Human t cell isolation kit

Autologous CD8⁺ T cells were obtained from frozen PBMCs from the same donor using CD8⁺ microbeads from a human T-cell isolation kit (Miltenyi Biotec). To monitor cell proliferation, cells were labeled with 1.25 µM CellTrace Violet (Invitrogen) according to the manufacturer’s instructions. The washed cells were resuspended in complete RPMI and used for the suppression assay.

Peripheral blood samples from SLE patients and healthy volunteers were collected in anticoagulant tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Hypaque density-gradient centrifugation for 30 min, and washed twice with phosphate buffered saline before T cell purification by negative selection with the human T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of T cells was > 95% as determined by flow cytometry.

To validate the biodistribution of Jurkat/CAR T-cells after injection, we performed ex vivo immunostaining of organs. The mouse organs (liver, spleen) were harvested on day 3 after injection of unlabeled Jurkat/CAR T-cells (2 × 10⁷/200 μl) into the NSG mice with tumors, via their tail veins (n = 2). The tissues were ground using a gentleMACS^™ Dissociator (Miltenyi Biotec, Germany) in accordance with the supplier’s protocol. A mouse cell depletion kit (Miltenyi Biotec, Germany) was used to separate the Jurkat/CAR T-cells from the mouse cells. After counting the live cells, the human T-cell isolation kit (Miltenyi Biotec, Germany) was used, according to the supplier’s protocol. Red blood cells were removed using an RBC lysing buffer (Sigma Aldrich, MO) for one minute, followed by washing and re-suspension in 1x HBSS containing 1% FBS. The separated cells were used with PE-conjugated anti-CD3. The analysis staining process was the same as that used in vitro. Data were acquired from the stained cells using BD FACS CantoII flow cytometry (BD Biosciences). The results were evaluated with FlowJo software (Treestar Inc., Ashland, OR).

Human peripheral blood CD3⁺ T lymphocytes were isolated using Human T-cell Isolation kit (Miltenyi, DEU). CD3/CD28 T-cell activation Dynabeads (Gibco, USA) was used for T cell stimulation in the presence of IL-2 (PeproTech, USA). The lentiviral plasmid containing sequences of 8H9S3.3-ScFv (or FMC63-ScFv), human CD8 transmembrane sequence, cytoplasmic domains of human 41BB and human CD3 complex ζ chain were synthesized by Sangon Bitech (Shanghai, China). The backbone for lentiviral plasmid (pCDH-CMV-MCS-EF1-GFP) and packaging plasmids (PMD2.G, PSPAX2) were purchased from addgene. 293T cells and Lip3000 were used for lentiviruses production. CD3⁺ T lymphocytes were infected with lentivirus and infection efficiency was verified according to GFP expression.