Angela M. Thornton

Expression of Helios, an Ikaros Transcription Factor Family Member, Differentiates Thymic-Derived from Peripherally Induced Foxp3+ T Regulatory Cells

Helios, a member of the Ikaros transcription factor family, is preferentially expressed at the mRNA level by regulatory T cells (Treg cells). We evaluated Helios protein expression using a newly generated mAb and demonstrated that it is expressed in all thymocytes at the double negative 2 stage of thymic development. Although Helios was expressed by 100% of CD4+CD8−Foxp3+ thymocytes, its expression in peripheral lymphoid tissues was restricted to a subpopulation (∼70%) of Foxp3+ T cells in mice and humans. Neither mouse nor human naive T cells induced to express Foxp3 in vitro by TCR stimulation in the presence of TGF-β expressed Helios. Ag-specific Foxp3+ T cells induced in vivo by Ag feeding also failed to express Helios. Collectively, these results demonstrate that Helios is potentially a specific marker of thymic-derived Treg cells and raises the possibility that a significant percentage of Foxp3+ Treg cells are generated extrathymically.

CD4+CD25+ Regulatory T Cells Can Mediate Suppressor Function in the Absence of Transforming Growth Factor β1 Production and Responsiveness

CD4+CD25+ regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4+CD25−T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact–dependent and cytokine independent. The role of TGF-β1 in CD4+CD25+ suppressor function remains unclear. While most studies have failed to reverse suppression with anti–transforming growth factor (TGF)-β1 in vitro, one recent study has reported that CD4+CD25+ T cells express cell surface TGF-β1 and that suppression can be completely abrogated by high concentrations of anti–TGF-β suggesting that cell-associated TGF-β1 was the primary effector of CD4+CD25+-mediated suppression. Here, we have reevaluated the role of TGF-β1 in CD4+CD25+-mediated suppression. Neutralization of TGF-β1 with either monoclonal antibody (mAb) or soluble TGF-βRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4+CD25+ T cells. Responder T cells from Smad3−/− or dominant-negative TGF-β type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-β1–induced growth arrest, were as susceptible to CD4+CD25+-mediated suppression as T cells from wild-type mice. Furthermore, CD4+CD25+ T cells from neonatal TGF-β1−/− mice were as suppressive as CD4+CD25+ from TGF-β1+/+ mice. Collectively, these results demonstrate that CD4+CD25+ suppressor function can occur independently of TGF-β1.

tTregs, pTregs, and iTregs: similarities and differences

Foxp3+ T‐regulatory cells (Tregs) are primarily generated in the thymus (tTreg), but also may be generated extrathymically at peripheral sites (pTreg), or induced in cell culture (iTreg) in the presence of transforming growth factor β (TGFβ). A major unresolved issue is how these different populations of Tregs exert their suppressive function in vivo. We have developed novel systems in which the function of Tregs can be evaluated in vivo in normal mice. Our studies demonstrate that one prominent mechanism of action of polyclonal tTregs is to inhibit T‐effector cell trafficking to the target organ, while antigen‐specific iTregs primarily prevent T‐cell priming by acting on antigen‐presenting dendritic cells (DCs). Interleukin‐10 (IL‐10) plays an important role in the suppressive function of antigen‐specific iTregs by controlling the expression of MARCH1 and CD83 on the DC. Activated tTregs may mediate infectious tolerance by delivery of cell surface‐expressed TGFβ to naive responder T cells to generate pTregs. Manipulation of Treg function will require the ability to differentiate tTregs from pTregs and iTregs. The expression of the transcription factor Helios has proven to be a useful marker for the identification of stable tTregs in both mouse and human.

Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion

Foxp3(+) regulatory T cells (Tregs) maintain self-tolerance and adoptive therapy, and using Foxp3(+) Tregs has been proposed as treatment for autoimmune diseases. The clinical use of Tregs will require large numbers of cells and methods for in vitro expansion of Tregs are being developed. Foxp3(+) Tregs can be divided into 2 subpopulations based on expression of the transcription factor, Helios. Foxp3(+)Helios(+) Tregs (70%) are thymic-derived, whereas Foxp3(+)Helios(-) Tregs (30%) are induced in the periphery. Foxp3(+)Helios(+) Tregs differ from Foxp3(+)Helios(-) Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression on days to weeks of expansion in vitro. Addition of a 25 mer DNA oligonucleotide of random composition for a short period during the expansion of Foxp3(+) Tregs in vitro results in prolonged stabilization of the Foxp3(+)Helios(+) subpopulation and yields an optimal population for use in cellular biotherapy.

Cutting Edge: CD4 T Cell-Mast Cell Interactions Alter IgE Receptor Expression and Signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4+CD25+ regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4+ T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcεRI expression. Despite the known inhibitory functions of IL-10 and TGFβ1, FcεRI suppression was independent of IL-10 and TGF-β1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C4 production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcεRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.

The Deubiquitinase CYLD Targets Smad7 Protein to Regulate Transforming Growth Factor β (TGF-β) Signaling and the Development of Regulatory T Cells

Background: CYLD is a deubiquitinating enzyme (DUB) that hydrolyzes Lys-63-linked polyubiquitin chains that are attached covalently to cellular proteins. Results: CYLD knock-out mice have increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. Conclusion: CYLD regulates lysine 63-linked ubiquitination of Smad7 to control the development of peripheral Tregs. Significance: TGF-β signaling in T cells is regulated by lysine 63-Linked ubiquitination. CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-β led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4+ cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-β signaling function in T cells and the development of Tregs through deubiquitination of Smad7.

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice.

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4+CD25+T regulatory cells are able to limit the effectiveness of CD4+T cells and are able to favor the maintenance and the progression of B. abortus infection.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos−/−) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos−/− Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos−/− mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4+ Tconvs. Eos−/− mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

CD4+ CD5+regulatory T cells render naive CD4+ CD25-T cells anergic and suppressive: Induced regulatory T cells

CD4+u2003CD25+u2003Foxp3+ naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4+u2003CD25– T cells from T‐cell receptor (TCR) transgenic mice on a RAG deficient (RAG–/–) background in the presence of anti‐CD3 and interleukin‐4 (IL‐4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non‐responsive to restimulation with anti‐CD3 or exogenous IL‐2, and could not up‐regulate IL‐2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti‐CD3 but responded to restimulation with IL‐2. The undivided population was capable of suppressing the response of fresh CD4+u2003CD25– T cells and CD8+ T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti‐transforming growth factor‐β partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4+u2003CD25– responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.