Purpose: The Vd1 þ subset of gd T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vd1 þ T-cell-based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. Experimental design: We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2-3 weeks) Vd1 þ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL).Results: We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinicalgrade cell culture bags) and differentiation of cytotoxic Vd1

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On the path to successful immunotherapy of hematopoietic tumors, ␥␦ T cells offer great promise because of their human leukocyte antigen (HLA)-unrestricted targeting of a wide variety of leukemias/ lymphomas. However, the molecular mechanisms underlying lymphoma recognition by ␥␦ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to ␥␦ T cell-mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all V␥9 ؉ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by ␥␦ T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to ␥␦ T cell-based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials. (Blood. 2010;115:2407-2411)