Fig. 1

Neo-2/15 is the optimal IL-2R agonist for NK cells. a Protein structures of natural human IL-2 and IL-15 as well as superkines H9, H9T, N72D and Neo-2/15 and their binding affinities to IL-2Rβγ predicted using the Swisse model. Kd: equilibrium binding dissociation value. b Heatmap showing the expansion of human primary NK cells obtained from healthy young males (age < 34 years) at indicated days after being stimulated with 1 nM indicated cytokine or superkine. The number 1, 2, and 3 indicate three different individuals for each group. c Heatmap of the expansion of primary NK cells from healthy young females (age < 41 years). d Heatmap of the expansion of primary NK cells from healthy elder individuals (age > 60 years). e Heatmap of the expansion of primary NK cells from cancer patients. f Heatmap of the expansion of primary NK cells from organ transplant recipients receiving immunosuppressants (FK506 plus Mycophenolate Mofetil). b-f n = 6. g The proliferation of NK-92 incubated with indicated cytokine or superkine for 1 week is quantified by cell expansion fold. Data were fitted with a logistic equation (smooth curves) to estimate the EC₅₀ value. h Schematic representation of testing the stability of IL-2R ligands in vitro (top). Different cytokine or superkine is individually added to the medium of AsPC-1 cell cultures, the culture supernatant is collected 5 days later and added to cytokine-free NK-92 culture medium at 1:10 ratio. NK-92 are harvested 1 hour later, then the total and the phosphorylated STAT5 in cell lysates are detected by immunoblotting (bottom). -: complete NK-92 culture medium with 1 nM indicated cytokine or supekine. +: cytokine-free NK-92 culture medium with 10% tumor-culture supernatant contained indicated cytokine or supekine. β-Actin served as loading control. i NK-92 stimulated with 1 nM indicated cytokine or superkine for 4 hours are subjected to immunoblotting using indicated antibodies. β-Actin served as the loading control. The expression of LAG3, TIGIT, TIM-3, PD-1, NKG2A (j), NKG2D and CD16 (k) in primary NK cells derived from healthy donors stimulated with 1 nM indicated cytokine or superkine for 4 hours were detected by flow cytometry. Data are presented as mean ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3). l Cytotoxicity of IL-2, IL-15, H9, H9T, N72D or Neo-2/15 stimulated human primary NK cells obtained from healthy young males (age < 35 years) on K562 target cells after co-culture for 4 hours at E:T ratios of 1:1 was detected by flow cytometry (ns, not significant, *P < 0.05, **P < 0.01, n = 3)