Megan K. Levings

Interleukin-10-secreting type 1 regulatory T cells in rodents and humans

Summary:  Interleukin‐10 (IL‐10)‐secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL‐10 and transforming growth factor‐β(TGF‐β), and by their ability to suppress antigen‐specific effector T‐cell responses via a cytokine‐dependent mechanism. In contrast to the naturally occurring CD4+CD25+ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL‐10‐dependent process in vitro and in vivo. Specialized IL‐10‐producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL‐10‐producing T‐cell populations that are induced by IL‐10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4+CD25+ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.

Type 1 T regulatory cells

Summary: Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell‐to‐cell interactions and/or the production of interleukin (IL)‐10 and transforming growth factor (TGF)‐β. Type‐1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL‐10 and TGF‐β. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL‐10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL‐15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL‐10 and TGF‐β. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

IFN-α and IL-10 Induce the Differentiation of Human Type 1 T Regulatory Cells

CD4+ T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-α, but not TGF-β, induces naive CD4+ T cells derived from cord blood to differentiate into Tr1 cells: IL-10+IFN-γ+IL-2−/lowIL-4−. Naive CD4+ T cells derived from peripheral blood require both exogenous IL-10 and IFN-α for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-β. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-α strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.

Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor β, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells

T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4+ T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β. The relationship between CD25+CD4+ T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25+CD4+ T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25−CD4+ T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25+CD4+ T cells. Cloned human CD25+CD4+ T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25+CD4+ T cell clones with suppressive function produce IL-10, but all produce TGF-β. Suppression mediated by CD25+CD4+ T cell clones is partially dependent on TGF-β, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25+CD4+ T cells are distinct from IL-10–producing Tr1 cells.

The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs

Little is known about the molecules that control the development and function of CD4+ CD25+ Tregs. Recently, it was shown that the transcription factor FOXP3 is necessary and sufficient for the generation of CD4+ CD25+ Tregs in mice. We investigated the capacity of FOXP3 to drive the generation of suppressive CD4+ CD25+ Tregs in humans. Surprisingly, although ectopic expression of FOXP3 in human CD4+ T cells resulted in induction of hyporesponsiveness and suppression of IL-2 production, it did not lead to acquisition of significant suppressor activity in vitro. Similarly, ectopic expression of FOXP3delta2, an isoform found in human CD4+ CD25+ Tregs that lacks exon 2, also failed to induce the development of suppressor T cells. Moreover, when FOXP3 and FOXP3delta2 were simultaneously overexpressed, although the expression of several Treg-associated cell surface markers was significantly increased, only a modest suppressive activity was induced. These data indicate that in humans, overexpression of FOXP3 alone or together with FOXP3delta2 is not an effective method to generate potent suppressor T cells in vitro and suggest that factors in addition to FOXP3 are required during the process of activation and/or differentiation for the development of bona fide Tregs.

The Role of IL-10 and TGF-β in the Differentiation and Effector Function of T Regulatory Cells

Suppression by T regulatory (Tr) cells is essential for the induction of peripheral tolerance. Many types of CD4+ Tr cells have been described in a number of systems, and although the precise mechanisms which mediate their effects remain to be defined, it is well established that they can suppress immune responses via cell-cell interactions and/or the production of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). Type 1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-β, and these cytokines mediate their ability to suppress pathological immune responses in the settings of transplantation, allergy and autoimmune disease. Tr1 cell activity is not necessarily beneficial, and they can also suppress immune responses to antigens from tumours and pathogens. In vivo, the differentiation of Tr1 cells is likely controlled by certain dendritic cells which promote IL-10 production and may express tolerogenic costimulatory molecules. Another subset of CD4+ Tr cells is defined by constitutive expression of CD25, and although these CD4+CD25+ Tr cells appear to suppress via mechanisms which are largely independent of cytokines, they may actively promote the differentiation of Tr1 cells. Many questions about the basic biology of Tr1 cells remain to be answered, but the development of therapeutic strategies designed to harness their immunoregulatory effects can already be contemplated.

The role of different subsets of T regulatory cells in controlling autoimmunity

T regulatory cells-in addition to clonal deletion and anergy-are essential for the downregulation of T cell responses to both foreign and self antigens, and for the prevention of autoimmunity. Recent progress has been made in characterising the different subsets of T regulatory cells, the factors that drive their differentiation, and their mode of action. The resolution of these mechanisms will make it possible to use T regulatory cells therapeutically in human autoimmune diseases.

Human CD4+ T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+ T Regulatory Cells

Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25− T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.

CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL‐23 receptor (IL‐23R), CCR6, CD161, and the transcription factor retinoic acid‐related orphan receptor C (RORC), and originate from a CD161+CD4+ naïve T‐cell precursor in response to the combined activity of IL‐1β and IL‐23. We show here that not only CD4+TCRαβ+, but also CD8+TCRαβ+, CD4−CD8− TCRαβ+, and CD4−CD8− TCRγδ+ circulating lymphocytes that produce IL‐17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8+ and CD4−CD8− umbilical cord blood T cells that already express RORC and IL‐23R mRNA and that can be induced to differentiate into IL‐17‐producing cells in the presence of IL‐1β and IL‐23. Finally, we provide evidence that umbilical cord blood naïve CD4+CD161− T cells, upon lentivirus‐mediated transduction with RORC2 can acquire the ability to express IL‐23R, IL‐1RI, and CD161, as well as to produce IL‐17. Taken together, these data allow to conclude that T‐cell subsets able to produce IL‐17, as well as precursors of IL‐17‐producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2‐dependent.

CD4+ T‐regulatory cells: toward therapy for human diseases

T‐regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over‐abundance of Tregs can inhibit anti‐tumor and anti‐pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4+ Tregs: forkhead box protein 3 (FOXP3)+ Tregs and interleukin‐10‐producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3+ Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg‐based therapies are effective in humans.