Figure 4.

Fewer Nedd4^−/−T cells produce IL-2. (a) T cells were isolated from Nedd4^+/+ and Nedd4^−/−fetal liver chimeras, stimulated or left unstimulated as indicated for 20 h, and intracellular IL-2 production was analyzed by FACS. Histograms are gated on CD4⁺ populations. Numbers indicate percentages of IL-2-producing T cells among total CD4⁺ T cells. (b) The percentages of IL-2-producing CD4⁺ T cells were quantified from multiple experiments and the data combined. *, P = 0.0092. (c) Nedd4^+/+ and Nedd4^−/−CD4+ T cells were isolated and labeled with CFSE. Cells were then cultured for 3 days in media (dashed line) or stimulated in the presence or absence of IL-2 (solid line). CFSE dilution was measured by flow cytometry. (d) D10 T cells were transfected with control siRNA or Nedd4-specific siRNA and then cultured in media or stimulated with anti-CD3 and anti-CD28. Top, after stimulation, IL-2 concentrations in the supernatants were measured by ELISA. Bottom, immunoblot of whole cell lysates to illustrate Nedd4 expression after siRNA treatment. These are representative data from four independent experiments. Error bars show s.e.m. **,P = 0.0002