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: bioRxiv

Article Title: Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

doi: 10.1101/2025.11.04.685127

Figure Lengend Snippet: (A) Experimental design. Mice were immunized intravenously with OVA-expressing rLCMV, rVSV or their reciprocal pseudotypes on d0 and d21, and blood as well as serum samples were collected over time. (B) Representative FACS plots of OVA-tetramer-binding CD8 T cells and their phenotype in blood at d20. (C) Frequencies of OVA-tetramer binding CD8 T cells in blood at d7 and d20. (D) Frequencies of OVA-tetramer-binding CD8 T cells with an effector (KLRG1 + CD127 - ) or memory precursor (KLRG1 - CD127 + ) phenotype in blood at d20. (E) Frequencies of OVA-tetramer binding CD8 T cells in blood after primary immunization (d20) and after boost (d28, d42) with rLCMV- or rVSV-based vectors. (F) Vector- neutralizing antibodies in serum of mice vaccinated with rLCMV- or rVSV-based vectors over time. (G) Cytopathic effect of EGFP-expressing rLCMV and rVSV after infection of BHK21 cells. (H) Luciferase activity in serum of WT mice immunized with sNluc-expressing vectors pseudotyped with VSVG or LCMVGP. Samples from mice immunized with rLCMV-OVA were used to determine technical backgrounds. Symbols in (C,D,E) represent individual mice with bars in (C,D) showing the mean±SEM. Symbols in (F) show the mean±SEM of n=5 (F) or n=3 (H) mice. One representative experiment of two similar ones is shown. Statistical analysis was performed by two-way ANOVA with Bonferroni’s post- test for multiple comparisons (C, D, E and H); ns: not significant; *: p < 0.05, **: p < 0.01, p > 0.05 was considered not statistically significant and is not indicated.

Article Snippet: Purification of CD8 T cells was performed with magnetic-activated cell sorting (naïve CD8 T+ T cells Isolation Kit, mouse, Miltenyi Biotec).

Techniques: Expressing, Binding Assay, Plasmid Preparation, Infection, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

doi: 10.1101/2025.11.04.685127

Figure Lengend Snippet: (A) Cytopathic effect of rVSV-EGFP and rVSVMq-EGFP vectors on Vero E6 cells at 0, 12 or 24 hours after infection. (B) Luciferase activity in serum of WT (top) and Ifnar -/- Ifngr -/- Rag -/- mice (bottom) immunized with sNluc- expressing vectors. Samples from mice immunized with rLCMV-OVA were used to determine technical backgrounds. (C) We immunized WT mice with rLCMV-S1, rVSVMq-S1 or rVSV-S1 on d0 and collected blood over time to analyze S1-tetramer-binding CD8 T cells. Representative FACS plots of S1-tetramer- binding CD8 T cells in blood at d14. (D) Frequencies of S1-tetramer-binding CD8 T cells in blood over time. (E) Gating strategy to identify S1-epitope-specific CD8 T cell subsets with an effector or memory precursor phenotype as judged based on KLRG1/CD127 (top) or CD27/CX3CR1 expression (bottom) at d14. (F) Frequencies of effector (KLRG1 + CD127 - ) and memory precursor (KLRG1 - CD127 + ) subsets (top) among S1-tetramer-binding CD8 T cells, and the abundance of subsets based on CX3CR1 expression levels in conjunction with CD27 (bottom) at d14 after immunization. (G) We immunized WT mice with rLCMV-OVA, rVSVMq-OVA or rVSV-OVA and 14 days later challenged them with OVA- expressing Listeria monocytogenes and collected spleens on d17. (H) Bacterial loads on d17 in spleen. Symbols in (B,D) represent the mean±SEM of n=3 (B) and n=5 (D) mice. Symbols in (F,H) represent individual mice with bars indicating the mean±SEM. One representative experiment of two similar ones is shown. Statistical analyses were performed by two-way ANOVA with Bonferroni’s post-test for multiple comparisons (B, D, F) or one-way ANOVA with Tukey’s post-test (H) *: p < 0.05, **: p < 0.01, p > 0.05 was considered not statistically significant and is not indicated.

Article Snippet: Purification of CD8 T cells was performed with magnetic-activated cell sorting (naïve CD8 T+ T cells Isolation Kit, mouse, Miltenyi Biotec).

Techniques: Infection, Luciferase, Activity Assay, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

doi: 10.1101/2025.11.04.685127

Figure Lengend Snippet: (A) We immunize WT mice with rLCMV, rVSVMq or rVSV expressing the S1 domain of the SARS-CoV-2 spike protein (Wuhan Hu-1 strain) and spleens were collected at d7 and d14. (B) Clustering of splenic S1 epitope-specific CD8 T cells from two mice per condition (vector used, time point), visualized using t-distributed stochastic neighbor embedding (t- SNE). Each cell is represented by a point and colored by cluster. (C) Dotplot of gene expression levels of selected genes among clusters. (D) Differential abundance of CD8 T cells in each cluster, individually displayed for each vector and time point. (E) Gene set enrichment analysis of cluster 1 cells from day 14, comparing rVSVMq-induced CD8 T cells compared against those induced by rVSV. Gene set enrichment analysis was conducted and gene sets with an FDR < 0.01 are displayed. (F) IFN-α levels in serum of mice immunized with rLCMV-S1, rVSVMq-S1 or rVSV-S1. (G-I) Luciferase activity in serum of WT and Ifnar -/- mice immunized with sNluc-expressing rLCMV (G), rVSVMq (H) or rVSV (I). Symbols in (F) represent individual mice (n=4 per group) with bars showing the mean±SEM. Symbols in (G-I) show the mean±SEM of 3 mice per group. One representative experiment of two similar ones is shown in (F, G, H and I). Statistical analyses in (G-I) were performed by two-way ANOVA with Bonferroni’s post-test for multiple comparisons, *: p < 0.05, **: p < 0.01, p > 0.05 was considered not statistically significant and is not indicated.

Article Snippet: Purification of CD8 T cells was performed with magnetic-activated cell sorting (naïve CD8 T+ T cells Isolation Kit, mouse, Miltenyi Biotec).

Techniques: Expressing, Plasmid Preparation, Gene Expression, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

doi: 10.1101/2025.11.04.685127

Figure Lengend Snippet: (A) We treated mice with either anti-IFNAR antibody or isotype control and immunized them with rVSV-S1, rVSVMq-S1 or rLCMV-S1 on d0. We analyzed blood over time and collected the spleen on d30. (B) Frequencies of S1 epitope-specific CD8 T cells in blood over time. (C) Splenic count of total S1 epitope- specific CD8 T cells in in spleen on d30. (D) Splenic count of S1 epitope-specific CX3CR1 high effector CD8 T cells in spleen on d30. (E) We transferred 2000 OT-1 or OT-1x Ifnar -/- cells i.v. at d-1 and immunized the recipients at d0 with rVSV-OVA, rVSVMq-OVA or rLCMV-OVA. NK cell-depleting antibody was administered on d-1 and d1. (F) Frequencies of OT-1 and OT-1x Ifnar -/- cells in blood over time. (G) Total numbers of OT-1 and OT-1x Ifnar -/- cells in spleen at day 30. (H) Splenic count of OT-1 and OT-1x Ifnar -/- cells with an effector phenotype (CX3CR1 high ) at day 30. Symbols in (C,D,G,H) represent individual mice with bars showing the mean±SEM. Symbols in (B,F) show the mean±SEM of 5 mice per group. One representative experiment of two similar ones is shown. Statistical analyses were performed with two-way ANOVA with Bonferroni’s post-test for multiple comparisons (B,C,D,F,G,H); ns: not significant; *: p < 0.05, **: p < 0.01.

Article Snippet: Purification of CD8 T cells was performed with magnetic-activated cell sorting (naïve CD8 T+ T cells Isolation Kit, mouse, Miltenyi Biotec).

Techniques: Control