Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner by Yoshinobu Koguchi, Timothy J. Thauland, Mark K. Slifka, and David C. Parker Blood Volume 110(7):2520-2527 October 1, 2007 ©2007 by American Society of Hematology

Antigen recognition induces the mobilization of preformed CD40L in effector CD4+ T cells. 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. Yoshinobu Koguchi et al. Blood 2007;110:2520-2527 ©2007 by American Society of Hematology

Preformed CD40L exists in lysosomal compartments. 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). Yoshinobu Koguchi et al. Blood 2007;110:2520-2527 ©2007 by American Society of Hematology

CD40L colocalizes more strongly with FasL than with CTLA-4. 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). Yoshinobu Koguchi et al. Blood 2007;110:2520-2527 ©2007 by American Society of Hematology

Effector/memory but not naive CD4+ T cells from unmanipulated animals mobilize preformed CD40L. Effector/memory but not naive CD4+ T cells from unmanipulated animals mobilize preformed CD40L. CD40L expression in naive and effector/memory CD4+ T cells from unmanipulated normal (A-C), CD40-deficient (D-F), and CD40L-deficient (G-I) animals was analyzed by conventional surface staining (A,D,G), intracellular staining (B,E,H), and the surface mobilization assay (C,F,I). (A,B,D, E,G,H) The levels of CD40L (shaded) and isotype control (line) staining for the indicated population of CD4+ T cells are shown. (C,F,I) Shaded indicates CD40L with stimulation. –– indicates CD40L without stimulation. –– indicates isotype control with stimulation. ------ indicates isotype control without stimulation. Data shown are representative of 4 independent experiments. Yoshinobu Koguchi et al. Blood 2007;110:2520-2527 ©2007 by American Society of Hematology

LCMV-specific effector and memory Th1 cells rapidly mobilize preformed CD40L upon antigenic stimulation. LCMV-specific effector and memory Th1 cells rapidly mobilize preformed CD40L upon antigenic stimulation. The surface mobilization assay was combined with intracellular cytokine staining of LCMV-infected splenocytes to detect mobilization of preformed CD40L on the cell surface of LCMV-specific effector (A) (day 9 after infection) and memory (B) (6 months after infection) Th1 cells 30 minutes after stimulation with LCMV GP61 peptide-pulsed APCs as described in “Materials and methods, Flow cytometric analysis of CD40L expression.” The levels of CD40L (shaded) and isotype control (line) for IFN-γ+ and IFN-γ− populations are shown. Numbers in plots indicate the percentage of that population among CD4+ cells in the sample. Data shown are representative of 3 independent experiments. Yoshinobu Koguchi et al. Blood 2007;110:2520-2527 ©2007 by American Society of Hematology