An In Vitro test battery using human whole blood for immunotoxicity hazard identification: Proof of concept studies with dexamethasone and benzo(a)pyrene

Abstract

Immunotoxicity assessment is nearing a crossroads predicated on mounting pressure for reduction/replacement of animals in toxicology. This has fueled the development of alternative New Approach Methodologies (NAMs) for hazard identification. This work details a comprehensive human whole blood NAM battery for immunotoxicity testing. The test system is a closed tube culture containing whole blood diluted 1:3 in culture media with or without an immune stimulant, anti-CD3/CD28 or viral peptide pool. Model immunotoxicants dexamethasone (DEX; 0 – 1.94 μM) and benzo(a)pyrene [B(a)P; 0–6.3 μM], were added to the test system for 24 hours. Immune cells were identified and counted by flow cytometric immunophenotyping and assessed for natural killer (NK) cell activity and T cell activation. Supernatants were interrogated for proinflammatory cytokine concentrations. In vitro treatment with DEX resulted in concentration-dependent suppression of cytokine production, NK cell activity, and T cell activation induced by anti-CD3/CD28, as well as viral-induced cytokine production. B(a)P caused suppression of cytokine production and a nonsignificant reduction in T cell activation but did not impact NK cell activity, however, immunosuppression by B(a)P only occurred following metabolic activation by S9 fraction enzymes. Advantages of this NAM battery include assessment of adaptive immunity and direct translation of immunotoxicity to viral host resistance. These results provide evidence of in vitro immunotoxicity that reflect known outcomes from in vivo studies. This multi-endpoint human whole blood NAM battery should be useful for screening compounds for immunotoxicity hazard identification without reliance on animal systems for increased translatability to humans.