Dynabeads human t activator cd3 cd28

HLACs, PBMCs, or SupT1-R5 (10⁶ cells/mL) were mock-treated or infected with F4.HSA at a final concentration of 1500–3000 ng/mL p24^Gag, and cultured for 3 days at 37˚C. Where appropriate, primary cells were pre-activated with 10 μg/ml PHA (Sigma) or with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher), using the appropriate media supplemented with 100 U/mL human interleukin-2 (IL-2) (Life Technologies). To assess infection rates, cells were subjected to flow cytometric analysis as detailed below. For experiments where samples were sorted after infection, cells were first enriched by negative selection for CD4+ T cells using the EasySep Human CD4+ T Cell Enrichment Kit (STEMCELL Technologies) to minimize sort time. Where indicated, sorted cells were mock-treated or stimulated for 2–3 days with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) at a ratio of 1 bead/cell in the presence of 60U/ml IL-2 (Life Technologies), 12 nM PEP005 (Ingenol 3-angelate, Sigma-Aldrich), 40 nM Romidepsin (Sigma-Aldrich), or 10 ng/ml human IL-7 (R&D System), and then harvested for flow cytometric analysis as detailed below.

PBMCs from healthy donors were activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies) in a 14 mL polypropylene round-bottom tube with a ratio of 1 : 1 and incubated for 6, 10, 15, 20, 24, and 48 hours in RPMI + 10% FCS at 5% CO₂. Cells were harvested, washed, and stained for 20 minutes with CD3 APC (Becton Dickinson) and CD30 PE (Becton Dickinson) or CD30L PE (R&D System) antibodies. Cells were acquired on a FACSCalibur cytometer (Becton Dickinson) and analysed with FlowJo 9.3.3 software (Tree Star).

For the first 2 weeks, TILs were stimulated with Dynabeads Human T Activator CD3/CD28 (11131D, Life Technologies) in the presence of high dose IL-2 (Proleukin, Prometheus) as previously described(5). Briefly, one 1-2mm tissue fragment per well was placed in a 24-well plate in 2 mL of complete X-VIVO 15 media supplemented with 1x10⁶/mL CD3/CD28 Dynabeads and 3000 IU/mL IL-2, and then cultured at 37°C in 5% CO₂. 3000 IU/mL IL-2 were added on alternate days. Cells were split to maintain a density of 1x10⁶ cells/ml. Negative control cultures lacked IL-2 and CD3/CD28 beads. After 7 days IL-2 was reduced to 1000 IU/mL. On attaining confluency cell were resuspended to obtain a more uniform distribution. After 14 days, the CD3/CD28 Dynabeads were removed with a Dynal MPC-S Magnet (A13346, Thermo Fisher) according to manufacturer instructions, and cells counted. If TILs did not expand after 14 days, the initial expansion was extended or TIL cultures were discarded.

Human primary CD4⁺ T cells were obtained from peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors through the Infectious Diseases BioBank at King’s College London (ethics reference MM2-220518) under overall permission from the Southampton and South West Hampshire Research Ethics Committee (REC; B) (REC reference 19/SC/0232). PBMCs were isolated using density gradient centrifugation in SepMate tubes (STEMCELL Technologies) with Lymphoprep density gradient medium (STEMCELL Technologies). Total CD4⁺ T cells were isolated using a Human CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). CD4⁺ T cells were cultured in RPMI-1640 medium with GlutaMAX and HEPES supplemented with 10% heat-inactivated autologous human serum and 1% penicillin-streptomycin (Life Technologies). Cells were then activated using Dynabeads Human T-Activator CD3/CD28 (Life Technologies) and recombinant human interleukin-2 (30 U/mL; Roche) for 48 h prior to infection.
Activated CD4⁺ T cells were transduced with lentiviral vectors (1−10 ng of p24^Gag of vector per 50,000 cells). 48 h post-transduction, the cells were fixed in 4% paraformaldehyde in DPBS before assessing transduction efficiency by flow cytometry measuring intracellular GFP expression.