Figure. 4. TCR affinity does not determine the activation status or effector function of divided CD4+ T cells. (A-C) CTV-labelled C7 and C24 Tg cells were stimulated with E6 peptide in the presence of recombinant IL-12p70 and anti-IFN-γ mAb for 72 h. (A) Flow cytometric analysis of the MFI of CD44, CD25, IRF4 and CXCR3 as well as FSC-A (cell size) in each division of C7 and C24 populations at 42 h after peptide stimulation. (B-C) Tg cells were stimulated with E6 peptide in the presence of the graded concentrations of recombinant IL-12p70 and T-bet expression determined by flow cytometry. (B) T-bet expression in C7 and C24 Tg cell populations 60 h after stimulation. Numbers outside and within parentheses represent the percentage of T-bet+ cells and the MFI of T-bet expression in T-bet+ populations, respectively. (C) MFI of T-bet in each division of CTV-labelled C7 and C24 cells 66 h after stimulation. Data shown are representative of two independent experiments with similar results. (D-E) Purified C7 and C24 CD4+ T cells were stimulated with E6 peptide under non-polarizing (anti-IL-12, anti-IFN-g and anti-IL-4 mAbs), Th1 (IL-12p70 with anti-IFN-g and anti-IL-4 mAbs) or Th2 (IL-4 with anti-IFN-g and anti-IL-12 mAbs) culture conditions for 96 h. Following expansion in IL-2-containing medium for 48 h, an equal number of T cells were re-stimulated with immobilized anti-CD3 in the presence (D) or absence (E) of BFA for 6 h. (D) Intracellular IFN-g and IL-4 expression under the indicated culture conditions was analyzed by flow cytometry. (E) Quantification of IFN-g and IL-4 produced by Th1 and Th2 Tg cells by Cytokine Bead Array. Data shown are the mean ± the sd of triplicate cultures. For all data the bars, lines, symbols or FACS plots in black and red represent C7 and C24 cells, respectively.
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