Figure 2.

Effect of galunisertib on TGFβ1-induced SMAD2 phosphorylation, down-regulation of cytotoxicity receptors and TRAIL, and inhibition of release of perforin and granzyme A in aNK cells. A, aNK cells cultured with IL-2 (10 ng/ml) were treated with galunisertib alone (5 µM), TGFβ1 alone (15 ng/ml), or TGFβ1 and galunisertib for 18 hours. Whole lysates from aNK cells were subjected to pSMAD2, total SMAD2, and β-actin immunoblot assay. B, down-regulation by TGFβ1 of DNAM-1, NKp30, NKG2D, and TRAIL, and reversal by galunisertib. aNK cells were pretreated for 48 hours with TGFβ1 (10 ng/ml) alone, with galunisertib (5 µM) alone, or with TGFβ1 and galunisertib. An additional group received TGFβ1 alone for 24 hours, followed by addition of galunisertib for another 24 hours. Using 9-color flow cytometry, viable aNK cells were identified according to CD56 and CD16 expression and absence of CD3, CD14, and CD19 expression, and then their levels of cytotoxicity receptors and of TRAIL were determined. Stain Index values are given in each histogram overlay. The presented results are similar to those for aNK cells propagated from three additional human donors. Red lines, specific antibody; black lines, isotype control. C, expression of ligands for DNAM-1 (CD112 and CD155), NKp30 (B7-H6), and NKG2D (MICA, MICB, ULBP1, ULBP2/5/6, and ULBP3) and of TRAIL-R2 on NB cell lines and on PDX cells (COG-N-415x). D, suppression of perforin and granzyme A secretion from aNK cells by TGFβ1 is inhibited by galunisertib. After 6 hours of co-culturing aNK cells with NB cells, perforin and granzyme A were quantified in the supernatant of each treatment condition using a Luminex multiplexed microbead assay (*, p < 0.05).