T cell activation and expansion kit

T cells derived from NHP F128 PBMCs were expanded with the T cell activation and expansion kit (Miltenyi Biotec). Biotinylated antibodies against non-human primate CD3 and human CD2 and CD28 were conjugated to anti-Biotin MACSiBead particles and were used to mimic antigen-presenting cells and activate resting T cells from peripheral blood mononuclear cells (PBMCs). T cells were expanded by adding IL-2 and fresh medium every 2–3 days. Genomic DNA was extracted using the QIAamp DNA blood kit (Qiagen). Flanking regions of the genes of interest were identified from ‘Ensembl’ genome database and primers were designed to amplify the VH regions of the genes by PCR (S1 Table). The amplifications were performed using a Taq DNA polymerase which adds a single deoxyadenosine to the 3′ end of the PCR product. Linearized vectors having complimentary 3´ deoxythymidine (T) residues allowed the VH gene amplifications to be ligated into the TA vector for subcloning (Life Technologies). The vectors were sequenced to identify the germline versions of the VH regions of the antibodies in this study. gL-reverted antibody constructs were then generated by synthesis after aligning antibodies LC and HCs to the corresponding germline V and J regions and reverting the nucleotide sequence to the putative gL configuration, while leaving the mature HCDR3 and LCDR3 unaltered.

To generate NY-ESO-1 TCR-specific T cells, human healthy donor PBMCs were thawed and washed in PBS. CD8 T cells were then isolated using the CD8 microbeads kit (Miltenyi, positive selection) according to the manufacturer’s instructions on an AutoMACS. Isolated cells were washed and resuspended in suppl. RPMI with 8% human serum as described above and plated at 1.5 Mio/ml. T cell activation and expansion kit (Miltenyi) anti-CD3+ anti-CD28 stimulatory magnetic beads were coated overnight at 4 °C on a rotator shaker according to the manufacturer’s instructions. These beads were washed in medium and added to the CD8 T cells at a 1:1 ratio together with 150 U/ml IL-2. 24 h later, NY-ESO-1 TCR lentiviral particles produced as described above were added at a multiplicity of infection (MOI) of 2. Cells were then expanded every 2 days with fresh medium and replenishing 50 U/ml IL-2 for 5 days. The percentage of transduced cells was then calculated by staining for TCR Vbeta13.1+ T cells in comparison to non-transduced cells. TCR Vbeta 13.1+ cells were sorted by staining of TCR-Vbeta13.1 antibody by flow cytometry (H131 clone PE-Cy7, 1:25, SorpAria) or using purified antibody (H131 clone purified, 1:50 dilution) followed by magnetic column purification (anti-mouse IgG Microbeads, Milentyi).

The healthy PBMC donors provided informed consent for the use of their samples for research purposes, and all procedures were approved by the Research Ethics Board of the Guangzhou Institutes of Biomedicine and Health. T cells were enriched from peripheral blood mononuclear cells (PBMCs) harvested from healthy donors using a pan T cell isolation kit (Miltenyi Biotec, 130-096-535) and then activated with a T cell activation and expansion kit (Miltenyi Biotec, 130-091-441) for 2 days in RPMI-1640 medium (Gibco, C11975500BT) supplemented with 10% fetal bovine serum (FBS) (Vigonob, XC6936T) and 1% penicillin/streptomycin (Gibco, 15140-122). CAR molecules were introduced to T cells through incubation with lentiviral supernatants in the presence of 8 μg/ml polybrene (Sigma-Aldrich, TR-1003-G) following T cell activation, and the medium was replaced with fresh medium containing IL-2 (300 IU/ml, PeproTech, AF-200-02) 12 h later. Subsequently, fresh medium was added every 1–2 days to maintain the cell density within the range of 0.5–1 × 10⁶/ml.