Anergic T cells inhibit parenchymal cell–mediated migration. (Panels A-B) EC monolayers (5 × 104/transwell) were incubated for 16 hours with either anergic (•; 5 × 104/transwell) or responsive (○; 105/well) alloreactive CD4+ T-cell lines or in medium alone (▵). T cells were subsequently removed and fresh T cells (7 × 105/well; panel A) or granulocytes (106/well; panel B) were seeded onto the EC monolayers. The results are expressed as percentage of migrated T cells at the given time points and reported as the average of 3 experiments of identical design. The bars show the standard deviations (SD). * indicates statistically significant (at least P < .01, panel A; at least P < .02, panel B) versus control cultures (EC monolayers cultured in medium). (Panel C) EC monolayers were incubated overnight with supernatants obtained from cultures of T cells in anergizing (•) or resting (○) conditions or in medium alone (▵). Following 2 washes with warm culture medium, fresh T cells (7 × 105/well) were seeded onto the EC monolayers. The results are expressed and reported as specified for panels A-B. (Panel D) TNF-α–treated RTEC monolayers (5 × 104/transwell) were incubated for 16 hours with either anergic (5 × 104/well), or responsive (105/well) CD4+ T-cell lines or clones, or in medium alone. T cells were subsequently removed and fresh T cells (7 × 105/well) were seeded onto the RTEC layers. The results are expressed and reported as specified for panels A-B. * indicates statistically significant (at least P < .05) versus control cultures (RTEC monolayers cultured in medium). (Panels E-G) EC monolayers (5 × 104/transwell) were incubated for 30 minutes at RT with an antihuman CD40 mAb (5 μg/mL, filled symbols) or with an isotype-matched mAb (open symbols). Excess antibody was then removed by gentle washing. Bound antibody was then cross-linked by addition of goat antimouse Miltenyi microbeads (30 μL/μg mAbs) during incubation with anergic (panel E) or responsive (panel F) 7P.61 T cells or in medium alone (panel G) for a further 16 hours. T cells were then removed and migration of freshly added alloreactive T cells (7 × 105/well) was monitored. In all these experiments, cell migration was monitored over the next 6 to 26 hours. The results are expressed and reported as specified for panels A-B. * indicates statistically significant (at least P < .01) versus control cultures (EC monolayers cultured in the presence of anergic T cells).
