Characterizing Engineered T Cell Responses

CD27 (BioLegend Cat #302830) CCR7 (BioLegend Cat #353218) labeled antibodies were used to stain the expanded T cell populations as previously described.⁵⁴ (link) Expression of the CD19 CARs was detected using the biotinylated F(ab′)2 fragment from goat anti-mouse immunoglobulin G (IgG) sera (specific for scFvs of murine origin; Jackson ImmunoResearch), followed by staining with streptavidin-PE (BD Biosciences/Pharmingen, Cat# 554061). Intracellular cytokine staining was performed as previously described.⁵⁵ (link) Briefly, after T cells stopped expanding, CD3/28 beads were removed by magnetic separation. The following day, the T cells were mixed with K562s expressing CD19 at a ratio of 2:1 and incubated for 5 hr with GolgiStop (BD Biosciences). Surface CD4 (BioLegend Cat# 317433) and CD8 (BD Biosciences Cat# 560273) staining was performed, samples were fixed, and the following day, intracellular staining was performed as previously described,⁵⁶ (link) with an LSR II flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR). The following antibodies were used for intracellular staining: -IL-2 (BD Biosciences Cat# 554567), interferon γ (IFNγ) (BD Biosciences Cat# 552882, TNF (BD Biosciences Cat# 557647), and MIP-1β (BD Biosciences Cat# 560688).