Blocking of CD99 inhibits TEM of SJL.PLP7 cells. T cells were allowed to transmigrate through the monolayer of bEnd.5 cells grown on transwell filters, either for 2 hours in the absence of the chemokine SDF-1 (A-C), or for 30 minutes in the presence of 100 ng/mL SDF-1 in the lower chamber (C-D). (A) Endothelial cells were incubated for 30 minutes prior to the start of the assay either without antibodies (w/o Ab) or with the indicated concentrations of preimmune control IgG (co-IgG), affinity-purified anti-CD99 IgG (anti-CD99), or a mAb against ICAM-1 (anti–ICAM-1). Antibodies remained present during the assay. P < .005, anti-CD99 (15 μg/mL) versus co-IgG (15 μg/mL); P < .0005, anti-CD99 (30 μg/mL) versus co-IgG (30 μg/mL) or anti–ICAM-1 (15 μg/mL) versus co-IgG (15 μg/mL). (B) Endothelial cells were pretreated with 30 μg/mL preimmune IgG (co-IgG), intact anti-CD99 antibodies (anti-CD99), F(ab′)2 fragments of control IgG or of anti-CD99 IgG, or with a mAb against ICAM-1 (anti–ICAM-1). As in panel A, antibodies remained present during the assay. P < .0005, co-IgG versus anti-CD99 or anti–ICAM-1; P < .0005, anti-CD99 F(ab′)2 versus co-IgG F(ab′)2. (C) Endothelial cells were pretreated with 30 μg/mL preimmune IgG (co-IgG), intact anti-CD99 antibodies (anti-CD99), Fab fragments of control IgG (co-IgG Fab) or of anti-CD99 IgG (anti-CD99 Fab), or with a mAb against ICAM-1 (anti–ICAM-1). As in panels A-C, antibodies remained present during the assay. P < .01, anti-CD99 versus co-IgG; P < .001, anti–ICAM-1 versus co-IgG. (D) After preincubation of the endothelial cells with 30 μg/mL of the indicated antibodies, the antibodies either remained present during the assay (first 3 bars) or were washed away before the addition of the lymphocytes (indicated by “pre-inc.” underneath the last 3 bars). P < .001, anti-CD99 versus co-IgG; P < .005, anti–ICAM-1 versus co-IgG; P < .005, co-IgG pre-inc. versus anti-CD99 preinc. or anti–ICAM-1 pre-inc. (E) Lymphocytes were preincubated with 30 μg/mL preimmune control IgG, anti-CD99 IgG or a mAb against LFA-1, or with F(ab′)2 fragments of anti-CD99 antibodies. Antibodies and F(ab′)2 fragments were washed away prior to adding the lymphocytes into the assay. Each panel is representative of at least 5 experiments; each measurement was done in triplicate. P < .001, co-IgG versus anti-CD99 or F(ab′)2 or anti–LFA-1. Results are presented as mean ± SEM, and are representative of at least 3 experiments.
