CD8a in human melanoma tissue microarrays. Scale bar 50 μm. f Correlation analysis between the number of TBC1D23 positive cells and the number of CD8 + T cells in human melanoma tissue microarrays. Human melanoma tissue microarrays were generated by SHANGHAI OUTDO BIOTECH CO.LTD. Data were analyzed by Pearson’s correlation

Article Snippet: CD8+ T cells were isolated from spleens and lymph nodes of wild-type C57BL/6 mice using CD8+ T cell isolation beads (MCE #HY-K0310), then activated by stimulation for 72 h on plates coated with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28, supplemented with 20 ng/mL mIL-2.

Techniques: Expressing, Staining, Generated

Journal: Molecular Biomedicine

Article Title: TBC1 domain family member 23 is essential for STING-mediated anti-melanoma effect

doi: 10.1186/s43556-026-00418-3

Figure Lengend Snippet: TBC1D23 is critical for STING-dependent infiltration of immune cells. a , b Representative FACS data of frequency ( a ) and quantification ( b ) of tumor infiltrating CD45 + T cells or CD8 + T cells (n = 5 per group) of the mice as in Fig. a. c B16F10 tumors were isolated as in Fig. a, then stained with anti-CD45-cy3, anti-F4/80-cy5, anti-CD8-spAqua or anti-GZMB-FITC, and counterstained with DAPI. F4/80 is marker of macrophages. Scale bar, 50 μm. d Quantitative analysis of the numbers of CD45 + cells, macrophages, CD8 + T cells and GZMB + CD8. + T cells within same area of tumors as in c. Data are representative of three independent experiments. means ± SEM. Data were analyzed by one-way ANOVA ( c and d )

Article Snippet: CD8+ T cells were isolated from spleens and lymph nodes of wild-type C57BL/6 mice using CD8+ T cell isolation beads (MCE #HY-K0310), then activated by stimulation for 72 h on plates coated with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28, supplemented with 20 ng/mL mIL-2.

Techniques: Isolation, Staining, Marker

Journal: Molecular Biomedicine

Article Title: TBC1 domain family member 23 is essential for STING-mediated anti-melanoma effect

doi: 10.1186/s43556-026-00418-3

Figure Lengend Snippet: Deletion of TBC1D23 in macrophages impairs STING-mediated antigen presentation. a , b Mean fluorescent intensity (MFI) of MHC-II (A) and CD86 (B) in BMDMs stimulated with or without DMXAA. WT and Tbc1d23 KO BMDMs were stimulated with or without DMXAA for 24 h, then subjected to flow cytometry analysis (n = 3 biologically independent samples). Left and right panels show representative histograms and quantitative data, respectively. c , d Flow cytometric analysis of expression of H2-k b and H2-k b -SIINFEKL on the surface of B16F10-OVA. WT and Tbc1d23 KO BMDMs were treated with or without DMXAA, supernatants were collected and applied to B16F10-OVA cells pre-treated with either isotype control or anti-Ifnar1. Expression of H2-k b and H2-k b -SIINFEKL on the surface of B16F10-OVA was analyzed by flow cytometry. Left and right panels show representative histograms and quantitative data, respectively. e–g Flow cytometric analysis of antigen-specific cytotoxicity in CD8⁺ T cells. WT and Tbc1d23 KO BMDMs were treated with or without DMXAA, supernatants were collected and then applied to B16F10-OVA cells. CD8 + T cells were isolated from the spleen and lymph node of OT-I mice and co-cultured with B16F10-OVA cells pretreated with supernatants for 48 h. Percentages of GZMB + CD8 + T cells in CD8 + T cells ( e ), and CD107a + CD8 + T cells in CD8 + T cells ( f ), Zombie NIR. + cells in tumor cells ( g ) were analyzed by flow cytometry. Data are representative of three independent experiments. Data were analyzed by one-way ANOVA ( a–d , g ) or by unpaired t test ( e–f )

Article Snippet: CD8+ T cells were isolated from spleens and lymph nodes of wild-type C57BL/6 mice using CD8+ T cell isolation beads (MCE #HY-K0310), then activated by stimulation for 72 h on plates coated with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28, supplemented with 20 ng/mL mIL-2.

Techniques: Immunopeptidomics, Flow Cytometry, Expressing, Control, Isolation, Cell Culture