Induction of apoptosis in macrophages by LZhuTRAIL. MDM from 11 HIV-1-negative donors were mock or HIV-1_Bal infected with or without GM-CSF. Fourteen days following infection, cells were treated with LZhuTRAIL and analyzed by TUNEL to determine the levels of apoptosis.

Sensitivity of PBL from HIV-1-infected donors to titrated doses of LZhuTRAIL. Cells were isolated from HIV-1-infected donors and incubated with increasing concentrations of LZhuTRAIL as indicated. Cell death was measured by Hoechst staining. Data are representative of three independent experiments. Spontaneous levels of apoptosis are indicated by asterisks.

LZhuTRAIL induces apoptosis in cells from HIV-1-infected patients. PBL from 26 randomly selected HIV-1-infected patients or 5 uninfected controls were treated with 1 μg of LZhuTRAIL and analyzed for apoptosis by Hoechst staining, as were CD4⁺ T cells and CD8⁺ T cells.

LZhuTRAIL reduces viral gene expression in infected cells. CD4/DR⁻ RO⁺ cells were isolated from seven HIV-1-infected patients, treated with LZhuTRAIL or agonistic TRAIL receptor antibodies (or isotype controls), and analyzed for p24 antigen production (A) and for the presence of integrated proviral DNA. (B) Viral RNA in culture supernatants. (C and D) Unfractionated cells from four HIV-1-infected patients with suppressed plasma viremia for more than 12 months were treated with or without LZhuTRAIL or agonistic MAbs to TRAIL-R2 (or isotype controls) and tested for p24 antigen (C) or viral RNA in culture supernatants (D).

(A) Analysis of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and TRAIL mRNA expression by RT-PCR. (Top) HIV-infected Jurkat T cells show increased message for TRAIL-R2, TRAIL-R3, and TRAIL/Apo2L compared to mock-infected controls (normalized to β-actin). (Center) HIV-1_Bal-infected MDM show increased message for all TRAIL receptors and TRAIL/Apo2L compared to mock-infected controls. (Bottom) PBL from four HIV-1-positive individuals show increased message for TRAIL-R2, -R3, and -R4 or TRAIL/Apo2L compared to PBL from four HIV-1-negative individuals. (B) Cell surface expression of TRAIL-R1, -R2, -R3, and -R4 in CD4⁺ T cells from HIV-infected patients and healthy controls. Open histograms with dotted lines, cells from HIV-infected patients stained with an isotype control MAb; shaded histograms, cells from HIV-1-infected patients stained with anti-TRAIL receptor MAbs; open histograms with solid lines, cells from healthy controls stained with anti-TRAIL receptor MAbs. (C) Jurkat T cells (top) or PBL (bottom) from HIV-negative patients were treated with gp120 or a control (BSA) as indicated and analyzed by flow cytometry for TRAIL receptor expression. Open histograms with dotted lines, isotype control staining; open histograms with solid lines, cells treated with BSA and stained with the indicated anti-TRAIL receptor antibody; shaded histograms, cells treated with gp120 and stained with the indicated anti-TRAIL receptor antibody.