foxp3 gfp cells  (Sony Biotechnology)

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: Effects of POH1 ablation on thymic Foxp3+ Treg cell generation. a Western blot analyzing the expression of POH1 and β-actin with or without stimulation of anti-CD3 and anti-CD28 in splenic CD4+ T cells. Data are representative of three independent experiments with similar results. b Flow cytometry analyzing the percentage of CD4Single+ and CD8Single+ T cells in thymocytes derived from 4-week-old poh1fl/flCd4cre and poh1+/+Cd4cre control littermates. c Percentages of various thymocyte subsets derived from 4-week-old poh1fl/flCd4cre and control littermates are presented (n = 9 per genotype). The results from four separate experiments were pooled. d BM cells from poh1fl/flCd4cre (CD45.2+) or poh1+/+Cd4cre mice (CD45.2+) were mixed 1:1 with BM from WT (CD45.1+) C57BL/6 mice, and these cells were injected into lethally irradiated CD45.1+ mice to generate bone marrow chimeras. Eight weeks later, the proportion of CD4Single+ and CD8Single+ T cells among the thymocytes from the same origin is shown. The results from three separate experiments were pooled. e Flow cytometry analyzing the percentage of Foxp3+ Treg cells among CD4Single+ thymocytes from 4-week-old poh1fl/flCd4cre and control mice. f Pooled data from e representing four separate experiments (n = 8 per genotype). g BM cells from poh1fl/flCd4cre (CD45.2+) or poh1+/+Cd4cre mice (CD45.2+) were mixed 1:1 with BM from WT (CD45.1+) C57BL/6 mice, and these cells were injected into lethally irradiated CD45.1+ mice to generate bone marrow chimeras. Eight weeks later, the proportion of Foxp3+ Treg cells among the CD4Single+ thymocytes from the same origin is shown. The results from three separate experiments were pooled. Numbers adjacent to outlined areas show the percent of cells in each. Each symbol represents a single mouse, and horizontal bars represent the mean. *P < 0.05; ***P < 0.001, two-tailed unpaired Student’s t test

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: Western Blot, Expressing, Flow Cytometry, Derivative Assay, Injection, Irradiation, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: POH1-deficiency impairs the transition of precursors into Treg cells. a Flow cytometry analyzing the percentage of CD25+Foxp3−, CD25−Foxp3+ and CD25+Foxp3+ cells among CD4Single+ thymocytes derived from 4-week-old poh1fl/flCd4cre or poh1+/+Cd4cre mice. b Pooled data from a representing three separate experiments. Each symbol represents a single mouse, and horizontal bars represent the mean. c, d Flow cytometry analyzing the expression of CD25 (c) and CFSE labeling (d) in sorted CD4Single+CD25−Foxp3-GFP−thymocytes from poh1fl/flCd4cre or poh1+/+Cd4cre mice. Cells were stimulated with plated bound anti-CD3 and anti-CD28 for 4 days. The numbers above bracketed lines indicate the percentage of proliferated cells. Data are representative of two independent experiments with similar results. e Flow cytometry analyzing the Foxp3 expression in purified thymic CD4Single+CD25+Foxp3-GFP−Treg cell precursors from poh1fl/flCd4cre mice and control littermates after stimulation with or without IL-2 for 24 h. Data are representative of three independent experiments with similar results. f Flow cytometry analyzing the p-STAT5 in purified thymic CD4Single+CD25+Foxp3-GFP− Treg cell precursors from poh1fl/flCd4cre mice and control littermates after stimulation with or without IL-2 for 15 min. Data are representative of two independent experiments with similar results. g Flow cytometry analyzing the expression of GITR and OX40 in CD4Single+CD25+Foxp3-GFP−Treg cell precursors from the thymus of 4-week-old poh1fl/flCd4cre or poh1+/+Cd4cre mice. Data are representative of four independent experiments with similar results. Numbers adjacent to outlined areas show the percent of cells in each. **P < 0.01; ***P < 0.001, two-tailed unpaired Student’s t test

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: Flow Cytometry, Derivative Assay, Expressing, Labeling, Purification, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: POH1 is required for Treg cell expansion. a, b Flow cytometry analyzing the expression of GITR, OX40, CTLA4 (a), CD44 and CD62L (b) in CD4Single+Foxp3+ Treg cells from the thymus of poh1fl/flCd4cre mice and control littermates. Data are representative of three independent experiments with similar results. c Flow cytometry analyzing the expression of Ki67 in CD4Single+Foxp3+ Treg cells from the thymus of poh1fl/flCd4cre mice and control littermates. d Pooled data from c representing three separate experiments. Each symbol represents a single mouse, and horizontal bars represent the mean. e Flow cytometry analyzing the incorporation of BrdU in CD4Single+Foxp3+ Treg cells from the thymus of poh1fl/flCd4cre mice and control littermates. f The percentage of BrdU+ cells in thymic Treg cells from poh1fl/flCd4cre mice and control littermates is shown. Data are shown as the mean + SD (n = 3 per genotype) and are representative of two independent experiments. g Flow cytometry analyzing Foxp3 expression in CD4Single+CD25−Foxp3-GFP− thymocytes from poh1fl/flCd4cre or oh1+/+Cd4cre mice. Cells were stimulated with or without TGF-β and IL-2 in the presence of plated bound anti-CD3 and anti-CD28 for 4 days. Data are representative of three independent experiments with similar results. **P < 0.01; ***P < 0.001, two-tailed unpaired Student’s t test

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: Treg-specific deficiency of POH1 leads to lethal autoimmune inflammation. a Typical littermate and poh1fl/flFoxp3Cre mice (left). Thymi, spleens and lymph nodes from 19-day-old poh1fl/flFoxp3Cre mice and poh1+/+Foxp3Cre control littermates (right). The images are representative of 20 independent mice with similar observations. b Survival of poh1fl/flFoxp3Cre and control mice monitored for 50 days (n = 9 mice per genotype). c Hematoxylin and eosin staining of kidney, lung and liver sections from 19-day-old poh1fl/flFoxp3Cre mice and control littermates. Data are representative of four independent experiments with similar results. d Flow cytometry analyzing the expression of CD44 and CD62L in CD4+ T cells from the spleens and lymph nodes of 19-day-old poh1fl/flFoxp3Cre mice and control littermates. Data are representative of five independent experiments with similar results. e Flow cytometry profiles of spleens and lymph nodes from poh1fl/flFoxp3Cre mice and poh1+/+Foxp3Cre control littermates showing expression of IFN-γ and IL-17 in CD4+ T cells stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A. Data are representative of four independent experiments with similar results. f Flow cytometry analyzing the CD80 and CD86 expression in B cells from spleens of 19-day-old poh1fl/flFoxp3Cre mice and control littermates. Data were pooled from two independent experiments. Each symbol represents a single mouse, and horizontal bars represent the mean. *P < 0.05; ***P < 0.001, two-tailed unpaired Student’s t test

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: POH1 maintains the pool size of generated Treg cells. a Flow cytometry analyzing the expression of Foxp3 among CD4+ T cells from the thymi, spleens and lymph nodes of 19-day-old poh1fl/flFoxp3Cre mice and control littermates. Data were pooled from three independent experiments. b Flow cytometry analyzing the expression of CD25 in Foxp3+ Treg cells from spleens of poh1fl/flFoxp3Cre mice and control littermates. Data are representative of four independent experiments with similar results. c Flow cytometry analyzing p-STAT5 in purified splenic Foxp3+ Treg cells from spleens of poh1fl/flFoxp3Cre mice and control littermates after stimulation with or without IL-2 for 15 min. Data are representative of two independent experiments with similar results. d–f Flow cytometry analyzing the expression of CD44, CD62L (d), GITR, CTLA4, OX40, PD1 (e), and Ki67 (f) in peripheral Foxp3+ Treg cells from poh1fl/flFoxp3Cre mice and control littermates. Data are representative of three independent experiments with similar results. g Flow cytometry analyzing the cell cycle of splenic Foxp3+ Treg cells from poh1fl/flFoxp3Cre mice and control littermates. h Cell cycle analysis of splenic Foxp3+ Treg cells from poh1fl/flFoxp3Cre mice and control littermates. Each symbol represents a single mouse, and horizontal bars represent the mean

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: Generated, Flow Cytometry, Expressing, Purification, Cell Cycle Assay

Journal: Cell Death and Differentiation

Article Title: Requirement for POH1 in differentiation and maintenance of regulatory T cells

doi: 10.1038/s41418-018-0162-z

Figure Lengend Snippet: RNA-seq analysis of POH1-deficient and POH1-sufficient Treg cells. a Gene expression (log2 normalized) in Treg cells from poh1fl/flFoxp3Cre mice (poh1−/− Foxp3+) or poh1+/+Foxp3Cre control littermates (poh1+/+ Foxp3+). b Empirical cumulative distribution function for the change in expression (log2 values) of all genes expressed in POH1-deficient Treg cells and for the subsets of genes upregulated (TCR up) in a TCR-dependent manner. Numbers in parentheses (key) indicate total genes in each group. c Expression of selected genes upregulated in a TCR-dependent manner in POH1-deficient or control Treg cells. d Expression of selected genes associated with cell cycle. e Validation of RNA-seq data. Genes were selected from the RNA-seq data, and the mRNA expression was analyzed by real-time PCR in Treg cells from 2-week-old poh1fl/flFoxp3Cre mice or poh1+/+Foxp3Cre control littermates. mRNA levels were normalized with GAPDH, and mRNA levels in control Treg cells were arbitrarily set to 1. Data are shown as the mean + SD and are representative of two independent experiments

Article Snippet: For the inducible Treg cell assay, CD4 Single+ CD25 − Foxp3-GFP − T cells were stimulated with plate-bound anti-CD3 (5 μg/mL) and anti-CD28 (3 μg/mL) in the presence or absence of TGF-β (2 ng/mL; R&D systems, Minneapolis, MN, USA) and IL-2 (20 U/mL; Peprotech NJ, USA) for 4 days.

Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction