Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A. Representative images of tissue microarray IHC, (n = 206) stained with AURKA/DAB-brown, hematoxylin-nuclei/blue. Scale bar-300 μm. Insets-x250 enlarged areas. B, C Quantification N-AURKA positive(+) cells as in A, 3 randomly-assigned fields, n = 100 cells/field. B Pathological stages (Normal = 32, DCIS = 24, IDC = 72, MIDC-LN = 32, ILC = 24) and C receptor-based subtypes (Normal = 32, TNBC = 39, HER2+ = 32, ER+/PR+ = 30). D WB analysis of nuclear/cytoplasmic fractionations, as indicated. E Quantification of AURKA in cytoplasm/nucleus, percent-of-total, normalized to controls. F Schematic outline of cell line production. G, H Immunofluorescence and WB analysis of AURKA-sublines produced in F stained with AURKA (green), RFP(red), DAPI-nuclei/blue; clones indicated as c1/c2/c3. Scale bar-10 μm. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Microarray, Staining, Immunofluorescence, Produced, Clone Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Experimental design of xenograft study, n = 3 mice/group. B Quantification of Fluorescent-IHC using phospho-AURKA-T288 antibody in tumors. n = 50 mitotic-cells/tumor. C Quantification of final tumor volume/mm3. D Representative H&E images of lung metastases and DAB-IHC images of liver metastases stained with anti-RFP-antibody. Metastases outlined by black line. Scale bar-50 μm. E, F Quantification of metastases: number and area of metastases normalized to total area. G Lung and liver metastasis penetrance in mice treated with vehicle or MLN8237. One-way ANOVA, ±S.E.M. Tukey’s test. H Model of Nuclear-AURKA-HIF1 mediated gene expression. Normoxia: HIF1A is hydroxylated/ubiquitinated by PHDs/VHL resulting in degradation. Hypoxia: low-oxygen stabilizes HIF1A leading to HIF1A/B dimerization/activation, transactivation of hypoxia-response genes. Normoxia+N-AURKA: HIF1A/B activity is increased leading to metastasis. MLN8237 inhibits AURKA activity decreasing metastasis.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Staining, Gene Expression, Activation Assay, Activity Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells with indicated antibodies, DMSO-vehicle or DMOG for 7 h. B Quantification of WB results as in A, fold of change over DMSO-control. C, D Representative images of Immunoprecipitation/WB analysis with indicated antibodies, WCL-whole cell lysate. E Quantification of ChIP qPCR against selected promoter region, normalized to total input. F WB analysis of ChIP (before de-crosslinking). G Mass-spectrometry analysis of AURKA-IP complexes: Venn diagram displaying the numbers of proteins found in the NLS- or NES-AURKA complexes. The 850 proteins/yellow were further filtered, nuclear/blue. Pie-chart showing distribution of N-AURKA binding partners based on functional Panther Gene Ontology terms. Tables showing selected nuclear protein classifications for H RNA-binding and I DNA-binding from PANTHER Gene Ontology. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control, Immunoprecipitation, ChIP-qPCR, Mass Spectrometry, Binding Assay, Functional Assay, RNA Binding Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Heat-map of differentially expressed genes (DEGs, mean values) in cells. Gene expression (n = 3167) is normalized log2 counts/million. B Principal Component Analysis of RNA-Seq libraries as in A. C Volcano-plot analysis of RNAseq data as in A NLS-AURKA vs. control, log2 fold changes of gene expression on the x-axis, FDR statistical significance (−log10 p value) on the y-axis; upregulated (red), downregulated (black) genes in NLS, all genes (blue). Genes with at least a >1.5 fold change and FDR < 0.01 are displayed. D Visualization of Gene Ontology terms for NLS-AURKA vs. control; up/downregulated GOterms (FDR < 0.1) are depicted as circles; the distance indicates the relationship between terms: closer distance means higher similarity. Color and size of circles indicate significance of differential expression of an individual GO term in log10 p value. E WB analysis of selected RNA-seq target proteins as in A. F Quantification of WB in E, fold change over control. G GSEA comparison NLS-AURKA vs. control for selected GOterm gene sets. H Heat-map of HIF-dependent DEGs. I Predicted upstream regulators: activated (yellow), inhibited (blue) using IPA activation z-score. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing, Control, Quantitative Proteomics, Comparison, Activation Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells as indicated, treated with siRNA: control-scr or anti-HIF1A/B. B Quantification of WB results in A, fold of change over siScr. C Individual cell movement tracking-plots toward chemoattractant. Cell lines as indicated. Graphs of D total distance, E cell-body directionality, F cell speed. G qPCR analysis of select genes in control and NLS-AURKA cells with siScr and siHIF1A/B, fold change over control-siScr. H qPCR analysis of FOXM1 in control and NLS-AURKA cells treated with siScr and siHIF1A/B, fold change over control-siScr. I, J qPCR analysis of FOXM1, HIF1A, and HIF1B in control and NLS-AURKA cells treated with siScr and siFOXM1, fold change over control-siScr. HIF1B: (unpaired t test: con-Scr vs NLS-AURKA-Scr). One-way ANOVA, ±S.E.M. Tukey’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control

Journal: PLoS ONE

Article Title: High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells

doi: 10.1371/journal.pone.0110741

Figure Lengend Snippet: (A, B) PBMC from healthy donors were stimulated for 14 h with recombinant CMV pp65, or (C) for increasing periods of time with the autologous lymphoblastoid cell line (LCL), then assessed for reactivity by IFN-γ capture assay. Cells were maintained at (A) too high a concentration during IFN-γ secretion, or (B) an optimal concentration. Cells are gated on viable, CD14– CD16– CD19– subsets. (C) The percentage of CD8+ and CD4+ T cells secreting IFN-γ, gated on viable CD3+ cells. Results are representative of (A, B) two, or (C) three independent experiments.

Article Snippet: Alternatively, PBMC were stimulated with 1/100 (vol/vol) recombinant human cytomegalovirus (CMV) phosphoprotein 65 (pp65; Miltenyi Biotec).

Techniques: Recombinant, Concentration Assay

Journal: PLoS ONE

Article Title: High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells

doi: 10.1371/journal.pone.0110741

Figure Lengend Snippet: IFN-γ production measured by the capture assay in (A) unstimulated PBMC samples and (B) PBMC from healthy donors stimulated with the autologous lymphoblastoid cell line (LCL) or CMV pp65 (top two panels), or PBMC from melanoma patients A02 or D14 stimulated with autologous melanoma cells (bottom two panels), as indicated. Cells were gated on the single cell, low scatter, viable, CD14–CD16–CD19– subpopulation. Percentages are shown in each quadrant; ND = not done.

Article Snippet: Alternatively, PBMC were stimulated with 1/100 (vol/vol) recombinant human cytomegalovirus (CMV) phosphoprotein 65 (pp65; Miltenyi Biotec).

Techniques:

Journal: PLoS ONE

Article Title: High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells

doi: 10.1371/journal.pone.0110741

Figure Lengend Snippet: Cloning efficiency and functional retention of T cell clones.

Article Snippet: Alternatively, PBMC were stimulated with 1/100 (vol/vol) recombinant human cytomegalovirus (CMV) phosphoprotein 65 (pp65; Miltenyi Biotec).

Techniques: Clone Assay, Functional Assay

Journal: PLoS ONE

Article Title: High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells

doi: 10.1371/journal.pone.0110741

Figure Lengend Snippet: ELISA was used to measure IFN-γ in supernatants of T cell clones (▴CD4+; • CD8+) stimulated with (A) autologous or allogeneic lymphoblastoid cell lines (LCL), (B) autologous LCL either unloaded or pre-incubated with recombinant pp65 for 1-2 h, or (C, D) autologous or allogeneic melanoma cells (as indicated in graph). The stimulator: T cell responder ratios were (A–C) 50,000∶1,000 or (D, patient D14) 100,000∶10,000. Dotted line indicates>100 pg/ml in excess of control, the cut-off for positivity used in , which summarises the data from this figure.

Article Snippet: Alternatively, PBMC were stimulated with 1/100 (vol/vol) recombinant human cytomegalovirus (CMV) phosphoprotein 65 (pp65; Miltenyi Biotec).

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Incubation, Recombinant

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) List of 19 test compounds (plus S-2HG) used in screen in described in . (B) Percentage viability of CD8 + T cells following 7 days of treatment with 400μM of test compound, as determined by flow cytometry. Ordinary one-way ANOVA relative to CT; n=4. (C) Percentage of CD62L hi /CD44 hi CD8 + T cells following 7 days of treatment with increasing concentrations of DEG (left) or S-2HG (right). Ordinary one-way ANOVA; n=4. (D) Number of viable CD8 + cells following 7 days of treatment with increasing concentration of DEG (right) or S-2HG (left), as determined by counting beads and flow cytometry. Ordinary one-way ANOVA relative to CT; n=4. All scatter plots show median and 95% CI where each dot represents one murine donor. *p<0.05, **p<0.01, ***p<0.001, #p<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A and B) Percentage of CD62L hi /CD44 hi and number of total murine CD8 + T cells following 7 days of treatment with 400μM of test compound. Ordinary one-way ANOVA relative to control treated cells (CT); n=4. (C) Representative flow cytometry counter plots representing the expression of CD44/CD62L in mouse CD8 + T cells and CD45RO/CCR7 in human CD8 + T cells treated +/− 500μM DEG for 7 days. Pre-gated on live, single, CD8 + events. (D) Heatmap representing proportion of mouse CD8 + T cells expressing CD62L/CD44 (left) and the proportion of human CD8 + T cells expressing CCR7/CD45RO (right) following 7 days of treatment with increasing concentrations of DEG. Two-way ANOVA relative to CT; n=4-5. (E) Cell surface expression of CD25 and CD95 on human CD8 + T cells following 7 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by Median Fluorescence Intensity (MFI). Paired test; n=8. (F) Expression of markers associated with exhaustion on human CD8 + T cells following 7-10 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by MFI. Paired t test; n= 4-8. (G) Expression of cytotoxic granules on human CD8 + T cells following 7 days of treatment +/− DEG 500μM, as determined by flow cytometry and shown by MFI. Paired t test; n=4. (H) IFNγ secretion by human CD8 + T cells, 3 days post activation and treatment +/− DEG 500μM, as measured by ELISA. Paired t-test; n=4 All scatter plots show median and 95% confidence interval (CI). Each dot represents one donor (human or murine as indicated). *p<0.05, **p<0.01, ***p<0.001, #P<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Control, Flow Cytometry, Expressing, Fluorescence, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Chromatograms of isotope tracing. Human CD8 + T cells were treated with 13 C5-DEG for varying time length and mass spectrometry for glutarate was performed. (B) Quantified 13 C5-Glutarate from cells treated with 13 C5-DEG for varying time lengths, as described and shown as chromatograms in . (C) Glutarate and 2HG levels in activated human CD8 + T cells. Wilcoxon test: n=5. (D) Glutarate levels in human CD8 + T cells following 7 days of treatment +/− DEG 500μM. Paired t test; n=6. (D) Glutarate levels in human CD8 + T cells following 7 days of treatment +/− 500μM DEG. Paired t test; n=6. (E) Gcdh levels in transcripts HIF1 OE (HIF1AAA) and HIF2 OE (HIF2AAA) – transduced mouse CD8 + T cells, relative to vector control ( Veliça et al ., 2021 ). (F) GCDH levels in T cells transduced with an shGCDH or GCDH overexpressing vector as determined by western blot analysis. Paired t test; n=3. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05, **p<0.01, ***p<0.001, #p<0.0001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Mass Spectrometry, Plasmid Preparation, Control, Transduction, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Pathway of lysine and tryptophan catabolism. (B) Glutarate levels in naïve and 72 hr activated mouse CD8 + T cells. Paired t test; n=3. (C) Glutarate levels in naïve or 72 hr activated CD8 + T cells isolated from Hif-1αfl/fl or Hif-1αfl/fl dlckcre mice (HIF ko). Two-way ANOVA; n=3. (D) Glutarate levels in mouse CD8 + T cells cultured at 21% or 1% oxygen for 24 h from day 5 post activation. Paired t test; n=3. (E) GCDH protein copy number in naïve or activated CD8 + T cells from P14 transgenic mice. Unaired t test; n=3. Data from ImmPRes database ( Howden et al ., 2019 ). (F) GCDH counts per million (CPM) in HIF overexpressing (HIF OE) mouse CD8 + T cells. Unpaired t test; n=3. ( Veliça et al ., 2021 ). (G) Expression of markers of T cell differentiation and exhaustion in human CD8 + T cells following 7-10 days treatment +/− DEG 500μM and/or transduction with either shGCDH or GCDH OE vector. Expression as determined by flow cytometry and shown as MFI FC relative to relative untreated control (black dotted line). One sample t test; n=6-12. (J All scatter plot shows median + 95% CI where each dot represents one donor (murine or human as indicated). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Isolation, Cell Culture, Activation Assay, Transgenic Assay, Expressing, Cell Differentiation, Transduction, Plasmid Preparation, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Illustration of alphaketoglutarate (αKG) dependent dioxygenase (αKGDD) reactions investigated in this study. (B) Representative western blot and log2 FC protein expression of H3K9me2 in human CD8 + T cells treated with +/− DEG and/or αKG for 7 days. Graph shows median + 95% CI. Each dot represents one human donor. Kruskal-Wallis test; n=3-9.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Cell free enzymatic inhibition assay for TET2. Inhibition was determined using increasing concentrations of glutarate. Graph shows Michaelis-Menten line of best fit. (B) 5hmC MFI, as determined by flow cytometry, in human CD8 + T cells following 7 days of treatment with increasing concentrations of DEG. Ordinary one-way ANOVA; n=4. (C) Cell free enzymatic inhibition assay for HIF-P4-H1. Values are means of at least 3 independent experiments. Graph shows Michaelis-Menten line of best fit. (D) HIF-PH activity luciferase reporter assay in MEFs treated for 16h with DEG. N=4-6. (E) qPCR analysis of CD8 + T cells treated with DEG for 7 days. Each dot represents one human donor relative to untreated donor control (dotted black line), normalised to HPRT. One sample t test; n=5-8. (F) Representative western blot and log 2 FC in protein expression of specific histone H3 (H3) methylation sites in human CD8 + T cells treated for 7 days +/− DEG 500μM. Each dot represents one human donor normalised to total H3, and relative to untreated control (dotted black line). One-sample Wilcoxon test; n=6-12. (G) Representative western blots and log2 FC protein expression of H3K27me3 and H3K9me3 in human CD8 + T cells treated with +/− DEG and/or αKG for 7 days. Kruskal-Wallis test; n=3-12. (H) Cell free enzymatic inhibition assay for KDM6A. Values are means of at least 3 independent experiments. Graph shows Michaelis-Menten line of best fit. All scatter plots show median + 95% CI, where each dot represents one human donor. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Inhibition, Flow Cytometry, Activity Assay, Luciferase, Reporter Assay, Control, Western Blot, Expressing, Methylation

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Model of protein lysine-glutarylation. (B) Representative western of naïve and activated human CD8 + T cells. N=3. (C) Representative western blot of activated human CD8 + T cells cultured at different oxygen tensions (21%, 5% or 1%). N=4. (D) Representative western of human CD8 + T cells cultured with increasing concentrations of DEG for 7 days. N=4. (E) Representative western of activated human CD8 + T cells cultured with DEG for various time lengths as indicated. All samples were harvested 7 days post activation. N=4. (F) Representative western of CD8 + T cells transduced with shNTC or shGCDH vectors. RQR8 + cells were purified by FACS prior to analysis. N=3. (G) Coomassie staining of proteins immunoprecipitated with a pan-k-glutarylation antibody and separated by SDA-PAGE. 30×10 6 mouse CD8 + T cells, 7 days post activation were used. (H) PDHc isolated from 30×10 6 mouse CD8 + T cells by immunoprecipitation. (I) k-glutarylation WB of immunoprecipitated PDHc as described in . (J) Mode of PDHc. (K) Lipoate and PDHE2 glutarylation levels in HeLa cells treated with DEG. (K) % lipoylation of DLAT (PDHE2) in HEK293 cells treated with DEG for 24h as determined by western blot analysis and relative to βactin. Unpaired t test; n=4. *p<0.05.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Cell Culture, Activation Assay, Transduction, Purification, Staining, Immunoprecipitation, Isolation

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of cells treated with increasing concentrations of DEG for 7 days (as described in ). Ordinary one-way ANOVA; n=3. (B) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of human CD8 + T cells 7 days post activation. Cells were treated with DEG 500μM for different time lengths as indication and harvested at the same time 7 days post activation (as described in ). Ordinary one-way ANOVA; n=3. (C) Quantified total protein lysine-glutarylation or lysine-glutarylation of 67-70kDa protein, as determined by western blot analysis and relative to PPIB, of human CD8 + T cells which were cultured at different oxygen tensions for 24hr prior to harvest (as in described in ). (D) Model of lysine-glutarylated protein isolation and identification by mass spectrometry. Lysine-glutarylation immunoprecipitation was performed on 30×10 6 mouse CD8 + T cells, 7 days post activation. Following protein separation by SDS-PAGE, gel was stained with Coomassie blue and stained bands were excised, gel was digested and mass spectrometry was performed. Data was analysed using the Mascot Server v.2.3.1 and the SwissProt protein database. 3 independent experiments and analysis performed. (E) Top hits for each excised band from mass spectrometry described in . (F) Oxoglutarate dehydrogenase (OGDH) complex isolated from 30×10 6 mouse CD8 + T cells by immunoprecipitation. (G) k-glutarylation WB of immunoprecipitated OGDH complex as described in S4I. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Western Blot, Activation Assay, Cell Culture, Isolation, Mass Spectrometry, Immunoprecipitation, SDS Page, Staining

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Schematic of glycolysis and TCA cycle. (B) PDHc activity of human CD8 + T cells treated with DEG for 30 min (acute) or 7 days (chronic), normalised to total protein concentration. Paired t test; n=9-14. (C) PDHc activity of CD8 + T cells transduced with shGCDH relative to NTC control. RQR8 + cells were purified by FACS before assay. Unpaired t test; n= 3. (D) Basal ECAR levels in CD8 + T cells as determined by Seahorse FX analysis following 30 min DEG treatment via seahorse injection. ECAR normalised to ECAR prior to DEG/CT injection. Paired t test; 30 min n=10. (E) Basal ECAR levels in CD8 + T cells following 7 days of treatment +/− DEG 500μM as determined by Seahorse FX analysis Paired t test; 30 min n=5. (F) Basal OCR levels in CD8 + T cells as determined by Seahorse FX analysis following 30 min DEG treatment via seahorse injection. During assay cells were plated in FX media +/− glucose and glutamine as indicated. OCR represented at FC relative to untreated control. One-sample t test; n=6-10. (G) Basal OCR levels in CD8 + T cells following 7 days of treatment +/− DEG 500μM as determined by Seahorse FX analysis. During assay cells were plates in FX media +/− glucose and glutamine as indicated. One sample t test; n=6-10. (H) Seahorse XF Analysis of ECAR levels in CD8 + T cells (7 days post activation). Injections 1-4: DEG/CT, Glucose (Glc), Oligomycin (O), 2-Deoxy-D-Glucose (2-DG), as indicated. Mean and Error with 95% CI; n=10. (I) ECAR measurements during a standard GST of CD8 + T cells treated with +/− DEG 500μM for 7 days. Mean and Error with 95% CI; n=6. (J) Seahorse XF Analysis of OCR levels in CD8 + T cells (7 days post activation). Injections 1-4: DEG/CT, Oligomycin (O), Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (F), Rotenone/Antimycin (R/A). Mean and Error with 95% CI; n=10. (K) ECAR measurements during a standard GST of CD8 + T cells treated with +/− DEG 500μM for 7 days. Mean and Error with 95% CI; n=6. (L) mtDNA copy number in cells treated with +/− DEG 500μM. Acute treatment of CD8 + T cells 7 days post activation for 24h. Chronic treatment of CD8 + T cells for 7 days, from activation. Paired t test; n=4 (M) Mitochondrial fatty acid (FA) and glucose (Glc) oxidative capacity of CD8 + T cells treated +/− DEG 500μM for 7 days, as determined by OCR measurement pre and post addition of BPTES (BP), UK5099 (UK) and etomoxir (ETO) as indicated in . Paired t test; n=6. (N) Protein expression of phosphorylated PDH (ser239) and PDHK1, as determined by western blot analysis and normalised to PPIB. Acute treatment of CD8 + T cells 7 days post activation for 30 min. Chronic treatment of CD8 + T cells for 7 days, from activation. Protein levels normalised to PPIB or complex V expression as indicate. Wilcoxon test; acute n=10, chronic n=5. All scatter plots show median + 95% CI where each dot represents one human. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Activity Assay, Protein Concentration, Transduction, Control, Purification, Injection, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A and B) Quantitative analysis of MST and GST following acute (30 min) (left) and chronic (7 days) (right) DEG exposure, as described in , shown as log2 FC relative to untreated control (black dotted line). One sample t test; n=6-10. (C) Model of mitochondrial fuel flexibility tests. (D) Glutamine oxidative capacity of CD8 + T cells treated +/− DEG 500 μM for 7 days, as determined by OCR measurement pre and post addition of BPTES (BP), UK5099 (UK) and etomoxir (ETO) as indicated. Paired t test; n=5. (E) Gene expression of CD8 + T cells following 7 days of treatment with DEG, as determined by qPCR analysis, and represented as fold change relative to untreated control (dotted black line). One sample t test; n=3-4. (F) Protein expression of PDHc in CD8 + T cells treated +/− DEG 500μM. Acute treatment of CD8 + T cells 7 days post activation for 30 min. Chronic treatment of CD8 + T cells for 7 days, from activation. Protein levels normalised to PPIB or complex V expression as indicated. One-sample t-test; n=4. (G) Representative western blot of CD8 + T cells 7 days post activation as described and shown in . Cells were treated with DEG 500μM for different time lengths as indication and harvested at the same time 7 days post activation. All scatter plots show median and 95% CI where each dot represents one human donor. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Control, Gene Expression, Expressing, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Model of in vitro cytotoxicity assays. (B) Cytotoxicity and IFNγ expression (left) of CD19-CAR T cells following co culture with CD19 + Raji cells. Cytotoxicity of OT-1 following co culture with B16F10-OVA cells (right). CAR-T/OT-1 T cells were treated +/− DEG 500μM for 7 days prior to assay. Paired t test; n=9-12. (C) Model of HER2-CAR-T_shRNA cell generation. (D) Percentage cytotoxicity of HER2-CAR T cells with embedded shGCDH or NTC as determined by alamar blue assay following co-culture with HER2 expressing SKOV3 cells. Paired t test; n=4. (E) Adoptive cell therapy (ACT) model with CAR-T cells. 1×10 6 SKOV3 cells were implanted subcutaneously (s.c.) in NSG animals that were injected 21 days later with human shNTC- or shGCDH-expressing CAR-T cells generated as shown in . Tumour growth was monitored every 2-3 days until day 70. (F) Tumor growth data. Thin lines represent tumor growth from individual animals and thick lines represent an exponential (Malthusian) growth curve. No ATC n=5, HER2-CAR-T_shNTC n=11, HER2-CAR-T_shGCDH n=12. (G) Survival curves using 1000mm 3 in tumor volume as the threshold. Log-rank (mantel-Cox) test. (H) Tumor growth model. 1.0×10 6 B16F10 cells were s.c. injected in C57BL/6j mice. From day 4 post tumor inoculation, mice were injected intraperitoneally with 10mg/kg DEG, or control, every 2-3 days. On day 14 after tumor inoculation, peripheral blood, tumor, spleen and tumor draining lymph node from some animals were processed to single cell suspensions and analysed by flow cytometry. Tumor growth was monitored until day 30. (I) Tumor growth data. Thin lines represent tumor growth from individual animals and thick lines represent an exponential (Malthusian) growth curve (n=16). (J) Survival curves using 1000mm 3 in tumor volume as the threshold. Log-rank (mantel-Cox) test; n=14. (K) Frequency of CD8 + T cells in the peripheral blood, 14 days post tumor inoculation. Mann-Whitney test; n=25. (L) Frequency of CD8 + T cells in the tumours. Mann-Whitney test; n=14-16. All scatter plots show median and 95% CI where each dot represents one donor (human or murine as indicated). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: In Vitro, Expressing, Co-Culture Assay, shRNA, Alamar Blue Assay, Injection, Generated, Control, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Glutarate regulates T cell function and metabolism

doi: 10.1101/2022.10.20.513065

Figure Lengend Snippet: (A) Cell count of human CAR-T cells treated +/− 500μM for 7 days. Paired t test; n=6. (B) Cell count of murine OT-1 CD8 + T cells treated +/− 500μM for 7 days. Paired t test; n=6. (C) Cell count of HER2-CAR-T_shRNA cell numbers 7 days post transduction. Paired t test; n=4. (D) Cell count of different cancer cell lines treated with increasing concentrations of DEG for 72hr. Cell count relative to appropriate untreated control. One way ANOVA; n=3. (E) In vitro B16F10 cell growth +/− DEG 500μM as determined by cell counting and represented as log2 FC. One way ANOVA; n=3. (F) Gating strategy used for analysis of and . (G) Frequency of cell types in peripheral blood, tumor, spleen and tumour draining lymph nodes, 14 days post tumor inoculation, represented as log2 FC relative to CD45 + count. Graph shows median + 95% CI where each dot represents one mouse. Unpaired t test; n=14-16. *p<0.05, **p<0.01.

Article Snippet: Incubation with Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-049-401) was followed by magnetic bead isolation on a MACS column.

Techniques: Cell Counting, shRNA, Transduction, Control, In Vitro

Journal: iScience

Article Title: Early expansion of CD38+ICOS+ GC Tfh in draining lymph nodes during influenza vaccination immune response

doi: 10.1016/j.isci.2021.103656

Figure Lengend Snippet: Annual influenza vaccination details

Article Snippet: , PepTivator Influenza A (H1N1) Matrix Protein 2 (MP2) , Miltenyi Biotec , 130-099-812 , 30nmol/mL , 50μg/mL; 12.5μg/250 μL culture.

Techniques: Injection, Virus

Journal: iScience

Article Title: Early expansion of CD38+ICOS+ GC Tfh in draining lymph nodes during influenza vaccination immune response

doi: 10.1016/j.isci.2021.103656

Figure Lengend Snippet: Functional T cell assay stimulants

Article Snippet: , PepTivator Influenza A (H1N1) Matrix Protein 2 (MP2) , Miltenyi Biotec , 130-099-812 , 30nmol/mL , 50μg/mL; 12.5μg/250 μL culture.

Techniques: Functional Assay, Concentration Assay

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a Expression of TF from RNA-seq data of ileal biopsies from Crohn’s Disease (CD); illeal (iCD, n = 162), colonic (cCD, n = 56), Ulcerative Colitis (UC, n = 62), and query IBD healthy control cohort (CTRL, n = 42) patients in the RISK study (GEO ID GSE57945). b Expression of coagulation and inflammation-associated genes from RNA sequencing of paediatric CD ( n = 11), UC ( n = 7), and inflamed query non-IBD biopsies ( n = 12). c TF, CD3 and CD4 co-staining and d the average number of TF + CD3 + CD4 + T cells per field in colonic biopsies from paediatric IBD patients (IBD, n = 4, non-IBD, n = 3). e – h Lamina propria cells were isolated from colonic biopsies from paediatric IBD patients ( n = 5) and inflamed query non-IBD biopsies ( n = 2). e – g The percentage of TF expressing CD3 + CD4 + TF + T cells and h their cell surface TF expression was measured by flow cytometry and compared. Student’s t -test (two-tailed) ( d , h ) or Mann–Whitney U Test ( a , g ) was used to determine statistical significance from a 322 biological donors (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42), d 7 biological donors ( n = 4 IBD, n = 3 inflamed non-IBD), g , h 7 biological donors ( n = 5 IBD, n = 2 inflamed non-IBD) and expressed as mean ± s.e.m. Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Expressing, RNA Sequencing, Control, Coagulation, Staining, Isolation, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a Schematic diagram of the T cell transfer model of colitis. CD4 + T effector cells were isolated from donor C57BL/6 wild-type (WT) mice and i.p. injected into host Rag1 −/− mice ( Rag1 −/− T cells). Rag1 −/− mice i.p. injected with PBS were used as a control ( Rag1 −/− vehicle). b Colitis developed over a 4-week period, and disease progression was measured by % weight loss compared to original weight. c This was confirmed by subsequent colon histology analysis. d TF staining and e Corrected total fluorescence (CTF) of mice colons following T cell transfer-induced colitis. f TF and CD3 co-staining of mouse colons following T cell transfer-induced colitis (reproduced n = 4 Rag1 −/− T cells and n = 3 Rag1 −/− vehicle). 2-way ANOVA ( b ) or two-tailed Student’s t -test ( c , e ) was used to determine statistical significance from 3–4 biological replicates, n = 4 male Rag1 −/− T cells and n = 3 male Rag1 −/− vehicle, expressed as mean ± s.e.m. Scale bars: 100 µm ( d , f ). Source data are provided in the Source Data file. Created in BioRender. Preston, R. (2025) https://BioRender.com/o91u731 .

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Injection, Control, Biomarker Discovery, Staining, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor human blood and plated at a density of 0.8 × 10 6 /ml with IL-2 for unactivated conditions (θ). Plated cells were activated by anti-CD3/anti-CD28 activation beads and stimulated with IL-2 for Th0 conditions, and differentiation cytokines (αIL-4 + IL-12) were added to skew cells to a Th1 lineage. a – d Following 5 days in culture, θ, Th0 and Th1 cells were washed with EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in normal pooled platelet-poor plasma. b Lagtime, c peak thrombin levels and d endogenous thrombin potential (ETP) were measured and compared between θ, Th0 and Th1 cells. e The rate of clot formation was measured in θ, Th0 and Th1 cells. f – i θ, Th0 and Th1 cell-mediated thrombin generation was analysed by calibrated automated thrombinography using FVII-deficient platelet-poor plasma. g Lagtime, h peak thrombin levels and i ETP were measured and compared between θ, Th0 and Th1 cells. j F3 gene expression, k , l the percentage of cells expressing TF, m cell surface TF expression and n T cell-dependent FXa generation was measured in θ, Th0 and Th1 cells. Student’s paired t -test (two-tailed) ( c – e , g – i , l – n ), Wilcoxon test (two-tailed) ( b ), or Mann–Whitney U Test (two-tailed) ( j ) was used to determine statistical significance. Data is expressed as mean ± s.d. ( b – e , g – i , l – n ) for 7 ( a – d ), 10 ( e ), 6 ( g – i ), 8 ( j ) and 4 ( l – n ) biological donors/group. Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Activation Assay, Clinical Proteomics, Gene Expression, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor human blood and skewed to θ (unactivated cells), Th0, and Th1 cell subtypes as described previously. a ASMase translocation to the cell surface, d PS exposure on the outer membrane leaflet, and g PDI recruitment to the cell surface were assessed. TF decryption pathways in θ, Th0 and Th1 were analysed by b , c ASMase cell surface expression, e , f fluorescently labelled lactadherin binding to exposed cell surface PS, and h , i cell surface PDI expression. Student’s paired t -test (two-tailed) ( c , f , i ) was used to determine statistical significance. Data is expressed as mean ± s.d. ( c , f , i ) for 5 ( c ), 8 ( f ), and 4 ( i ) biological donors/group. Source data are provided in the Source Data file. Created in BioRender. Preston, R. (2025) https://BioRender.com/x11c079 .

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Translocation Assay, Membrane, Expressing, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor adult human peripheral blood, inflamed non-IBD paediatric peripheral blood and paediatric IBD peripheral blood were plated at a density of 1 × 10 6 /ml and incubated for 24–48 h at 37 °C in AIM-V media supplemented with immune replacement serum. a – d The percentage of cells expressing TF and e their cell surface TF expression was measured by flow cytometry and compared between groups. f – j Plated cells were washed in EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in FXII-deficient plasma. g Lagtime, h peak thrombin levels and i ETP were measured. Furthermore, the ability of isolated T cells to facilitate FXa generation in which the T cells were the sole source of TF, was measured ( j ). Mann–Whitney U Test (two-tailed) ( d , e , g ) or Student’s t -test (two-tailed) ( j , h , i ) was used to determine statistical significance. Data is expressed as mean ± s.e.m. ( d , e , g – j ) for d , e , j 17 biological donors ( n = 12 IBD, n = 2 inflamed non-IBD, N = 3 healthy adult), g – i 21 biological donors ( n = 13 IBD, n = 2 inflamed non-IBD, N = 6 healthy adult) and j 18 biological donors ( n = 12 IBD, n = 2 inflamed non-IBD, N = 4 healthy adult). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Incubation, Expressing, Flow Cytometry, Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: a PROC (Protein C; PC) expression in colonic biopsies from patients in the RISK study (GEO ID GSE57945) (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42). b , c Following cell culture, T cells were washed and incubated with fluorescently labelled activated PC. Binding was measured by flow cytometry. Following activated PC pre-treatment, activated PC was removed, θ (unactivated cells) and Th0 cells were washed with EDTA-containing PBS, and their capacity to initiate clotting was analysed by d – g thrombin generation and h clot formation assays. Activated PC pre-treated Th1 cell-dependent thrombin generation was analysed by i – l thrombin generation assays and m clot formation assays. Following activated PC pre-treatment, Th0 procoagulant activity was analysed by n FXa generation assay, o F3 gene expression and r , s PDI cell surface expression by flow cytometry. Similarly, Th1 cell thrombogenicity was analysed by p FXa generation assay, q F3 gene expression and t , u PDI cell surface expression by flow cytometry. The contribution of PDI-mediated TF decryption was assessed by treating Th0 ( v , w ) and Th1 ( x , y ) cells with 10 mM rutin for 1 h. Cells were washed with EDTA-containing PBS, and their capacity to initiate clotting was analysed by v – y thrombin generation. Student’s t -test (two-tailed) ( c ), Mann–Whitney U Test (two-tailed) ( a , n – q ), Student’s paired t -test (two-tailed) ( e – g , j – l , m , s , u , w , y ), or Wilcoxon test ( h ) was used to determine statistical significance. Data is expressed as mean ± s.e.m. ( a , c , e – h , j – l , m – q , s , u , w , y ) for a 322 biological donors (iCD, n = 162, cCD, n = 56, UC, n = 62, control non-IBD group, n = 42), c 5–10 biological donors ( n = 5 θ, n = 10 Th0 and n = 9 Th1), e – g , j – l 6 biological donors, h , m 10 biological donors, n , p , s , u 4 biological donors, o , q 8 biological donors and w , y 3–5 biological donors ( n = 3 θ, n = 5 Th0/Rutin and n = 5 Th1/Rutin). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Expressing, Control, Cell Culture, Incubation, Binding Assay, Flow Cytometry, Coagulation, Activity Assay, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling

doi: 10.1038/s41467-025-57001-7

Figure Lengend Snippet: CD4 + T cells were isolated from donor adult human peripheral blood, inflamed non-IBD paediatric peripheral blood and paediatric IBD peripheral blood, plated at a density of 1 × 10 6 /ml and incubated at 37 °C in AIM-V media supplemented with immune replacement serum, +/- 20 nM of activated PC for 24–48 h. a – d Cells were washed in EDTA-containing PBS, and their ability to initiate thrombin generation was analysed by calibrated automated thrombinography in FXII-deficient plasma. b Lagtime, c ETP and d peak thrombin levels were measured and compared, as was their ability to facilitate FXa generation ( e ). Mann–Whitney U Test (two-tailed) ( b – d ) or Student’s t -test (two-tailed) ( e ) was used to determine statistical significance for all conditions except IBD vs IBD + activated PC. For these conditions, either the Wilcoxon test (two-tailed) ( b – d ) or the Student’s paired t -test (two-tailed) ( e ) was used as the data is matched. Data is expressed as mean ± s.e.m. for b – d 21 biological donors ( n = 13 IBD/IBD activated PC, n = 2 inflamed non-IBD, N = 6 healthy adult) and e 19 biological donors ( n = 13 IBD, n = 2 inflamed non-IBD, N = 4 healthy adult). Source data are provided in the Source Data file.

Article Snippet: Isolated CD4 + T cells were plated at a density of 0.8 × 10 6 /ml in AIM-V media (#12055091, ThermoFisher) supplemented with CTS Immune Cell SR (#A2596101 Gibco, ThermoFisher), activated with anti-CD3/anti-CD28 activation beads per manufacturer’s instructions (#11131D, Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation, ThermoFisher), stimulated with IL-2 (#202-IL-050, R&D), and/or T cell differentiating cytokines and antibodies (Th1: anti-IL-4 (#130-095-753), IL-12 (#130-096-704) (Miltenyi Biotec), Treg: TGFβ (#11343160, Immunotools), Th17: TGFβ, IL-1β (#201-LB-005, R&D), IL-23 (#1290-IL-010, R&D), IL-6 (#206-IL-010, R&D), anti-IFNγ (#130-095-743, Miltenyi Biotec)) +/- activated PC (Cambridge Bioscience).

Techniques: Isolation, Incubation, Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles for all-trans retinoic acid in TGF-β–mediated induction of Foxp3 and Il10 genes in developing regulatory T cells

doi: 10.1084/jem.20080950

Figure Lengend Snippet: The combination of TGF-β– and CpG-activated BMDC induces IL-10–expressing CD4 T cells . (A) 10 5 D10 BMDC that were either left unactivated or preactivated with the indicated agonists were used in combination with 1 µg/ml OVA peptide to stimulate 5 × 10 5 naive CD4 T cells from 10BiT. Foxp3 gfp .OT-II mice, in the absence (top) or presence (bottom) of 5 ng/ml of recombinant human TGF-β1. On day 3, CD4 T cells were examined for expression of Thy1.1 and GFP. Numbers represent the percentage of CD4 T cells in the respective quadrants. (B) Naive CD4 T cells were stimulated as described in A with CpG-BMDC in the presence of TGF-β only or TGF-β plus anti–IFN-γ and anti–IL-4 (each at a final concentration of 10 µg/ml). On day 5, cells were restimulated for 5 h with PMA/ionomycin in the presence of brefeldin A, and viable CD4 T cells were analyzed for expression of Thy1.1, Foxp3, IL-17A, and IFN-γ. (C) Day-5 Thy1.1 + T cells (induced in the presence of CpG-BMDC and TGF-β) were MACS sorted and cultured for 24 h with Il10 −/− splenic DC in the absence (−) or presence (+) of 1 µg/ml OVA peptide. IL-10 protein in culture supernatants was quantitated by cytometric bead array. Mean cytokine concentrations ±SEM are shown. (ND, not detected). (D) 5 × 10 5 CFSE-labeled OT-II CD4 T cells were stimulated with 1.0 µg/ml OVA peptide and 10 5 IL-10–deficient splenic DC in the lower chamber of a transwell plate. In some cases, an equal number of DC or DC plus 5 × 10 5 day-5 Thy1.1 + T cells (differentiated as in C) were similarly activated in the upper chamber. Where indicated, anti–IL-10R antibody was added to the lower chamber at a final concentration of 10 µg/ml. After 4 d, cells were harvested from the bottom chamber and the CFSE profile of the CD4 T cells was analyzed by FACS. Numbers in A and B indicate the percentages of CD4 T cells in the given quadrants. All data are representative of at least two experiments.

Article Snippet: PE-conjugated anti-CD44 (IM7), anti-α 4 β 7 (LPAM), anti-CD90.1 (OX-7), anti-CD103 (M290), anti-IL-17 (TC11-18H10), PerCP (peridinin chlorophyll-a protein)-conjugated anti-CD90.1 (OX-7), and functional grade anti-IL-10R (1b1.3a) were purchased from BD; allophycocyanin-conjugated anti-CD4 (GK1.5), anti-IL-10 (JES5-16S3), anti–IFN-γ (XMG1.2), PE-conjugated and anti–IL-10 (JES5-16S3), PE-Cy7–conjugated anti-CD4 (GK1.5), affinity-purified anti-CCR9 (CW-1.2), and functional grade anti–IL-6 (MP5-20F3) were purchased from eBioscience.

Techniques: Expressing, Recombinant, Concentration Assay, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles for all-trans retinoic acid in TGF-β–mediated induction of Foxp3 and Il10 genes in developing regulatory T cells

doi: 10.1084/jem.20080950

Figure Lengend Snippet: CpG-BMDC–mediated induction of IL-10 in CD4 T cells is Myd88 and ICOSL independent but IL-6 and IL-21 dependent. (A) Naive T cells were activated on CpG-BMDC from WT, Myd88 −/− , or Icosl −/− mice in the presence of TGF-β and analyzed on day 3 for expression of Thy1.1 and GFP. (B) CD4 T cells were cultured with WT CpG-BMDC plus TGF-β in the presence of the additives indicated and analyzed on day 3 for expression of Thy1.1 and GFP. (C) D10 BMDC (left) were left unstimulated or stimulated for 24 h with the indicated TLR/NOD agonists. MACS purified CD4 + Thy1.1 + cells (right), induced in the presence of CpG-BMDC plus TGF-β, were cultured for 24 h with Il10 −/− splenic DC in the absence or presence of OVA peptide. IL-6 was quantified by cytometric bead array and IL-21 by ELISA. Mean cytokine concentrations ±SEM are shown (ND, not detected; *, P < 0.05; ***, P < 0.001). All data are representative of at least three experiments.

Article Snippet: PE-conjugated anti-CD44 (IM7), anti-α 4 β 7 (LPAM), anti-CD90.1 (OX-7), anti-CD103 (M290), anti-IL-17 (TC11-18H10), PerCP (peridinin chlorophyll-a protein)-conjugated anti-CD90.1 (OX-7), and functional grade anti-IL-10R (1b1.3a) were purchased from BD; allophycocyanin-conjugated anti-CD4 (GK1.5), anti-IL-10 (JES5-16S3), anti–IFN-γ (XMG1.2), PE-conjugated and anti–IL-10 (JES5-16S3), PE-Cy7–conjugated anti-CD4 (GK1.5), affinity-purified anti-CCR9 (CW-1.2), and functional grade anti–IL-6 (MP5-20F3) were purchased from eBioscience.

Techniques: Expressing, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles for all-trans retinoic acid in TGF-β–mediated induction of Foxp3 and Il10 genes in developing regulatory T cells

doi: 10.1084/jem.20080950

Figure Lengend Snippet: at-RA inhibits TGF-β–mediated induction of IL-10 in CD4 T cells. (A) Analysis of gut-homing receptor expression on CD4 T cells activated on CpG-BMDC in the presence of TGF-β. Shaded histograms, isotype control; open histograms, antibody to indicated marker. (B) Naive CD4 T cells were stimulated with CpG-BMDC and TGF-β only or TGF-β plus 100 nM at-RA for 3 d before being analyzed for expression of GFP and Thy1.1. (C) Naive CD4 T cells were stimulated on unactivated or preactivated BMDC alone or in the presence of TGF-β only, TGF-β plus 100 nM at-RA, or TGF-β plus 1 µM LE540. On day 5, cells were restimulated for 5 h with D10 BMDC and 1 µg/ml OVA peptide in the presence of brefeldin A and analyzed for expression of Thy1.1, GFP, and IL-17A. All FACS plots are gated on CD4 T cells and numbers represent the percentages of cells in the respective quadrants. (D) The polarized T cell populations were restimulated on day 5 with anti-CD3 and anti-CD28 antibodies for 24 h before the IL-10 content of culture supernatants was determined by cytometric bead array. Mean cytokine concentrations ± SEM are shown. *, P < 0.05. All data are representative of at least three experiments.

Article Snippet: PE-conjugated anti-CD44 (IM7), anti-α 4 β 7 (LPAM), anti-CD90.1 (OX-7), anti-CD103 (M290), anti-IL-17 (TC11-18H10), PerCP (peridinin chlorophyll-a protein)-conjugated anti-CD90.1 (OX-7), and functional grade anti-IL-10R (1b1.3a) were purchased from BD; allophycocyanin-conjugated anti-CD4 (GK1.5), anti-IL-10 (JES5-16S3), anti–IFN-γ (XMG1.2), PE-conjugated and anti–IL-10 (JES5-16S3), PE-Cy7–conjugated anti-CD4 (GK1.5), affinity-purified anti-CCR9 (CW-1.2), and functional grade anti–IL-6 (MP5-20F3) were purchased from eBioscience.

Techniques: Expressing, Marker

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles for all-trans retinoic acid in TGF-β–mediated induction of Foxp3 and Il10 genes in developing regulatory T cells

doi: 10.1084/jem.20080950

Figure Lengend Snippet: RA inhibits transcription but not translation of IL-10. (A and B) CD4 T cells differentiated as described in were reactivated for 24 h with anti-CD3 and anti-CD28 antibodies, and IL-10 (A) and IL-21 (B) production was quantified by cytometric bead array. (C) On day 5 after activation, CD4 T cells differentiated in the presence of CpG-activated BMDC plus the additives indicated were harvested for analysis of Il10 mRNA by real-time PCR. Data are normalized to cells activated with CpG-activated BMDC and antigen only and are from one of two experiments with similar results. (D) CD4 + Thy1.1 + cells were isolated from naive 10BiT mice as shown (top left) and left unstimulated or stimulated for 24 h with anti-CD3 and anti-CD28 antibodies only or with the addition of 100 nM at-RA or 1 µM LE540 before IL-10 protein in supernatants was quantified by cytometric bead array. Mean cytokine concentrations ±SEM are shown in A, B, and D, and all data are representative of three independent experiments. **, P < 0.005.

Article Snippet: PE-conjugated anti-CD44 (IM7), anti-α 4 β 7 (LPAM), anti-CD90.1 (OX-7), anti-CD103 (M290), anti-IL-17 (TC11-18H10), PerCP (peridinin chlorophyll-a protein)-conjugated anti-CD90.1 (OX-7), and functional grade anti-IL-10R (1b1.3a) were purchased from BD; allophycocyanin-conjugated anti-CD4 (GK1.5), anti-IL-10 (JES5-16S3), anti–IFN-γ (XMG1.2), PE-conjugated and anti–IL-10 (JES5-16S3), PE-Cy7–conjugated anti-CD4 (GK1.5), affinity-purified anti-CCR9 (CW-1.2), and functional grade anti–IL-6 (MP5-20F3) were purchased from eBioscience.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Isolation