Abstract 3032Poster Board II-1008Gamma delta T cells (GDTCs), a small subset of T-lymphocytes (<10%) involved in tumor immune surveillance, are promising candidates for adoptive immunotherapy demonstrated by their ability to elicit cytolytic responses against many tumors. We have isolated and expanded GDTCs as a first step in developing a clinical protocol (Siegers, GM et al., ASH 2008). GDTCs exist in subsets whose specificity and function are determined by receptor rearrangement and tissue localization. The Vdelta2 (Vd2) subset in blood recognizes small phosphate containing non-peptide antigens and has been shown to kill myeloma and Burkitt lymphoma cells, whereas Vdelta1 (Vd1) GDTCs are typically found in tissue mucosae and provide defense against epithelial cancers. Although circulating GDTCs are predominantly of the Vdelta2 (Vd2) subset, we found that in 59% of GDTC cultures derived from the peripheral blood of healthy donors (n=17), the Vdelta1 (Vd1) subset was preferentially expanded, comprising 70.5% ± 14.7% (mean ± standard deviation) as determined by flow cytometry. In the remaining cultures, Vd2 GDTCs comprised 75.9 ± 14.2%. Preferential expansion of Vd1 did not correlate with a higher percentage of this subset in donor blood prior to GDTC isolation. In one expanded culture, Vd1 and Vd2 were equally present (40.3% and 41.3% respectively, on day 17). To determine activation status of Vd1 and Vd2 subsets simultaneously when co-incubated for 3 hours at a 1:5 effector:target ratio (E:T) with EM2eGFPluc, Ph(+) leukemic target cells, exposure of the degranulation-induced marker CD107 was determined by flow cytometry. Assays performed on culture days 10 to 17 (n=8) revealed that only 3.4 ± 2.7% Vd1 cells were activated, whereas Vd2 cells exhibited ten-fold activation with 34.1 ± 4.7% expressing CD107. To further investigate the different cytotoxic potential of these GDTC subsets, we generated 3 Vd2 clones from Donor 1 and 7 clones (3 Vd1 and 4 Vd2) from Donor 2. 3 clones were obtained from 200 Vd1-sorted cells, and 4 clones from 600 Vd2-sorted cells, suggesting superior clonogenicity of Vd1. Indeed, Vd1 clones grew faster than Vd2 from this donor. After 40 days in culture, we obtained 57 ± 37 × 106 Vd1 and 37 ± 23 × 106 Vd2 cells from a single cell on day 0. The enhanced growth of Vd1 explains how this subset predominates in most polyclonal GDTC cultures, despite donors having more Vd2 than Vd1 in their blood (Vd2:Vd1 = 5.7±3.2, n=7). Polyclonal expansion of GDTCs from Donor 2 yielded 11.2 × 106 cells on day 20, from 1.7 × 106 on day 0, a 6.7-fold expansion compared to 107-fold achieved with clones from the same donor. Vd2 clones were screened for their ability to lyse EM2eGFPluc in vitro. In a flow-cytometric assay based on propidium iodide staining, Vd2 clones exhibited cytotoxicities ranging 4.5%-10.6% for a 4-hour co-incubation at 2.6:1 E:T. Clones from Donor 1 were tested again and ranking confirmed in a 4-hour cytotoxicity assay at 10:1 E:T, with a range of 23.5%-35.4% for clones A1, B3 and C6, respectively. When C6 was compared to polyclonal GDTCs from the same donor, it was found to be more cytotoxic (9.0% versus 2.0% at 10:1 for 4 hours). Vd2 clones and polyclonal GDTC from Donor 2 were compared; clone E5 exhibited 10-fold (49.2%) and E3 1.4-fold (7.6%) cytotoxicity of polyclonal GDTCs (5.3%). Published reports describe an increase in Vd1 in B-CLL patients, hence we used MEC1, an EBV-positive B-cell line derived from B-CLL, as a target. At a 1.9:1 ratio over 4 hours, % cytotoxicity ranged 7.0% - 13.8% (D3 most cytotoxic). Vd1 clones were compared with polyclonal GDTC cultures derived from Donors 2 and 3, which exhibited 57% and 52% Vd1, respectively. Clone D3 again proved most cytotoxic at 10:1 E:T over 4 hours, with 40.8% compared to 18.6% (Donor 3) and 6.8% (Donor 2). Immunophenotyping indicates phenotypic stability in clones over time that is not evident in polyclonal populations. We conclude that the increased cytotoxicity, superior expansion potential and extended culture duration as well as phenotypic stability of GDTC clones make them a more attractive therapeutic agent than polyclonal cultures for the treatment of hematological malignancies. Our study reveals the potential importance of selecting specific and potent GDT effector cells for treating Ph(+) and B-CLL leukemias with GDTCs. We next plan to test this approach in our established pre-clinical xenogeneic leukemia mouse model.(Dhamko H was the recipient of an ASH Summer Trainee Research Award). DisclosuresNo relevant conflicts of interest to declare.