Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Generated, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Flow Cytometry, Expressing, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Injection, Control, 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: 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