Development of functionally active recombinant form of mouse IL-35 (57.11)

Abstract

Abstract As a novel heterodimeric cytokine and one of the IL-12 family members, IL-35 is composed of IL-12p35 subunit and Epstein-Barr virus-induced gene 3 (EBI3) protein. IL-35 is known to play an essential role in immune regulation of the CD4+CD25+ Treg cells, alleviating inflammatory responses. We have developed the recombinant fusion construct of mouse IL-35 (mIL-35)-hIgG1 Fc (hFc), in which EBI3 and IL-12p35 are joined by a flexible linker of (Gly4Ser)3. The mIg k-chain leader sequence at the amino terminus also allows secretion of the fusion protein. The mIL-35 construct was stably expressed in HEK 293 cells, and culture supernatants of single clones were screened by ELISA using a combination of anti-hFc and anti-mEBI3 antibodies, among which the most positive clone 35-5 was selected and adapted in a serum-free culture system. Functional activity of the secreted mIL-35 from this clone was tested using the [3H]thymidine-based cell proliferation assay. When CD4+ T cells purified from TCR-transgenic DO11.10 mice were stimulated with a combination of syngeneic irradiated APCs and OVA peptide in the presence of the mIL-35 culture supernatants, the mIL-35 significantly suppressed CD4+ T cell proliferation. Furthermore, the mIL-35-mediated inhibition of CD4+ T cell proliferation was reversed by anti-mIL-35 antibody. Taken together, these results demonstrate that the recombinant mIL-35 is functionally active and can be a useful research tool to study T cell-based immune regulations.