Basaal I - P31 - TNFR2-agonist facilitates high purity expansion of human Treg starting from low purity isolated Treg
X. He, S. Landman, S. Bauland, J. van den Dolder, H.J.P.M. Koenen, I. Joosten
Chair(s): dr. Ron W.F. de Bruin, Erasmus MC, Rotterdam
Thursday 10 march 2016
12:30 - 13:00h
at Foyer
Categories: Postersessie
Parallel session: Postersessies XI - Opgesplitst in 3 tijdblokken en 3 categoriëen (klinisch, basaal, donatie)
Due to the central role of T cell mediated regulation in the maintenance of allograft tolerance, Foxp3+ Treg are thought to be the most promising cell type for use as a tolerance inducing therapy. However, naturally occurring FOXP3+ Treg represent only a small fraction (<5%) of human peripheral blood CD4+ T cell. Thus, Treg must be isolated and expanded ex vivo for obtaining sufficient cells for therapeutic application in solid organ transplant patients. Although therapeutic Treg flow-sorting is feasible, most clinic centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNg and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNg producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Re-stimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either high purity FACS-sorted or low purity MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.
