The role of lncRNA bound to IκBα and p65 in the sensitivity to AICD of tumor-specific T cells

Analysis of p65 and IκBα binding lncRNA profile in T cells primed by MDA-MB-231-lysate pulsed DCs for 6 days. Results provide insight into the molecular mechanisms underlying tumor-mediated AICD in T cells. Mononuclear cells were obtained from peripheral blood of three HLA-A2+ healthy donors and cultured for 14 d in serum-free X-VIVO 20 medium (BioWhittaker, Walkersville, Maryland) with human GM-CSF (50 ng/ml; Peprotech) and IL-4 (20 ng/ml; Peprotech). Non-adherent DCs were enriched by depletion of contaminating T and B lymphocytes and pulsed for 20 h with lysates (200 μg protein/1 × 10^6 cells/ml) from MDA-MB-231 (HLA-A2+) that were lysed by 5 freeze/thaw cycles. T cells were incubated for 13 d in RPMI-1640 with 10% human AB serum (PromoCell) and IL-2 (25 U/ml; Peprotech) followed by incubation in the same medium with antigen-specific DCs (5:1) for 6 days. T cells were retrieved from the cocultures at day 6.T cells were lysed with RIP lysis buffer supplemented with protease inhibitor cocktail and RNase Inhibitor (1000U/ml, Ambion), followed by incubation with p65, IκBα or control IgG antibody-coated microbeads at 4℃ overnight.After the immunoprecipitation, the coprecipitated RNA were extracted. The quality of extracted RNA was assessed using the NanoDrop ND-2000 spectrophotometer.