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: Life Science Alliance

Article Title: The expansion in lymphoid organs of IL-4 + BATF + T follicular helper cells is linked to IgG4 class switching in vivo

doi: 10.26508/lsa.201800050

Figure Lengend Snippet: (A) Immunofluorescence staining of CD4 (red), ICOS (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. The yellow broken line demarcates the area within GCs. The white broken line demarcates the area outside GC. (B) Immunofluorescence staining of CD4 (red), ICOS (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. (C) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain tissue from an IgG4-RD patient, normal tonsils, and normal mesenteric lymph nodes. (D) CD4 + ICOS + IL-4 + (red) and CD4 + ICOS + IL-4 − (green) cells were quantified in tissue from 12 tonsils. (E) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain normal tonsils and mesenteric lymph nodes.

Article Snippet: These specimens were incubated with the following antibodies: anti-AID (clone: ZA001; Invitrogen), anti-IgG4 (clone: ab109493; Abcam), anti-ICOS (clone: 89601; Cell Signaling Technology), anti-IL-4 (clone: MAB304; R&D Systems), GATA3 (clone: CM405A; Biocare), CXCR5 (clone: MAB190; R&D Systems), Bcl-6 (clone: CM410A,C; Biocare), BATF (clone: 10538; Cell Signaling Technology), CD4 (clone: CM153A; Biocare), and CD19 (clone: CM310 A,B; Biocare), followed by incubation with secondary antibodies using a SuperPicTure Polymer Detection Kit (Invitrogen) and an Opal 3-Plex Kit (Fluorescein, Cyanine3, and Cyanine5).

Techniques: Immunofluorescence, Staining, Fluorescence, Software

Journal: Life Science Alliance

Article Title: The expansion in lymphoid organs of IL-4 + BATF + T follicular helper cells is linked to IgG4 class switching in vivo

doi: 10.26508/lsa.201800050

Figure Lengend Snippet: (A) Immunofluorescence staining of CD4 (red), Bcl-6 (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. Quantification of CD4 + Bcl-6 + IL-4–positive T FH and CD4 + Bcl-6 + IL-4–negative T FH in 12 tonsils. (B) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G1) and normal tonsils. Quantification of CD4 + CXCR5 + IL-4 + and CD4 + CXCR5 + T FH cells in 17 IgG4-RD SMGs, 12 tonsils, and 7 SS salivary glands. The P -value is based on the Mann–Whitney U test. (C) Almost all IL-4–expressing CD4 + T cells in IgG4-RD lymph nodes were CXCR5 + T FH cells. Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD lymph node (LN1). The quantification of CD4 + IL-4 + CXCR5–positive T FH and CD4 + IL-4 + CXCR5–negative non-T FH cells in lymph nodes from two patients with IgG4-RD (LN1 and LN2) is shown. (D) CD4 + IL-4 + BATF + T cells were enriched in IgG4-RD SMGs. Immunofluorescence staining of CD4 (red), IL-4 (green), BATF (magenta), and DAPI (blue) in IgG4-RD SMGs (G12). White arrows indicate CD4 + IL-4 + BATF + T cells. (E) Quantification of CD4 + BATF + IL-4 + T cells and CD4 + T cells in 17 IgG4-RD SMGs and 12 normal tonsils. The P -value is based on the Mann–Whitney U test. (F) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain normal tonsils and IgG4-RD lymph node (LN1). (G) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G3). (H) Immunofluorescence staining of ICOS (green), BATF (magenta), and IL-4 (Red) in IgG4-RD LNs (LN1). (I) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD LNs (LN1). The quantification of ICOS + BATF + IL-4–positive T and ICOS + BATF + IL-4–negative T cells in lymph nodes from six patients with IgG4-RD (LN1–LN6). (J) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G3).

Article Snippet: These specimens were incubated with the following antibodies: anti-AID (clone: ZA001; Invitrogen), anti-IgG4 (clone: ab109493; Abcam), anti-ICOS (clone: 89601; Cell Signaling Technology), anti-IL-4 (clone: MAB304; R&D Systems), GATA3 (clone: CM405A; Biocare), CXCR5 (clone: MAB190; R&D Systems), Bcl-6 (clone: CM410A,C; Biocare), BATF (clone: 10538; Cell Signaling Technology), CD4 (clone: CM153A; Biocare), and CD19 (clone: CM310 A,B; Biocare), followed by incubation with secondary antibodies using a SuperPicTure Polymer Detection Kit (Invitrogen) and an Opal 3-Plex Kit (Fluorescein, Cyanine3, and Cyanine5).

Techniques: Immunofluorescence, Staining, Fluorescence, Software, MANN-WHITNEY, Expressing

Journal: Life Science Alliance

Article Title: The expansion in lymphoid organs of IL-4 + BATF + T follicular helper cells is linked to IgG4 class switching in vivo

doi: 10.26508/lsa.201800050

Figure Lengend Snippet: (A) Gating strategy used to sort IL-4–secreting and nonsecreting tonsillar CD45RA + CXCR5 + T FH cells following anti-CD3/anti-CD28 stimulation. CD45RA + CXCR5 − cells and CD45RA − CXCR5 − IL-4 + cells were sorted as additional controls. (B) A heatmap of differentially expressed genes across all four conditions depicting the Z -scores for normalized expected read counts. (C) A correlation matrix of differentially expressed genes. (D) Expression pattern of individual ILs is depicted on a log scale. (E) Expression of CD molecules, cytokines, and transcription factors clustered into patterns using k -means. Z -scores of the expected read counts for each cluster are shown.

Article Snippet: These specimens were incubated with the following antibodies: anti-AID (clone: ZA001; Invitrogen), anti-IgG4 (clone: ab109493; Abcam), anti-ICOS (clone: 89601; Cell Signaling Technology), anti-IL-4 (clone: MAB304; R&D Systems), GATA3 (clone: CM405A; Biocare), CXCR5 (clone: MAB190; R&D Systems), Bcl-6 (clone: CM410A,C; Biocare), BATF (clone: 10538; Cell Signaling Technology), CD4 (clone: CM153A; Biocare), and CD19 (clone: CM310 A,B; Biocare), followed by incubation with secondary antibodies using a SuperPicTure Polymer Detection Kit (Invitrogen) and an Opal 3-Plex Kit (Fluorescein, Cyanine3, and Cyanine5).

Techniques: Expressing

Journal: Life Science Alliance

Article Title: The expansion in lymphoid organs of IL-4 + BATF + T follicular helper cells is linked to IgG4 class switching in vivo

doi: 10.26508/lsa.201800050

Figure Lengend Snippet: (A) CD4 + CXCR5 + IL-4 + T cells were enriched around TLOs in IgG4-RD SMGs. Immunofluorescence staining of CD4 (red), IL-4 (magenta), CXCR5 (green), and DAPI (blue) in IgG4-RD SMGs (patient G3). Quantification of CD4 + CXCR5 + IL-4 + T FH cells and CD4 + CXCR5 + T FH cells comparing those outside and within GCs from each of five different areas in TLOs of 17 patients with IgG4-RD (G1–G17). The P -value is based on the Mann–Whitney U test. (B) IgG4 + B cells were enriched outside GC, especially some these cells express AID. Immunofluorescence staining of CD4 (red), CD19 (blue), and Bcl6 (green) in IgG4-RD SMGs (patient G3). Immunofluorescence staining of AID (red), Bcl6 (magenta), IgG4 (green), and DAPI (blue) in IgG4-RD SMGs (patient G3). The numbers of IgG4 + cells (per square micrometer) outside and within GCs were quantified from each of five different areas in TLOs of 17 patients with IgG4-RD (G1–G17). The P -value is based on the Mann–Whitney U test. (C) AID-expressing B cells outside GCs in IgG4-RD lymph nodes were abundant. Immunofluorescence staining of Bcl6 (red), AID (green), and ICOS (orange) in a normal tonsil and IgG4-RD lymph node. (D) Immunofluorescence staining of ICOS (green), IL-4 (red), and AID (magenta) in IgG4-RD SMGs. (E) Immunofluorescence staining of IgG4 (green), ICOS (magenta), IL-4 (red), and DAPI (blue) in TLOs with an IgG4-RD patient (G3). Magenta arrows indicate ICOS + IL-4 + T FH cells. Green arrows indicate IgG4 + B cells. A number of IL-4–expressing T FH cells and IgG4+ B cells formed close and extensive intercellular plasma membrane contacts. The distance was measured between the edge of each IgG4 + B cell nucleus to the edge of the closest IL-4–expressing T FH -cell nucleus, and an internuclear distance of less than 0.4 μm was indicative of a T-B conjugate. (F) Immunofluorescence staining of ICOS (green), IgG4 (magenta), BATF (red), and DAPI (blue) in the lymph nodes of an IgG4-RD patient. Green arrows indicate ICOS + BATF + T FH cells. Magenta arrows indicate IgG4 + B cells. A number of BATF + ICOS + T FH cells and IgG4 + B cells formed close and extensive intercellular plasma membrane contacts. The distance was measured between the edge of each IgG4 + B-cell nucleus to the edge of the closest BATF + ICOS + T FH -cell nucleus. All internuclear distances in this figure were <0.2 μm. LZ, light zone; DZ, dark zone.

Article Snippet: These specimens were incubated with the following antibodies: anti-AID (clone: ZA001; Invitrogen), anti-IgG4 (clone: ab109493; Abcam), anti-ICOS (clone: 89601; Cell Signaling Technology), anti-IL-4 (clone: MAB304; R&D Systems), GATA3 (clone: CM405A; Biocare), CXCR5 (clone: MAB190; R&D Systems), Bcl-6 (clone: CM410A,C; Biocare), BATF (clone: 10538; Cell Signaling Technology), CD4 (clone: CM153A; Biocare), and CD19 (clone: CM310 A,B; Biocare), followed by incubation with secondary antibodies using a SuperPicTure Polymer Detection Kit (Invitrogen) and an Opal 3-Plex Kit (Fluorescein, Cyanine3, and Cyanine5).

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Expressing, Membrane

Journal: Life Science Alliance

Article Title: The expansion in lymphoid organs of IL-4 + BATF + T follicular helper cells is linked to IgG4 class switching in vivo

doi: 10.26508/lsa.201800050

Figure Lengend Snippet: (A) Quantification of CD4 + CXCR5 + IL-4 + and CD4 + CXCR5 + T FH cells in 17 IgG4- RD SMGs, seven SS LSGs, 12 tonsils, two cervical lymph nodes, and three mesenteric lymph nodes. The P -value is based on the Mann–Whitney U test. (B) Correlations of the proportion of CD4 + CXCR5 + IL-4 + T FH cells and CD4 + BATF + IL-4 + T cells in SMGs from patients with IgG4-RD and their serum IgG, IgA, IgE, IgM, or IgG4 levels. The r and P -value were determined using Spearman’s rank correlations. (C) Correlations of the proportions of CD4 + CXCR5 + IL-4 + T FH cells in SMGs from patients with IgG4-RD and their ratio of IgG4 + /IgG + cells (n = 17). The r and P -value were determined using Spearman’s rank correlations. (D) The frequency of CD4 + CXCR5 + IL-4 + T FH cells in SMGs from patients with IgG4-RD (n = 17) correlated with the frequency of CD4 + BATF + IL-4 + T cells and serum IgG4 concentrations. The r and P -value were determined using Spearman’s rank correlations.

Article Snippet: These specimens were incubated with the following antibodies: anti-AID (clone: ZA001; Invitrogen), anti-IgG4 (clone: ab109493; Abcam), anti-ICOS (clone: 89601; Cell Signaling Technology), anti-IL-4 (clone: MAB304; R&D Systems), GATA3 (clone: CM405A; Biocare), CXCR5 (clone: MAB190; R&D Systems), Bcl-6 (clone: CM410A,C; Biocare), BATF (clone: 10538; Cell Signaling Technology), CD4 (clone: CM153A; Biocare), and CD19 (clone: CM310 A,B; Biocare), followed by incubation with secondary antibodies using a SuperPicTure Polymer Detection Kit (Invitrogen) and an Opal 3-Plex Kit (Fluorescein, Cyanine3, and Cyanine5).

Techniques: MANN-WHITNEY