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: 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: