Cd45ro pe cy7

Directly after Ficoll-Paque isolation, between 5∙10⁷ and 20∙10⁷ PBMCs were stained with CD95-FITC, CD4-APC-eF780 (both eBioscience), CD3-PerCP, CCR7-BV421, CD8a-BV510, and CD45RO-PE-Cy7 (all from BioLegend), and either CD56-APC, CD56-PE, or CD56-PE/Dazzle-594 (the first purchased from BD Biosciences and the latter two from BioLegend). T-cell subpopulations were defined as follows: truly naive, T_TN (CCR7⁺CD45RO^-CD27⁺CD95⁺), central memory, T_CM (CD45RO⁺CD27⁺), effector memory, T_EM (CD27^-CD45RO⁺) and effector memory re-expressing RA, T_EMRA (CD27^-CD45RO^-). The full gating strategy can be found in S1 Fig. Cells were sorted on a FACSAria II or FACSAria III sorter (BD Biosciences), and data was analyzed with the BD FACSDiva v8.0.1 and FlowJo V10 software.

For analysis of B7-H3 surface expression and B7-H3xCD3 binding, cells were stained with a parental murine B7-H3 antibody (10 µg/mL) carrying the same B7-H3 binding clone as our construct (7C4), B-H3xCD3 or the corresponding isotype controls followed by a goat anti-mouse-PE conjugate (Dako, Glostrup, Denmark) or a donkey anti-human-PE conjugate (Jackson ImmunoResearch, West Grove, USA), respectively. T cell activation, degranulation and proliferation were determined using CD69-PE, CD107a-PE (BD Pharmingen) as well as CD4-APC, CD8-FITC, CD62L-PB and CD45ro-PeCy7 (BioLegend, San Diego, CA) fluorescence conjugates. For flow cytometric analysis of target cell lysis, tumor cells were loaded with 2.5 µM CellTrace™ Violet (Thermo Fisher Scientific, Waltham, MA) and cultured with monocyte-depleted PBMC (E:T 4:1) in the presence or absence of B7-H3xCD3 or MOPCxCD3 (1 nM each). Standard calibration beads (Sigma-Aldrich, St. Louis, MO) were used to ensure analysis of equal assay volumes and to account for the number of target cells that had vanished from the culture. 7AAD (Biolegend) was used for live- and dead-cell discrimination. Measurements were performed using a FACS Canto II or FACS Fortessa (BD Biosciences, San Diego, CA) and data was analyzed using the software FlowJo (FlowJo LCC, Ashland, OR).