IP-FCM detects the specific inhibition of CD25:IL-2 interaction by the first-generation SMIPPI, Ro-26-4550.

(A) Recombinant human CD25 (rhCD25) was immunoprecipitated with anti-CD25 antibody-conjugated CML beads. Recombinant human IL-2 (1000 U/ml) and one of several small-molecules were mixed with the beads for one hour, followed by probing with an anti-IL-2 antibody. Inhibition of the IL2:CD25 interaction was observed only upon addition of 10 mM R0-26-4550 (R26, blue shaded histogram). (B) Median fluorescence intensity from three experiments shows R26 significantly reduced IL-2:CD25 binding compared to either vehicle (DMSO) or PP2 negative controls (ANOVA: F_3,10 = 10.82, p = 0.0051; **p<0.01, *p,0.05) (C) Endogenous CD25 was immunoprecipitated from lysates from PMA/Ionomycin-stimulated human PBMCs, and analyzed as in (A) using 10,000 U/ml IL-2. (D) Median fluorescence intensity from four experiments including (C) shows R26 significantly reduced IL-2:CD25 binding compared to either vehicle (DMSO) or PP2 negative controls (ANOVA: F_3,15 = 10.07, p = 0.0013; **p<0.01) (E) Experiments for (C–D) included IL-2 and CD25 ELISAs that were performed. Compared to negative controls, R26 did not change the amount of IL2 or CD25 detected (N.S, not significant by Student’s T-test, p>>0.05), indicating that the difference in (C–D) was due altered IL-2:CD25 interaction.