Human t cell enrichment kit

Human peripheral blood mononuclear cells were obtained from blood following centrifugation in Lymphocyte Separation Solution (Wisent Inc., St-Bruno, Canada) as previously described (24 (link)). Blood donors were male and female subjects between 18 and 65 years of age who had indicated that they had no known health issues and who had fasted for 12 h prior to the blood draw. T-cells were then isolated by negative selection using the human T-cell enrichment kit from Stem Cell Technologies (Vancouver, Canada) following the manufacturer’s instructions. Primary T-cells and the Jurkat cell line (ATCC, Manassas, VA) were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, 10 µg/ml streptomycin, 10 mM HEPES, d-glucose (up to 25 mM), and 1 mM sodium pyruvate at 37°C in a 5% CO₂ atmosphere. Jurkat cells are a human leukemic T-cell lymphoblast cell line that is often used to study T-cell functions. T-cells were stimulated with anti-CD3/anti-CD28 Dynabeads (1 × 10⁶ beads/ml) (Invitrogen) according to the manufacturer’s instructions in the presence of 30 U/ml IL-2 (Sigma-Aldrich) for up to 72 h before all experiments. HepG2 cells (ATCC) were cultured in Eagle’s minimal essential medium supplemented with 10% FBS, 100 U/ml penicillin, and 10 µg/ml streptomycin at 37°C in a 5% CO₂ atmosphere.

CD3 T cells were isolated from total PBMCs obtained from healthy adult subjects (pooled two subjects) by negative isolation using a human T-cell enrichment kit (Stem cell). Prior to CD3 enrichment, PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) for subsequent assessment of T-cell proliferation. For agonist-stimulated PBMCs, CD3 T cells were removed from PBMCs by positive selection using a human T-cell enrichment kit (Stem cells). Harvested PBMCs (minus CD3 T cells) were adjusted to a concentration of 2x10⁶ cells mL⁻¹ with cultured RPMI medium and cultured with 0.1 μg mL⁻¹ LPS or 1 μg mL⁻¹ CLO97 or without stimuli in a 37 °C, 5% CO₂ atmosphere for 20 hrs. Agonist-stimulated PBMCs (minus CD3 T cells) from adult and old subjects were washed three times with cultured RPMI medium and co-incubated 1:1 with T cells from young subjects in a 37 °C, 5% CO₂ atmosphere for 5 days. For the gating of CFSE^lowCD3+ T cells, frequencies with a gating of > 20 events were considered significant. Peripheral blood mononuclear cells used for these experiments were obtained from leukapheresis of healthy adult and old subjects.

Human Mo-DCs were generated by differentiation of CD14⁺ monocytes (EasySep Human CD14+ positive selection kit (Stemcell)) in the presence of 100 ng/ml GM-CSF and 100 ng/mL IL-4 for 6 days in X-Vivo15 with 2% human AB serum. Mo-DCs were harvested, seeded, and stimulated with MEDI9197 for 18 h. Allogeneic human CD3⁺ T cells were isolated using the Human T cell enrichment kit (Stemcell), then added to the mo-DCs at a ratio of 10:1 and co-cultured for 3–5 days. IL-2 was measured by DELFIA ELISA (R&D) in day 3 supernatants and IFNγ (BD) in day 5 supernatants.