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: PLoS ONE

Article Title: Expanded Polyfunctional T Cell Response to Mycobacterial Antigens in TB Disease and Contraction Post-Treatment

doi: 10.1371/journal.pone.0011237

Figure Lengend Snippet: A) PBMC were stimulated overnight and cells were analysed by flow cytometry for expression of CD107a, TNF-α, IL-2 and IFN-γ. Following lymphocyte gating, CD4+ and CD8+ T cells were analysed separately for combinations of all 4 factors. B) Representative dot plots illustrating cytokine production from CD4+ and CD8+ T cells following PHA, PPD, EC and no stimulation.

Article Snippet: Following centrifugation the supernatant was carefully removed and 20 μl of cytokine cocktail added (IFN-γ-APC, TNF-α-PECy7 and IL-2-PE; BDPharmingen).

Techniques: Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Dectin-1 diversifies Aspergillus fumigatus –specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

doi: 10.1084/jem.20100906

Figure Lengend Snippet: Enhanced expression of T-bet in Af3.16 TCR-tg cells primed in vitro with Dectin-1 −/− DCs. Naive Af3.16 TCR-tg cells were cultured in vitro with FLT3L-induced DCs isolated from Dectin-1 −/− or control C57BL/6J mice in the presence or absence of A. fumigatus hyphae. (A) T-bet expression was assessed by intracellular staining. Histograms shown are representative of 10 individual wells analyzed and are gated on CD4 + Vβ8.3 + Af3.16 TCR-tg cells. Isotype control staining is shown. (B) The amount of T-bet expressed by Af3.16 TCR-tg cells primed with WT or Dectin-1 −/− DCs as determined by T-bet mean fluorescence intensity (MFI) in gated Af3.16 TCR-tg cells. Each symbol represents one individual well per culture condition. Bars represent the mean with SEM. (C–E) Secreted IFN-γ (C) and IL-17A (D) at day 5 of culture or IL-2 (E) at day 2 of culture was measured by ELISA in 10 individual wells. Af3.16 TCR-tg cells were cultured with WT or Dectin-1 −/− DCs with or without hyphae as indicated. Data shown are for one experiment representative of two separate experiments. Values shown are mean ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001 compared with control WT DCs. N/D, no cytokine detected.

Article Snippet: The production of IFN-γ (BD), IL-17A (eBioscience), and IL-2 (BD) was assessed by ELISA according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Cell Culture, Isolation, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay