Blockade of CD154 aborts germinal center formation and inhibits T- and B-cell activation. (A) At 5 to 7 weeks after skin grafting, spleens were harvested form animals treated with anti-CD154 mAb alone, anti-αβ TCR mAb alone, and both mAbs. Untreated B6 mice served as controls. Spleens were suspended in OCT, frozen in 2-methyl-butane, sectioned, and fixed with acetone. The spleen sections were blocked using Tris-saline/3% BSA (Sigma Chemical, St Louis, MO) and incubated with the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). The sections were stained with HRP-PNA (1:100; Sigma) and developed with the AEC substrate kit (Vector Laboratories). Germinal center counts were performed using a Nikon Eclipse E400 microscope (Nikon Instruments, Westchester, OH). The images were captured by Nikon Act-1 for L1 software and further processed by Adobe Photoshop version 9.0.2 (Adobe Systems, San Jose, CA). Representative immunohistochemistry of germinal center analyses at 4×, 10×, and 20× for each of the treatment groups is shown. (B) B6 recipients were treated with anti-CD154 or its isotype hamster IgG around the time of skin grafting from BALB/c, and the number of T cells (CD4⁺ and CD8⁺), total B cells (CD19⁺), immature B cells (CD19⁺CD24^highCD23^low), and follicular B cells (CD19⁺CD24^lowCD23^high) in spleens were enumerated at days 7, 15, and 25 after skin grafting by 4-color flow cytometry. Data from a representative time point, day 15, are presented. (C) The percentages of alloreactive T cells (CD71⁺) in CD8⁺ or CD4⁺ gate and activated B cells (CD69⁺) in CD19⁺ gate were detected. (D) CD8 or CD4 T cells from anti-CD154 treated mice or from mice treated with control IgG were assessed with intracellular staining for their ability to express IFN-γ or IL-10. Single-cell suspension of spleen cells was prepared and cultured in complete tissue culture media (RPMI 1640 [Invitrogen, Carlsbad, CA] supplemented with 10% fetal bovine serum [Valley Biomedical, Winchester, VA], 1 mM sodium pyruvate [Invitrogen], 100 U/mL penicillin, 100 μg/mL streptomycin [Invitrogen], and 2 mM l-glutamine [Invitrogen]) for 18 hours. During the final 6 hours of culture, phorbol 12-myristate 13-acetate (50 ng/mL), ionomycin (500 ng/mL), and brefeldin A (10 μg/mL) (Sigma Chemical) were added to the culture. Cells were stained for CD4 or CD8 for 30 minutes at 4°C and then washed and fixed in 2% formaldehyde (Polysciences, Warrington, PA) for 15 minutes at 37°C, followed by ice-cold methanol at 4°C overnight. The cells were washed and permeabilized with 1% Triton X-100 (Roche Diagnostic Corp, Indianapolis, IN) in fluorescence-activated cell sorter (FACS) buffer. PE-conjugated anti–IFN-γ, anti–IL-10, or rat-IgG1 mAbs were added to the cells for 30 minutes, followed by washing in Triton X-100–FACS buffer. Significant P values are indicated above the respective data bars (*P < .05). Data are presented as averages plus or minus SD.