γδ T lymphocytes from NHL patients proliferate in response to lymphoma cells. (A) PBMCs from FL NHL patients were stained with CFSE and cocultured with ULBP⁺ H9 or ULBP⁻ C1R lymphoma cells. At day 2, 25 U/mL rIL-2 was added (arrow). In some experiments 5 μg/mL anti–HLA-I or anti-NKG2D mAb was added at the onset of the culture. At days 1, 5, and 7, cells were recovered and stained with PE-conjugated anti-γδ mAb and samples were run on a FACS gated on γδ T cells. Results are expressed as percentage of proliferating γδ T cells, identified as red cells with decreased green fluorescence intensity; mean ± SD of experiments performed with PBMCs from 10 FL NHL patients. (B) Proliferating Vδ1 (i) or Vδ2 (ii) T cells from 10 FL lymphoma patients, stained with CFSE, cultured as in panel A, and identified with PE-conjugated anti-Vδ1 or anti-Vδ2 mAb as red cells with decreased green fluorescence intensity. Data were analyzed with ModFit LT computer program and results expressed as percentage of Vδ1 or Vδ2 cells in second, third, fourth, or fifth generation, compared to the parental nonproliferating population, at different time points, as indicated. (C) Vδ1 or Vδ2 T cells were purified by negative immunodepletion with magnetic microbeads as described¹⁴  and cultured with irradiated PBMCs alone (none) or with H9 lymphoma cells (+H9). After 7 days cells were recovered, counted, checked for Vδ1 or Vδ2 expression, and the results expressed as mean fold-increase in cell number ± SD of experiments performed with cells obtained from 6 FL patients. Asterisk indicates P < .01 versus Vδ2.