American Society of Hematology

Figure 2

$Figure 2. Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells. (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28. (B) Th1 cells were anergized with plate-bound anti-CD3 and, after a 3-day resting period, stimulated with anti-CD3 and anti-CD28. IL-2 expression (ELISA) and BrdU incorporation were measured. Error bars show mean ± SEM of 3 experiments. (C) ChIP assays were performed with antiacetylated H4 antibodies to assess acetylation of the il2 promoter. Control cells were analyzed and compared with cells anergized using either ionomycin (Iono) or anti-CD3. In some cells, TSA (10 nM) was added together with ionomycin to inhibit the activity of HDACs. Experiments were also performed with a control IgG. PCR products are shown for precipitated fractions and sample inputs. One experiment of 4 is shown. Numbers below input bands indicate relative amount (to control cells) quantified using qPCR. Graph shows relative intensity of immunoprecipitated complexes from all experiments (mean + SEM). (D) ChIP experiments were performed in control (Ctrl) and anergized (Iono) cells (± TSA) using anti-H4 antibodies. PCR reactions were carried out with different amounts of sample (2-fold increases) to confirm linearity of the amplification. (E) Samples from panels C and D were also analyzed using primers for the CD3ϵ promoter. (F) Th1 cells were anergized by stimulation with anti-CD3 for 16 hours in the presence or the absence of TSA, washed, and left resting for 3 days. After that, ChIP assays were performed using antiacetylated H4 antibodies and primers specific for the IL-2 promoter. The gels show PCR amplifications of 3 different amounts of chromatin (2-fold increases) for the anti-CD3–treated and control cells. PCR amplifications of the inputs from the different samples are also shown. (G) Control cells or cells anergized with ionomycin were restimulated with anti-CD3 and anti-CD28 for 6 hours and then used to immunoprecipitate chromatin complexes using an antiacetylated H4 antibody. Amplified products from different amounts of each sample (2-fold increases) and from the different inputs are shown.$

Anergizing stimuli induce H4 deacetylation of the il2 promoter in anergic Th1 cells. (A) Th1 cells were treated with 1 μM ionomycin (Iono) for 16 hours and analyzed by intracellular IL-2 staining following stimulation with anti-CD3 and anti-CD28. (B) Th1 cells were anergized with plate-bound anti-CD3 and, after a 3-day resting period, stimulated with anti-CD3 and anti-CD28. IL-2 expression (ELISA) and BrdU incorporation were measured. Error bars show mean ± SEM of 3 experiments. (C) ChIP assays were performed with antiacetylated H4 antibodies to assess acetylation of the il2 promoter. Control cells were analyzed and compared with cells anergized using either ionomycin (Iono) or anti-CD3. In some cells, TSA (10 nM) was added together with ionomycin to inhibit the activity of HDACs. Experiments were also performed with a control IgG. PCR products are shown for precipitated fractions and sample inputs. One experiment of 4 is shown. Numbers below input bands indicate relative amount (to control cells) quantified using qPCR. Graph shows relative intensity of immunoprecipitated complexes from all experiments (mean + SEM). (D) ChIP experiments were performed in control (Ctrl) and anergized (Iono) cells (± TSA) using anti-H4 antibodies. PCR reactions were carried out with different amounts of sample (2-fold increases) to confirm linearity of the amplification. (E) Samples from panels C and D were also analyzed using primers for the CD3ϵ promoter. (F) Th1 cells were anergized by stimulation with anti-CD3 for 16 hours in the presence or the absence of TSA, washed, and left resting for 3 days. After that, ChIP assays were performed using antiacetylated H4 antibodies and primers specific for the IL-2 promoter. The gels show PCR amplifications of 3 different amounts of chromatin (2-fold increases) for the anti-CD3–treated and control cells. PCR amplifications of the inputs from the different samples are also shown. (G) Control cells or cells anergized with ionomycin were restimulated with anti-CD3 and anti-CD28 for 6 hours and then used to immunoprecipitate chromatin complexes using an antiacetylated H4 antibody. Amplified products from different amounts of each sample (2-fold increases) and from the different inputs are shown.

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