N-803 hastens T-cell activation and allogenic rejection in mixed lymphocyte reactions. (A) MLR experimental design. Briefly, PBMCs were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and incubated with unmatched, irradiated PBMCs with or without IL-2 and increasing concentrations of N-803. NK cells from stimulator donor were 12/15/18-activated and allowed to differentiate into ML-NK in parallel. (B-C) CD8 T cells were examined by flow cytometry from days 4 through 11 after incubation with allogenic stimulators. (B) Representative histograms depicting proliferation (CFSE dilution) and activation markers CD25 and CD38. (C) Summary data from panel B. (D) Summary data from CD4 T cells as examined in panels B and C. (E-I) ⁵¹CR-release killing assays using MLR-stimulated PBMCs (R; responder) as effectors against allogeneic in vitro differentiated ML NK cells as targets. (E) Killing assay schema. PBMCs were harvested from MLR on day 10 and cocultured with ⁵¹Cr-pulsed ML NK cells (matched to original allogenic stimulator [S] cells; S-ML NK) and killing assessed. (F) Summary data from panel E. (G-I) PBMCs incubated with IL-2 and 35 ng/mL N-803 were incubated with anti-MHC-I (G), anti-FAS-L (H), or anti-TRAIL (I) before addition of labeled ML-NK targets and allogenic killing assessed in the presence of blocking antibodies. N = 6 normal donor responders, 4 normal donor stimulator/targets in 2 independent experiments. Data were analyzed using 2-way analysis of variance, *P < .05, **P < .01, ***P < .001. Unless indicated (purple asterisk), statistics are for each condition, compared with IL-2 only condition. Purple asterisk indicates significance for 35 ng/mL N-803 + IL-2 compared with IL-2 only. Mean is depicted with error represented as standard error of the mean.