CD95-induced block of store-operated Ca2+ entry requires
CD95 and ASM. (A) Coculture with CD95L+
glioblastoma (LN18) blocks store-operated Ca2+ entry in T
lymphocytes. Similar results were obtained with a second
CD95L+ glioblastoma cell line (LN229). Fura-2 loaded Jurkat
T cells were cocultured with CD95L+ glioblastoma cells
(LN18) for 1.5 h and Ca2+ entry was stimulated with TG
(1 μM for 10 min). Pseudocolored ratio images of lymphocyte
fluorescence encode intracellular free Ca2+ concentrations
according to the color bar shown in C. Fluorescence
ratio images of lymphocytes were digitally overlaid on transmission
images obtained simultaneously with differential interference contrast.
(Bars = 20 μm.) (B) Neutralization of CD95L with
a CD95-Fc-fusion protein (3 μg/ml) prevents block of
Ca2+ entry in T cells following coculture with LN18 tumor.
(C) Ca2+ block requires a functional CD95
receptor. In gld thymocytes, coculture blocks
Ca2+ entry. In contrast, in CD95-defective
lpr thymocytes, influx of Ca2+ is not
blocked. Frequency distribution histograms of
[Ca2+]i in single TG-stimulated thymocytes
cocultured with LN18 tumor are shown. [Ca2+]i
was measured in 104 cells by using flow cytometry. The
color bar shows the pseudocolor scale for panels A and
B. (D) ASM-deficient cells respond to
CD95 stimulation (100 ng/ml anti-CD95 Ab for 1 h) with an
increased Ca2+ plateau, whereas the
[Ca2+]i rise evoked by TG in control cells is
inhibited. Without CD95 triggering, the store-operated
[Ca2+]i rise is higher in ASM-deficient cells
(NPDA) compared with control (JY) cells.
[Ca2+]i was measured with imaging microscopy
in fura-2 loaded cells. Reconstitution of ASM into NPDA cells restores
the Ca2+ block by CD95. Stimulated Ca2+ entry
is increased after 1 h of CD95 stimulation in control-transfected
NPDA cells. ASM transfection reverses this effect, leading to a block
of Ca2+ entry by CD95. ASM transfection without CD95
stimulation has no effect on [Ca2+]i. Bars
indicate mean [Ca2+]i levels 5 min after TG
stimulation in 104 transfected NPDA cells measured by flow
cytometry. Two independent experiments were performed.