Significance: Natural regulatory T cells (nTregs) play important roles in preventing autoimmune diseases, but they may be unstable in the presence of inflammation. Here we report that all - trans RA (atRA) but not rapamycin prevents human nTregs from converting to Th1/Th17 cells and sustains their suppressive function in inflammatory environments. Adoptive transfer of nTregs pretreated with atRA enhances their suppressive effects on xenograft-vs. - host diseases. Moreover, we show that atRA suppresses IL-1 receptor upregulation, accelerates IL-6 receptor downregulation, and affects the epigenetic modifications in Foxp3 locus in nTregs following inflammatory stimulation. We suggest that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated diseases.

Objective: Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow–derived mesenchymal stem cells (BM‐MSCs) may have the potential to control or even prevent RA, but BM‐MSC–based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen‐induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva‐derived mesenchymal stem cells (G‐MSCs) would significantly improve the therapeutic effects.Methods: CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G‐MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti‐CD25 antibody) was used to deplete Treg cells in arthritic mice.Results: Infusion of G‐MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down‐regulated the production of inflammatory cytokines (interferon‐γ and interleukin‐17A). Infusion of G‐MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios‐negative cells. When Treg cells were depleted, infusion of G‐MSCs partially interfered with the progression of CIA. Pretreatment of G‐MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G‐MSCs on CIA.Conclusion: The role of G‐MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G‐MSCs provide a promising approach for the treatment of autoimmune diseases.

Objective: Transferred CD4+CD25+FoxP3+ Treg cells can prevent autoimmune disease, but generally fail to ameliorate established disease. This study was undertaken to compare the effects of antigen‐specific Treg cells induced with interleukin‐2 (IL‐2) and transforming growth factor β (TGFβ) ex vivo (induced Treg [iTreg] cells) to the effects of equivalent expanded thymus‐derived natural Treg (nTreg) cells on established collagen‐induced arthritis (CIA).Methods: CIA was induced in DBA/1 mice by immunization with type II collagen (CII), and before or shortly after immunization, mice were treated with iTreg or nTreg cells that were generated or expanded in vitro. Clinical scores were determined. Inflammatory responses were determined by measuring the levels of anti‐CII antibody in the serum and examining the histologic features of the mouse joints. The Th1/Th17‐mediated autoreactive response was evaluated by determining the cytokine profile of the draining lymph node (LN) cells of the mice by flow cytometry.Results: Following transfer, nTreg cells exhibited decreased FoxP3 and Bcl‐2 expression and decreased suppressive activity, and many converted to Th17 cells. In contrast, transferred iTreg cells were more numerous, retained FoxP3 expression and their suppressive activity in the presence of IL‐6, and were resistant to Th17 conversion. Notably, 10 days after the transfer of donor iTreg cells, predominance was shifted from Th17 cells to Treg cells in the draining LNs of recipient mice.Conclusion: These findings provide evidence that transferred TGFβ‐induced iTreg cells are more stable and functional than nTreg cells in mice with established autoimmunity. Moreover, iTreg cells can have tolerogenic effects even in the presence of ongoing inflammation. The therapeutic potential of human iTreg cells in subjects with chronic, immune‐mediated inflammatory diseases should be investigated.

It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4⁺Foxp3⁺ regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs. Addition of atRA to naïve CD4⁺CD25⁻ cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3⁺ iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4⁺ cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4⁺ cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4⁺ cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4⁺ cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered. We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Background Although natural regulatory T cells (nTregs) can suppress osteoclastogenesis, the role of TGF-β-induced CD4+Foxp3+ Tregs (iTregs) in osteoclastogenesis remains unknown. Objective To determine the effects of iTregs on osteoclastogenesis in vitro and on bone erosion in vivo in collagen-induced arthritis (CIA). Methods Osteoclastogenesis was induced in bone marrow CD11b+ cells with receptor activator of nuclear factor κ B (NF-κB) ligand (RANKL) and macrophage colony stimulating factor. Graded doses of Tregs were added to inhibit osteoclastogenesis. Transwell and antibody blockade experiments were performed to assess the roles for cell contact and soluble cytokines. NF-κB activation was determined by western blot. iTregs or nTregs were adoptively transferred to mice with CIA to assess in vivo effects on disease incidence and bone erosion, the latter determined by CT scanning. Results Both nTregs and iTregs greatly suppressed osteoclastogenesis in vitro, but only iTregs sustained this effect when interleukin-6 was present. iTregs, but not nTregs, significantly suppressed development of CIA. Bone erosions in iTregs-treated mice were diminished compared with untreated mice or nTregs-treated mice. Treatment with iTregs, but not with nTregs, dramatically decreased NF-κB p65/p50 levels in osteoclasts in vitro and p65/50 and RANKL expression by synovial tissues in vivo. Conclusion iTregs may be therapeutically beneficial in rheumatoid arthritis and related diseases associated with bone erosions.

Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4⁺FoxP3⁺) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.