CD40L colocalizes more strongly with FasL than with CTLA-4. (A, B) In vitro–primed effector CD4⁺ T cells were used to analyze the degree of colocalization between CD40L and FasL (A) or CTLA-4 (B) as described in “Flow cytometric analysis of CD40L expression.” (C,D) The specificity of the polyclonal anti-CD40L Ab was tested with in vitro–primed effector CD4⁺ T cells from CD40L-sufficient (C) or -deficient (D) mice. DIC indicates differential interference contrast image. Scale bar equals 4 μm. (E) The Pearson correlation coefficient between CD40L and FasL or CTLA-4 was obtained as described in “Materials and methods, Fixed cell microscopy, data processing, and analysis of colocalization.” A total of 25 cells and 30 to 50 Z sections for each cell from 2 experiments were analyzed. Each circle represents data from a single cell. Median (bar in the box), interquartile ranges (boxes), and maximum and minimum (whiskers) are also shown. The Pearson correlation coefficient for CD40L-FasL is significantly higher than that for CD40L–CTLA-4 (P < .001; Wilcoxon rank-sum test).