Figure 5. IL-15 promotes NKG2D-mediated cytolytic degranulation, cPLA2 activation, and AA release in primary IE-CTLs. (A) IL-15 up-regulates cPLA2 expression, and induces ERK, JNK, and cPLA2 activation in fresh IE-CTLs. (left) IL-15 induces ERK, JNK, and cPLA2 phosphorylation in a time-dependent manner. Fresh IE-CTLs were starved overnight and stimulated with 20 ng/ml IL-15 for indicated time periods. Cell lysates were immunoblotted with anti-p-ERK, -p-cPLA, and -p-cPLA2 antibodies. Total ERK and total cPLA2 are shown to assess equal loading. (right) Fresh IE-CTLs were cultured with IL-15 or IL-7 for 48 h. cPLA2 expression was determined by Western blot. Equal loading was assessed using an anti-CD3ζ mAb. (B) IL-15 synergizes with NKG2D to induce cPLA2 phosphorylation in a dose- (left) and time-dependent (right) manner. Fresh IE-CTLs (left) or TALL-104 CTLs (right) were stimulated with IL-15 alone, anti-NKG2D mAb alone, or IL-15 in combination with anti-NKG2D mAb for 15 min at the indicated concentration (left), or for the indicated time at saturating concentrations (4 µg/ml anti-NKG2D mAb and 20 ng/ml IL-15) (right). For each panel one out of 2 representative experiments is shown. (C and D) IL-15 stimulation plays a critical role in NKG2D-mediated cPLA2 activity (C) and cytolytic granule release (D) in primary IE-CTLs. Freshly purified IE-CTLs were cultured with or without IL-15 (20 ng/ml) for 24–48 h and pretreated for 30 min with vehicle control or CF3 before stimulation with anti-NKG2D mAb or IgG1 isotype control. Rescue experiments were performed by adding 50 µM of AA after 1 h of stimulation. Data are means ± SD of six experimental points from two independent experiments. P values are from a Tukey-adjusted pairwise comparison. (E) IL-15 enhances NKG2D-mediated AA release. Fresh IE-CTLs were cultured with or without IL-15 (10 ng/ml) for 24 h and analyzed for AA release upon 1 h stimulation. (left) IE-CTLs were not stimulated (NS) or stimulated with plate-bound anti-NKG2D mAb (4 µg/ml). (right) IE-CTLs were stimulated with control EL-4 cells or MICA-transfected EL4 cells (MICA/EL-4) at a IE-CTLs to target cell ratio of 1:0.5. Data are means ± SD of three independent experiments. P values are from a Tukey-adjusted pairwise comparison.
Figure 5.

IL-15 promotes NKG2D-mediated cytolytic degranulation, cPLA2 activation, and AA release in primary IE-CTLs. (A) IL-15 up-regulates cPLA2 expression, and induces ERK, JNK, and cPLA2 activation in fresh IE-CTLs. (left) IL-15 induces ERK, JNK, and cPLA2 phosphorylation in a time-dependent manner. Fresh IE-CTLs were starved overnight and stimulated with 20 ng/ml IL-15 for indicated time periods. Cell lysates were immunoblotted with anti-p-ERK, -p-cPLA, and -p-cPLA2 antibodies. Total ERK and total cPLA2 are shown to assess equal loading. (right) Fresh IE-CTLs were cultured with IL-15 or IL-7 for 48 h. cPLA2 expression was determined by Western blot. Equal loading was assessed using an anti-CD3ζ mAb. (B) IL-15 synergizes with NKG2D to induce cPLA2 phosphorylation in a dose- (left) and time-dependent (right) manner. Fresh IE-CTLs (left) or TALL-104 CTLs (right) were stimulated with IL-15 alone, anti-NKG2D mAb alone, or IL-15 in combination with anti-NKG2D mAb for 15 min at the indicated concentration (left), or for the indicated time at saturating concentrations (4 µg/ml anti-NKG2D mAb and 20 ng/ml IL-15) (right). For each panel one out of 2 representative experiments is shown. (C and D) IL-15 stimulation plays a critical role in NKG2D-mediated cPLA2 activity (C) and cytolytic granule release (D) in primary IE-CTLs. Freshly purified IE-CTLs were cultured with or without IL-15 (20 ng/ml) for 24–48 h and pretreated for 30 min with vehicle control or CF3 before stimulation with anti-NKG2D mAb or IgG1 isotype control. Rescue experiments were performed by adding 50 µM of AA after 1 h of stimulation. Data are means ± SD of six experimental points from two independent experiments. P values are from a Tukey-adjusted pairwise comparison. (E) IL-15 enhances NKG2D-mediated AA release. Fresh IE-CTLs were cultured with or without IL-15 (10 ng/ml) for 24 h and analyzed for AA release upon 1 h stimulation. (left) IE-CTLs were not stimulated (NS) or stimulated with plate-bound anti-NKG2D mAb (4 µg/ml). (right) IE-CTLs were stimulated with control EL-4 cells or MICA-transfected EL4 cells (MICA/EL-4) at a IE-CTLs to target cell ratio of 1:0.5. Data are means ± SD of three independent experiments. P values are from a Tukey-adjusted pairwise comparison.

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