Lack of T-Cell Receptor–Induced Signaling Is Crucial for CD95 Ligand Up-regulation and Protects Cutaneous T-Cell Lymphoma Cells from Activation-Induced Cell Death

Abstract

Restimulation of previously activated T cells via the T-cell receptor (TCR) leads to activation-induced cell death (AICD), which is, at least in part, dependent on the death receptor CD95 (APO-1, FAS) and its natural ligand (CD95L). Here, we characterize cutaneous T-cell lymphoma (CTCL) cells (CTCL tumor cell lines and primary CTCL tumor cells from CTCL patients) as AICD resistant. We show that CTCL cells have elevated levels of the CD95-inhibitory protein cFLIP. However, cFLIP is not responsible for CTCL AICD resistance. Instead, our data suggest that reduced TCR-proximal signaling in CTCL cells is responsible for the observed AICD resistance. CTCL cells exhibit no PLC-;1 activity, resulting in an impaired Ca 2+ release and reduced generation of reactive oxygen species upon TCR stimulation. Ca 2+ and ROS production are crucial for up-regulation of CD95L and reconstitution of both signals resulted in AICD sensitivity of CTCL cells. In accordance with these data, CTCL tumor cells from patients with Sézary syndrome do not up-regulate CD95L upon TCR-stimulation and are therefore resistant to AICD. These results show a novel mechanism of AICD resistance in CTCL that could have future therapeutic implications to overcome apoptosis resistance in CTCL patients. [Cancer Res 2009;69(10):4175-83]

Deficiency of SATB1 expression in Sézary cells causes apoptosis resistance by regulating FasL/CD95L transcription

“…42 Recently, several reports consistently demonstrated that CTCL cells, including Hut78 cells, are highly resistant to apoptosis, in particular AICD, which has been postulated to be responsible for the development and progression of CTCLs. 6,43,44 The SATB1 expression-restored Hut78 cells in our study demonstrated increased spontaneous apoptosis, compared with control Hut78 cells. When stimulated with anti-CD3 antibody or PMA/ionomycin in the presence of IL-2, which simulates the AICD process in vitro, the SATB1-restored cells showed higher rates of apoptosis.…”

“…6,21 For FasL/CD95L-induced apoptosis, cells were seeded at 5 ϫ 10 5 /mL in 1 mL RPMI 1640 culture media. Super FasL (40 ng/mL; Alexis Biochemicals) was added into the cell culture and incubated for 16 hours.…”

“…Specific apoptosis was calculated as (percentage of induced apoptosis Ϫ percentage of spontaneous apoptosis)/(100 Ϫ percentage of spontaneous apoptosis). 6 …”

“…5 In particular, the resistance to activation-induced cell death (AICD) is characteristic in Sézary cells. 6 During the course of an immune response, antigen-reactive T cells clonally expand and then are removed by apoptosis to maintain immune homeostasis. The majority of effector T cells are eliminated by AICD.…”

“…15 For AICD, isolated primary CD4 ϩ cells from healthy volunteers and primary Sézary cells purified from patients were stimulated with 1g/mL phytohemagglutinin for 16 hours, followed by culturing for 6 days in the presence of 25 U/mL recombinant human interleukin-2 (IL-2), added every 2 days of culture, as described previously. 6 …”