IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function

• DOI: 10.3389/fimmu.2021.783282
• Publication Date: 2021-12-23T06:30:41Z
• Authors: Nikolaos Skartsis; Yani Peng; Leonardo M. R. Fe...; Vinh Nguyen; Emilie Ronin; Yannick D. Muller; Flavio Vincenti; Qizhi Tang
• Keywords: Adult; Male; CD28 signaling; Immunology; Primary Cell Culture; Graft vs Host Disease; Tregs; Immunotherapy, Adoptive; T-Lymphocytes, Regulatory; Etanercept; Ikaros Transcription Factor; Mice; 03 medical and health sciences; TNFα; Animals; Humans; Cells, Cultured; Aged; Cell Proliferation; IL-6; 0303 health sciences; Interleukin-6; Forkhead Transcription Factors; RC581-607; Middle Aged; metabolomics; Healthy Volunteers; 3. Good health; Disease Models, Animal; inflammation; Female; Immunologic diseases. Allergy

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregsex-vivowith pro-inflammatory cytokines IL-6 and TNFαand observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion ofTNF receptor 2using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFαwithout aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functionsin vitroandin vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.