TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNFα and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNFα may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. ### Competing Interest Statement QT is a co-founder and scientific advisor of Sonoma Biotherapeutics. QT, NS and FV are coinventors of a patent on manufacturing Tregs based on results from this work. The remaining authors have no relevant competing interests to disclose. * aCD3/28 : anti-CD3 and anti-CD28 dynabeads CD28SA : CD28 super agonist CNS : Central Nervous System FAO : Fatty acid oxidation FOXP3 : Forkhead box protein P3 GVHD : Graft-versus-host-disease GSH : glutathione GSSG : glutathione disulfide LTα : lymphotoxin α NSG : NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ OXPHOS : oxidative phosphorylation RORγT : Retinoic acid-related orphan receptor γT Tconv : conventional CD4+ T cell Teff : effector T cell TNFR1 : Tumor necrosis factor receptor 1 TNFR2 : Tumor necrosis factor receptor 2 TNFSF : Tumor necrosis factor superfamily TCR : T cell receptor TSDR : Treg-specific demethylated region