NKG2D down-regulation is not prevented by blocking MICA, MICB, and ULBP3. PBMCs were incubated with or without TB40/E for 3 days. (A) Samples were analyzed by 3-color flow cytometry with a panel of mAbs specific for MICA (BAM195), MICB (MAB1599), ULBP-1 (MAB1380), ULBP-2 (MAB1298), and ULBP-3 (MAB1517) in combination with anti-CD3 and anti-CD56. Histograms correspond to the isotype controls (thin lines), mock-treated (gray profiles), and TB40/E-infected (bold lines) samples. Data from 1 representative donor of 3 tested is shown. (B) Anti-MICA, -MICB, and -ULBP3 blocking Abs were included in the PBMC cultures with or without TB40/E, and surface NKG2D expression (1D11) on CD56⁺CD3⁻ cells was monitored by flow cytometry. Geometric mean values for mean fluorescence intensity are included. Data correspond to 1 representative donor of 4 tested. (C) PBMCs were incubated overnight with 50 U/mL IL-2 and were used as effector cells in degranulation assays with MICA-, MICB-, and ULBP-3–transfected cells in the presence or absence of specific blocking mAbs. Bar graphs present the percentage of CD107a⁺ CD56⁺CD3⁻ cells in each condition.