Figure 2.

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.