Date Published: May 25, 2016
Publisher: Public Library of Science
Author(s): Xuehui He, Sija Landman, Stijn C. G. Bauland, Juliette van den Dolder, Hans J. P. M. Koenen, Irma Joosten, Gilles Marodon.
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.
Following identification of Treg, the immunomodulating role of Treg was demonstrated in a variety of preclinical autoimmunity and transplantation models. Their clinical relevance was highlighted by demonstrating that the immunosuppressive function of Treg was hampered in autoimmunity and allergy. Clinical application of Treg has been hampered by the paucity of Treg cell numbers and the fact that standard methods of ex vivo Treg expansion produce heterogeneous cell populations . For clinical application of Treg-based immunotherapy isolation of Treg using a good manufacturing practice (GMP) system is required. Clinical grade flow-sorting which retrieves highly pure Treg is restricted to a few clinic centers worldwide. In contrast, magnetic bead isolation of CD4+CD25+ Treg using a GMP compliant closed system, such as CliniMACS, that results in lower Treg purity  is more generally used. For Treg expansion most centers apply polyclonal expansion protocols making use of anti-CD3 plus anti-CD28 mAb stimulation in the presence of rhIL-2 together with or without rapamycin [2–8]. This results in a heterogeneous Treg population revealing inadvertent pro-inflammatory (IL-17A, IFNγ) cytokine producing potential . The fact that human Treg could lose FOXP3 expression and suppressive functions and acquire the capacity to produce pro-inflammatory cytokines under pro-inflammatory micro-environmental conditions [10, 11] might have important implication for Treg-based clinical therapy. Therefore, it is essential to develop highly efficacious expansion protocols that promote strong Treg proliferation whilst maintaining or promoting Treg stability and suppressor function. We and others have evidence that pharmaceutical agents influence Treg phenotype and functional capacity [12–14], 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]. In contrast to effector T cells (Teff), Treg are less sensitive to mTOR inhibition by rapamycin since Treg proliferation and survival preferentially depends more on the STAT5  and Pim kinase pathways .
An important issue for Treg-based immunotherapy is to maintain stability and suppressive function of Treg during and after ex vivo expansion and following their transfer into patients. Although clinical grade high purity Treg isolation by GMP flow cytometry is available in a few medical centres worldwide, most clinic centres use GMP qualified magnetic bead based isolation techniques that result in limited Treg purity. Hence, in this study we focused on optimizing an ex vivo Treg expansion protocol that produces high numbers of stable potent human Treg starting with low purity magnetic bead isolated Treg. We found that the combined use of 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.