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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Clin Immunol. 2010 May 23;136(3):338–347. doi: 10.1016/j.clim.2010.04.013

Fig.3.

Fig.3

Functional analyses of the DMF5 TCR transduced T cells. (A). Cytokine synthesis by the DMF5 TCR transduced CD4+CD25− (i) and CD8+ (ii) T cells. The TCR-transduced T cells were stimulated with either the MART-127–35 cognate peptide (M1) or MAGE-3271–279 control peptide (M3) and cytokine secreted in the supernatant were quantified 16 hr post co-culture set up. Data represents composite analysis of results (mean ± SEM) of 5 separate experiments with TCR-transduced T cells from 5 different donors. (B). Multiparametric intra-cellular cytokine staining of DMF5 TCR bearing CD4+CD25− (left column) and CD8+ (right column) cells. The TCR-transduced T cells were stimulated with either the MART-127–35 cognate peptide (M1) or MAGE-3271–279 control peptide (M3) and cells were stained for IL-2, IFN-γ, TNF-α, MIP-1β and CD107a by intra-cellular staining. Selected combinations are shown as indicated. (C). Analysis of polyfunctional response exhibited by the TCR-transduced CD4+CD25− and CD8+ T cells. The TCR-transduced T cells were stimulated with either the MART-127–35 cognate peptide (M1) or MAGE-3271–279 control peptide (M3) and analyzed for multiple functional parameters shown on the X-axis by flowcytometry. The data were analyzed with FlowJo (TreeStar, USA) and polyfunctionality was assessed with PESTLE and SPICE softwares (provided by Mario Roederer, NIH, Bethesda, MD). Each slice of the pie chart shows fraction of total responsive cells were positive for given number of functions (color coded groups below the bar plot). (D). (i) CD4+CD25− TCR transduced T cells were stimulated with MART-127–35 cognate peptide (M1) or MAGE-3271–279 control peptide (M3) and cells were stained with anti-FoxP3-APC by intracellular staining. (ii) Freshly isolated human peripheral blood CD4+CD25+ T cells were stained for FoxP3 as a positive control. (iii) TCR transduced CD4+CD25− T cells were stimulated for 16hr with either T2 alone, M3 or M1 pulsed T2 and supernatant were tested for TGF-β and TNF-α by ELISA. Representative of three separate experiments is shown.