Human t cell activation expansion kit

Peripheral blood mononuclear cells (PBMCs) were collected from healthy voluntary donors and isolated from the whole blood via Ficoll-Paque PLUS density gradient centrifugation (cat# 17144002, cytiva). CD3⁺ T cells were isolated using the Pan T cell isolation kit (cat# 130-096-535, Miltenyi Biotec). Next, the isolated CD3⁺ T cells were stimulated for 48 h with the human T cell activation/expansion kit (cat# 130-091-441, Miltenyi Biotec) in accordance with the manufacturer’s instructions. For this process, the T cells were cultured in X-VIVO™ 15 media (cat# 02-060Q, Lonza) supplemented with 10% inactivated FBS, 10 ng/mL human IL-7 (cat# 200-07, Peprotech), and 10 ng/mL human IL-15 (cat# 200-15, Peprotech) at a final concentration of 1 × 10⁶ cells/mL.

Human CD8⁺ T cells (HLA-A2-positive, STEMCELL, Vancouver, BC, Canada) were purchased. The cells were expanded by a 1:10 ratio of beads from the human T Cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Every three days, the media, containing 20 units/ml of recombinant interleukin -2 (CORNING, Corning, NY, USA), was changed. CD8⁺ T cells will, hereafter, refer to human CD8⁺ T cells. Both CD8⁺ T cells and Jurkat cells were cultured with high glucose RPMI1640 (GIBCO, Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum, i.e., FBS (GIBCO), 1% Penicillin-Streptomycin; PS (GIBCO), L-glutamine, HEPES, and ß-mercaptoethanol. HCT116 (HLA-A2-positive), Jurkat cells, and NF-κB-luciferase reporter Jurkat cells were cultured with RPMI1640 media containing L-glutamine (GIBCO), 10% FBS, and 1% PS. NF-κB-luciferase reporter Jurkat cells were kindly provided by Dr. Park, Yoon from Korea Institute of Science and Technology. Jurkat cells (2~5 × 10⁵ cells/well) were activated using a T Cell Activation/Expansion Kit (Miltenyi Biotec); the ratio of cell-to-bead was 2:1.

Blood from anonymous healthy donors (buffy coats) was generously provided by Dr. Michael Lenardo and the National Institutes of Health Blood Bank. PBMC were isolated using Ficoll density gradient centrifugation, and CD8⁺ T cells were purified from PBMC using the EasySep Human CD8⁺ T-cell enrichment kit (Stem Cell Technologies, Vancouver, BC, Canada). CD8⁺ T cells (>95% purity) were stained for 30 min on ice with anti-CD45RO-APC and anti-CD62L-FITC antibodies (BioLegend, San Diego, CA, USA). Memory subsets were sorted on a BD FACSAria cell sorter (BD Biosciences, San Jose, CA, USA); CM T cells were gated as CD45RO^hi and CD62L^hi, EM T cells were gated as CD45RO^hi and CD62L^lo. Sorted subsets were activated 1:1 with beads coated with anti-CD3/CD2/CD28 antibodies (Human T-cell Activation/Expansion Kit, Miltenyi Biotec Inc, San Diego, CA, USA) in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA)+10% fetal calf serum (FCS) (Sigma-Aldrich, St Louis, MO, USA) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland) for 3 days. Activated T cells were washed in PBS and subsequently cultured in media as described above with 100 U/ml rIL-2 (PeproTech, Rocky Hill, NJ, USA) at 1×10⁶ cells/ml for ≥10 days, changing media every 3 days.

Long-term CD8 + T cell culture was applied according to the manufacturer’s instructions and as described by Petersen et al.^{30 (link)}. Briefly, CD8 + T cells were maintained at a concentration of 1 × 10⁶ cells/mL in culture media supplemented with 20U/mL IL-2 and the respective test compounds (BPA or 0.01% DMSO). Cells were split every three to seven days with addition of 20U/mL IL-2 and test compounds; each 14 days, cells were re-stimulated using the human T Cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured for a total of 49 days; during this time, aliquots were taken on a regular basis from each cell culture for determination of effect parameters. At the end of the long-term culture cell pellets were stored at -80 °C until further processing.

Culture medium consisted of RPMI-1640 supplemented with 10% fetal bovine serum and antibiotic/antimycotic at 1X (all from Gibco, Life Technologies, Carlsbad, CA). T cell activation was performed with a Human T-cell Activation/Expansion kit containing anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany), used according to the manufacturer’s protocol. Briefly, anti-biotin MACSiBead particles were resuspended thoroughly and 25 μL transferred onto a 1.8 mL Eppendorf tube. Fresh medium (200 μL) was added and then centrifuged at 300 X g for 5 min, the supernatant aspirated and the pellet resuspended in 100 μL fresh medium. Cells where suspended in 900 μL of medium, and added to the MACSiBead particles for a total volume of 1000 μL. The resulting mixture was distributed at a density of 250,000 cells/well in a 96-well plate (Greiner Bio-one, Monroe, NC) and incubated at 37°C for 24 h in a 5% CO₂ atmosphere.

Total CD3+ T lymphocytes were isolated with magnetic cell sorting using a human-specific CD3+ microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) from restored peripheral blood mononuclear cells (PBMCs). Sorted CD3+ T cells were subject to culture using TexMACS immune cell-culturing medium (Miltenyi Biotec, Bergisch Gladbach, Germany). Activation and expansion was induced by using human T-cell activation/expansion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with addition of 100 U/mL of IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany).

Naïve T cells were isolated from the PBMCs using the Naïve CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Subsequently, the naïve T cells were activated with anti-CD2/anti-CD3/anti-CD28 beads to mimic antigen-presenting cells by using the Human T Cell Activation/Expansion Kit (Miltenyi Biotec). Activated T cells were then seeded at a concentration of 2.5 × 10⁶ cells per ml into a 24-well plate. Then, tofacitinib was added at concentrations of 100 nM or 600 nM for 30 min before TGF-β1 (20 ng/ml), IL-6 (20 ng/ml), IL-23 (10 ng/ml), and anti-IL4 (1 μg/ml) were added and incubated for 3 days. At day 3, the cell clumps were homogenized, and the cell suspension was split and supplemented with 5 ng/ml IL-2. Cell analysis was done after another 4 days of incubation.