The Effect of Various Stimulants on Cytokine Secretion Profiles in Freshly Isolated Peripheral Blood Mononuclear Cells (PBMC)

Abstract

Abstract Cytokines play critical roles in in cancer, in regulating autoimmune diseases and in chemically induced tissue damage repair. In response to antibody treatment or as a result of corona virus disease (COVID), a cytokine storm may occur where high levels of inflammatory cytokines are produced leading to organ damage. To understand how to regulate cytokine levels, primary cell in vitro assays may assess the effects of small molecule compounds or antibodies. We evaluated OKT3, Polyinosinic-polycytidylic acid (Poly I:C), Resiquimod, CpG oligodeoxynucleotides (CpG ODN), DynaBeads with aCD3/aCD28, Phytohemagglutinin (PHA)-M and lipoplolysaccharide (LPS) on cytokine secretion from 6 normal donors. PBMCs isolated from fresh blood using a density gradient medium were plated at 1 × 105 cells per well in the presence and absence of the test compounds for 48 hours. Supernatants were assessed for interleukin 2 (IL-2), tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). The remaining cells were assessed for viability to evaluate cytotoxicity. Flow cytometric profiles of T cell subsets were determined. The data generated from the 6 normal donors varied greatly, though the effects of the stimulants followed the same trends. The Dynabeads (CD3/28) generated the highest values of IL-2, IFNγ and TNFα in all donors. OKT3 and PHA-M, Resiquimod and LPS generated lesser amounts of the cytokines whereas Poly I:C and CpG ODN did not induce these cytokines over background levels. Only PHA-M caused a decrease in cell viability. Additionally, the release of cytokines was not associated with activation of T cells. Cytokine release assays provide a robust readout with certain stimuli and may facilitate evaluation of novel test compounds for functional activity.