Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Abstract

Treg1 cell therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs2 and induced (i)Tregs3 has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA)4. Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of Rapamycin (rapa)5 retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell (Tconv)6 proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors (HD)7. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the pro-inflammatory cytokine TNF-α, and were more efficient in suppressing synovial Tconv proliferation compared to their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step.

Epigenetic dynamics during CD4+ T cells lineage commitment

“…Several studies have shown that stable FoxP3 expression can be induced by several molecules such as TGF-␤, all-trans retinoic acid or rapamycin (Battaglia et al, 2005;Lu et al, 2014;Mucida et al, 2007;Rossetti et al, 2015;Zheng et al, 2007b) but it remains to be determined which extracellular stimuli and chromatin remodeling enzymes can be used to induce epigenetic changes that contribute to the stability of the Treg lineage.…”

Extra‐thymically induced T regulatory cell subsets: the optimal target for antigen‐specific immunotherapy

“…Pre‐clinical studies have revealed that adoptive transfer of CD4 + Treg cells can provide effective immune suppression. For example, transferring ex vivo expanded Foxp3 + t Treg cells can suppress inhibitory antibody formation in haemophilia, delay allograft rejection and protect against autoimmune cholangitis and rheumatoid arthritis . Focusing on Treg cells differentiated in vitro , adoptive transfer of IL‐10 Treg cells has been shown to protect against colitis, rheumatoid arthritis and CNS autoimmune disease, whereas Foxp3 + i Treg cells have been shown to suppress colitis, graft rejection, spontaneous abortion, graft‐versus‐host disease and CNS autoimmune disease (Table ) .…”

Characterization of proinsulin‐specific regulatory T cells in type 1 diabetes at different ages of onset

“…Another major limitation of our study was the lack of information on the stability of PI‐specific Tregs. The Tregs may exhibit plasticity in the proinflammatory environment and demethylation of the TSDR region of FOXP3 locus correlates with the expression of FOXP3 under proinflammatory conditions . Thus, methylation analysis of FOXP3‐TSDR locus of expanded PI‐specific Tregs could provide significant information on the stability of PI‐specific Tregs.…”