Dynabeads human t expander cd3 cd28

Human peripheral blood mononuclear cells (PBMCs) were isolated as described [18 (link)] from heparinized peripheral blood obtained from healthy donor leukapheresis products or discard kits containing residual blood components of healthy donors that had undergone apheresis at the COHNMC with the approval of the COHNMC IRB and Office of Human Subjects Protection. Blood samples from healthy donor leukapheresis products were obtained with written informed consent. The COHNMC IRB waived the need for written informed consent of blood samples from healthy donor leukapheresis discard kits as these were de-identified and obtained from discard material. T_CM isolation (using CD14- and CD45RA-depletion followed by CD62L-selection), anti-CD3/CD28 bead stimulation, and lentiviral-mediated transduction were then done as previously described [27 (link)] using MOIs ranging from 1 to 3. The Dynabeads Human T Expander CD3/CD28 (Invitrogen) were removed between days 10 and 14, and bead-free T cells were expanded at a cell density of 0.5–1 x10⁶ cells/mL with cytokines [27 (link)]. In some cases the cells transduced with CE7R28Z-T2A-CD19tDHFR^FS_epHIV7 underwent further expansion (Rapid expansion method; REM) as previously described [18 (link)] with or without DHFR^FS-mediated selection using 0.1 μM methotrexate (MTX) [24 (link)].

T cells were stimulated with Dynabeads Human T-Expander CD3/CD28 (Invitrogen) at a 1:3 ratio (T cell:bead) overnight in X-VIVO-15 (Lonza) supplemented with 10% FBS, 2 mM L-glutamine, and IL2/IL15 [50 U/mL IL2 (Novartis), 0.5 ng/mL IL15 (CellGenix)]. Stimulated T cells were then transduced with lentiviral vector (M.O.I. of 1.0) encoding the Fabrack-CAR. Mock and CAR transduced T cells were cultured with indicated cytokines three times a week for 18 days before subsequent analyses. Efficiency of CAR transduction in human T cells was 40%–60% (online supplemental figure S6).

Tn/mem cells were stimulated with Dynabeads Human T-Expander CD3/CD28 (catalog 11141D, Invitrogen) at a 1:3 cell-to-bead ratio and transduced with lentivirus at a multiplicity of infection of 1.5 to 3 in X-Vivo15 containing 10% FBS and 100 μg/mL protamine sulfate (catalog NDC 63323-0229-15, APP Pharmaceuticals), 50 U/mL rhIL2 (catalog NDC 76310-0022-01, Clinigen), and 0.5 ng/mL rhIL15 (catalog 1013-050, CellGenix). Cultures were then maintained at 37 °C and 5% CO₂, with addition of X-Vivo15 media containing 10% FBS as required to keep cell density around 6 × 10⁵ cells/mL, with cytokine supplementation 3 times a week. On day 7 of culture, the CD3/CD28 Dynabeads were removed from cultures using a DynaMag 5 magnet (catalog 12303D, Invitrogen). T cell lines were enriched with EasySep CD19 selection kit II (catalog 17854, Stemcell) around day 14 and propagated for 19 to 24 days prior to cryopreservation.

For lentivirus package, CAR expression plasmid, together with pMD2.G and psPAX2 packaging plasmids (Addgene) were transfected into HEK-293T cells by HighGene transfection reagents (ABclonal). Lentiviral supernatant was harvested 48 h and 72 h after transfection. Lymphoprep™ (Axis-Shield) was used to isolate and purify peripheral blood mononuclear cells (PBMCs) by gradient centrifugation. Then CD3⁺ T cell populations were isolated by CD3 MicroBeads (Miltenyi Biotec) according to the manufacturer’s instruction, and culured in X-VIVO 15 medium, supplemented with 10% human AB serum, 10 mM N-acetyl-cysteine, 10 mM glutamine, and 10 mM MEM amino acid (all purchased from Sigma-Aldrich), as well as 100 IU/ml rhIL-2, 5 ng/ml rhIL-7, and 5 ng/ml rhIL-15 (all purchased from Novoprotein). After stimulation by Dynabeads^TM Human T-Expander CD3/CD28 (Gibco) for 24 h, virus was added to activated T cells in RetroNectin-coated plate and centrifuged for 2 h at 1000 × g. Twenty-four hours after transfection, T cells were replaced with fresh medium. 3 days after transduction, CAR expression was detected by flow cytometry.