Preformed CD40L exists in lysosomal compartments. (A) In vivo–primed TCR transgenic CD4⁺ effector T cells were enriched by negative selection and identified based on Vβ3 staining by fluorescent microscopy (data not shown) as described in “Flow cytometric analysis of CD40L expression.” These cells were subsequently analyzed for colocalization of CD40L with compartment markers. Merged pictures between CD40L (green) and the compartment markers, Lamp-2, cathepsin D, β₂-microglobulin (β2MG), EEA-1, and giantin (red) are shown. Representative pictures of 1 Z-section from a stack are shown. Scale bar equals 4 μm. (B) The Pearson correlation coefficient between CD40L and each compartment marker was obtained as described in “Fixed cell microscopy, data processing, and analysis of colocalization.” A total of 20 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 maximums and minimums (whiskers) are also shown. The Pearson correlation coefficient for CD40L–Lamp-2 and CD40L–cathepsin D are significantly higher than those for CD40L-β2MG, CD40L–EEA-1, and CD40L-giantin (P < .005; Wilcoxon rank-sum test). There is no statistical difference between CD40L–Lamp-2 and CD40L–cathepsin D (P = .20; Wilcoxon rank-sum test).