title: A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells.
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.
authors.affiliation: Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Xuehui
authors.identifier: http://orcid.org/0000-0002-3801-4091
authors.initials: X
authors.lastname: He
authors.affiliation: Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Sija
authors.initials: S
authors.lastname: Landman
authors.affiliation: Sanavisie Bodyclinic, Mill, The Netherlands.
authors.forename: Stijn C G
authors.initials: SC
authors.lastname: Bauland
authors.affiliation: Hycult Biotech, Uden, The Netherlands.
authors.forename: Juliette
authors.initials: J
authors.lastname: van den Dolder
authors.affiliation: Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Hans J P M
authors.initials: HJ
authors.lastname: Koenen
authors.affiliation: Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Irma
authors.initials: I
authors.lastname: Joosten
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000819:agonists*; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D016207:Cytokines; D047889:Receptors, Tumor Necrosis Factor, Type II; C415902:TNFRSF1B protein, human; D020123:Sirolimus