Fig 6.

MMQO enhances TCR/CD3 stimulation of HIV-1 reactivation from latency and prevents TCR-induced proliferation in primary T cells. (A and B) MMQO synergizes with CD3 to reactivate HIV-1 from latency. Jurkat-LAT-GFP cells were stimulated for 18 h on 96-well plates with coated anti-CD3 (1 μg/ml), soluble anti-CD28 (0.5 μg/ml), and MMQO (160 μM) separately or in combination. (B) Jurkat-LAT-GFP cells were stimulated with coated anti-CD3 and increasing concentrations of MMQO as indicated for 18 h. GFP expression was measured by flow cytometry, and results indicate the percentage of GFP⁺ cells. (C) Jurkat-LAT-GFP cells were stimulated with increasing concentrations of coated anti-CD3 and MMQO at 80 μM for 18 h. (D) Differential effects of MMQO on IL-2 and TNF-α promoters and HIV-LTR transactivation. Jurkat cells were transiently transfected with the plasmids IL-2-Luc, TNF-α-Luc, and LTR-Luc, and 24 h later they were stimulated with coated anti-CD3 (1 μg/ml) in the absence or the presence of either soluble anti-CD28 (0.5 μg/ml) or MMQO (160 μM) or with MMQO alone for 24 h. The results are expressed as the percentage of activation considering CD3-induced transactivation as 100% activation. (E) Activity of MMQO on NF-κB-, NFAT-, AP-1-, and Sp1-responsive minimal promoters. Jurkat cells were transfected with NF-κB, NFAT, AP-1, and Sp1 luciferase reporter plasmid DNA and stimulated as described for panel C. Luciferase activity was measured 24 h later after protein normalization. The results are expressed as the percentage of activation considering CD3-induced transactivation as 100% activation. (F) Effect of MMQO on α-CD3-induced NF-κB and MAPK activation. Jurkat cells were stimulated with anti-CD3 and MMQO (160 μM) separately or in combination for 15 min, and the levels of phospho-IκBα, phosho-p65 (Ser536), phospho-JNK, phospho-ERK, and phospho-p38 were assessed by immunoblotting with specific antibodies and anti-tubulin MAb as a loading control. (G) MMQO antagonizes HIV-1 latency through the JNK pathway. Jurkat-LAT-GFP cells were treated with the ERK inhibitor PD98059 (25 μM), IKK2 inhibitor (25 μM), and JNK inhibitor SP600125 (1 μM) or left untreated, and 30 min later they were stimulated with anti-CD3 plus MMQO (80 μM) or with MMQO alone for 18 h. HIV-GFP reactivation was assessed by flow cytometry, and results indicate the percentage of GFP⁺ cells. (H) Effects of MMQO on T-cell proliferation. Human PBMCs were stimulated with SEB (1 μg/ml) in the presence or the absence of increasing concentrations of MMQO for 72 h. [³H]thymidine incorporation was measured by liquid scintillation counting and is represented as disintegrations per minute (DPM). Throughout the figure data are represented as means ± SEM (n = 3).