Ox40 apc

AIM assays were performed as previously described ^{43 (link)}. Briefly, cryopreserved PBMC were thawed, washed, resuspended in R10, and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and subsequently treated with CD40 blocking antibody (Miltenyi Biotec, cat. no. 130–094-133) for a final concentration of 0.5ug/mL for 15 minutes at 37°C. Cells were incubated in the presence or absence of our 10-LRA panel, as previously described. After an 18hr incubation, cells were harvested and stained for 50 minutes at 4°C with the surface staining monoclonal antibodies (mAb); (PD-L1-PE/Cy7 (Biolegend, cat. no. 329717), CD40L-PE (BD Biosciences [BD], cat. no. 561720), OX40-APC (BD cat. no. 563473), CD69-BV650 (Biolegend cat. no. 310933), CD3-BV605 (Biolegend cat. no. 317322), CD4-BV421 (BD cat. no. 562424), CD8-PerCp-Cy5.5 (BD cat. no. 560662), CD25- BUV395 (BD cat. no. 564034) and LIVE/DEAD Near-IR stain (ThermoFisher cat. no. L34975), washed, fixed and permeabilized (BD cytofix fixation buffer, cat. no. 554655), and then stained for 30 minutes at 4°C with intracellular mAb Acetyl-histone H3-Alexa Fluor 488 (Cell Signaling Technology, cat. no. 9683S) in 1x perm/wash buffer (BD, cat. no. 554723). The cells were washed and fixed (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis.

CpG ODN 1826 (5ʹ-tccatgacgttcctgacgtt-3ʹ) and isotype control were purchased from InvivoGen (San Diego, CA, USA). Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin solution, phosphate-buffered saline (PBS) and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Matrigel was purchased from Corning (Corning, NY, USA). The TUNEL Apoptosis Detection Kit was purchased from KeyGen BioTECH (Nanjing, Jiangsu, China). The T Cell Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies including CD45-PerCP-Cy5.5, CD3e-BV510, CD4-FITC, CD25-PE, OX40-APC, CD8a-FITC, CD11b-BV421, Gr-1-APC, F4/80-PE, CD8a-APC, CD44-PE-Cy7, CD62L-PE, IFN-γ-PE, and FoxP3-eFluor450 were purchased from BD Biosciences (San Jose, CA, USA) and Biolegend (San Diego, CA, USA).