Extended Data Fig. 5: Serine-dependent sphingolipid metabolism promotes human NK cell cytotoxicity.
From: Species-specific serine metabolism differentially controls natural killer cell functions
(a) Percent specific lysis of A375 melanoma cells by NTC-edited or PHGDHCRISPRa human NK cells cultured in Ser+ or Ser- media for 48 h followed by tumour cell coculture with IL-2/15 for 16 h at a 2:1 E:T ratio. (b) MFI of LDLR in human NK cells cultured in media containing the indicated serine concentrations for 48 h with IL-2/15. (c, d) MFI of LDLR (c) and pS6 (d) of NTC-edited or PHGDHCRISPRa human NK cells cultured in Ser+ or Ser- media for 48 h. (e) Transcript expression of genes encoding sphingolipid synthesis enzymes in naïve or IL-2/15-stimulated human NK cells. (f) Percent specific lysis of K562 cells by human NK cells cultured for 48 h in Ser+ media, Ser- media with 1% ethanol vehicle control, or Ser- media supplemented with the indicated ceramide species (50 μM). (g) MFI of BODIPY 493/503 of human NK cells cultured for 48 h in Ser+ media, Ser- media with 1% ethanol vehicle control, or Ser- media supplemented with sphingomyelin (d18:1/24:0) (5 μM). (h) Viable cell yield of human NK cells cultured for 48 h in Ser+ media, Ser- media with 1% ethanol vehicle control, or Ser- media supplemented with sphingomyelin (d18:1/24:0) (5 μM). (i) MFI of pS6 in Ser + , Ser-, or Ser- + sphingomyelin (d18:1/24:0) cultured human NK cells. (j, k) Quantification of percent IFN-γ+ and IFN-γ MFI of cytokine-producing cells (j) or MFI of GzmB and perforin (k) of Ser + , Ser-, or Ser- + sphingomyelin (d18:1/24:0) cultured human NK cells stimulated with IL-2/15, K562 cells, and/or IL-12 or IL-18 where indicated. (l) Percent CD107a+ Ser + , Ser-, or Ser- + sphingomyelin (d18:1/24:0) cultured human NK cells stimulated with K562 cells. (m) MFI of filipin III staining of Ser + , Ser-, or Ser- + sphingomyelin (d18:1/24:0) cultured human NK cells treated with or without methyl-β-cyclodextrin. Data represent individual paired donors or mice. Data are representative of (a-d) n = 5, (e-h, l, m) n = 6, (i, j) n = 4, and (k) n = 4-6 independent human donors. *p < 0.05, **p < 0.01, ****p < 0.0001 by (a, c, d, f,-i, k) one-way ANOVA, (b) two-sided paired t-test, or (e, j, l, m) two-way ANOVA. Exact p-values: (a) (left) p = 0.0091, (right) p = 0.0441; (b) p = 0.0135; (c) p = 0.0255; (d) p = 0.0065 NTC ser+ vs NTC ser-, p = 0.0237 NTC ser+ vs PHGDHCRISPRa ser-; (e) (left to right) p = 0.0197, 0.0097, 0.0082, 0.0316, 0.0182; (f) p = 0.0031; (i) p = 0.0418; (j) p < 0.0001; (k) p = 0.0251; (l) p = 0.0439; (m) p = 0.0243, p = 0.0317.
