Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia

Abstract

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus (Ad)-CD154 (CD40L) gene therapy experience reductions in leukemia cell counts and lymph node size associated with induction of the death receptor Fas (CD95). CD4 T cell lines can induce apoptosis of CD40-activated CLL cells via a CD95 ligand (CD95-L)-dependent mechanism. To examine whether CD95-L was sufficient to induce cytolysis of CD40-activated CLL cells, we used Chinese hamster ovary cells transfected with CD95-L as cytotoxic effector cells. CD40-activated CLL cells were initially resistant to CD95-mediated apoptosis despite high-level expression of CD95. However, after 72 h, CLL cells from seven of seven patients became increasingly sensitive to CD95-mediated apoptosis. This sensitivity correlated with a progressive decline in Flice-inhibitory protein (FLIP), which was induced within 24 h of CD40 ligation. Down-regulation of FLIP with an antisense oligonucleotide or a pharmacologic agent, however, was not sufficient to render CLL cells sensitive to CD95-mediated apoptosis in the 24-72 h after CD40 activation. Although the levels of pro-Caspase-8 appeared sufficient, inadequate levels of Fas-associated death domain protein (FADD) and DAP3 may preclude assembly of the death-inducing signaling complex. Seventy-two hours after CD40 ligation, sensitivity to CD95 and a progressive increase in FADD and DAP3 were associated with the acquired ability of FADD and FLIP to coimmunoprecipitate with the death-inducing signaling complex after CD95 ligation. Collectively, these studies reveal that CD40 ligation on CLL B cells induces a programmed series of events in which the cells initially are protected and then sensitized to CD95-mediated apoptosis through shifts in the balance of the anti- and proapoptotic proteins FLIP and FADD.

Figure 1

CD4 CTL induce CD95-dependent apoptosis of CD40-activated CLL cells. CTL activity of CD4 T cells expanded from the blood of patients with CLL was tested against autologous and nonrelated, allogeneic CLL target cells. CLL target cells were mock (hatched bars) or CD40-activated (black bars), and replated for an additional 24 h before use. CD4 effector and CLL target cells were cocultured at an effector-to-target cell ratio of 10:1, incubated for 4 h, and stained with DiOC₆ for 15 min at 37°C before flow cytometric analysis. To test for specificity, CD4 effectors or CLL target cells were preincubated with monoclonal antibodies specific for CD95L or MHC class I (W6/32), respectively. Target cells also were preincubated with 50 μM ZVAD to test for caspase dependence.

Figure 2

CD95-mediated apoptosis of CD40-activated CLL cells. CLL cells were CD40-activated and harvested 1, 3, or 5 days later for use as targets in a DiOC₆ apoptosis assay. CHO (open bars), CHO-CD95-L (black bars), or anti-CD95L mAb pretreated CHO-CD95-L (hatched bars) effector cells were cocultured with target cells at an effector-to-target cell ratio of 10:1 for 8 h at 37°C. DiOC₆ dull or apoptotic target cells were measured by flow cytometric analysis. Data represent the mean ± SD from experiments testing seven different CLL patient samples.

Figure 3

Time course of CD95 expression on CLL cells after CD40 activation. Flow cytometric analysis of CD95 expression before (pre) and at indicated timepoints after 24-h stimulation on HeLa (open squares) or HeLa-CD154 transfected cells (filled squares). Data shown are the mean fluorescence intensity ratio (MFIR) from two different CLL patient samples stained with phycoerythrin (PE)-labeled anti-CD95 or isotype control mAb.

Figure 4

Time course of FLIP expression in CLL cells after CD40 activation. (A) Representative immunoblot analysis of FLIP_L and FLIP_S isoforms. Lysates were prepared from CLL cells harvested before (pre) and at days 1, 3, and 5 (D1, D3, D5) after CD40 activation. For Western analysis, 12.5 μg of protein was subjected to 12% SDS/PAGE and immunoblotted with FLIP mAb. Blots were stripped and reprobed with antiactin mAb to ensure equal protein loading. (B) FLIP immunoblots were scanned and quantitated by using NIH image software. Data were normalized to actin bands and represent the mean ± SD of seven separate experiments.

Figure 5

Down-regulation of FLIP in CLL cells. (A) CLL cells were electroporated with PBS, FLIP antisense, or control scrambled oligo as described. Cells were then CD40 activated for 24 h and harvested for FLIP immunoblot analysis, or (B) CD95 apoptosis assays, as described. Bars indicate the % apoptosis of control-treated CLL cells (open bars), FLIP-antisense-treated CLL cells (black bars), or JURKAT cells (hatched bars) when incubated with CHO cells or CHO cells that express CD95-L (CHO-CD95-L).

Figure 6

Down-regulation of FLIP with CDDO. (A) CLL cells were CD40 activated and replated in media alone for an additional 24 h with (−) or without (+) 1 μM CDDO. Cells were then harvested for immunoblot analysis of FLIP and (B) FADD or (C) CD95 apoptosis assays. Data shown are the mean CD95-mediated apoptosis of four patient samples up to 72 h after CD40 activation in the absence (open bar) or presence (black bar) of 1 μM CDDO treatment, ±SD. In one sample (gray bar), sensitivity to CD95-mediated apoptosis was tested at day 5 after CD40 activation to confirm that CDDO-treated CLL cells still acquired sensitivity to CD95. Apoptosis of Jurkat cells demonstrates activity of CHO-FasL (hatched bars).

Figure 7

Time course analysis of apoptosis regulatory protein expression after CD40 activation of CLL cells. Cells were CD40 activated for 24 h and immunoblot analysis performed for the proteins (A) FADD and tumor necrosis factor–receptor-associated factor 1, (B) pro-caspase-8, and (C) DAP3 and xIAP. (D) Immunoblots for pro-caspase-8 and FADD were scanned, and the mean normalized expression calculated (±SD, n = 3). * indicates significant difference from pre-CD40-activated CLL cells as calculated by the Bonferroni t test.

Figure 8

Immunoprecipitation of DISC. CLL cells were CD40-activated and harvested at days 1 and 5 for DISC immunoprecipitation. BJAB- or CD40-activated CLL cells were either untreated or stimulated with 2 μg/ml of APO-1 mAb for 15 min before the preparation of cell lysates. CD95 DISC was immunoprecipitated by using protein A-Sepharose beads (+). Unstimulated controls were lysed before immunoprecipitation with APO-1 and protein A-Sepharose beads (−). Immunoprecipitates were then subjected to immunoblot analysis with anti-FLIP mAb (Upper) or anti-FADD (Lower). A431- and CD40-activated CLL cell lysates were loaded as controls for the migration position of FADD and FLIP.