Fig 1: IL-4 and IL-21 activated JAK/STAT pathways and stimulated downstream gene expressions in Ramos B cells. Ramos and Jurkat were treated with γc cytokines for 15 mins and harvested for western blot analyses. (A) IL-21 and IL-4 significantly induced STAT3 and STAT6 phosphorylation respectively. No STAT5 phosphorylation was observed in Ramos cells after any cytokine treatments. (B) IL-2, IL-9 and IL-21 treatments triggered STAT3 and STAT5 phosphorylation in Jurkat cells. Ramos cells were starved in serum-free RPMI1640 medium for 24 hours and treated with either IL-4 or IL-21 for 6 and 24 hours. (C) IL-4 significantly reduced CASP7 and induced CD23 expressions dose dependently at 6 and 24 hours respectively. (D) IL-21 significantly induced PRDM1 and IRF4 expressions dose dependently at 24 hours. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to vehicle controls art respective time points by one-way ANOVA, N = 3.
Fig 2: Anti-γc antibody attenuated NK activation only while maintained NK cell survival. PBMCs were pretreated by anti-γc (5 µg/ml) for 1 hr, followed by IL-2 (50 ng/ml) and IL-15 (50 ng/ml) stimulation for 3 days. (a and b) Anti-γc attenuated IL-2- and IL-15-induced proliferation on PBMC culture. Representative images of FACS analysis showing that anti-γc suppressed IL-2- and IL-15-induced (c) CD3−CD56+ NK cells and (d) FasL+ NK cell population. (e and g) Quantification demonstrated that anti-γc did not alter γc cytokines-induced CD3CD56+ NK cell population, but significantly suppressed (f) IL-2 and (h) IL-15 induced FasL+ NK cell population. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were compared to isotype control by Student's t-test, N = 4. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were compared to isotype control with cytokine treatment using Dunnett's multiple comparisons test following one-way ANOVA. N = 4 for all groups.
Fig 3: Long-term γc cytokine treatment shaped autoimmune phenotype in purified NK cells. (a and b) Percentage of T cells and NK cells after expansion with IL-2 and IL-21. ∗∗∗p < 0.001 as compared to controls using student t-test, N = 2 for all groups. (c) Purified NK cell cytotoxicity assay to K562 cell line after priming and expansion with differential γc cytokines for 14 days. (d) Representative histogram of FACS analysis showing differential levels of activating and inhibitory receptors after 3-day γc cytokine (IL-2 : 50, IL-15 : 50, IL-21 : 25 ng/ml) treatment. (e) IL-2/IL-15 and IL-2/IL-21 combinatorial treatment significantly upregulated activating to inhibitory receptor ratio (NKp46/KIR). ∗p < 0.05 and ∗∗∗p < 0.001 as compared to controls using Dunnett's multiple comparisons test following one-way ANOVA, N = 3 for all groups.
Fig 4: IL-2 and IL-15 contributed to NK cell activation and K562 cytotoxicity. Supernatants were harvested for ELISA, while PBMC cells were harvested for FACS analysis after 3-day cytokine (IL-2 : 20, IL-15 : 20, and IL-21 : 25 ng/ml) incubation. Differential or combinatorial treatment of IL-2- and IL-15-induced production of (a) IFN-γ, (b) IL-6, (c) perforin, (d) granzyme A, and (e) granzyme B, respectively. Representative images of intracellular (f) IFN-γ and (g) granzyme B in gated NK cells after 3-day cytokine challenges. (h) Schematic diagram for NK cell-mediated cytotoxicity assay with FACS analysis. A 3-day cytokines (IL-2 : 20, IL-15 : 20, and IL-21 : 25 ng/ml) stimulated PBMCs or supernatants were introduced to K562 for studying cell-mediated or soluble factor-mediated cytotoxicity assay, respectively. (i) All studied groups, except IL-21, enhanced K562 lysis significantly compared to control. (j) In E : T ratio of 25 : 1, IL-15, IL-2/IL-15, IL-2/IL-21, and IL-15/IL-21 treated PBMCs significantly lysed K562 lysis cells. (k) Supernatants harvested from IL-15 and IL-2/IL-15 treated PBMCs significantly lysed K562 lysis cells. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were compared to medium or control groups using Dunnett's multiple comparisons test following one-way ANOVA for (a–e) and (j and k). ∗p < 0.05 and ∗∗∗p < 0.001 were compared to control using Tukey's multiple comparisons test following two-way ANOVA for (i). ##p < 0.01 was compared to indicated group by Student's t-test. N = 3 for all groups.
Fig 5: Anti-γc reversed cytokines-induced autoimmune phenotype in purified NK cells. Purified NK cells were long-term stimulated with IL-2 + IL-21 (IL-2 : 50, IL-21 : 25 ng/ml) for 3 weeks, followed by cytotoxicity assay. (a) Representative images of FACS analysis demonstrating long-term stimulated NK cells with γc cytokines exhibited cytotoxicity against K562 and MSC. Optimal cytotoxicity to MSC was obtained at E : T ratio 10 : 1. (b) Quantification demonstrated 3-day IL-2/IL-15 and IL-2/IL-21 (IL-2 : 50, IL-15 : 50, IL-21 : 25 ng/ml) combinatorial treatment promoted MSC lysis. (c) Representative FACS images showing anti-γc reversed IL2-/IL-15-induced cytotoxicity to MSCs. (d) Quantification confirmed this result. ∗p < 0.05 and ∗∗∗p < 0.01 were compared to control using Dunnett's multiple comparisons test following one-way ANOVA. N = 3 for all groups.
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