AUTHOR=Urbano Paulo C. M. , Koenen Hans J. P. M. , Joosten Irma , He Xuehui 

TITLE=An Autocrine TNFα–Tumor Necrosis Factor Receptor 2 Loop Promotes Epigenetic Effects Inducing Human Treg Stability In Vitro

JOURNAL=Frontiers in Immunology

VOLUME=Volume 9 - 2018

YEAR=2018

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2018.00573

DOI=10.3389/fimmu.2018.00573

ISSN=1664-3224

ABSTRACT=A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28-superagonist stimulation in vitro, in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3+CD28 stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro. Combined stimulation with rapamycin and TNFR2-agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2-agonist mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28-superagonist, in the presence of rapamycin and a TNFR2-agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα - TNFR2 underlying the maintenance of Treg stability in vitro. Addition of rapamycin to CD28-superagonist stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2-agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28-superagonist. Interestingly, rapamycin seemed to downregulate CD28-superagonist induced EZH2 expression, which could be rescued by the additional addition of TNFR2-agonist antibody. This process appeared TNFα dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα - TNFR2 loop plays an important role in endorsing Treg stability.