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. 2015 Mar 25;5:9482.
doi: 10.1038/srep09482.

ADP-ribose/TRPM2-mediated Ca2+ Signaling Is Essential for Cytolytic Degranulation and Antitumor Activity of Natural Killer Cells

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Free PMC article

ADP-ribose/TRPM2-mediated Ca2+ Signaling Is Essential for Cytolytic Degranulation and Antitumor Activity of Natural Killer Cells

So-Young Rah et al. Sci Rep. .
Free PMC article

Abstract

Natural killer (NK) cells are essential for immunosurveillance against transformed cells. Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel gated by ADP-ribose (ADPR). However, the role of TRPM2-mediated Ca(2+) signaling in the antitumor response of NK cells has not been explored. Here, we show that ADPR-mediated Ca(2+) signaling is important for cytolytic granule polarization and degranulation but not involved in target cell recognition by NK cells. The key steps of this pathway are: 1) the activation of intracellular CD38 by protein kinase A following the interaction of the NK cell with a tumor cell results in the production of ADPR, 2) ADPR targets TRPM2 channels on cytolytic granules, and 3) TRPM2-mediated Ca(2+) signaling induces cytolytic granule polarization and degranulation, resulting in antitumor activity. NK cells treated with 8-Br-ADPR, an ADPR antagonist, as well as NK cells from Cd38(-/-) mice showed reduced tumor-induced granule polarization, degranulation, granzyme B secretion, and cytotoxicity of NK cells. Furthermore, TRPM2-deficient NK cells showed an intrinsic defect in tumoricidal activity. These results highlight CD38, ADPR, and TRPM2 as key players in the specialized Ca(2+) signaling system involved in the antitumor activity of NK cells.

Figures

Figure 1
Figure 1. Critical role of TRPM2 for cytotoxicity of NK cells against tumor cells via Ca2+-dependent degranulation.
(a) Tumor cell-induced Ca2+ signals in NK cells from TRPM2+/+ or TRPM2−/− mice. Arrow indicates the time of addition of B16F10 tumor cells. Data are mean ± SEM of three independent experiments. *P < 0.001 vs basal; #P < 0.05. (b) Impairment of degranulation in TRPM2−/− NK cells upon stimulation with B16F10 cells. TRPM2+/+ or TRPM2−/− NK cells were stimulated with target cells for 2 h at 37°C and then stained with FITC-conjugated anti-CD107a mAb and PE-conjugated anti-NK1.1 mAb. NK cells were gated on forward scatter/side scatter characteristics. (c) Reduced granzyme B release in tumor cell-stimulated TRPM2−/− NK cells. The amount of granzyme B released into the media was measured by ELISA after incubation with NK cells and B16F10 cells for 30 min. (d) Decrease in cytolytic activity against B16F10 cells in TRPM2−/− NK cells. TRPM2+/+ or TRPM2−/− NK cells were assessed for cytolytic activity against B16F10 target cells in a 4-h calcein-release assay. Data shown in b, c, and d are representative of three independent experiments. *P < 0.001. (e) B16F10 cells (1 × 105) were injected into the flanks of TRPM2+/+ and TRPM2−/− mice (n = 10 per cohort), and tumor growth was monitored. *P < 0.001. (f) Kaplan-Meier plot of TRPM2+/+ and TRPM2−/− recipient mice after s.c. injection with B16F10 cells (1 × 105) (n = 10 per cohort). (g) Antitumor effect of TRPM2+/+ or TRPM2−/− NK cells in the lung metastasis model of B16F10 cells. TRPM2−/− mice (n = 4 per cohort) were injected i.v. with 1 × 105 B16F10 melanoma cells. IL-2 activated NK cells (2 × 106 cells) from TRPM2+/+ or TRPM2−/− mice were injected i.v. into B16F10-bearing TRPM2−/− mice 2 d later. Lungs were harvested 14 d after and fixed with 4% paraformaldehyde, and metastatic nodules were counted. Representative images of lungs are shown. *P < 0.01.
Figure 2
Figure 2. Tumor cell-induced NK cell granule polarization and degranulation requires CD38-mediated Ca2+ signals.
(a) Cd38+/+ or Cd38−/− NK cells stimulated with B16F10 tumor cells. The percentage of conjugation formation at indicated time was analyzed by flow cytometry. Data are mean ± SEM of three independent experiments. (b) Cd38−/− NK cells were inhibited tumor-triggered translocation of perforin and granzyme B towards immunological synapses. Cd38+/+ or Cd38−/− NK cells were stimulated with B16F10 cells at 1:1 ratio at 37°C for 20 min, and then stained with perforin or granzyme B. The dashed line indicates the immunological synapse. (Scale bar, 5 μm). All images were representative of at least three independent experiments. (c) Degranulation of Cd38+/+ or Cd38−/− NK cells upon stimulation with B16F10 cells for 2 h at 37°C. Shown is a representative of three independent experiments. (d and e) Ca2+ signals in Cd38+/+ or Cd38−/− NK cells upon stimulation with B16F10 cells. NK cells were loaded with Fluo-4 AM for 40 min at 37°C and Ca2+ levels in NK cells were measured following treatment with B16F10 cells labeled with cell tracker orange CMRA (red). Arrow indicates the time of addition of B16F10 cells. (Scale bars, 10 μm). Shown is a representative of three independent experiments.
Figure 3
Figure 3. ADPR-mediated Ca2+ signals are required for cytolytic degranulation of NK cells.
(a) 8-Br-ADPR inhibits tumor cell-induced sustained Ca2+ signals in NK cells. NK cells were pretreated with 100 μM 8-Br-ADPR, 100 μM 8-Br-cADPR, 10 μM Ned19, or 2 μM XeC for 20 min. Arrow indicates the time of addition of B16F10 cells. (b) Inhibition of tumor cell-induced NK cell degranulation by 8-Br-ADPR. Data shown in a and b are representative of three independent experiments. *P < 0.001 vs basal; #P < 0.05. (c) 8-Br-ADPR inhibits tumor cell-induced translocation of perforin and granzyme B towards immunological synapses. NK cells were added to B16F10 cells at 1:1 ratio at 37°C for 20 min and stained with perforin or granzyme B. The dashed line indicates the immunological synapse. (Scale bars, 5 μm). All images were representative of at least three independent experiments. (d) NK cells were treated with PME (30 μg) for 20 min, and homogenized and fractionated on a continuous 0.4 M to 1.6 M sucrose density gradient using ultracentrifugation. Fractions were analyzed by immunoblotting to identify cellular organelles and identified using antibodies against each marker: early endosome (EEA1), lysosomes (LAMP1), plasma membrane (Na+K+-ATPase). Distributions of granzyme B and perforin were assessed by immunoblotting. Shown is a representative of three independent experiments. The gels have been run under the same experimental conditions. The full-length blots and the cropped blots are presented in Supplementary Figure 2. (e and f) 8-Br-cADPR inhibits tumor-induced granzyme B secretion (e) and cytotoxicity activity (f) of NK cells. 100 μM 8-Br-ADPR, 100 μM 8-Br-cADPR, 10 μM Ned19, or 2 μM XeC was preincubated for 20 min. Data are mean ± SEM of three independent experiments. #P < 0.001.
Figure 4
Figure 4. Tumor-induced ADPR production by intracellular CD38 is responsible for sustained Ca2+ signals in NK cells.
(a and b) Tumor cells induce ADPR production but cADPR in Cd38+/+ NK cells. Levels of ADPR and cADPR were determined after treatment of NK cells with 30 μg PME prepared from B16F10 tumor cells for 40 s. Data are mean ± SEM of three independent experiments. *P < 0.001; #P < 0.05. ns, not significant. (c–e) Cibacron blue 3GA (CB) inhibits of tumor-stimulated ADPR induction (c), sustained Ca2+ signal (d), cytotoxicity (e). CB (100 μM), ara-2′-F-NAD (200 nM), bafilomycin A1 (200 nM) or GPN (50 μM) was preincubated for 30 min. Data shown in c–e are representative of three independent experiments. *P < 0.001 vs basal; #P < 0.05.
Figure 5
Figure 5. Tumor cells produce ADPR by activation of PKA but not PKG in NK cells.
(a) ADPR levels were determined after treatment of NK cells with 30 μg PME for 40 s. Rp-8-Br-cAMPS (100 μM) or Rp-8-pCPT-cGMPS (20 μM) was preincubated for 30 min. 100 μM N6-benzoyl-cAMP (PKA activator, 40 s) was used for ADPR measurement. Data are mean ± SEM of three independent experiments. *P < 0.001 vs basal; #P < 0.05. (b) cAMP levels were determined after treatment of NK cells with 30 μg PME. Data are mean ± SEM of three independent experiments. (c) Inhibition of tumor cell-induced sustained Ca2+ increase by a PKA inhibitor, Rp-8-Br-cAMPS, and an adenylate cyclase inhibitor, SQ 22536. Rp-8-Br-cAMPS (100 μM) or SQ 22536 (250 μM) was preincubated for 30 min. (d) SOCE induced by bafilomycin A1. Bafilomycin A1 (1 μM)-induced SOCE was inhibited with 50 μM SK96365 but not by 20 μM ACA. SK96365 or ACA was pre-incubated for 30 min. Data shown in c and d are mean ± SEM of three independent experiments. n = 10.
Figure 6
Figure 6. Tumor cell increases the interaction of CD38 with TRPM2 in NK cells.
(a) Increased co-localization of CD38 with TRPM2 in NK cells upon tumor cell treatment. NK cells were incubated with B16F10 cells for 20 min and then stained with CD38 and TRPM2. (b) Tumor cell PME increases association of CD38 with TRPM2 in NK cells. NK cells were treated with PME for 15 s. Cells were extracted with a lysis buffer and then subjected to immunoprecipitation (IP) using an anti-CD38 mAb. Immunoprecipitated proteins were analyzed by western blotting (WB) with anti-CD38 pAb or anti-TRPM2 pAb. The gels have been run under the same experimental conditions. The full-length blots and the cropped blot are presented in Supplementary Figure 3. (c) Defect of tumor cell-stimulated translocation of perforin towards immunological synapses in TRPM2−/− NK cells. TRPM2+/+ or TRPM2−/− NK cells were added to B16F10 target cells at 1:1 ratio at 37°C for 20 min and then stained with perforin and TRPM2. DAPI was used as a nuclear stain (blue). The dashed line indicates the immunological synapse. (Scale bar, 5 μm). All images were representative of at least three independent experiments.
Figure 7
Figure 7. CD38 is critical for the antitumor effects of NK cells via cytolytic granule exocytosis.
(a) Antitumor effects of Cd38+/+ and Cd38−/− NK cells in a lung metastasis model of B16F10 cells. C57BL/6 mice (n = 4 per cohort) were injected i.v. with 1 × 105 B16F10 melanoma cells. After 2 d, IL-2 activated NK cells (2 × 106 cells) from Cd38+/+ or Cd38−/− mice were injected i.v. into B16F10-bearing wild-type mice. Lungs were harvested 14 d later and metastatic nodules were counted. nd, not detected. Data are mean ± SEM of three independent experiments. Representative images of lungs are shown. *P < 0.001. (b) Comparison of cytolytic activity of Cd38+/+ and Cd38−/− NK cells against various target cell lines. Cd38+/+ or Cd38−/− NK cells cultured for 10 d were assessed for cytolytic activity as indicated, over 4 h. Data are mean ± SEM of three independent experiments. *P < 0.001; #P < 0.05. (c and d) Cd38+/+ and Cd38−/− NK cells are expressed equivalent amounts of perforin and granzyme B at mRNA (c) and protein levels (d). The gels have been run under the same experimental conditions. The full-length blots and the cropped blots are presented in Supplementary Figure 4. Data are mean ± SEM of three independent experiments. ns, not significant. (e) Comparison of the amount of granzyme B released into the media by Cd38+/+ and Cd38−/− NK cells on stimulation with B16F10 cells for 30 min. Data are mean ± SEM of three independent experiments. *P < 0.001 vs basal; #P < 0.05.

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