MLR assay

Monocytes were purified from fresh human blood. Briefly, human PBMCs were isolated using a Ficoll gradient and allowed to adhere to the plate for 2 h, after which cells in suspension were removed. Fresh AIM V medium (Thermo Fisher Scientific) was used without activation or stimulation reagents. mAb1, mAb2, or isotype control antibodies were separately incubated with monocytes at 10 µg/ml for 48 h. Treated monocytes were then cocultured with viably thawed CD4 T cells (Biological Specialty Corporation) at a ratio of 10:1 (T cells to monocyte-derived dendritic cells) in an MLR. The MLR was cultured for 5 d, after which the cells were analyzed by flow cytometry using an LSRFortessa X-20 instrument (BD Biosciences) to determine cell numbers and functional cytokine (IFNγ) responses. Secreted IFNγ was analyzed using a human IFNγ AlphaLISAγ Detection Kit per the manufacturer’s recommendations (PerkinElmer).