Stimulated Cytokine Production (PMBC)
Mitogen-stimulated production of cytokines was used to assess T-cell function.
Blood samples for the assessment of stimulated cytokine production were collected on two occasions: 2 months before Quarantine, during the screening physical; and on Quarantine Day 0, prior to viral challenge.
The Luminex 100 multiplex bead-based immunoassay system, which is based on the principle of solid phase sandwich immunoassays, was used to measure stimulated production of 10 cytokines: Interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and granulocyte-macrophage colony stimulating factor (GM-CSF). Multiplex analysis permits simultaneous quantification of different cytokines in a small sample volume. Luminex multiplex technology has been demonstrated to be a valid alternative method for the quantification of cytokines1. Peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) for production of IL-1β or phytohemagluttenin (PHA) for production of the remaining 9 cytokines at a final concentration of 20 µg/mL and incubated at 37oC for 48 hours in the atmosphere of 5% CO2 in air. PHA-stimulated and unstimulated plasma samples were analyzed using Biosource multiplex immunoassay kits (Biosource International, Camarillo, CA), which contained combined antibodies for 10 different cytokines.
The kits were validated by the manufacturer and determined to be free of antibody cross-reactivity. All reagents, working standards, and samples were prepared as per the manufacturer’s specifications and were run in duplicates. The plates were read within 24 hours using the Bio-plex Reader (Luminex 100, Luminex Corporation, Austin, TX). Stimulated cytokine concentrations were determined using Bio-Plex Manager Software (Bio-rad Corporation, Hercules, CA), interpolating from the standard curve (Logistic-5PL curve fit, Brendan Technologies, Carlsbad, CA). Pooled plasma controls also were included on all plates to further assure assay reliability. In all cases, stimulated cytokine production was quantified by subtracting cytokine levels in unstimulated samples from the stimulated levels.
Cytokine assays were performed at the Immunologic Monitoring and Cellular Products Laboratory (IMCPL), University of Pittsburgh Cancer Institute. The IMCPL has an established quality assurance/quality control program for cytokine determinations and a long-standing expertise in cytokine measurements for both clinical and research programs.
1 DuPont, N.C., Wang, K., Wadhwa, P.D., Culhane, J.F., & Nelson, E.L. (2005). Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: Determinants of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology, 66, 175–191.