Figure 2.

Effects of Cryopreservation on the Cytotoxicity of Vγ9Vδ2 T Cells Modified with NKG2D CAR

(A) Flow cytometric analysis of GFP and NKG2D expression on Vγ9Vδ2 T cells (γδ T) and Vγ9Vδ2 T cells modified with NKG2D CAR (NKG2Dz) before freezing (24 h post-electroporation) and 24 h after thawing. The results of one representative experiment out of three independent experiments with three different donors are shown. (B) Delfia EuTDA cytotoxicity assay (2 h EuTDA culturing) to assess tumor cell lysis efficiency. Fresh: NKG2D CAR mRNA electroporated cells were analyzed before freezing (24 h post-electroporation). Thawed: the electroporated cells were cryopreserved, stored in a liquid nitrogen tank for 1 month, and analyzed 24 h after thawing. The results of one representative experiment out of three with different donors are shown. The differences between NKG2Dz-γδ T and mGFP-γδ T are statistically significant (p < 0.001) in all tested groups. (C) Thawed Vγ9Vδ2 T cells modified with NKG2D CAR produce robust levels of cytokines in response to SKOV3-Luc tumor cells when compared with mock-γδ T (∗∗∗) or mGFP CAR-modified γδ T (+++). Data represent the mean ± SD of Vγ9Vδ2 T cells from three different PBMC samples. ∗∗∗p < 0.001 and +++p < 0.001, statistical significance between NKG2Dz-γδ T and mock-γδ T and between NKG2Dz-γδ T and mGFP-γδ T, respectively.