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. 2016 Apr 14;6:24090. doi: 10.1038/srep24090

Figure 4. In vitro rNef-mediated Akt-dependent hyperactivation of T cells favors increased IL-2 production and T-cell proliferation.

Figure 4

(A,B) Exogenous HIV-1 Nef triggers IL-2 production in TCR-stimulated T-cells. (A), Five million PBLs were stimulated with anti-CD3-mAb (5 μg/ml), anti-CD3-mAb + rNef, anti-CD28-mAb (5 μg/ml), anti-CD28-mAb + anti-CD3-mAb or rNef (100 ng/ml) alone for 24 h. Post treatment cells were fixed, permeabilized and expression of intracellular IL-2 was determined by flow cytometry. Flow cytometric data indicating the mean fluorescence intensity (MFI) for intracellular IL-2 production. Data are representative of 5 independent experiments. (B), Five million PBLs were either left untreated or treated with rNef (100 ng/ml), anti-CD3-mAb (5 μg/ml), anti-CD28-mAb (5 μg/ml), anti-CD3-mAb + rNef, anti-CD28-mAb + rNef, and antiCD3-mAb + antiCD28-mAb for 24 h. Post 24 h supernatants were collected and IL-2 production was measured in supernatants by ELISA. Means ± s.d. (n = 3). (C), Exogenous HIV-1 Nef triggers cell proliferation in TCR-stimulated T cells. Five million PBLs were either left untreated or treated with rNef (100 ng/ml), anti-CD3-mAb (5 μg/ml), anti-CD28-mAb (5μg/ml), anti-CD3-mAb + rNef, anti-CD28-mAb + rNef and antiCD3-mAb+ antiCD28-mAb in the absence of Akt I and PI3K inhibitor for 24 h. Cell proliferation was measured using MTT cell proliferation assay. Means ± s.d. (n = 3). (D,E), Exogenous HIV-1 Nef triggers IL-2 production and T cell proliferation in CD3 stimulated cells in PI3K/Akt dependent manner. Five million PBLs were either left untreated or treated with Akt I (25 μM, 50 μM) and PI3K inhibitor LY294002 (25 μM, 50 μM) for 2 h followed by treatment with rNef (100 ng/ml), heat denatured rNef (100 ng/ml), anti-CD3-mAb (5 μg/ml) and anti-CD3-mAb + rNef, for 24 h. After 24 h supernatants were collected and IL-2 production and T cell proliferation was determined by ELISA and MTT assay respectively. Means ± s.d. (n = 3). LY: LY294002; AktI: Akt inhibitor VIII.