Anti human cd3 and anti human cd28 antibodies

For the human one-way MLR, 1 × 10⁴ human immature Mo-DCs were seeded in a round-bottomed 96-well plate and then incubated in the presence or absence of wogonin (1 μM) in 100 μL of RPMI 1640 medium containing 10% human serum for two days. Following incubation, the Mo-DCs were washed once and then cocultured with 1 × 10⁵ HLA-DR nonshared allogeneic CD4⁺ naive T cells in RPMI 1640 medium supplemented with 10% human serum in a round-bottomed 96-well plate for seven days. At seven days after incubation, the cells were washed twice and stimulated with anti-human CD3 and anti-human CD28 antibodies (BD Pharmingen, San Diego, U.S.A.) for 24 h. The supernatant was then collected for the IFN-γ, IL-4, IL-5, and IL-17 ELISA.

The infectious HIV-1 clone (pNL4-3) was obtained through the AIDS Research and Reference Reagent Program, NIAID, NIH, USA. The let-7i promoter reporter (pGL4-let-7i) was kindly bestowed by Dr. Ashish Lal (Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA). pRL-TK renilla luciferase control reporter vector was from Promega (USA). A perfect binding sequence of let-7i was inserted into the 3′ UTR of luciferase gene of the pMIR-REPORT™ miRNA Expression Reporter Vector (Promega, USA) to generate pMIR-REPORT-let-7i_bindingsite construct.
Anti-human CD3 and anti-human CD28 antibodies were from BD Biosciences (Palo Alto, CA). The anti-IL-2 and anti-CD95 antibodies for flow cytometry analysis were from BD Biosciences (Palo Alto, CA). Annexin-V cell apoptosis assay kit was from Keygen (Nanjing, China).
The let-7i mimics and negative control (mm-NC) were purchased from RiboBio (Guangzhou, China). let-7i mimic sequence: 5′-UGAGGUAGUAGUUUGUGCUGUU-3′; NC mimic sequence: 5′-UUUGUACUACACAAAAGUACUG-3′. The let-7i antisense inhibitor and control were purchased from GenePharma (Shanghai, China). Small interfering RNAs (siRNAs) smart pool used to specifically target human IL-2 mRNA were synthesized by RiboBio (Guangzhou, China).