Loss of IRF-4–binding protein leads to the spontaneous development of systemic autoimmunity
J. Clin. Invest. Jessica C. Fanzo, et al. 116:703 doi:10.1172/JCI24096 [Go to this article.]

Figure 4
T cells are resistant to apoptosis. (A) Proliferation of T cells from WT and IBP mutant mice. Cells were stimulated with plate-bound anti-CD3ε (2C11) (1 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies or with PMA (50 ng/ml) plus ionomycin (1 μM) for 48 hours. The culture was then pulsed with [³H]thymidine for 18 hours. This experiment is representative of 5 independent experiments. The data were analyzed using 2-tailed Student’s t test. A statistical probability of P < 0.05 was considered significant. *P < 0.05; **P < 0.005 (IBP^trap/trap versus WT). (B) Apoptosis of IBP^+/+ and IBP^trap/trap T cells upon CD3 restimulation. Purified naive CD4⁺ T cells from IBP^+/+ (black bars) or IBP^trap/trap (white bars) mice were stimulated for 3 days and then harvested. The cells were then restimulated with either IL-2 alone or IL-2 with anti-CD3 mAbs at the indicated doses for 24 hours. Cells were then stained with propidium iodide, and the percentage of apoptotic cells was determined by quantification of the sub-G₀ population by FACS. Each assay was conducted in duplicate. The experiment is representative of 3 separate experiments. (C) SEB-specific deletion of T cells in IBP^trap/trap mice. IBP^+/+ (n = 4) (filled circles) and IBP^trap/trap (n = 4) (open circles) mice were injected i.p. with SEB on day 0. Peripheral blood cells were stained with antibodies against Vβ8 and CD4 (left panel) or Vβ6 and CD4 (right panel) and analyzed by FACS at the indicated time points. Results are expressed as a mean ± SD. Statistical differences were determined using 2-tailed Student’s t test. (D) Loss of mitochondrial potential. Purified naive CD4⁺ T cells from IBP^+/+ (black bars) or IBP^trap/trap (white bars) mice were stimulated for 3 days and then harvested. The cells were then restimulated with either IL-2 alone or IL-2 with anti-CD3 mAbs at 10 μg/ml for 24 hours. Cells were then stained with Mitotracker Deep Red 633 to assess loss of mitochondrial potential. The percentage of cells that displayed a decrease in intensity of staining (Mitotracker lo T cells) is indicated. The experiment is representative of 3 separate experiments. Results are expressed as mean ± SD. Statistical differences were determined using 2-tailed Student’s t test.