Figure 1. Enhanced responses of previously activated T cells in PAG-deficient mice.

(A). Purified CD4⁺ T cells from wild-type (WT) or PAG knock-out (KO) mice were activated in vitro with anti-CD3 plus anti-CD28 and expanded in IL-2. Expression of PAG was verified by immunoblotting of total cell lysates. (B). As in (A) except that cells were re-stimulated with anti-CD3 (0.05 µg/ml), anti-TCR (0.05 µg/ml) plus anti-CD28 (1 µg/ml) or P+I. Thymidine incorporation (expressed hereafter as CPM) and production of IFN-γ, IL-2 or IL-4 were monitored. Means with standard deviations (SD) of triplicate values are shown. (C). Same as in (B), except that cells were re-stimulated with the indicated concentrations of SEB and APCs. (D). Purified CD4⁺ T cells from TCR AND transgenic mice were first activated in vitro with anti-CD3 plus anti-CD28 and then re-stimulated with the indicated concentrations of PCC peptide and APCs, or with P+I. (E). Mice (two mice in each group) were immunized with OVA and adjuvant. After 9 days, CD4⁺ T cells were isolated and re-stimulated with the indicated concentrations of OVA and WT splenocytes, or P+I. After 4–5 days (for OVA re-stimulation) or 2 days (for P+I), production of IFN-γ was monitored, as detailed in (B). (F,G). CD4⁺ T cells activated in vitro with anti-CD3 plus anti-CD28 were infected with retroviruses encoding green fluorescent protein (GFP) alone, or in combination with WT PAG or PAG Y314F. After sorting of GFP-positive cells, expression of PAG was verified by immunoblotting of total cell lysates (F). Responsiveness to SEB was also examined, as detailed in (C) (G). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Representative of n > 25 (A), n = 4 (B), n = 6 (C), n = 4 (D), n = 3 (E) and n = 2 (F,G). Related to Figure S1.