il-1ra cytokine  (ProSpec)

Journal: bioRxiv

Article Title: IL-33 drives polyfunctionality and antitumor activity of a unique ST2+ NK cell population

doi: 10.1101/2023.02.14.528486

Figure Lengend Snippet: (A) Quantification of IFN-γ secretion by healthy donors’ blood NK cells upon stimulation with IL-33, IL-18, IL-1α or IL-1β alone (10 ng/mL) or in combination with IL-12 (10 ng/mL) for 24 h. Histogram bars represent the median (n = 4 individual experiments). Friedman test with Dunn’s multiple comparisons test was performed. (B) NK cells were activated for 24 h with IL-33, IL-18, IL-1α or IL-1β alone (10 ng/mL) or in combination with IL-12 (10 ng/mL) and then co-cultured with calcein-loaded K562 target cells for 4 h at a 5:1 effector to target ratio. Calcein release in the supernatant was quantified by fluorometry to calculate lysis percentage. Results are expressed as mean + SEM (n = 9 individual experiments). One-way repeated measures ANOVA with Dunnett’s multiple comparisons test against levels in medium was performed. (C) NK cells were activated for 24 h with IL-33, IL-18, IL-1α or IL-1β alone (10 ng/mL) or in combination with IL-12 (10 ng/mL) and then stained for CD25 and CD69 (black line) or corresponding isotypic control (grey). Representative histogram plots (left) and quantification (%) (right) of CD25 + or CD69 + NK cells after cytokine stimulation. Symbols represent paired individual experiments (n=7). Results are expressed as mean + SEM. One-way repeated measures ANOVA with Dunnett’s multiple comparisons test against levels in IL-12 was performed. (D) IFN-γ secretion by healthy donors’ blood NK cells upon stimulation for 24 h with IL-33 and IL-12 (10 ng/mL each) in the presence of anti-ST2 (10 µg/mL), anti-IL-18 (1 µg/mL) blocking antibodies, IL-1RA antagonist (100 ng/mL) or mIgG1 control antibody (1 or 10 µg/mL as control for anti-IL-18 or anti-ST2, respectively). Results are expressed as mean + SD and are representative of three individual experiments. One-way repeated measures ANOVA with Tukey’s multiple comparisons test was performed. (E) Real-time quantitative PCR (RT-qPCR) analysis of IL1RL1 (ST2) and IL1RAcP mRNA expression in resting or activated NK cells with IL-12, IL-33, or the combination (10 ng/mL each for 24 h). Results are expressed as mean + SEM of three individual experiments. Each symbol represents one donor. Two-tailed paired Student t- test was performed. (F) NK cells were activated with IL-12 (10 ng/mL) in the presence or not of lisofylline (500 μM) for 24 h prior to ST2 surface expression analysis by flow cytometry. Representative dot plots (left) and quantification (%) (right) of ST2 + NK cells after cytokine stimulation. One-way repeated measures ANOVA with Tukey’s multiple comparisons test was performed; n = 3. (G) p-STAT4 was immunoprecipitated in NK cells following activation or not with IL-12 (10 ng/mL) for 24 h and p-STAT4 enrichment in ST2 promoter region was measured by RT-qPCR analysis. PERFORIN-1 (PRF1) and negative control sets 1 and 2 were used respectively as positive and negative controls for p-STAT4 binding. Results are expressed as mean + SEM of two individual experiments.

Article Snippet: For blocking experiments, cells were incubated with anti-ST2 (10 μg/mL, R&D), anti-IL-18 (1 μg/mL, MBL), mIgG1 (1 or 10 μg/mL, R&D) antibodies or IL-1RA (100 ng/mL, Peprotech).

Techniques: Cell Culture, Lysis, Staining, Control, Blocking Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Two Tailed Test, Flow Cytometry, Immunoprecipitation, Activation Assay, Negative Control, Binding Assay