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Ivy Dambuza, Chengrong Yu, Rashid Mahdi, Renxi Wang, Sung-Hye Kim, Charles E. Egwuagu, ; IL-12p35 Inhibits Th1 proliferation and promotes the expansion of Th17 and Treg. Invest. Ophthalmol. Vis. Sci. 2013;54(15):101.
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The IL-12 family cytokines have emerged as an important regulator of host immunity and in particular T-helper lineage commitment. It is comprised of IL-12, IL-23, IL-27, IL-35 and each member consists of an α (p19, p28, p35) and a β (p40, Ebi3) subunit. Although the bioactivities of each heterodimer cytokine are well characterized, little is known about functions of the independent α and β subunits. In this study we addressed the effects of IL-12p35 subunit in T and B cell activation and differentiation.
Full-length mouse IL-12p35 was cloned and expressed in High Five insect cells. The secreted recombinant IL-12p35 (rIL-12p35) was purified on His-Trap columns and size exclusion HPLC chromatography; characterized by non-reducing PAGE and western blotting. Naïve CD4+ T cells were cultured under Th0, Th1, Th2, Th17, or Treg polarizing condition in presence or absence of rIL-12p35. CD19+ B cells were sorted and activated with LPS in the presence or absence of rIL-12p35. Proliferation and effector functions of T or B cells were analyzed by [3H]-thymidine incorporation assay, FACS and ELISA.
Two forms of rIL-12p35 were generated, a 35 kDa monomeric rIL-12p35 and a 70 kDa rIL-12p35 homodimer. Both forms were immuno-reactive to an IL-12p35-specific antibody on Western blot gels. Both rIL-12p35 forms inhibited proliferation of TCR-activated CD4 T cells and LPS-activated CD19+ B cells, with rIL-12p35 monomer displaying more suppressive activity. Their suppressive activity correlated with up-regulation of IL-10 secretion in the culture supernatants. Interestingly, while rIL-12p35 suppressed proliferation of Th1 cells, it promoted the expansion of Th17 and Treg with no effect on Th2 cells.
Data presented here show that rIL-12p35 has biological activities that extend beyond its role as a component of the heterodimeric cytokine, IL-12 or IL-35. The ability of rIL-12p35 to suppress proliferation of activated T and B cells and induce lymphocytes to secrete the anti-inflammatory cytokine, IL-10, makes it an attractive therapeutic candidate for use in restraining the excessive inflammatory responses that occur during autoimmune diseases, such as uveitis.
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