Figure 3
IMiDs regulate ex vivo induced SOCS1 expression and STAT activation. (A) SOCS1 protein expression was shown by immunoprecipitation-Western blot in effector cells, incubated in the presence of aCD3 Ab and lenalidomide (1μM) or pomalidomide (1μM) for 16 hours, and then stimulated with SOCS1 inducing cytokine IFNγ (100 ng/mL) for 15 minutes to 4 hours (top panel). Expression of SOCS1 protein relative to IgH chain was demonstrated (fold) using ImageJ 1.37v (http://rsb.info.nih.gov/ij/) densitometric analysis (bottom panel). One representative of 3 independent experiments is shown. Intracellular expression of STAT1 pTyr701 and STAT1 (B) and STAT3 pTyr705 and STAT3 (C) are shown in PBMCs from healthy donors (n = 3) by flow cytometric analysis. aCD3 Ab stimulated PBMCs were incu-bated in the absence or presence of lenalidomide and pomalidomide for 16 hours, and then stimulated with IFNγ for 15 minutes. Expression of phosphorylated STATs is shown as a percent expression relative to control baseline levels in aCD3 Ab–stimulated cells. Top panels demonstrate phosphorylated STATs expression in CD4 T cells as representative plots, and bottom panels demonstrate phosphorylated STATs expression in all effector cells as percent expression relative to control baseline levels in aCD3 Ab stimulated cells. Numbers indicate percentage of positive cells. One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05).

IMiDs regulate ex vivo induced SOCS1 expression and STAT activation. (A) SOCS1 protein expression was shown by immunoprecipitation-Western blot in effector cells, incubated in the presence of aCD3 Ab and lenalidomide (1μM) or pomalidomide (1μM) for 16 hours, and then stimulated with SOCS1 inducing cytokine IFNγ (100 ng/mL) for 15 minutes to 4 hours (top panel). Expression of SOCS1 protein relative to IgH chain was demonstrated (fold) using ImageJ 1.37v (http://rsb.info.nih.gov/ij/) densitometric analysis (bottom panel). One representative of 3 independent experiments is shown. Intracellular expression of STAT1 pTyr701 and STAT1 (B) and STAT3 pTyr705 and STAT3 (C) are shown in PBMCs from healthy donors (n = 3) by flow cytometric analysis. aCD3 Ab stimulated PBMCs were incu-bated in the absence or presence of lenalidomide and pomalidomide for 16 hours, and then stimulated with IFNγ for 15 minutes. Expression of phosphorylated STATs is shown as a percent expression relative to control baseline levels in aCD3 Ab–stimulated cells. Top panels demonstrate phosphorylated STATs expression in CD4 T cells as representative plots, and bottom panels demonstrate phosphorylated STATs expression in all effector cells as percent expression relative to control baseline levels in aCD3 Ab stimulated cells. Numbers indicate percentage of positive cells. One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05).

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