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: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: JHU083 inhibited T cells differentiation in vivo . Mice of Vehicle-AIH and JHU083-AIH group were intravenously injected with 8ml/kg ConA, and Vehicle-WT group was injected with normal saline of equal volume. JHU083-AIH group was gavaged with 0.3mg/kg JHU083 24h and 1h before ConA injection, and Vehicle-WT and Vehicle-AIH group were treated with equal vehicle at the same time points. (A) Flow cytometry was applied to analyze the Th1 cell percentage via IFNγ expression, the Th2 cell percentage via IL4 expression, and the Th17 cell percentage via IL17A expression. (B) ELISA was applied to analyze serum IFNγ (Th1 cells) and IL17 (Th17 cells) secretions. Flow cytometry was also applied to analyze the differentiation of CD8(+) T cells into cytotoxic T lymphocytes, evidenced by secreting IFNγ (C) and Granzyme (D) . Data were expressed as means ± SEM. * P < 0.05, ** P <0.01, and *** P <0.001. ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vivo, Injection, Saline, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: DON treatment suppressed T cells differentiation in vitro . Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5μM DON, and cells were analyzed 24h after ConA stimulation. (A) The differentiation markers of CD4+ T cells (IFNγ for Th1 cells, IL4 for Th2 cells and IL17 for Th17 cells) were detected using flowcytometry. (B) qRT-PCR was applied to analyze IFN-γ, IL4 and IL17 mRNA levels. (C) ELISA was applied to analyze supernatant IFN-γ and IL17 secretions. Flow cytometry was also applied to detect the production of activation markers of CTL, such as IFNγ (D) and Granzyme B (E) . Data were expressed as means ± SEM. ** P <0.01 and *** P <0.001, ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activation Assay