Journal: Scientific Reports

Article Title: Interferon Regulatory Factor 4 controls T H1 cell effector function and metabolism

doi: 10.1038/srep35521

Figure Lengend Snippet: WT and IRF4 −/− mice were i.v. infected with 5 × 10 4 LmOVA. Eight days post infection, spleen cells were stimulated for 4 h with 10 −5 M LLO 189-201 and intracellular expression of IFN-γ, TNF-α and CD40L was determined by flow cytometry. ( a ) Representative dot plots for CD4 + CD40L + spleen cells from infected mice stimulated with LLO 189-201 . ( b ) Frequencies of CD40L + , IFN-γ + and TNF-α + cells among CD4 + T cells from spleens of infected mice following stimulation. Bars represent mean ± SEM from 4 individually analyzed mice per group. The result is representative for 4 independent experiments.

Article Snippet: Fluorochrome-labelled antibodies anti-CD4 (clone RM4-5), anti-CD8α (53-6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD71 (R17217.1.4), anti-CD90.1 (His51), anti-CD90.2 (53-2.1), anti-CD127 (A7R34), anti-CD154/CD40L (MR1), anti-KLRG1 (2F1), anti-TCR Vα2 (B20.1), anti-CXCR3 (CXCR3-173), anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-T-bet (eBio4B10) and anti-IRF4 (3E4) were purchased from BD Biosciences, BioLegend or eBioscience.

Techniques: Infection, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Interferon Regulatory Factor 4 controls T H1 cell effector function and metabolism

doi: 10.1038/srep35521

Figure Lengend Snippet: Purified CD4 + T cells from CD90.1 + CD90.2 − WT and CD90.1 − CD90.2 + IRF4 −/− mice were mixed in a 1:1 ratio and 2 × 10 6 cells were i.v. transferred into RAG1 −/− mice, which had been infected with 10 5 Lm 4 h earlier. Eight days post infection, spleen cells of mice were analyzed. ( a ) Representative CD90.1/CD90.2 dot blots of CD4 + T cells at the time point of transfer (input) and at d8 post infection. ( b ) Normalized ratio of WT and IRF4 −/− CD4 + T cells. The figure shows results for individual mice combined from 3 experiments and the mean. ( c ) Surface expression of CD44, CD62L, KLRG1 and CXCR3 on transferred CD4 + T cells. ( d ) CD40L, TNF-α, and IFN-γ expression of transferred CD4 + T cells from spleens of infected mice following stimulation with PMA/ionomycin. ( e ) Representative intracellular T-bet staining for transferred cells (MFI mean fluorescence intensity). ( f ) T-bet expression in WT and IRF4 −/− CD4 + T cells following transfer and with or without infection. ( c,d,f ) Show mean ± SEM for 4 individually analyzed mice per group. Results are representative for 3 independent experiments (with the exception of results for CXCR3 which are derived from one experiment only).

Article Snippet: Fluorochrome-labelled antibodies anti-CD4 (clone RM4-5), anti-CD8α (53-6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD71 (R17217.1.4), anti-CD90.1 (His51), anti-CD90.2 (53-2.1), anti-CD127 (A7R34), anti-CD154/CD40L (MR1), anti-KLRG1 (2F1), anti-TCR Vα2 (B20.1), anti-CXCR3 (CXCR3-173), anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-T-bet (eBio4B10) and anti-IRF4 (3E4) were purchased from BD Biosciences, BioLegend or eBioscience.

Techniques: Purification, Infection, Expressing, Staining, Fluorescence, Derivative Assay

Journal: Scientific Reports

Article Title: Interferon Regulatory Factor 4 controls T H1 cell effector function and metabolism

doi: 10.1038/srep35521

Figure Lengend Snippet: Purified CD4 + T cells from WT CD90.1 + CD90.2 + or IRF4 −/− CD90.1 − CD90.2 + OT-II mice were mixed in a 1:1 ratio. 10 6 cells were i.v. transferred into CD90.1 + CD90.2 − WT mice which had been i.v. infected with 10 5 LmOVA one day before. On d4 post transfer, spleen cells from infected recipients were incubated for 4 h with 10 −6 M of OVA 323-339 peptide or with PMA/ionomycin and stained intracellularly for CD40L, TNF-α, and IFN-γ. Figures show frequencies of CD40L + , IFN-γ + and TNF-α + cells among transferred OT-II T cells following stimulation with OVA 323-339 ( a ) and PMA/ionomycin ( b ). Bars represent mean ± SEM from 6 individually analyzed mice per group. The result is representative for 4 independent experiments.

Article Snippet: Fluorochrome-labelled antibodies anti-CD4 (clone RM4-5), anti-CD8α (53-6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD71 (R17217.1.4), anti-CD90.1 (His51), anti-CD90.2 (53-2.1), anti-CD127 (A7R34), anti-CD154/CD40L (MR1), anti-KLRG1 (2F1), anti-TCR Vα2 (B20.1), anti-CXCR3 (CXCR3-173), anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-T-bet (eBio4B10) and anti-IRF4 (3E4) were purchased from BD Biosciences, BioLegend or eBioscience.

Techniques: Purification, Infection, Incubation, Staining

Journal: Scientific Reports

Article Title: Interferon Regulatory Factor 4 controls T H1 cell effector function and metabolism

doi: 10.1038/srep35521

Figure Lengend Snippet: CD4 + T cells from WT (CD90.1 + ) and IRF4 −/− mice (CD90.2 + ) were mixed with a 1:1 ratio. Cells were stimulated in vitro with anti-CD3 mAb, anti-CD28 mAb and IL-2 in the presence of IL-12 and anti-IL-4 mAb to induce T H1 differentiation. ( a ) After 4d, cells were stimulated for 4 h with PMA/ionomycin and IFN-γ and CD40L expression was determined by intracellular staining. Spleen cells from naive WT and IRF4 −/− mice were stimulated and stained in parallel (d0). ( b ) T-bet expression in WT and IRF4 −/− CD4 + T cells without stimulation and at d4 of stimulation (numbers give the MFIs). ( c ) Ratio of WT and IRF4 −/− CD4 + T cells in co-culture at indicated days of stimulation. ( d ) Loss of CFSE staining intensity on viable WT and IRF4 −/− CD4 + T cells. ( e ) Expression of Cdkn2a at d0 and d2 of purified WT and IRF4 −/− CD4 + T cells stimulated in individual cultures. Blots and histograms in ( a,b ) show representative results for CD4-gated cells. Results are representative for 3 ( a–c ) or 2 ( d ) independent experiments. Bars in ( e ) represent the mean ± SEM of 4 individual samples from 2 independent experiments.

Article Snippet: Fluorochrome-labelled antibodies anti-CD4 (clone RM4-5), anti-CD8α (53-6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD71 (R17217.1.4), anti-CD90.1 (His51), anti-CD90.2 (53-2.1), anti-CD127 (A7R34), anti-CD154/CD40L (MR1), anti-KLRG1 (2F1), anti-TCR Vα2 (B20.1), anti-CXCR3 (CXCR3-173), anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-T-bet (eBio4B10) and anti-IRF4 (3E4) were purchased from BD Biosciences, BioLegend or eBioscience.

Techniques: In Vitro, Expressing, Staining, Co-Culture Assay, Purification

Journal: Journal of Immunology Research

Article Title: Common Variable Immunodeficiency and Circulating T FH

doi: 10.1155/2016/4951587

Figure Lengend Snippet: Expression of costimulatory molecules (CD40L and ICOS) in PHA+IL-2 stimulated PBMC from CVID patients and N.

Article Snippet: Anti-CD4 (clone RPA-T4), CD25 (clone M-A251), CD45RO (clone UCHL1), CXC-chemokine receptors, 5 (CXCR5) (CD185, clone RF8B2) and 7 (CCR7, Clone 3D12), CD19 (clone HIB19), CD27 (clone M-T271), inducible T cell costimulator (ICOS) (CD278, clone DX29), CD40 ligand (CD40L) (CD154, clone TRAP1), and programmed-death 1 (PD-1) (CD279, clone MIH4) antibodies were purchased from BD Pharmingen, BD, and eBioscience (San Diego, CA, USA).

Techniques: Expressing