A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats
J. Clin. Invest. Nora Müller, et al. 118:1405 doi:10.1172/JCI32698 [Go to this article.]

Figure 4
Analysis of cytoskeletal rearrangements after stimulation by JJ316 in vitro by confocal microscopy. (A) CD4⁺ T cells, CD4⁺CD25⁺ T cells, CD8⁺ cells, and B cells were purified from naive rats and stimulated with JJ316 on microscopic slides for the indicated time periods. F-actin polymerization was visualized using phalloidin–Alexa Fluor 594, and CD4⁺ T cell populations were additionally stained for FoxP3 expression. Scale bar: 5 μm. (B) The diameters of at least 150 individual cells per subtype and the time point were quantified by a computer-aided method; mean ± standard error of the mean. Statistical analysis by 2-way ANOVA. The asterisks refers to the CD4⁺FoxP3⁺ and the CD4⁺CD25⁺FoxP3⁺ Tregs and the pound signs to the CD4⁺FoxP3^– Th and the CD8⁺ cells (both shown on the top of the graph). Changes in the size of B cells are not significant (shown below the graph). (C) Magnetically purified CD4⁺CD25^– and CD4⁺CD25⁺ T cells were stimulated as in A. The percentage of FoxP3⁺ cells in both cell populations was determined before (0 hours) and 7 hours after JJ316 stimulation in vitro. Mean ± standard error of the mean. Statistical analysis by Student’s t test. (D) CD4⁺ T cells were stimulated with JJ316 (JJ) for 30 minutes in the absence or presence of the PI3K inhibitor LY294002 (LY) or the MAPK inhibitor U0126 (U) and stained with phalloidin and an anti-FoxP3 antibody as in A. The relative number of polarized cells is depicted in each case as a measure for activation by JJ316. Treatment and 150 individual cells per subtype were quantified by a computer-aided method; mean ± standard error of the mean. ***P < 0.001; ^###P < 0.001.