Abstract
Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most 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 IFNγ 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 IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation 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 FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.
Highlights
Following identification of Treg, the immunomodulating role of Treg was demonstrated in a variety of preclinical autoimmunity and transplantation models
We found that the combined use of Tumour necrosis factor receptor 2 (TNFR2)-agonist and rapamycin promotes Treg proliferation rates, enhances TSDR demethylation and increases both Treg stability and function in vitro
Low purity Treg expanded in the presence of TNFR2-agonist plus rapamycin suppressed in vivo inflammation in a humanized mouse model
Summary
Following identification of Treg, the immunomodulating role of Treg was demonstrated in a variety of preclinical autoimmunity and transplantation models. We and others have evidence that pharmaceutical agents influence Treg phenotype and functional capacity [12,13,14], indicating that by delicate selection of pharmaceutical agents it is possible to further support the stability of human Treg In this respect, the mTOR inhibition by rapamycin is an interesting example, since it has been shown to promote preferential outgrowth of highly suppressive Treg [4, 14, 15]. We show that expansion of low purity MACS-isolated human Treg in the presence of TNFR2-agonist and rapamycin results in a stable homogenous FOXP3+, Helios+, CD127low Treg population that shows profound suppressor potential both in vitro, and in vivo in a preclinical humanized mouse model. A TNFR2-agonist based expansion protocol shows great potential for ex vivo Treg expansion for clinical purposes
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