Efficient recognition of the capillary tube comprising HLA-A2⁺ aECs by K288.E5. (A) Comparison of T-cell recognition of aECs cultivated under different conditions. HUVEC2 cells (2 × 10⁴/well) plated in tissue-culture wells (forming a monolayer) or in polymerized Matrigel (10 mg/mL, assembling into tubular structures) were mixed with K288.E5 at a 1:2 ratio and were cocultured overnight. IFN-γ release from CTLs was measured by IFN-γ ELISA. (B) Destruction by K288.E5 of capillary tubes formed in Matrigel. Tube-forming GFP⁺ HUVEC2 cells (1 × 10⁴/well) were cultivated overnight with CTLs (3 × 10⁴/well) in the presence of AZT (10 μM). Fluorescent structures were imaged before and after cocultivation using an inverted microscope. (C) Capillary destruction by K288.E5 was blocked by anti-HLA 1 mAb and Granzyme B inhibitor. T-cell assays were performed exactly as described in panel B except that, before cocultivation, tubes and CTLs were treated for 1 hour with anti-HLA 1 mAb (W6/32, 20 μg/mL) and a Granzyme B inhibitor (zAAD, 100 μM; Enzyme Systems), respectively. All experiments were repeated twice, with similar results. Original magnification, × 20 for B and C.