Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo

Abstract

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter.

Keywords: Pichia Pastoris expression; Tregs; diphtheria toxin; fusion toxin; murine IL-2.