Stimulated human peripheral T cells produce high amounts of IL-35 protein in a proliferation-dependent manner.
The p35 subunit of IL-12 and the Epstein-Barr virus-induced gene 3 (EBI3) have been shown to form a heterodimeric cytokine, named interleukin-35 (IL-35). Recently, mRNA expression of both IL-12p35 and EBI3 was clearly shown in stimulated human T effector cells. Here, we investigated the production of IL-35 protein in human anti-CD3/CD28-stimulated pan T cells as well as T cell subpopulations using a specific human IL-35 ELISA system. We measured high concentrations of IL-35 (up to 3 ng/ml) in cell culture supernatants of stimulated pan T cells as well as CD4(+), CD8(+) and CD4(+)CD25(-) T cell subpopulations at 72 h after stimulation. Very low amounts of IL-35, in the range of 100pg/ml, were detectable in supernatants of resting T cells. These observations could be confirmed using a dot-blot assay for IL-12p35 and EBI3. High concentrations of IL-35 could be also measured in cell culture supernatants of both, resting and stimulated CD4(+)CD25(+) T cells. In order to learn more about the regulation of IL-35 production, we studied the effect of dexamethasone, cyclosporine A and rapamycin on IL-35 production of anti-CD3/CD28-stimulated human pan T cells as well as CD4(+) and CD8(+) T cell subpopulations. All three drugs significantly suppressed IL-35 production of these cells in a proliferation-dependent manner. In summary, we could show that stimulated human peripheral blood T cells of healthy donors produce high amounts of IL-35 protein. However, the biological function of this cytokine remains to be elucidated.