Antigen recognition induces the mobilization of preformed CD40L in effector CD4⁺ T cells. (A) In vitro–generated effector CD4⁺ T cells from CD40L-sufficient and -deficient (CD40L KO) TCR transgenic mice were stimulated or left unstimulated with PMA plus ionomycin for 2 hours. Levels of CD40L (shaded) and isotype control (line) staining on gated effector CD4⁺ T cells after surface staining at 4°C are shown. (B) In vitro–generated effector CD4⁺ T cells from CD40L-sufficient and CD40L KO TCR transgenic mice were fixed, permeabilized or left unpermeabilized, and stained for intracellular CD40L at 4°C without acute stimulation. Levels of CD40L (shaded) and isotype control (line) staining on gated effector CD4⁺ T cells are shown. (C) The surface mobilization assay was conducted as described in “Materials and methods, Surface mobilization assay” by addition of anti-CD40L mAb at 37°C for 30 minutes (middle and right panels), and was compared with the surface staining at 4°C following 30 minutes of stimulation (left panel). CD40L KO effector CD4⁺ T cells were used as a specificity control for the mobilization assay. Shaded histogram indicates levels of CD40L with stimulation. –––– indicates levels of CD40L without stimulation. –––– indicates levels of isotype control with stimulation. -------- indicates levels of isotype control without stimulation. (D) The surface mobilization assay was conducted for CD40L, FasL, CTLA-4, and CD107a. Histograms are assigned as in panel C. (E) The kinetics of surface mobilization of CD40L were assessed by the mobilization assay. ■ indicates CHX-pretreated and PMA plus ionomycin-stimulated; □, CHX-untreated and PMA plus ionomycin-stimulated; ●, CHX-pretreated and unstimulated; and ○, CHX-untreated and unstimulated. Raw geometric mean fluorescent intensity of CD40L staining are shown. (F) IFN-γ levels in the supernatants from CHX-pretreated (■) or -untreated (□) effector CD4⁺ T cells cultured in the presence of PMA plus ionomycin for the indicated periods of time were measured by ELISA. (G) The mobilization of CD40L and CTLA-4 on in vitro effector CD4⁺ T cells was induced with antigen-pulsed CH12 B cells for 30 minutes. Histograms are assigned as in panels C and D. (H) Day-14, in vivo–primed TCR transgenic effector CD4⁺ T cells were analyzed for intracellular CD40L without stimulation and surface mobilization of CD40L upon PMA plus ionomycin stimulation as described in panels B and C. The levels of CD40L staining gated on CD4⁺, Vα11⁺, and Vβ3⁺ T cells are shown. See the descriptions in panels B and C for the histogram assignment. Experiments were repeated at least 5 times with similar results.