Fig 2: IL-15 maintains NK cell survival through a PDGF-D−PDGFRβ autocrine pathway. (A) 1 × 105 primary NK cells were cultured in vitro for 7 d in the presence of IL-15 (10 ng/mL) and PDGF-D (50 ng/mL). Cells were counted by trypan exclusion assay on days 3, 5, and 7 (n = 3). (B) 1 × 105 primary NK cells were cultured in vitro for 7 d in the presence of IL-15 (10 ng/mL) and anti-PDGF-D (10 μg/mL) or control IgG (10 μg/mL). Cells were counted by trypan exclusion assay on days 3, 5, and 7 (n = 3). (C) 1 × 105 PDGFRβ-transduced NK cells were cultured in vitro for 7 d in the presence of IL-2 (10 ng/mL) and PDGF-D (50 ng/mL). The cells were counted by trypan exclusion assay on days 3, 5, and 7 (n = 3). (D) 1 × 105 primary NK cells were cultured in vitro for 7 d in the presence of IL-15 (10 ng/mL) and PDGF-D (50 ng/mL) as well as anti-PDGFRβ (10 μg/mL). Cells were counted by trypan exclusion assay on days 3, 5, and 7 (n = 3). (E) 1 × 105 primary NK cells were cultured in vitro for 7 d in the presence of IL-15 (10 ng/mL) and PDGF-D (50 ng/mL) as well as anti-NKp44 (10 μg/mL). Cells were counted by trypan exclusion assay on days 3, 5, and 7 (n = 3). (F–I) Primary NK cells were cultured in the presence of IL-15 (10 ng/mL) and PDGF-D (50 ng/mL) as well as anti-NKp44 (10 μg/mL) or anti-PDGFRβ (10 μg/mL) for 48 h. The cultured NK cells were then harvested for immunoblotting to determine protein levels of BCL-2, BCL-XL, and MCL-1 (n = 3). (J and K) 1 × 106 sorted PDGFRβ+ (Pos) and PDGFRβ− (Neg) NK cells pretreated with IL-15 (10 ng/mL) for 24 h were cultured in the presence of PDGF-D (50 ng/mL) for 48 h without IL-15. Cell apoptosis and proliferation were analyzed by annexin V staining (J) and Ki67 staining (K), respectively. Data shown are representative histograms and summary data (n = 3). (L and M) 5 × 106 sorted PDGFRβ+ (Pos) and PDGFRβ− (Neg) NK cells overexpressing IL-15 were injected into NOD/SCID/IL-2rg (NSG) mice. The mice were injected intravenously with PDGF-D (1 μg per mouse) or phosphate-buffered saline daily for 3 d. After they were killed, adoptively transferred NK cells (CD45+CD56+) in their peripheral blood were identified by flow cytometry. The cells were stained for annexin V (L) and Ki67 (M). Data shown are representative histograms and summary data (n = 4). Data represent three independent experiments. Data shown are means ± SD. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3: PDGFRβ promotes IL-15–mediated NK cell survival in vitro and in vivo. (A) 5 × 105 sorted PDGFRβ+ (Pos) and PDGFRβ− (Neg) NK cells were cultured in vitro for 7 d in the presence of IL-15 (10 ng/mL). The cells were counted via trypan blue exclusion assay on days 3, 5, and 7 (n = 5). (B and C) On day 7, Ki67 levels in PDGFRβ+ and PDGFRβ− NK cells were determined by flow cytometry (n = 5). (D and E) Annexin V levels in PDGFRβ+ and PDGFRβ− NK cells on day 7 were determined by flow cytometry (n = 5). (F and G) Immunoblotting showing protein levels of BCL-2, BCL-XL, and MCL-1 in PDGFRβ+ (Pos) and PDGFRβ− (Neg) NK cells purified by fluorescence-activated cell sorting (FACS) (n = 3). (H–J) 1 × 107 sorted PDGFRβ+ or PDGFRβ− IL-15-transduced NK cells were injected into NOD/SCID/IL-2rg (NSG) mice. Blood samples were collected for analysis at the indicated time after adoptive transfer. Data shown are representative dot plots on day 9 after adoptive transfer (H), percentages (I), and absolute numbers (J) of PDGFRβ+ and PDGFRβ− NK cells on days 3, 6, and 9 after adoptive transfer (n = 5). Data represent three independent experiments. Data shown are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 4: IL-15–induced PDGFRβ expression is mediated by PI3K/AKT signaling. (A and B) Primary NK cells were treated with IL-15 (10 ng/mL) for the indicated times. mRNA levels of PDGFRB at different time points (A) (n = 3) or at 1 h (B) (n = 10) were examined by qPCR. (C and D) Primary NK cells were pretreated with ActD (5 μg/mL) (C) or cycloheximide (CHX, 20 μg/mL) (D) for 1 h, washed twice with RPMI-1640, and then treated with IL-15 (10 ng/mL) for 24 h. Dimethyl sulfoxide (DMSO) was used as a control. Expression levels of PDGFRβ were examined by flow cytometry (n = 5). Data shown are representative histograms and percentage of inhibition with the following equation: % inhibition = 100 × [1 − (DMSO-inhibitor)/DMSO]. (E) Primary NK cells were pretreated with wortmannin (1 μM), afuresertib (10 μM), TPCA-1 (1 μM), rapamycin (10 μM), torin1 (10 μM), decernotinib (10 μM), C118-9 (10 μM), STAT5-IN-1 (10 μM), AZD6244 (10 μM), or CI-1040 (10 μM) for 1 h, washed twice with RPMI 1640, and then treated with IL-15 (10 ng/mL) for 24 h. DMSO was used as control. Data shown are percent of inhibition (n = 5). The mean value of the inhibitory rate is shown. (F) Luciferase reporter assay shows that p65 activates PDGFRB gene transcription. (G–I) Binding of p65 to the PDGFRB promoter in IL-15–treated NK cells (G) or resting NK cells (H) as determined by ChIP-qPCR or PCR (I) (n = 3). Data represent three independent experiments. Data shown are means ± SD. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 5: IL-15 induces PDGFRβ expression in human NK cells. (A–C) NK cells were purified from PBMCs of healthy donors and stimulated with IL-15 (10 ng/mL) for 24 h. Expression levels of PDGFRα and PDGFRβ were examined by flow cytometry. Data shown are representative dot plots (A), percentages (B), and mean fluorescence intensity (MFI) of PDGFRβ in NK cells (C) (n = 20). Resting NK cells were used as the control. (D and E) NK cells were treated with various doses of IL-15 for 24 h (D) or with the same dose of IL-15 (10 ng/mL) at the indicated times (E). Data shown are the percentages of PDGFRβ+ NK cells among total NK cells (n = 5). (F) Immunofluorescence analysis of PDGFRβ expression on resting and IL-15–treated NK cells. Sodium-potassium ATPase was used as a cell membrane marker. (Scale bar, 20 μm.) (G) Expression of PDGFRβ in the cytoplasmic, nuclear, and cell membrane fractions of NK cells determined by immunoblotting. (H–J) CD56dim and CD56bright NK cells were sorted from PBMCs of healthy donors and treated with IL-15 (10 ng/mL) for 24 h. Expression levels of PDGFRβ were then determined by flow cytometry. Data shown are representative dot plots (H), percentages (I), and MFI (J) of PDGFRβ on NK cells (n = 6). Data represent three independent experiments. Data shown are means ± SD. NS, not significant. *P < 0.05 and ****P < 0.0001.