Song Guo Zheng

Natural and Induced CD4+CD25+ Cells Educate CD4+CD25− Cells to Develop Suppressive Activity: The Role of IL-2, TGF-β, and IL-10

Thymus-derived, natural CD4+CD25+ regulatory T cells can educate peripheral CD4+CD25− cells to develop suppressive activity by poorly understood mechanisms. TGF-β has IL-2-dependent costimulatory effects on alloactivated naive, human CD4+ T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4+CD25+ cells are the targets of the costimulatory effects of IL-2 and TGF-β. These cells do not divide, but, instead, greatly increase the numbers of CD4+CD25− cells that become CD25+ cytokine-independent suppressor cells. These CD4+CD25+ regulatory cells, in turn, induce other alloactivated CD4+CD25− cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-β and IL-10. The suppressive effects of these secondary CD4+CD25+ cells depend upon TGF-β and IL-10. Moreover, both the naive CD4+ cells induced by IL-2 and TGF-β to become suppressor cells, and the subsequent CD4+CD25− cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4+CD25+ regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4+ regulatory T cells in vivo.

IL-2 Is Essential for TGF-β to Convert Naive CD4+CD25− Cells to CD25+Foxp3+ Regulatory T Cells and for Expansion of These Cells

IL-2 and TGF-β both have important roles in the induction and maintenance of immunologic tolerance, but whether these cytokines act separately or together to achieve this effect is poorly understood. Although others have reported that IL-2 can directly enhance forkhead box protein P3 (Foxp3) transcription factor expression by natural CD4+CD25+ regulatory T cells, in this study, we report that the role of IL-2 on the generation of peripheral regulatory CD4+ cells is indirect. Ab neutralization studies and experiments with IL-2-deficient mice have revealed that IL-2 is required for TGF-β to induce naive CD4+CD25− cells to become CD25+ and express Foxp3, and develop the characteristic properties of CD4+CD25+ regulatory cells. This effect of IL-2 on the generation and expansion of these adaptive Foxp3+ regulatory cells is nonredundant, but IL-4, IL-7, and IL-15, other common γ-chain cytokines, could sustain Foxp3 expression. Because subjects with autoimmune diseases often have defects in the production of IL-2 and/or TGF-β, the generation of autologous T regulatory cells ex vivo with these cytokines for transfer in vivo may have considerable therapeutic potential.

Generation Ex Vivo of TGF-β-Producing Regulatory T Cells from CD4+CD25− Precursors

Previously we reported that TGF-β has an important role in the generation and expansion of human “professional” CD4+CD25+ regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF-β induces activated CD4+CD25− T cells to become Th3 suppressor cells. While stimulating CD4+ cells with TGF-β modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF-β the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF-β-primed CD4+ T cells to fresh CD4+ cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF-β and was also partially contact dependent. Increased TGF-β production was associated with a decreased production of IFN-γ and IL-10. Depletion studies revealed that the precursors of these TGF-β-producing CD4+ suppressor cells were CD25 negative. These studies provide evidence that CD4+CD25+ regulatory cells in human blood consist of at least two subsets that have TGF-β-dependent and independent mechanisms of action. TGF-β has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4+ and CD8+ cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection.

Natural and TGF-β–induced Foxp3+CD4+ CD25+ regulatory T cells are not mirror images of each other

Foxp3(+) CD4(+) CD25(+) regulatory cell (Treg) subsets that maintain immunologic homeostasis have been considered to be a homogeneous population of naturally occurring, thymus-derived CD4(+)CD25(+) cells (nTregs). However, similar Foxp3+ Tregs can be induced from CD25(-) precursors in vivo, and ex vivo with interleukin 2 (IL-2) and transforming growth factor beta (TGF-beta) (iTregs). These two subsets differ in their principal antigen specificities and in the T-cell receptor signal strength and co-stimulatory requirements needed for their generation. However, whether iTregs have any unique functions in vivo has been unclear. Although IL-6 can convert nTregs to Th17 cells, iTregs induced by IL-2 and TGF-beta are resistant to this cytokine and thereby might retain suppressive function at inflammatory sites. Thus, nTregs and iTregs may have different roles in the adaptive immune response.

Cutting Edge: Foxp3+CD4+CD25+ Regulatory T Cells Induced by IL-2 and TGF-β Are Resistant to Th17 Conversion by IL-6

TGF-β has pleiotropic effects on T cell differentiation that are determined by other cytokines in the local environment. Whereas IL-2 and TGF-β induce naive T cells to become forkhead/winged helix transcription factor (Foxp3) positive regulatory cells (iTregs), the combination of IL-6 and TGF-β induces IL-17-producing cells (Th17). Moreover, IL-6 can use TGF-β produced by thymus-derived natural regulatory T cells (nTregs) to convert them to Th17 cells. In this study, we report a major difference between iTregs and nTregs. Treatment of iTregs with IL-6 did not affect Foxp3 expression, and their suppressive activity in vitro and in vivo was intact. To explain this difference between nTregs and iTregs, we found that IL-2 and TGF-β down-regulate IL-6 receptor expression and IL-6 signaling. The resistance of iTregs to Th17 conversion suggests that they can function more effectively than nTregs in an inflammatory milieu and emphasizes the central role of IL-2 in combination with TGF-β to maintain immunologic homeostasis.

TGF-β Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-β could not induce CD4+CD25− cells from CTLA-4−/− mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-β to induce FoxP3-expressing mouse suppressor cells. TGF-β accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-β to induce CD4+CD25− cells to express FoxP3 and develop suppressor activity. TGF-β also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4−/− mice, the lack of TGF-β-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.

CD4+ and CD8+ Regulatory T Cells Generated Ex Vivo with IL-2 and TGF-β Suppress a Stimulatory Graft-versus-Host Disease with a Lupus-Like Syndrome

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 × C57BL/6) F1 mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-β induced both CD4+ and CD8+ cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-β-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-β-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.

The role of the combination of IL-2 and TGF-β or IL-10 in the generation and function of CD4+ CD25+ and CD8+regulatory T cell subsets

Recently, considerable attention has been focused on thymus‐derived CD4+ regulatory T cells that constitutively express CD25 and have a contact‐dependent, cytokine‐independent mechanism in vitro. However, peripheral CD4+ and CD8+ T cells can also be induced to become regulatory T cells. Here we review our studies using the combination of IL‐2 and transforming growth factor β (TGF‐β) to generate regulatory T cell subsets ex vivo, and the work of others using IL‐10 to induce suppressive activity. Under certain conditions, the autocrine effects of TGF‐β and IL‐10 induce peripheral T cells to produce immunosuppressive levels of each of these cytokines. This effect of TGF‐β is IL‐2 dependent. Under other conditions IL‐2 and TGF‐β can induce CD4+ cells to develop potent contact‐dependent, cytokine‐independent regulatory activity. At present, there is considerable confusion concerning the mechanism of action of CD4+ CD25+ cells because cytokine‐producing regulatory T cells generated in the periphery can express CD25 and other markers displayed by naturally occurring, thymus‐derived regulatory T cells. We, therefore, propose a nomenclature that identifies thymus‐derived and peripheral regulatory cells, and that also differentiates T regulatory cells from T helper cells. Because T regulatory cells broadly control T helper cell reactivity, the mechanisms that control regulatory cell function are also reviewed. Finally, the potential use of regulatory T cells generated ex vivo as an adoptive immunotherapy for certain autoimmune diseases, to prevent organ graft rejection, or to prevent pathologic host responses to infectious agents is discussed.

Cutting Edge: All-Trans Retinoic Acid Sustains the Stability and Function of Natural Regulatory T Cells in an Inflammatory Milieu

Recent studies have demonstrated that plasticity of naturally occurring CD4+Foxp3+ regulatory T cells (nTregs) may account for their inability to control chronic inflammation in established autoimmune diseases. All-trans retinoic acid (atRA), the active derivative of vitamin A, has been demonstrated to promote Foxp3+ Treg differentiation and suppress Th17 development. In this study, we report a vital role of atRA in sustaining the stability and functionality of nTregs in the presence of IL-6. We found that nTregs treated with atRA were resistant to Th17 and other Th cell conversion and maintained Foxp3 expression and suppressive activity in the presence of IL-6 in vitro. atRA decreased IL-6R expression and signaling by nTregs. Of interest, adoptive transfer of nTregs even from arthritic mice treated with atRA suppressed progression of established collagen-induced arthritis. We suggest that nTregs treated with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.

Role of SMAD and Non-SMAD Signals in the Development of Th17 and Regulatory T Cells

Whereas TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-β–dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.