ULBP1 is required for Vγ9Vδ2 T-cell recognition of leukemia/lymphoma cells and displays a highly heterogeneous expression in cancer patients. (A) Lentiviral shRNA-mediated knockdown of ULBP1 and MICA in Molt-4 or Jurkat leukemia cells was confirmed by quantitative RT-PCR using GUSB and PSMB6 as endogenous references. Cells were infected with 10 μL of high-titer virus (10⁷ CFU/mL) in media containing polybrene, submitted to selection 48 hours later, and collected for analysis 96 hours after infection. siSCRAM is an shRNA of scrambled (unspecific) sequence, used as an infection control. Error bars represent SD (n = 3). **P < .01. (B) Molt-4 or Jurkat leukemia cells, subjected to ULBP1 or MICA shRNA knockdown (as in panel A), were used in in vitro killing assays either in the presence (+) or absence (−) of γδ-PBLs (as in Figure 1A). Nontransduced (NT) and siSCRAM-transduced cells were used as controls. (C) Raji lymphoma cells were lentivirally transduced with ULBP1 (or control vector), and surface expression of ULBP1 was assessed by flow cytometry (left). In vitro killing assays were then performed either in the presence (+) or absence (−) of γδ-PBLs (right). (D-E) Quantitative RT-PCR analysis of mRNA expression of NKG2DLs in 8 follicular lymphoma (FL) and 4 diffuse large B-cell lymphoma (DLBCL) biopsies, normalized to housekeeping genes (GUSB and PSMB6) and to a reference sample (reactive follicles) obtained through the same procedure and indicated by the dashed line (D); and in 8 T acute lymphoblastic leukemia (T-ALL) and 7 B acute lymphoblastic leukemia (B-ALL) PBMC samples, normalized to housekeeping genes (GUSB and PSMB6) and to reference PBMCs from healthy persons, indicated by the dashed line (E).