Cell proliferation and cytotoxicity assay

CAR-transduced T cells were labeled with 10 μM eFluor 450 cell proliferation dye (CPD, eBioscience) according to the manufacturer’s instructions. 5 × 10⁵ CPD-labeled transduced CAR T cells were plated on 24 well/plates coated with recombinant human Siglec-6/Fc chimera protein (R&D System, Minneapolis, MN). Proliferation of T cells was analyzed by flow cytometry.

CAR T cell cytotoxicity assay was performed as described (20) with some modifications. Briefly, 2-week expanded, CAR T cells were co-cultured with target cells at the indicated ratios for 4-6 hours and analyzed by flow cytometry. %specific lysis=[(negative target-experimental target)/negative target x100]. The cytotoxic assay on primary CLL cells was performed by mixing a similar number of CAR-transduced T cells and primary CLL cells followed by co-culture for 5hs. Remaining CLL cells in culture were identified by flow cytometry as CD5⁺CD19⁺ cells.