Anti human cd28 functional grade purified

T effector cells, Tregs and HLA-DR-/CD14⁺ MDSC were isolated from Ficoll-separated PBMC by stepwise magnetic separation (MACS columns, Miltenyi Biotec Inc., Auburn, CA, USA). Teffector cells were isolated by CD4⁺ negative depletion (Miltenyi Biotec Inc..) and were together with Tregs separated by CD4⁺CD25⁺ Regulatory T Cell Isolation Kit, also from Miltenyi Biotec Inc.. For MDSCs, PMBCs were first incubated with Anti-HLA-DR Micro Beads and the HLA-DR negative population was further incubated with Anti-CD14 Micro Beads, both from Miltenyi Biotec Inc.. To evaluate the suppressive function of MDSCs, CFSE stained CD3⁺CD4⁺CD25⁻ (T-effectors) from MDS patients were stimulated by anti-human CD3 (OKT1, eBioscience) and anti-human CD28 (Functional grade purified, eBioscience) and cultured for 5 d under the following conditions: T-effectors alone, T-effectors, and Tregs (CD3⁺/CD4⁺/CD25^high) in 2:1 ratio; T-effectors and Tregs and MDSCs in 2:1:1ratio. To evaluate Tregs proliferation, the CD25²⁺ cells were stained with Violet proliferation dye (VPD). Cells were cultured in flat bottom wells in final concentration of 1 × 10⁶ cells/mL in Stem Span culture medium supplemented with GM-CSF (75 ng/mL). GM-CSF was added to support MDSCs in culture (personal communication with Professor Dmitri Gabrilovich, Section of Dendritic Cell Biology, H. Lee Moffitt Cancer Center).

The PBMCs (Allcells, Cat: PB005F) were resuspended in CTS^TM AIM V^TM SFM medium (GIBCO, Cat: A3021002) at a density of 1 × 10⁶ cells/ml, while 50 ng/ml anti-human CD3 functional grade purified (eBioscience, Cat: 160037–85) and 50 ng/ml anti-human CD28 functional grade purified (eBioscience, Cat: 160288–85) were added to activate T lymphocytes. The cells were then incubated at 37°C with 5% CO2 for 48 h. Concentrated lentiviruses were added to the cultured cells at a multiplicity of infection of 5, while 200 unit/ml of IL-2 (Shandong Quangang Pharmaceutical, Cat: S20020004) and 4 µg/ml polybrene (Sigma, Cat: h9268-5 g) were added and mixed well. The mixture was then incubated at 37°C with 5% CO₂ for 6–8 h, followed by centrifugation at 300 g for 5 min and replacement of the medium with IL-2-containing fresh CTS^TM AIM V^TM SFM medium. Fresh CTS^TM AIM V^TM SFM medium (with IL-2) was added every 2–3 days. Cell density was maintained at approximately 1 × 10⁶ cells/ml and amplified for 14 days.