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Ren-Xi Wang, Cheng-Rong Yu, Rashid Mahdi, Charles E. Egwuagu; Genetically Engineered Novel Il12p40/Il27p28 Heterodimeric Protein Inhibits Lymphocyte Proliferation and Th1 effector Functions. Invest. Ophthalmol. Vis. Sci. 2011;52(14):410.
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IL-12 family cytokines have emerged as an important family of proteins in host immunity. They are comprised of related α chain (p19, p28 or p35) and β chain (p40 or EBI3) proteins and combinatorial pairing resulting from covalent and non-covalent binding between α and β subunits gave rise to heterodimeric cytokines such as IL-12 (IL12p35/IL12p40); IL-23 (IL12p40/IL23p19); IL-27 (IL27p28/EBI3) or IL-35 (IL12p35/EBI3). Thus, generation of new combinations of IL-12 heterodimeric proteins may form the basis of a new class of therapeutic cytokines. In this study, we have genetically engineered a novel IL12p40/IL27p28 heterodimeric protein with intrinsic immunoregulatory properties.
Mouse IL27p28 and IL12p40 full-length cDNA fragments were cloned into a bicistronic vector containing FLAG-IRES and V5-His sequences and the IL12p40/IL27p28 heterodimeric protein of approximately 75kDa was expressed in insect cells and purified on Ni-NTA column and by centrifugation on centricon filtration units. Functional characterization of IL12p40/IL27p28 was accomplished by a variety of techniques including non-reducing polyacrylamide gel electrophoresis, western blot, RT-PCR, Thymidine incorporation, Annexin-V staining and intracellular cytokine staining assays.
We show that IL12p40/IL27p28 inhibited the proliferation of Conconavalin A or TCR-stimulated CD4+T cells. IL12p40/IL27p28 also inhibited transcription of genes coding for ICE, TNF-α, IFN-γ and to a lesser extent IL-17 and Foxp3 expression. The inhibitory effects on TCR-activated T cells were mediated through selective targeting of STAT1 but not STAT3 or STAT4.
Inhibition of lymphocyte proliferation and T cell effector functions by the novel ~75 kDa IL12p40/IL27p28 heterodimeric protein is an important proof-of-principle experiment that establishes that novel pairing of α and β IL-12 family protein subunits may lead to development of a new class of therapeutic cytokines.
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