CCR5 activation induces a caspase-dependent death of CD4 T cells. (a) CD4 T cells were incubated with 293T cells transfected with empty vector or 293T cells expressing the HIV_JRFL-env (R5env), either untreated or treated with soluble CD4 at different concentrations (sCD4). 293T cells were lysed and blotted by duo, tropic anti-gp160 to verify envelope expression (left panel). CD4 T cells were left untreated, pretreated with Z-IETD (a caspase 8 inhibitor) or Z-LHED (a caspase 9 inhibitor) before incubation with 293T cells expressing empty vector or R5env (right panel). (b) CD4 T cells treated or not treated with Z-VAD, SDF1α, or MIP1β were incubated with 293T cells expressing R5env or empty vector pretreated or not pretreated with sCD4, respectively. (c) CD4 T cells were treated with IgG-matched isotype control, anti-CD4 Leu-3a (αCD4 Ab), or anti-CCR5 MAB183 (αCCR5 Ab), followed by their cross-linking with goat anti-mouse Ab’s. Each point was assessed for caspase-dependent death (Z-VAD) as well as for Fas susceptibility (αFas). (d) CD4 T cells from a healthy donor with the CCR5 Δ32 mutation or from a control donor lacking the Δ32 CCR5 deletion were pretreated or not pretreated with Z-VAD and coincubated with 293T cells expressing R5env or empty vector, pretreated or not pretreated with sCD4.

CXCR4/CD4 receptor ratio determines the mode of CD4 T-cell death. (a) Untreated, IL-4 alone, or IL-4– and SDF1α-treated CD4 T cells were incubated or not incubated (0) with soluble X4 gp120. Cells were assessed for Fas (αFas) susceptibility and caspase-dependent death (Z-VAD). (b) CD4 T cells from a were simultaneously stained for CD4 and CXCR4 and analyzed using flow cytometry. CD4 T cells stained for CXCR4 were also analyzed using confocal microscopy.

Infected CD4 T-cell blasts die by chemokine receptor–dependent mechanisms. (a) PHA and IL-2–stimulated cells were infected with X4 HIV_(Lav-Bru), or R5 HIV_(JRFL), or mock (NI) virus. Cells were counted every 2–3 days and analyzed for cell death or by using flow cytometry changes in FSC consistent with apoptosis and for PI cell cycle. On day 6 of infection the percentage of cells infected with the R5 HIV strain shown to be apoptotic by using PI staining were 25% (– Z-VAD) and 15% (+ Z-VAD) or by FSC were 29% (– Z-VAD) and 19% (+ Z-VAD). On day 9 of infection the percentage of cells infected with X4 HIV shown to be apoptotic by PI staining were 71% (– Z-VAD) and 69% (+ Z-VAD) or by FSC were 68% (– Z-VAD) and 64% (+ Z-VAD). p24 values from the supernatants were: R5, 293(+)Z-VAD and 178(–)Z-VAD (P < 0.1); X4, 41(+)Z-VAD and 40(–) Z-VAD. SD = <10% at each data point. (b) The procedure was the same as in a, except cells were incubated in the presence or absence of caspase 8 inhibitor (Z-IETD) or caspase 9 inhibitor (Z-LEHD). (c) PHA and IL-2 stimulated and infected with R5 HIV_(JR-FL), or X4 HIV_(Lav-Bru), or mock infected were treated with AZT for 6 hours on day 3 of X4 HIV infection and on day 6 of R5 HIV infection. Thereafter, cells were extensively washed and incubated in the presence or absence of SDF1α or MIP1β, and cell death was analyzed 24 hours later. (d) Chemokine-receptor activation mediates death of directly HIV-infected and -uninfected CD4 T cells. PHA and IL-2–stimulated cells were infected with R5 HIV_(JR-FL), or X4 HIV_(Lav-Bru), or mock infected (NI). On day 4 for X4 HIV infection and day 6 for R5 HIV infection, cells were treated with AZT, and half the population was treated with Z-VAD; 18 hours later cells were stained for intracytoplasmic p24. The percentage of p24⁺ cells and apoptotic cells, as determined changes by FSC, were analyzed by using flow cytometry. The percentage of p24⁺ in the R5 HIV infection of T cells was 11% and 15% in the X4 HIV infection (+). SD = <10% at each data point.

CXCR4 activation induces a caspase-independent death of CD4 T cells. (a) CD4 T cells treated or not pretreated with Z-VAD, SDF1α, or MIP1β were incubated with 293T cells transfected with empty vector (vector) or HIV_HXB2-env (X4env), and untreated or pretreated soluble CD4 (sCD4) (left panel). CD4 T cells were untreated or pretreated with Z-IETD (a caspase 8 inhibitor) or Z-LEHD (a caspase 9 inhibitor) before incubation with 293T cells expressing empty vector or X4env (right panel). (b) CD4 T cells were untreated or treated with soluble gp120 (X4 gp120) alone or in combination with soluble CD4 (sCD4). Some cells were pretreated with SDF1α to block CXCR4 receptors. Caspase-dependent death was analyzed using Z-VAD (Z-VAD), and Fas susceptibility was analyzed using treatment with anti-Fas Ab’s (αFas). (c) CD4 T cells were preincubated or not preincubated with SDF1α and treated or not treated with soluble primary (92Ug20.9) X4 gp120 that was preincubated or not preincubated with sCD4. (d) CD4 T cells were cross-linked with anti-CD4 Leu-3a (αCD4 Ab), anti-CCR5 MAB 183 (αCCR5 Ab), anti-CXCR4 12G5 (αCXCR4 Ab), or matched isotype control (IgG) and assessed for death and/or Fas susceptibility as in b in the presence or absence of the pancaspase inhibitor (Z-VAD), caspase 8 inhibitor (Z-IETD), and caspase 9 inhibitor (Z-LEHD). (e) CD4 T cells were preincubated with 293T expressing or not expressing X4env preincubated or not preincubated with sCD4. Cell death was analyzed using changes in the light scatter or by TUNEL analysis using FACS.

CD8 T cells die by HIVenv-CXCR4 infection. (a) Purified CD4 and CD8 T cells were untreated (0) or treated with soluble X4 (LAI) gp120 and assessed for Fas susceptibility (αFas) or caspase-dependent death (Z-VAD). Ten percent of untreated cells, 23% of cells treated with X4 gp120, and 24% of cells treated with Z-VAD and X4 gp120 (P < 0.006) demonstrated changes in FSC compatible with apoptosis in flow cytometry. (b) CD8 T cells were untreated (0) or treated with SDF1α before incubating with X4 (LAI) gp120 and analyzed for caspase-dependent death (Z-VAD) or susceptibility to Fas-mediated apoptosis (αFas). CD8 T cells were simultaneously analyzed using flow cytometry for CXCR4 expression with and without SDF1α treatment (inset). (c) CD8 T cells were cross-linked with anti-CXCR4 12G5 Ab (αCXCR4 Ab) or matched isotype control (IgG) and assessed for Fas susceptibility (αFas) or caspase-dependent death (Z-VAD). (d) CD4 and CD8 T cells were mixed with 293T cells expressing empty vector (vector), HIV_JRFL-env (R5env), or HIV_HXB2-env (X4env). Cells were assessed for caspase-dependent death (Z-VAD). Transfected 293T cells were lysed and blotted by duotropic anti-gp160 Ab to verify envelope expression (inset).