Figure 3. T cells lacking Ndfip1 can produce IL-2 and proliferate in the absence of CD28 co-stimulation.

(A–D) Naïve (CD44^loCD62L^hiCD25⁻) CD4⁺ T cells were isolated from Ndfip1^−/− mice or Ndfip1^+/+ littermate controls and stimulated in vitro with anti-CD3 and anti-CD28 (A) or anti-CD3 only (B–E). (A and B) Cells were analyzed for levels of IL-2Rα by flow cytometry at the indicated time points. Plots are representative of three independent experiments from 3 mice of each genotype. (C) Supernatants were analyzed for IL-2 by ELISA at time points indicated. Graphs are of representative data from three independent experiments, showing a single comparison of genotypes performed in triplicate. White bars represent cultures of Ndfip1^+/+ T cells and black bars show Ndfip1^−/− T cells. Bars represent the mean and lines show the standard deviation of the triplicate samples. (D) Cells described in panel B were labeled with CFSE immediately prior to stimulation and analyzed at day three for proliferation. Histograms showing CFSE dilution are representative of at least three independent experiments. (E) IL-2 levels in cultures of Ndfip1^+/+CD28^−/− and Ndfip1^−/−CD28^−/− T cells stimulated with various concentrations of anti-CD3 as noted and analyzed at day three. White bars are Ndfip1^+/+CD28^−/− T cells and black bars are Ndfip1^−/−CD28^−/− T cells. Bars represent the mean and lines show the standard deviation of the triplicate samples.