Miltenyi automacs

Leukapheresis products were obtained from healthy donors under protocols approved by the City of Hope Institutional Review Board. Central memory T cells defined as CD45RO+CD62L+ were isolated on Miltenyi autoMACS (Miltenyi Biotec Inc). Briefly, PBMC were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and incubated with anti-CD14 and anti-CD45RA microbeads (Miltenyi Biotec Inc) for 30 minutes at room temperature (RT). CD14+ and CD45RA+ cells were then immediately depleted using the autoMACS. The unlabeled negative fraction of cells was labeled with biotinylated-DREG56 mAb (COHNMC CBG) followed by incubation with anti-biotin microbeads (Miltenyi Biotec Inc). The CD62L+ T_CM were purified with positive selection on auotMACS. Immediately after selection, the T_CM were activated, transduced, and maintained as described (13 (link)). Cultures were supplemented with lenalidomide every 48 hours for 21 days before in vitro analysis.

CAR T cells were produced using a cGMP production protocol(30 (link)) for indicated experiments. Leukapheresis products were obtained from healthy donors using protocols approved by the COH Institutional Review Board (IRB: 15283). Blood from different healthy donors was used for each experiment as indicated. Naïve and central memory T cells (Tn/mem) were defined as CD62L+CD14-CD25- and isolated by Miltenyi AutoMACS (Miltenyi Biotec, Inc., Bergisch Gladbach, Germany). Briefly, PBMCs were incubated with anti-CD14 and anti-CD25 microbeads and CD14+and CD25+ cells were immediately depleted. The unlabeled fraction was incubated with anti-CD62L microbeads and CD62L+ cells were purified with positive selection. Isolated Tn/mem or Tn cells were activated, transduced, and expanded for 14 days as described with 50U/mL IL2 and 0.5ng/mL IL15(30 (link), 33 (link)).