Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: IL-12 but not IL-21 induced the differentiation of human naive CD4+ (T)cells into Th1 and Tfh co-expressing cells. Human naive CD4+ T cells from cord blood were stimulated for 5 d with or without anti-CD3 and anti-CD28 mAbs in the presence or absence of IL-12, IL-21 or IL-12 plus IL-21. The cells were harvested and rested for 2 d with IL-2. The cells were re-stimulated for 6 h with PMA and ionomycin in the presence of BFA. The expression of IL-21 and IFN-γ was detected by FACS. A representative result of different cytokines on the development of Th1 and Tfh cells was shown (A). Statistical data of percentage of IL-21+CD4+ (B), IFN-γ+CD4+ (C), IFN-γ−IL-21+ (D), IFN-γ−IL-21+ (E) and IFN-γ+IL-21+ (F) CD4+ T cells were mean ± SD from 5 independent experiments as described in A. Cell surface expression of CXCR5, PD-1, ICOS and CXCR3 was assessed by surface staining and a representative result was shown (G). P < 0 .05 was considered significant.

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing, Staining

Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: IL-12 induced the differentiation of polyfunctional CD4+ (T)cells. Naive CD4+ T cells were stimulated for 5 d with anti-CD3 and anti-CD28 mAbs in the presence of IL-12. The cells were rested and re-stimulated for 6 h with PMA and ionomycin in the presence of BFA. The expression of IL-21, IFN-γ, TNF-α and IL-2 was detected by FACS. Most of IL-21-expressing CD4+ T cells co-expressed Th1 cytokine IFN-γ, TNF-α or IL-2. The representative dot plots were shown (A). Gated on IL-21− and IL-21+CD4+ T cells or IFN-γ− and IFN-γ+CD4+ T cells, the expression of IFN-γ or IL-21 was analyzed. The representative histogram graphs and statistical data of mean MFI were shown (B). Gated on IL-21−IFN-γ−, IL-21+IFN-γ+, IL-21+IFN-γ− and IL-21−IFN-γ+ CD4+ T cells, the expression of TNF-α and IL-2 in the 4 different subsets were analyzed. The representative dot plots (C) and statistical data of mean percentages (D) of TNF-α−IL-2−, TNF-α−IL-2+, TNF-α+IL-2− and TNF-α+IL-2+ were shown.

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing

Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: Kinetic studies of the expression of cytokines and transcription factors after IL-12 stimulation. Naive CD4+ T cells were stimulated for 1 to 5 d with anti-CD3 and anti-CD28 mAbs in the presence or absence of IL-12, and the cells and supernatants were harvested at different time-points. The levels of IFN-γ and IL-21 in supernatants were determined by ELISA (A). The cells were re-stimulated for 48 h with PMA and ionomycin, and levels of IFN-γ and IL-21 in supernatants were determined by ELISA (B). The cells were re-stimulated for 6 h with PMA and ionomycin in the presence of BFA, and the expression of IFN-γ, IL-21, T-bet and BCL−6 was detected by FACS. The representative results were shown (C, D).

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: Characteristic phenotypes of Th1 and Tfh cells. Naive CD4+ T cells were stimulated for 5 d with anti-CD3 and anti-CD28 mAbs in the presence of IL-12. The cells were harvested, rested and re-stimulated for 6 h with PMA and ionomycin in the presence of BFA. The expression of cytokines and surface markers were detected by FACS. A representative result of surface expression of CXCR5, CXCR3, PD-1, ICOS and CD40L (A), and expression of transcription factor T-bet and BCL−6 (B) on IFN-γ−IL-21−, IFN-γ+IL-21−, IFN-γ−IL-21+ and IFN-γ+IL-21+ CD4+ T cells were shown in histogram, statistical data of MFI (B) were mean±SD from 5 independent experiments. A representative result of IL-21 and IFN-γ expression gated on T-bet−BCL−6−, T-bet−BCL−6+, T-bet+BCL−6+ and T-bet+BCL−6−CD4+ T cells was shown in histogram graph and dot plots (C). The statistical data were mean ± SD from 5 independent experiments (D).

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing

Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: Effect of IFN-γ on modulating the expression and production of IL-21 and IFN-γ. Naive CD4+ T cells were stimulated for 3 d with anti-CD3 and anti-CD28 mAbs plus IL-12, and anti-IFN-γ or IFN-γ was added into the culture in the presence of IL-12 at day 0 and day 3, the expression of IL-21 and IFN-γ production were detected by FACS after re-stimulated with PMA and ionomycin at day 5, a representative dot plots were shown (A). Transcription factors phosphorylated STAT-1 and STAT-4, T-bet and BCL−6 were detected by FACS at day 3, a representative histogram graphs were shown (B). Naive CD4+ T cells were stimulated for 3 d in the presence of IL-12, IL-21 or IL-12 plus IL-21 with or without anti-IFN-γ. The cells were harvested, rested and re-stimulated for 6 h with PMA and ionomycin in the presence of BFA. IFN-γ and IL-21 production were analyzed by FACS (C). The cells were re-stimulated for 48 h with PMA and ionomycin, the levels of IFN-γ and IL-21 in supernatants were determined by ELISA (D). The cells were re-stimulated for 48 h with PMA and ionomycin in the presence of anti-IFN-γ. The levels of IFN-γ and IL-21 in supernatants were determined by ELISA (E). The expression of phenotypic markers (F) and transcription factor T-bet and BCL−6 (G) were analyzed by FACS under the conditions of IL-12 or IL-12 plus anti- IFN-γ. The representative histogram graphs and statistical data from 5 independent experiments were shown.

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Cycle

Article Title: IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells

doi: 10.1080/15384101.2015.1093703

Figure Lengend Snippet: Transcription factors regulating the expression and production of IFN-γ and IL-21. Naive CD4+ T cells (CD4+CD45RA+CD45RO−) and memory CD4+ T cells (CD4+CD45RA−CD45RO+) were stimulated with anti-CD3 and anti-CD28 plus IL-12 in the presence or absence of inhibitors for transcription factor for 5 d. The cells were harvested and re-stimulated with PMA and ionomycin for 6 h in the presence of BFA, and the expression of IFN-γ and IL-21 were detected by FACS (A). Cells were re-stimulated with PMA and ionomycin for 48 h, the levels of IFN-γ and IL-21 in the supernatants were detected by ELISA (B). Memory CD4+ T cells were stimulated with anti-CD3 and anti-CD28 plus IL-12 in the presence or absence of inhibitors for transcription factor for 5 d. The cells were harvested and re-stimulated with PMA and ionomycin for 6 h in the presence of BFA, and the expression of IFN-γ, IL-21, CXCR5, CXCR3, ICOS and PD-1 were detected by FACS (C). Expression of Tfh and Th1 cell phenotypes (D) and transcription factors (E) was detected by FACS.

Article Snippet: Human ELSIA kits for cytokine IFN-γ was purchased from BD Biosciences, IL-21 was purchased from eBioscience.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Memory, but Not Naive, CD4 + T Cells Expressing Transcription Factor T-bet Might Drive Rapid Cytokine Production *

doi: 10.1074/jbc.M114.608745

Figure Lengend Snippet: Kinetics of cytokine secretion by activated naive and memory CD4+ T cells. Naive and memory CD4+ T cells were isolated from CD4+ T cells by MACS microbeads, and the purity of naive or memory CD4+ T cells was more than 97% as determined by flow cytometry (a). Naive and memory CD4+ T cells were stimulated with (open histogram) or without (shaded histogram) PMA and ionomycin in the presence of BFA for 6 h. Cells were stained by intracellular staining and analyzed by FACS (b). Purified naive and memory CD4+ T cells were stimulated with immobilized anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 0 to 72 h, and the concentrations of cytokines IFN-γ, IL-2, and TNF-α were detected by ELISA (c). Data are representative of three separate experiments with similar results.

Article Snippet: Human ELISA kits for cytokines IFN-γ, IL-2, and TNF-α were purchased from BD Biosciences.

Techniques: Isolation, Flow Cytometry, Staining, Purification, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: A De- O -acylated Lipooligosaccharide-Based Adjuvant System Promotes Antibody and Th1-Type Immune Responses to H1N1 Pandemic Influenza Vaccine in Mice

doi: 10.1155/2016/3713656

Figure Lengend Snippet: Th1-type-predominant immune response to CIA06-adjuvanted influenza vaccine. Splenocytes were isolated from the mice that had been immunized as described in and cultured for 72 h in the absence (■) or presence of Greenflu-S (■). The levels of IFN- γ (a) and IL-5 (b) in the culture media were determined by sandwich ELISA, and IFN- γ : IL-5 ratios were calculated for each group (c). Statistical differences were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Results are expressed as the means ± SD of values obtained from triplicate cultures that used two spleens each. Data shown are representative of three independent experiments. ∗ P < 0.05; ∗∗ P < 0.01; and ∗∗∗ P < 0.001. NS, not significant.

Article Snippet: Recombinant mouse IL-2 was acquired from R&D Systems (Minneapolis, MN, USA), and IFN- γ and IL-5 cytokine ELISA kits were obtained from BD Biosciences (San Diego, CA, USA) and R&D Systems.

Techniques: Isolation, Cell Culture, Sandwich ELISA, Comparison

Journal: BioMed Research International

Article Title: A De- O -acylated Lipooligosaccharide-Based Adjuvant System Promotes Antibody and Th1-Type Immune Responses to H1N1 Pandemic Influenza Vaccine in Mice

doi: 10.1155/2016/3713656

Figure Lengend Snippet: CIA06-adjuvanted influenza vaccine stimulates both CD4 + and CD8 + T cell responses. Splenocytes were isolated from the mice ( n = 6) that had been immunized twice at a 3-week interval with nonadjuvanted or adjuvanted Greenflu-S (0.2 μ g) and stimulated with the vaccine for 72 h in the absence or presence of anti-CD4 and/or anti-CD8 antibodies. IFN- γ levels in the culture media were determined by sandwich ELISA. Statistical differences were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Results are expressed as the means ± SD of values obtained from triplicate cultures that used two spleens each. Data shown are representative of three independent experiments. ∗ P < 0.05; ∗∗ P < 0.01; and ∗∗∗ P < 0.001. NS, not significant.

Article Snippet: Recombinant mouse IL-2 was acquired from R&D Systems (Minneapolis, MN, USA), and IFN- γ and IL-5 cytokine ELISA kits were obtained from BD Biosciences (San Diego, CA, USA) and R&D Systems.

Techniques: Isolation, Sandwich ELISA, Comparison

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency results in excessive activation of NLRP3 inflammsome in macrophages. Two groups of macrophages, i.e., peritoneal macrophages from conditional SHP2 knockout (cSHP2-KO) and wild-type (WT) mice a–c , or PMA-differentiated THP-1 cells with shRNA-Ctrl or shRNA-SHP2 lentivirus d , f , were primed with 100 ng ml −1 LPS for 3 h, and then stimulated with ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h) or Nigericin (10 µM, 2 h), respectively. a , b , d , e Enzyme-linked immunosorbent assay (ELISA) of IL-1β and IL-18 in culture supernatants. ND represents not detectable. c , f Immunoblot analysis of cell lysates. g ELISA of IL-1β in supernatants of THP-1-derived macrophages left untreated or treated with SHP2 inhibitor PHPS1 (10 μM) or NSC87877 (10 μM) for 1 h, followed by LPS treatment and ATP, MSU or Nigericin stimulation. h Immunoblot analysis of proteins immunoprecipitated with anti-ASC from lysates of ATP-treated SHP2 knockdown THP-1-derived macrophages. i Immunoblot analysis of ASC in cross-linked pellets (upper panels) and cell lysates (lower panels) from ATP-treated SHP2 knockdown THP-1-derived macrophages. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , d , e , g )

Article Snippet: ELISA kits for murine or human IL-18 were purchased from Raybiotech (Norcross, GA).

Techniques: Activation Assay, Knock-Out, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: ANT1 knockdown suppresses activation of NLRP3 inflammasome. Two groups of THP-1-derived macrophages, i.e., with shRNA-Ctrl or shRNA-ANT1, were primed with 100 ng ml −1 LPS for 3 h, and then stimulated with ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h), or Nigericin (10 µM, 2 h), respectively. a , b ELISA of IL-1β and IL-18 in the culture supernatant. c Immunoblot analysis of cell lysates from THP-1-derived macrophages. d Immunoblot analysis of Co-IP from THP-1-derived macrophages. e , f Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining e or mitochondrial ROS by MitoSOX staining f from THP-1-derived macrophages with shRNA-Ctrl or shRNA-ANT1 lentivirus, followed by LPS treatment and ATP or Nigericin stimulation. g Quantitative real-time PCR analysis of mtDNA released from ANT1-knockdown THP-1-derived macrophages and left unstimulated (medium) or primed with LPS and stimulated with ATP, MSU or Nigericin. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , e–g )

Article Snippet: ELISA kits for murine or human IL-18 were purchased from Raybiotech (Norcross, GA).

Techniques: Activation Assay, Derivative Assay, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Co-Immunoprecipitation Assay, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction