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

Nikolaos Skartsis,Yannick D Muller,Leonardo M R Ferreira,Yani Peng,Emilie Ronin,Qizhi Tang,Vinh Nguyen,Flavio Vincenti

doi:10.3389/fimmu.2021.783282

Nikolaos Skartsis, Yannick D Muller + Show 6 more

Open Access

https://doi.org/10.3389/fimmu.2021.783282

Copy DOI

Abstract

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 Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and 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 of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without 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 functions in vitro and in 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.

Highlights

Tregs are a subset of CD4+ T cells that prevent unwanted immune activation in steady state and promote resolution of immune response at the site of inflammation [1,2,3]
To determine the direct effects of TNFa and IL-6 on human Tregs, we challenged FACS purified CD4+CD25+CD127lo/- Tregs with IL-6 and/or TNFa, during in vitro activation
Combination of IL-6 and TNFa resulted in significantly improved Treg expansion, which mainly manifested during the later stage of culture (Figure 1B)

Summary

Introduction

Tregs are a subset of CD4+ T cells that prevent unwanted immune activation in steady state and promote resolution of immune response at the site of inflammation [1,2,3]. It has been suggested that Tregs may be destabilized under certain inflammatory conditions and there has been debate on the IL-6 and TNFa Promote Tregs impact of proinflammatory signals on Treg function [7]. A and IL-6 promote T cell activation [12, 13] and increase resistance to Treg-mediated suppression [14,15,16], both of these functions contribute to mounting an effective immune response. While there is consensus that TNFa and IL-6 heighten effector T cell (Teff) resistance to suppression by Tregs [17, 18], their direct impact on Tregs, especially on human Tregs, is unclear

Methods

Results

Conclusion