Figure 3
Role of β-chemokines in HIV-1 restriction in CD4 T cells from subjects W278 and B195. A. CD4 T cells from subjects W278 and B195 were challenged with the HIV-BaL pseudotype in the absence (white bars) or presence of a combination of neutralizing anti-RANTES (5 μg/ml), anti-MIP1α (15 μg/ml) and anti-MIP1β (25 μg/ml) (R&D systems, France) mAbs (black bars) or with an isotype control antibody (45 μg/ml) (patterned bars). The antibodies were added 30 minutes before challenge and maintained throughout the time course of infection. Results are expressed as relative luciferase activity, compared to the maximal activity found in the presence of the neutralizing anti-β-chemokines in each case, and are the mean of three independent infections ± standard deviation. ** Significant difference (P < 0.001 and P = 0.008 for W278 and B195, respectively, independent sample t-test). B. Challenge with the HIV-BaL pseudotype (n = 3, mean ± SD) of CD4 T cells from EUs W278 and B195, stimulated with PHA three days before (white bars) or two hours after (black bars) challenge. Luciferase activity in cell lysates from a representative control challenged in the same conditions was attributed a value of 100%. C. Sensitivity of CD4 T cells to recombinant chemokines. Non mitogen-stimulated CD4 T cells from EU B195 (filled circles) and one control (open squares) were exposed to various concentrations of a mixture of the recombinant β-chemokines RANTES, MIP-1α and MIP-1β (R&D systems, France) for 30 minutes prior to and during infection. The mixtures contained the three chemokines at concentrations ranging from 500 ng to 2 ng each. Results (n = 3, mean ± SD) are expressed as luciferase activity per second in cell lysates. Points were fitted to a four-parameter logistic curve (r2 were 0.845 and 0.826 for B195 and control, respectively). Statistical analysis and curve-fitting were performed with Sigmaplot software (Systat Software, Inc, CA, USA).