NKG2D mediates Vγ9Vδ2 T-cell recognition of hematopoietic tumors that endogenously overexpress ULBP1 and MICA. (A) Susceptibility of leukemia and lymphoma cell lines (described in supplemental Table 2) to HMB-PP–activated γδ-PBL (> 90% Vγ9+) cytotoxicity was assessed by coincubating 3 × 104 tumor cells (prelabeled with 1mM DDAO-SE) with 3 × 105 γδ-PBLs from 3 independent donors for 3 hours, then staining with annexin V–fluorescein isothiocyanate and analyzing by flow cytometry. (B) γδ-PBLs were incubated with saturating amounts of anti-NKG2D/clone 1D11 and anti-TCRγδ/clones B1.11 or IMMU5102 blocking antibodies, or both anti-NKG2D and anti-TCRγδ,1 for 1 hour at 4°C. γδ-PBLs were then cocultured either with Jurkat or Molt-4 leukemia lines, and tumor cell lysis was assessed as in panel A. Error bars represent SD (n = 3). *P < .05. (C) Quantitative RT-PCR quantification of mRNA levels of NKG2D ligands in cells lines of panel A, normalized to glucuronidase-β (GUSB) and proteasome subunit β type 6 (PSMB6) housekeeping genes. (D) Flow cytometric analysis of cell-surface expression of ULBP1 and MICA in the leukemia/lymphoma lines of panel A. Data presented in this figure (A-D) are representative of at least 3 independent experiments with consistent results.
