Activation of cofilin in resting CD4 T cells cultured in vitro and stimulated with HIV-1 and gp120. Resting CD4 T cells were purified from uninfected donors by antibody-mediated negative depletion using Dynalbeads as previously described [20]. Cells were cultured overnight in the absence of cytokines or activation, and then stimulated with HIV-1 (10 ng p24) (A) or with gp120 (50 nM) for various times at 37°C as indicated. Stimulated cells were lysed and analyzed by western blot using antibodies against the phosphorylated cofilin (P-cofilin) or total cofilin or GAPDH for controls.

Activation of cofilin in resting CD4 T cells of HIV-1-infected patients. (A) Resting CD4 T cells were purified by antibody-mediated negative depletion using Dynalbeads. Cells purified from HIV-1-infected and uninfected donors were cultured overnight in the absence of cytokines or activation, and then lysed and analyzed by western blot using antibodies against P-cofilin or total cofilin. The relative ratios of P-cofilin to total cofilin were measured and plotted. HIV-1-infected patients had statistically-significant lower ratios of P-cofilin/cofilin (0.535 versus 1.142, p = 0.002), suggesting higher levels of active cofilin. For statistic analysis, a two-tailed Student's t-test on the means resulted in a p-value of p = 0.002. At a pre-determined significance level of 0.05, this shows that the difference in the mean ratios of the two sample groups is statistically significant. A standard power computation showed that the t-test was very well powered (95.2%) for this study. (B) Shown are the longer exposures of the western blots used in (A). The results were confirmed by NEPHGE-western blot to directly separate P-cofilin to active cofilin, and then probed with an anti-cofilin antibody [20]. Shown are the absolute ratios of P-cofilin to active cofilin in HIV-1 negative donors (C) and HIV-1-infected donors (D).

Lack of detection of T cell activation markers on the surface of resting CD4 T cells from HIV-1-infected and uninfected donors. Resting CD4 T cells were purified by antibody-mediated negative depletion using Dynalbeads. Cells purified from HIV-1-infected and uninfected donors were cultured overnight in the absence of cytokines or activation, and then stained for surface expression of HLA-DR or CD69. Shown are flow cytometry analyses of cells stained with a PE-labelled anti-human HLA-DR antibody (A and C, right panel) or a similarly labelled isotype control antibody (A and C, left panel). Cells were also stained with a PE-labelled anti-human CD69 antibody (B and D, right panel) or a similarly labelled isotype control antibody (B and D, left panel). Cells from the HIV negative donor (HIV^-) were used in (A) and (B), and cells from the HIV-1-infected donor (HIV⁺) were used in (C) and (D).