Human cd3 t cell isolation kit

Peripheral blood was obtained from the healthy donors. Blood sampling was performed following the required ethical procedures. Lymphocytes were isolated by density gradient centrifugation following the manual of the Human Lymphocyte Separation Medium (DAKEWE). Human T cells were purified using the human CD3 T cell isolation kit (BioLegend), stimulated with CD3/CD28 T cell Activator Dynabeads (Gibco) and cultured at 10⁶/mL in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza), supplemented with 5 ng/mL human IL-7 (PeproTech) and 5 ng/mL human IL-15 (PeproTech)^{40 (link),65 (link)}, with slight modification. 48 h after T cell stimulation, T cells were transduced with lentiviral supernatants from 293 T cell line in the presence of polybrene (6 μg/mL, Yeasen) by centrifugal infection. T cells were analyzed by flow cytometry 4 days after transduction and were used for further experiments. All human subjects were informed and signed informed consent prior to inclusion in the study and all human cell isolation and related experiments were approved by the Ethics Committee for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine (2019 NO.388).

Mouse T cells were isolated from the spleens and lymph nodes of Balb/c, C57BL/6, FGL2^−/−, and LAG3^−/− mice and purified with a Mouse CD3 T cell Isolation Kit (480031; BioLegend). Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE Healthcare Biosciences AB) and SepMate-50 (Stemcell Technologies, Inc.) according to the manufacturers’ protocols. Human T cells were purified from PBMCs with a Human CD3 T Cell Isolation Kit (19051; BioLegend). Mouse T cells and human T cells were activated with Dynabeads Mouse T-Activator CD3/CD28 (0077118, Gibco) or Dynabeads Human T-Activator CD3/CD28 (00805147, Gibco) per the manufacturer’s instructions and then cultured in RPMI-1640 (Gibco), 10% fetal bovine serum (Hyclone), 2 mM GlutaMAX (Gibco), 100 μM β-mercaptoethanol (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Corning) with 100 U interleukin (IL)−2 (Biolegend) and IL-15 (5 ng/mL) (Fisher). Twenty-four hours after activation, T cells were transfected with a lentivirus containing a control vector or an FGL2-blocking scFv vector. Cells were spun down at 1500 × g for 2 h with 8 μg/mL polybrene (MilliporeSigma) in RPMI medium in a retronectin (T100B; Takara)-coated 24-well plate. T cells were used for in vivo or in vitro experiments 2–4 days after transfection.

Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood drawn from healthy volunteers and patients with NMOSD through Ficoll gradient centrifugation (GE Healthcare), according to the supplier's instructions. Subsequently, T cells were isolated using a human CD3+ T‐cell isolation kit (BioLegend). Briefly, non CD3+ T cells were depleted by incubation with a biotin antibody cocktail including anti‐CD14, ‐CD15, ‐CD16, ‐CD19, ‐CD36, ‐CD56, ‐CD123, and ‐CD235, followed by incubation with magnetic streptavidin nanobeads.