Pitfalls in determining the cytokine profile of human T cells

Secretion of cytokines by T cells in vitro can be influenced by the methods chosen for T cell activation. However, the awareness of this fact appears insufficient. Two of the most widely applied methods for activation of T cells are phorbol 12-myristate 13-acetate (PMA) together with Ionomycin or anti-CD3/anti-CD28 stimulation. We analyzed production of IL-4, IFN-γ, IL-17 and IL-10 by a panel of human CD4 T-cell clones isolated from intestinal biopsies using the Bio-Plex™ assay and also flow-cytometry for the latter three cytokines. Higher levels of IL-17 and IFN-γ were produced by stimulation with PMA/Ionomycin compared to anti-CD3/anti-CD28. Some T-cell clones which were assigned to produce both cytokines by stimulation with PMA/Ionomycin, were only assigned to produce IFN-γ by anti-CD3/anti-CD28 stimulation. IL-10 production was higher after anti-CD3/anti-CD28 stimulation. Furthermore the dose response curve for PMA/Ionomycin differed for IL-10 compared to IL-17 and IFN-γ as it was biphasic with no IL-10 production at higher PMA/Ionomycin concentrations. These results demonstrated that the cytokine profile may be differently determined depending on the assay and conditions used and illustrate that care should be taken when designing and interpreting studies of cytokine production by T cells.

► Cytokine production from CD4 T-cell clone was dependent on stimulation method.
► Anti-CD3/anti-CD28 induced high amounts of IL-10.
► High concentration of PMA/Ionomycin induced large amounts of IL-17 and IFN-γ.
► IL-10 production after PMA/Ionomycin stimulation had a biphasic dose–response curve.