Journal: Immunity, Inflammation and Disease

Article Title: TRPM2 Deficiency Attenuates Allergic Rhinitis‐Like Inflammation With Altered Ca 2+ ‐NFAT Signaling, Treg Responses, and sIgE Production

doi: 10.1002/iid3.70490

Figure Lengend Snippet: TRPM2 deficiency is associated with attenuated Ca 2+ ‐NFAT signaling during T cell activation. (A) Intracellular Ca 2+ flux in splenic CD4 + T cells from WT and TRPM2 −/− mice, measured by flow cytometry using Fluo‐4 AM after stimulation with anti‐CD3/CD28 antibodies (5 μg/mL each). The graph shows the mean fluorescence intensity (MFI) over time from one representative experiment of three independent replicates. (B) Nuclear translocation of NFATc1 and IL‐2 production were assessed by Western blot from WT and TRPM2 −/− mice.

Article Snippet: Naïve CD4 + T cells were then isolated by negative selection using a commercial mouse CD4 + T Cell Isolation Kit (Cat. No. 130‐104‐454, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions.

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Translocation Assay, Western Blot

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: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ 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: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

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: BOLT activates T cell PI3K-AKT-mTOR pathway to enhance T cell anti-tumor effect. (A) Flow cytometry plots compare IFN-γ and IL-2 expression at pH 7.8 and 6.0 along with various doses of BOLT in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (B, C) Bar graphs show IFN-γ and IL-2 expression in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (D) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 antibodies and incubated for 3 days with cell culture media of different pH levels. Western blot was performed to determine the phosphorylation of Akt and S6 under acidic conditions (pH 6.5) and alkaline pH (7.8). (E) Activated CD4 + T cells were treated with 0, 0.25, and 0.5 mg/mL doses of BOLT following CD4 + T cells activation at pH 7.8. Western blot analysis showing the phosphorylation of Akt and S6 were observed. (F) CD4 + T cells were activated and treated with BOLT at acidic pH. Western blot analysis was performed to determine the phosphorylation of Akt and S6. Two-way ANOVA was used for multiple comparisons. Experiments were conducted in triplicate. Data are mean ± SEM, ∗ 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: Flow Cytometry, Expressing, Incubation, Cell Culture, Western Blot, Phospho-proteomics, Activation Assay

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: 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: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ 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: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

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: BOLT activates T cell PI3K-AKT-mTOR pathway to enhance T cell anti-tumor effect. (A) Flow cytometry plots compare IFN-γ and IL-2 expression at pH 7.8 and 6.0 along with various doses of BOLT in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (B, C) Bar graphs show IFN-γ and IL-2 expression in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (D) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 antibodies and incubated for 3 days with cell culture media of different pH levels. Western blot was performed to determine the phosphorylation of Akt and S6 under acidic conditions (pH 6.5) and alkaline pH (7.8). (E) Activated CD4 + T cells were treated with 0, 0.25, and 0.5 mg/mL doses of BOLT following CD4 + T cells activation at pH 7.8. Western blot analysis showing the phosphorylation of Akt and S6 were observed. (F) CD4 + T cells were activated and treated with BOLT at acidic pH. Western blot analysis was performed to determine the phosphorylation of Akt and S6. Two-way ANOVA was used for multiple comparisons. Experiments were conducted in triplicate. Data are mean ± SEM, ∗ 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: Flow Cytometry, Expressing, Incubation, Cell Culture, Western Blot, Phospho-proteomics, Activation Assay

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: Characterization of BCATc and BCATm in differentiated CD4 + T cells. (A–E) CD4 + T cells, isolated from spleens and lymph nodes of WT mice, were activated and treated with skewing cytokines to compare the expression of BCATc and BCATm upon subset differentiation. (A) Bcat1 and Bcat2 mRNA ( n = 9 mice/variant) and (B) BCATc and BCATm protein levels ( n = 6–9 mice/variant) in different T-cell subsets. (C) Time course of BCATc and BCATm expression in activated but undifferentiated cells ( n = 12 mice). (D) Time course of BCATc and BCATm expression in activated cells in the absence or the presence of individual T-cell skewing cytokines ( n = 5–12 mice/cytokine/time point). (E) mRNA levels of T-cell lineage transcription factors in activated and differentiated cells treated with 10 mM NALA ( n = 6–9 mice/variant). (F, G) Comparison of BCAT1 and BCAT2 expression between lymphocytes from healthy (H) human donors and patients under disease (“D”) state. (F) BCAT1 and BCAT2 expression in CD4 + and CD8 + T cells from healthy donors ( n = 5 donors/cell type) and patients with HCV ( n = 10 patients/cell type), accompanied by a heat map showing the R values of BCAT1 or BCAT2 correlation with respective T-cell lineage transcription factors. (G) BCAT1 and BCAT2 expression in CD4 + T cells ( n = 10) and B cells ( n = 9) from healthy donors and patients with RA/SLE ( n = 14–16 patients/cell type), accompanied by a heat map showing the R values of BCAT1 or BCAT2 correlation with respective T-cell lineage transcription factors. In A–E panels, data represent 2 to 3 independent experiments with n = 3 to 6 pooled mouse spleens and lymph nodes/experiment, mixed sex. The Western blot images are representative of three independent experiments. Average ± SEM. Statistical significance as determined by a two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Isolation, Expressing, Variant Assay, Comparison, Western Blot, Two Tailed Test

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: Characterization of mice carrying a single deletion of Bcat1 or Bcat2 in T cells. (A, B) Genomic DNA from mouse ear showing presence of floxed (fl) alleles of Bcat1 (encodes BCATc, refer to “3” and “4”) or Bcat2 (encodes BCATm, refer to “7” and “8”). The CD4Cre allele in “4” and “8” was maintained in a hemizygous state. A separate WT band was amplified to confirm this state ( WTCD4Cre ). In addition, “4” and “8” contained the WT alleles of Bcat1 or Bcat2 , respectively. (C, D) cDNA produced by a reverse transcriptase, confirming the presence of floxed Bcat1 or Bcat2 in CD4 + T cells (“3” and “7”) or brain tissues (“3-4”,”7-8”, positive control). In contrast, floxed Bcat1 or Bcat2 transcripts were absent from activated CD4 + T cells isolated from T-BCATc KO or T-BCATm KO mice, respectively (refer to “4” and “8” in C, D). Eukaryotic translation elongation factor ( EF1α ) was used as a loading control. (E, F) Western blotting confirming the loss of expression of BCATc (“4”) or BCATm (“8”) from activated CD4 + T cells and spleen (negative control for BCATc), but not brain (positive control). β-Tubulin was used as a loading control. CD4 + T cells were activated as described under Methods. In all panels, at least three independent experiments ( n = 3–6 mice/variant/experiment, mixed sex) were performed. The images are representative of DNA or protein gels of tissue-specific samples. Variant names and genotypes: “1” and “5” is WT mouse [ Bcat1 +/+ Bcat2 +/+ CD4 Cre - ], “2” is Heterozygous mouse by BCATc [ Bcat1 fl/+ Bcat2 +/+ CD4 Cre - ] or “6” by BCATm [ Bcat1 +/+ Bcat2 fl/+ CD4 Cre - ], “3” is T-BCATc fl/fl mouse [ Bcat1 fl/fl Bcat2 +/+ CD4 Cre - ], “7” is T-BCATm fl/fl mouse [ Bcat1 +/+ Bcat2 fl/fl CD4 Cre - ], “4” is T-BCATc KO mouse [ Bcat1 fl/fl Bcat2 +/+ CD4 Cre +/0 ] with T cell: [ Bcat1 −/− Bcat2 +/+ CD4 Cre +/0 ] and “8” is T-BCATm KO mouse [ Bcat1 +/+ Bcat2 fl/fl CD4 Cre +/0 ] with T cell: [ Bcat1 +/+ Bcat2 −/− CD4 Cre +/0 ].

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Amplification, Produced, Reverse Transcription, Positive Control, Isolation, Control, Western Blot, Expressing, Negative Control, Variant Assay

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: BCATc, but not BCATm, enhances T H 1 subset differentiation and function. Mice from respective groups were either used as a source of CD4 + T cells for in vitro differentiation into iT H 1 cells or were challenged with OVA-producing EL4 mouse lymphoma cells for 10 days to induce T H 1 immune response in vivo. (A–C) Results obtained using T-BCATc KO and T-BCATc fl/fl mice. (A) Tbx21 mRNA expression and secretion of IFN-γ from splenic/lymphatic iT H 1 cells in the absence or the presence of 10 mM NALA ( n = 3–10 mice/variant). (B,C) Representative flow charts along with average bar graphs showing T-bet expression and IFN-γ production in OVA-induced splenic (B) and lymphatic (C) T H 1 (CXCR3 + CD4 + ) cells ( n = 6 mice/variant, a representative experiment is shown). (D–F) Results obtained using T-BCATm KO and T-BCATm fl/fl mice. (D) Tbx21 mRNA expression and secretion of IFN-γ from splenic/lymphatic iT H 1 cells in the absence or the presence of 10 mM NALA ( n = 3–6 mice/variant). (E,F) Representative flow charts along with average bar graphs showing T-bet expression and IFN-γ production in OVA-induced splenic (E) and lymphatic (F) T H 1 (CXCR3 + CD4 + ) cells ( n = 6 mice/variant, a representative experiment is shown). For all panels, average ± SEM or ± SD of mixed sex. Statistical significance as determined by a two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, or ns = no significance.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vitro, In Vivo, Expressing, Variant Assay, Two Tailed Test

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: T H 2 and T H 17 function is dependent on leucine but not leucine degradation. Mice from respective groups were either used as a source of CD4 + T cells for in vitro differentiation into iT H 2 and iT H 17 cells or were challenged with house dust mites (HDM, T-BCATm KO , and control mice only) for 4 weeks to induce T H 2 immune response in vivo. (A) Gata3 mRNA expression and secretion of IL-13 from iT H 2 cells. T-BCATc mouse colony, n = 6–9 mice/variant, T-BCATm mouse colony, n = 3 mice/variant. (B) Rorc mRNA expression and secretion of IL-17 from iT H 17 cells. T-BCATc mouse colony, n = 6 mice/variant, T-BCATm mouse colony, n = 3–6 mice/variant. (C) Representative flow charts along with average bar graphs showing IL-13 production and GATA3 expression by CD44 + CD4 + T cells isolated from the lungs of HDM sensitized T-BCATm KO and littermate control T-BCATm fl/fl mice ( n = 6 mice/variant). (D-E) IL-13 and IL-17 secretion from iT H 2 and iT H 17 cells in the absence or the presence of 10 mM NALA. T-BCATc mouse colony, n = 6 to 9 mice/variant, T-BCATm mouse colony, n = 6 mice/variant. In all panels, average ± SEM or ± SD of mixed sex. Statistical significance as determined by a two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, or ns = no significance.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vitro, Control, In Vivo, Expressing, Variant Assay, Isolation, Two Tailed Test

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: BCATc deletion promotes a T H 1-phenotype in mouse Tregs. T-BCATc KO and littermate controls were either used as a source of CD4 + T cells for in vitro differentiation into iTregs or were challenged with OVA-producing EL4 mouse lymphoma cells for 10 days to induce Treg immune response in vivo. (A) Foxp3 and Tgfb , mRNA expression and secretion of IL-10 from iTregs in the absence or the presence of 10 mM NALA. (B) Tbx21 and Ifng mRNA expression and secretion of IFN-γ from iTreg in the absence or the presence of 10 mM NALA. In A-B, n = 10 to 12 mice/variant. (C) Representative flow charts with an average bar graph of CD4 + T cells co-expressing Foxp3 + T-bet + following differentiation to iTregs ( n = 3 mice/variant). (D) Representative flow charts and average bar graphs showing expression of Foxp3 + in CD25 + CD4 + T cells or Tregs co-expressing Foxp3 + T-bet + following EL4-OVA stimulation in vivo ( n = 6 mice/variant, a representative experiment is shown). For all panels, average ± SEM or ± SD of mixed sex. Statistical significance as determined by a two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, or ns = no significance.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vitro, In Vivo, Expressing, Variant Assay, Two Tailed Test

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: BCATm deletion causes a similar shift in Tregs toward a T H 1-like phenotype as a loss of BCATc. T-BCATm KO and littermate controls were either used as a source of CD4 + T cells for in vitro differentiation into iTregs or were challenged with OVA-producing EL4 mouse lymphoma cells for 10 days to induce Treg immune response in vivo. (A) Foxp3 and Tgfb , mRNA expression and secretion of IL-10 from iTregs in the absence or the presence of 10 mM NALA. (B) Tbx21 mRNA expression and secretion of IFN-γ from iTregs in the absence or the presence of 10 mM NALA. In A and B, n = 3–8 mice/variant. (C) Representative flow charts with an average bar graph of CD4 + T cells co-expressing Foxp3 + T-bet + following differentiation to iTregs ( n = 3 mice/variant). (D) Representative flow charts and average bar graphs showing expression of Foxp3 + in CD25 + CD4 + T cells or Tregs co-expressing Foxp3 + T-bet + following EL4-OVA stimulation in vivo ( n = 6 mice/variant, a representative experiment is shown). For all panels, average ± SEM or ± SD of mixed sex. Statistical significance as determined by a two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, or ns = no significance.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vitro, In Vivo, Expressing, Variant Assay, Two Tailed Test

Journal: Immunometabolism (Cobham, Surrey)

Article Title: A loss of the cytosolic branched-chain aminotransferase, BCATc, enhances T h 1 differentiation and skews Tregs to acquire a T h 1-like phenotype

doi: 10.1097/IN9.0000000000000084

Figure Lengend Snippet: KEGG pathway analysis of genes correlated with BCAT1 or BCAT2 in human tonsils. Positive and negative gene correlations based on KEGG pathway analysis of (A) activated T helper cells and (B) Tregs. Genes that significantly correlated with BCAT1 or BCAT2 , but appeared associated with more than one KEGG pathway, are presented in bold. n = 5 specimens, correlation P value cutoff <0.01.

Article Snippet: CD4 + T cells were isolated via negative magnetic separation using CD4 + T-cell isolation kit (Cat # 130-104-454, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by culture in RPMI-1640 medium (Cat # 10-040-CV, Corning, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Cat # 25-011-CV, Corning, New York, NY, USA), 0.1 mg/mL Pen/Strep (Cat # 15140-122, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM Glutamine (Cat # 25030-081, Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mg/mL Gentamycin (Cat # 120-098-661, Quality Biological, Gaithersburg, MD, USA), and 55 μM β-mercaptoethanol (Cat # 21985, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: