T cell enrichment kit

hPBMCs were obtained from human peripheral blood provided by Shanghai Liquan Hospital and hPBMCs cell isolation service provided by Milestone Biotechnologies. CD3⁺ T cells were enriched according to the instructions of the T Cell Enrichment Kit (Stemcell Technologies, Vancouver, BC, Canada, 19051), activated with anti-CD3/anti-CD28 Dynabeads (ThermoFisher, Waltham, MA, USA, 11131D) added at a ratio of 2:1, and proliferated. The cells were maintained in X-VIVO15 medium (Lonza, Basel, Switzerland, 04-418Q) supplemented with 5% (v/v) inactivated FBS (GIBCO, Grand Island, NY, USA, 1914970), 100 U/mL penicillin/streptomycin (GIBCO, Grand Island, NY, USA, 15140-122), and 300 IU/mL interleukin (IL)-2 (Sino Biological Inc., Chesterbrook, PA, USA, GMP-CD66).

T-cell proliferation studies were performed as described before with minor modifications (24 (link), 26 (link)). Briefly, whole blood from patients was cultured for 2 h at 37°C. Next, samples were washed and centrifuged at 250×g for 5 min. Cell pellets were then resuspended in red blood cell lysis buffer for 10 min/4°C, treated using DNase for 5 min/37°C, then filtered through a 70-µm nylon mesh filter and washed. CD15⁺ cells were enriched using a positive selection kit from Stemcell Technologies, Vancouver, Canada (#18651) following the manufacturer’s protocol, then cultured in the presence of 20% matched patient’s plasma and 500 nM of CpG-STAT3ASO or control CpG-scrON or with PBS. The following day, CD3⁺ T cells were then enriched using a T-cell enrichment kit (Stemcell Technologies, #17951) from healthy donor PBMCs obtained using density centrifugation over Histopaque-1077 at 1,500 rpm/20 min. T cells were then labeled with CFSE dye according to the manufacturer’s protocols (Thermo-Fisher Scientific, #C34554) and co-cultured with CD15⁺ cells and CD3/CD28 beads in round-bottom 96-well plates (Thermo Fisher, #11131D) at a ratio of 1:6 of T cells to CD15⁺ myeloid cells. After 3 days, flow cytometric analysis was performed to assess T-cell proliferation using CFSE dilution using antibodies specific to CD8a (RPA-T8, #25-0088-42).

T cells were isolated from cryopreserved PBMCs from three healthy HLA-A*02 donors using T cell enrichment kit (StemCell, Vancouver, Canada) following manufacturer’ instructions and stimulated using Dynabeads (Gibco, Waltham, MA). After 24 h, NY-ESO-1 lentivirus (A12 Lentivirus, Lentigen, Gaithersburg, MD) was added to T cells at a multiplicity of infection (MOI) of 10 in presence of Lentiboost A and Lentiboost B (Sirion Biotech, Cambridge, MA). After transduction, T cells added to the GRex system (WilsonWolf, Saint Paul, MN) in hTCM media for expansion and were harvested at day 11 after confirming the NYESO-1 TCR expression on CD3+ cells by flow cytometry using a NY-ESO-1 tetramer (MBL International, Waltham, MA). hTCM media was prepared by adding human IL-2, 50 Iµ/ml (Roche, Basel, Switzerland) and human IL-7, 2.5 ng/ml (Biolegend, San Diego, CA) to media containing RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Corning, NY), 10% FBS (Gemini, Sacramento, CA), HEPES 10 Mm (Gibco), Penicillin-Streptomycin 50 µ/ml (Gemini, Sacramento, CA), MEM Non-Essential Amino Acids Solution 1X, MEM Amino Acids Solution 1X, B2-mercaptoethanol 50 Mm (Gibco).