Cd3 cd28 t activator beads

^AlloHSC-iNKT cells were generated in the 2-Stage HSC-iNKT culture as described above, followed by an additional Stage 3 CAR engineering culture. At one week into Stage 2 culture, ^AlloHSC-iNKT cells were collected and stimulated with CD3/CD28 T-activator beads (ThermoFisher Scientific) in the presence of recombinant human IL-15 (10 ng/ml) and human IL-7 (10 ng/ml) for two days; the cells were then spin-infected with frozen-thawed Retro/BCAR-tEGFR retroviral supernatants supplemented with polybrene (10 μg/ml, Sigma-Aldrich) at 660 g at 30°C for 90 min. Retronectin (Takara) could be pre-coated on plate to increase transduction efficiency. After transduction, the resulting ^AlloBCAR-iNKT cells were expanded for another 1-2 weeks in C10 medium supplemented with recombinant human IL-15 (10 ng/ml) and IL-7 (10 ng/ml), and then were collected and cryopreserved for future use.

Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood from consenting healthy donors (protocol approved by the NRES Committee West Midlands ethical board; REC reference 14/WM/1254). Briefly, blood was layered over lymphoprep (Stem Cell Technologies) with resulting PBMC used for subsequent experiments. Plasma was also harvested for CMV IgG (ELISA kit) measurement. For repertoire analysis, all T-cell populations were sorted directly into RNAlater (Sigma) or RLT buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma) on a MoFlo Astrios Cell Sorter (Beckman Coulter). For activation and proliferation of T cells, either PBMC or isolated CD3⁺ T cells, obtained by positive magnetic bead isolation (Miltenyi), were labelled or not with 0.3 μM CFSE (eBioscience) and cultured with cytokines, 25 ng ml⁻¹ IL-7 (Peprotech), 100 IU ml⁻¹ IL-2, 25 ng ml⁻¹ IL-15, 5 ng ml⁻¹ IL-12, 5 ng ml⁻¹ IL-18 (all Miltenyi), and/or antibodies directed against CD3 (OKT3; eBioscience), CD28 (28.2), mIgG1κ (MOPC-21), TCR γδ (B1; all Biolegend) or CD3/CD28 T activator beads (Invitrogen), where indicated and for up to 7 days in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 1% sodium pyruvate, 50 μg ml⁻¹ penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma).

PBMC were isolated from heparinised venous blood by lymphoprep© (Stem Cell Technologies) density gradient centrifugation as per the manufacturers instructions. For proliferation of T cells, PBMC were labelled with 0.3 µM carboxyfluorescein succinimidyl ester (CFSE) (eBioscience) and cultured with 10 nM HMB-PP (Sigma) or CD3/CD28 T activator beads (Invitrogen) for 7 days in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma).

B16-ova tumors from C57BL/6 mice were harvested and CD8⁺ cells isolated using MACS beads (Miltenyi) as described above. Cells were stimulated with CD3/CD28 T activator beads (10 μL/mL, Gibco) for 12 hours in the presence of anti-CD107a antibody (AF488, Biolegend) to mark degranulating cells. Brefeldin A (10 μg/mL) was added during the last 4-6 hours of culture to block secretion of intracellular cytokines.