Fresh peripheral blood mononuclear cells (PBMCs) were extracted in
Ficoll-Paque PLUS (GE Healthcare) density media using SepMate™ Isolation
Tubes (STEMCELL Technologies). PBMCs were cultured overnight at a density of
>106 cells/mL in OpTmizer CTS T-cell Expansion media
(ThermoFisher) with 5% human AB serum (Sigma-Aldrich). Nonadherent PBMCs were
collected and either used for flow cytometric analysis or enriched for T cells
by using Dynabeads Human T-Cell Expander CD3/CD28 (ThermoFisher) at a 2:1 bead:T
cell ratio in combination with OpTmizer media supplemented with recombinant
human 12.5 ng/ml IL7 and 12.5 ng/ml IL15 (all cytokines from Peprotech). These T
cells were expanded and maintained at constant density until lentiviral
transduction of the ICAM1-CAR construct. For in vivo studies, a
frozen leukopak was used for donor T cells which were cultured using the
CliniMACS Prodigy (MIltenyi biotec) according to a protocol previously reported
(31 (link)).
Ficoll-Paque PLUS (GE Healthcare) density media using SepMate™ Isolation
Tubes (STEMCELL Technologies). PBMCs were cultured overnight at a density of
>106 cells/mL in OpTmizer CTS T-cell Expansion media
(ThermoFisher) with 5% human AB serum (Sigma-Aldrich). Nonadherent PBMCs were
collected and either used for flow cytometric analysis or enriched for T cells
by using Dynabeads Human T-Cell Expander CD3/CD28 (ThermoFisher) at a 2:1 bead:T
cell ratio in combination with OpTmizer media supplemented with recombinant
human 12.5 ng/ml IL7 and 12.5 ng/ml IL15 (all cytokines from Peprotech). These T
cells were expanded and maintained at constant density until lentiviral
transduction of the ICAM1-CAR construct. For in vivo studies, a
frozen leukopak was used for donor T cells which were cultured using the
CliniMACS Prodigy (MIltenyi biotec) according to a protocol previously reported
(31 (link)).
