Journal: Immunology

Article Title: Insufficient interleukin-12 signalling favours differentiation of human CD4 + and CD8 + T cells into GATA-3 + and GATA-3 + T-bet + subsets in humanized mice

doi: 10.1111/imm.12304

Figure Lengend Snippet: Preferential type 2 differentiation of human CD4+ and CD8+ T cells in humanized mice. HIS (human immune system) mice were generated by transplantation of human fetal liver-derived CD34+ haematopoietic stem cells into newborn NRG mice. Twelve weeks post transplantation type 1 versus type 2 differentiation of human CD3+ CD4+ and CD3+ CD8+ T cells present in the peripheral blood of HIS mice was compared with that of human peripheral blood mononuclear cell (PBMC) -derived T cells. (a) Intracellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) production of human PBMC (n = 8) and HIS (n = 20) derived CD4+ and CD8+ T cells upon unspecific stimulation with PMA/ionomycin. (b) Representative FACS plots showing IFN-γ and IL-4 production of human and HIS CD8+ T cells. (c) Expression levels (mean fluorescence intensity) of the transcription factors GATA-3 and T-bet in CD4+ and CD8+ T cells isolated from human (n = 8) or HIS (n = 15) peripheral blood. (d) Representative FACS plots showing the expression of GATA-3 in human and HIS mouse-derived CD3+ T cells. (e,f) signal transducer and activator of transcription 6 (STAT-6) (e) and STAT-5 (f) phosphorylation of human (n = 8) and HIS mouse (n = 10) derived CD4+ and CD8+ T cells after 15 min of stimulation with human IL-4 and IL-2, respectively. Group data are shown on the left and representative histograms on the right. Data represent mean ± SEM. Summarized data of n = 8 human donors and n = 10 to n = 20 HIS mice acquired in at least three independent experiments are shown. Unpaired Student's t-test: *P ≤ 0·05; **P ≤ 0·005; ***P ≤ 0·0002.

Article Snippet: Human CD3 + T cells were isolated from blood and spleen of humanized mice by magnetic bead isolation (CD3 + Positive Selection Kit, Stem Cell Technologies).

Techniques: Generated, Transplantation Assay, Derivative Assay, Expressing, Fluorescence, Isolation, Phospho-proteomics

Journal: Immunology

Article Title: Insufficient interleukin-12 signalling favours differentiation of human CD4 + and CD8 + T cells into GATA-3 + and GATA-3 + T-bet + subsets in humanized mice

doi: 10.1111/imm.12304

Figure Lengend Snippet: Co-expression profile of lineage transcription factors and cytokines in T cells of humanized mice. HIS (human immune system) mouse-derived peripheral CD4+ and CD8+ T cells (12 weeks post human CD34+ haematopoietic stem cells) were simultaneously analysed for the expression of the transcription factors GATA-3, T-bet, Foxp3 and Eomes and the production of the cytokines interferon-γ (IFN-γ) and interleukin-4 (IL-4). (a) Representative FACS plots showing Foxp3 (upper left) and Eomes (upper right) expression within the CD3+ T-cell population of HIS mice. Cells gated on Foxp3+ and Foxp3− CD4+ T cells (lower left) and Eomes+ and Eomes− CD8+ T cells (lower right) were then compared for co-expression of T-bet and GATA-3. (b) GATA-3 and T-bet co-expression patterns of CD4+ Foxp3+ (left) and CD8+ Eomes+ (right) T cells (n = 8). (c) Cells were stimulated ex vivo with PMA/ionomycin before intracellular staining for GATA-3, T-bet, IFN-γ and IL-4. Representative FACS plots show the expression of IFN-γ and IL-4 in CD8+ (upper panel) or CD4+ (lower panel) GATA-3+ T-bet– and GATA– 3+T-bet+ subsets. (d) Frequencies of IFN-γ+, IL-4+ and IFN-γ+/IL-4+ cells within the GATA-3+ T-bet– and GATA-3+ T-bet+ subsets of HIS CD4+ and CD8+ T cells (n = 15). Data represent mean ± SEM. Summarized data of n = 8 to n = 15 HIS mice acquired in at least two independent experiments are shown. Unpaired Student's t-test: *P ≤ 0·05; ***P ≤ 0·0002.

Article Snippet: Human CD3 + T cells were isolated from blood and spleen of humanized mice by magnetic bead isolation (CD3 + Positive Selection Kit, Stem Cell Technologies).

Techniques: Expressing, Derivative Assay, Ex Vivo, Staining

Journal: Immunology

Article Title: Insufficient interleukin-12 signalling favours differentiation of human CD4 + and CD8 + T cells into GATA-3 + and GATA-3 + T-bet + subsets in humanized mice

doi: 10.1111/imm.12304

Figure Lengend Snippet: Co-expression of GATA-3 and T-bet in single human CD4+ and CD8+ T cells is stable in vitro. (a) Concentrations of human and murine interleukin-12p70 (IL-12p70) and IL-4 and human interferon-γ (IFN-γ) in the serum of NRG mice (n = 10) and HIS (human immune system) mice (n = 18). (b, c) Purified HIS mouse-derived T cells were stimulated with anti-CD3/CD28-coated beads and cultured in vitro for 14 days without the addition of exogenous cytokines or in the presence of 10 ng/ml human recombinant IL-12p70. (b) Representative FACS plots show GATA-3 and T-bet expression patterns of purified CD4+ and CD8+ T cells directly post purification and at day 14 of in vitro culture. (c) Left: representative histograms show differences in GATA-3 expression levels of CD8+ T cells cultured ± human IL-12p70 for 14 days (−IL-12: grey solid; +IL-12 black open). Right: expression levels of GATA-3 in CD8+ and CD4+ cultured ± human IL-12p70 for 14 days. (d) Concentrations of human IL-4 and IFN-γ in supernatants of cultured T cells. Data represent mean ± SEM. Representative data of three independent in vitro experiments are shown. Unpaired Student's t-test: *P ≤ 0·05.

Article Snippet: Human CD3 + T cells were isolated from blood and spleen of humanized mice by magnetic bead isolation (CD3 + Positive Selection Kit, Stem Cell Technologies).

Techniques: Expressing, In Vitro, Purification, Derivative Assay, Cell Culture, Recombinant