Cross-linking of P-selectin glycoprotein ligand-1 induces death of activated T cells by Shu-Ching Chen, Chiu-Chen Huang, Chung-Liang Chien, Chung-Jiuan Jeng, Ho-Ting Su, Evelyn Chiang, Meng-Ru Liu, C. H. Herbert Wu, Chung-Nan Chang, and Rong-Hwa Lin Blood Volume 104(10):3233-3242 November 15, 2004 ©2004 by American Society of Hematology

TAB4 triggers cell death of activated mouse T cells and recognizes mouse PSGL-1. TAB4 triggers cell death of activated mouse T cells and recognizes mouse PSGL-1. (A) Activated mouse T cells on day 5 from stimulation with ConA (2 μg/mL) were incubated with TAB4 or control antibodies plus R anti-H overnight. Following double staining with Annexin-V FITC and propidium iodide, cells were analyzed by flow cytometry to determine the percentage of dead cells. Without gating, 10% of 10 000 total events were displayed. (B) TUNEL assays of control cells (left) and TAB4-treated cells (right). Few TUNEL-positive cells could be found in the activated T-cell (AT) population with IL-2 treatment (left). Plenty of TUNEL-positive cells were observed in AT treated with TAB4 for 6 hours. Arrows: cell nuclei with TUNEL-positive staining. Scale bar: 20 μm. (C) PSGL-1 transfectant cells, 10A, were stained with 5 μg/mL TAB4 or control antibody, hamster Ig, and analyzed by flow cytometry. (D) Total cell extract of 5 × 107 activated mouse T cells at late activated stages were immunoprecipitated by anti–PSGL-1 antibodies, TAB4, and 2PH1, respectively, and followed by immunoblotting for TAB4. (E) Total cell extracts of 3 × 107 activated mouse T cells at late activated stages were depleted for PSGL-1 by 2 successive rounds of incubation with affinity-purified anti–PSGL-1 antibodies using TAB4 or 2PH1. Residual proteins of the depleted extracts were immunoblotted with 2PH1 under reducing conditions. The Zeiss Axioskop was equipped with a 40×/0.75 Zeiss Plan-Neofluar objective lens (Carl Zeiss, Oberkochen, Germany) and a Nikon D1× digital camera (Nikon, Tokyo, Japan). Images were acquired with Nikon Capture 2 software and processed in Adobe Photoshop 7.0 (Adobe, San Jose, CA). Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

Cross-linking of PSGL-1 induces activated mouse T-cell death. Cross-linking of PSGL-1 induces activated mouse T-cell death. Mouse splenocytes of B6 mice stimulated with ConA (A-B) and those of AND TCR transgenic mice stimulated with specific antigen, PCC (C), were used. At the indicated time, activated mouse T cells were incubated with rabbit antihamster antibody plus TAB4 or control antibodies. After 6 hours, the cell death was assayed by flow cytometry with Annexin-V FITC and propidium iodide staining. Error bars in panels B and C indicate standard deviation. (D) PSGL-1 expression on T cells at different activation stages was detected by staining with TAB4 (coarse line) or hamster Ig (fine line), followed by antihamster FITC and analyzed by flow cytometry. The data represent at least 3 independent experiments and were analyzed by ANOVA test with Bonferroni correction for multiple comparisons. *Difference of cell death between TAB4-treated and negative control antibody–treated groups, P < 0.01. ∧ Significant increase of TAB4-induced cell death with time, P < .01 Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

Immobilized P- or E-selectin triggers activated mouse T-cell death. Immobilized P- or E-selectin triggers activated mouse T-cell death. ConA-stimulated activated mouse T cells were added to a 96-well plate precoated with (A) E-selectin or (B) P-selectin in titrated concentrations. After shear (80 rpm) for 30 minutes, the cells were incubated for a total of 5 hours. The cell death was assayed by cellular ELISA with biotinated Annexin, followed by streptavidin-β-gal. Color development by substrate ONPG was read at OD 420 nm. OD difference was shown as compared with BSA-coated wells. (C) Cell viability was assayed by fluorescence of Calcein am and analyzed by fluorescence microplate reader systems (excitation at 494 nm; emission at 530 nm). After incubation for 16 hours, the fluorescence of activated T cells in BSA-coated wells displayed at 756.4 and relative reduction in fluorescence of viable cells in wells coated with P- or E-selectins (5 μg/mL) compared with those in BSA-coated wells was shown. These data represent at least 3 independent experiments and were analyzed by general linear model with Bonferroni correction for multiple comparisons. Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

PSGL-1 mediates a caspase-independent cell death. PSGL-1 mediates a caspase-independent cell death. Mouse T cells at late activation stages were incubated with TAB4 in the presence of caspase inhibitors, Z-VAD-FMK (A) or Z-DEVD-FMK (B) in titrated concentration (0.3-100 μM). Jurkat cells were treated with anti-Fas antibody (CH11) as control (A, bottom panel). Positive Annexin V binding was analyzed by flow cytometry. Error bars indicate standard deviation. (C) Oligonucleosomal DNA fragmentation was checked in cells treated with TAB4 or control antibodies (left). Pulsed-field gel electrophoresis of genomic DNA prepared from TAB4-treated or control cells is shown (right). Similar results were obtained in 3 independent experiments. Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

Ultrastructural characteristics of PSGL-1–mediated cell death. Ultrastructural characteristics of PSGL-1–mediated cell death. Activated T cells were incubated with TAB4 or control antibodies for 6 hours. Semithin sections (1 μm) were obtained and stained with Toluidine blue (A, R anti-H; B, HIg; C, TAB4; D, anti-CD3). Cells with dark-stained nuclei were then prepared for the electronic microscopy (E, HIg; F, anti-CD3; G-H, TAB4). Arrowhead indicates apoptotic body; arrow, peripheral condensation; scale bar, 5 μm in Figure 5D. Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

Cross-linking of PSGL-1 triggered release of AIF Cross-linking of PSGL-1 triggered release of AIF. AIF translocation during activated T-cell death induced by TAB4. Cross-linking of PSGL-1 triggered release of AIF. AIF translocation during activated T-cell death induced by TAB4. Activated T cells were incubated with TAB4 for indicated time, 0 hours (A, untreated cells), 3 hours (B), 4 hours (C), and 6 hours (D). Following fixation, cells were stained with anti-AIF antibody and Hoechst 33342. Subcellular localization of AIF (green) and cell nuclei (blue) were visualized by a Leica SP2 confocal microscope. Arrowhead indicates apoptotic body; arrow, AIF translocation to nucleus; scale bar, 5 μm. Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology

Activated T-cell death triggered by PSGL-1 cross-linking involves release of mitochondrial mediators. Activated T-cell death triggered by PSGL-1 cross-linking involves release of mitochondrial mediators. Differential subcellular localization of mitochondrial mediators was analyzed in activated mouse T cells on death triggered by cross-linking of PSGL-1. Immunoblotting with anti-AIF antibody and anti–cytochrome c antibody, respectively, revealed that AIF translocation was first detected in the 3-hour TAB4-treated cytosolic fraction (A). Nuclear fraction of activated T cells at indicated time points following TAB4 treatment was immunoblotted by anti-AIF (B). (C) Cytochrome c release from mitochondria was detected in cytosolic fraction of 6-hour treated cells. The data represent 3 independent experiments. Shu-Ching Chen et al. Blood 2004;104:3233-3242 ©2004 by American Society of Hematology