Cd19 fc fusion protein

Analysis was performed using a BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FCS Express 6 software. Antibodies used included anti-CD5 PerCP/Cy5.5, anti-CD3 BV421, anti-γδ T cell receptor (TCR) phycoerythrin (PE) and anti-CD69 APC-Cy7 (BD Biosciences, San Jose, CA). CD5-Fc fusion protein (G&P Biosciences, Santa Clara, CA) and CD19-Fc fusion protein (ACROBiosystems, Newark, DE) were used to detect anti-CD5 constructs and anti-CD19 constructs, respectively, with a secondary anti-IgG Fc antibody (Jackson Immunoresearch Laboratories, West Grove, PA), as previously described.⁶ (link) Violet Proliferation Dye 450 (VPD450) was used to label the target cells in the cytotoxicity and co-culture studies, and cell death was assessed using eFluor 780 (described below). Degranulation of γδ T cells was detected using anti-CD107a APC (BD Biosciences, San Jose, CA).

γδ T cells were either electroporated fresh on day 12 of expansion or from thawed cells that were frozen on day 12 of expansion. Cells were thawed in 5% HSA in PBS and were centrifuged at 250 × g for 10 min at room temperature. The cells were cultured at 4 × 10⁶ cells/mL for 2 h in complete OpTmizer media with 1,000 IU/mL IL-2. Cells were then counted and the appropriate cell number for each reaction was aliquoted, washed twice with PBS, and resuspended in 100 μL OptiMEM (Life Technologies). The appropriate amount of mRNA was added to the tube and the mix was transferred to a 4-mm cuvette (Fisher Scientific). Electroporations using the BioRad’s Gene Pulser Xcell Electroporator were conducted at 500 V for 5 ms using a square wave. Cells were collected from the cuvette and cultured overnight at 2 × 10⁶ cells/mL in complete OpTmizer media with 1,000 IU/mL IL-2. Flow cytometry was used to confirm and analyze CAR expression after electroporation by labeling cells with a CD19-Fc fusion protein (AcroBiosystems) and an anti-IgG Fc secondary antibody (Jackson Immunoresearch Laboratories).