Megan E. Himmel

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.

Helios+ and Helios− Cells Coexist within the Natural FOXP3+ T Regulatory Cell Subset in Humans

FOXP3-expressing T regulatory cells (Tregs) can be divided into two distinct subsets: naturally occurring Tregs (nTregs) that develop in the thymus, and induced Tregs (iTregs) that differentiate in peripheral tissues upon exposure to Ag in a tolerogenic environment. Recently it has been proposed that expression of Helios, an Ikaros family transcription factor, may specifically identify nTregs, allowing specific tracking of Tregs from different origins in health and disease. Surprisingly, we found that Helios- cells can be readily identified within naive (CD45RA+CD31+CCR7+CD62L+) FOXP3+ Tregs, a finding inconsistent with the notion that lack of Helios expression identifies Ag-experienced iTregs that should express memory markers. To investigate the phenotype and function of naive Helios+ and Helios− Tregs within the nTreg population, we isolated single-cell clones from each subset. We found that both Helios+ and Helios− nTreg clones have a similar suppressive capacity, as well as expression of FOXP3 and cell surface proteins, including CD39 and CTLA-4. Helios− nTregs, however, produced significantly more CCL3 and IFN-γ compared with Helios+ nTregs. Despite increased cytokine/chemokine production, Helios− FOXP3+ nTreg clones were demethylated at the FOXP3 Treg-specific demethylated region, indicative of Treg lineage stability. When cultured under Th1-polarizing conditions, Helios+ and Helios− nTreg clones had an equal ability to produce IFN-γ. Collectively, these data show that a lack of Helios expression does not exclusively identify human iTregs, and, to our knowledge, the data provide the first evidence for the coexistence of Helios+ and Helios− nTregs in human peripheral blood.

The role of T-regulatory cells and Toll-like receptors in the pathogenesis of human inflammatory bowel disease.

Two related chronic inflammatory diseases, Crohn’s disease and ulcerative colitis, are together often referred to as inflammatory bowel disease (IBD). Current treatment options are not curative, and patients face lifelong therapy and debilitation. IBD is thought to be the product of a combination of genetic and environmental factors that result in the abnormal regulation of immune responses. Experimental models have demonstrated that normal CD4+ T‐regulatory (Treg) cell responses and commensal bacteria are required for the maintenance of gut immune homeostasis. Recent evidence that CD4+ T cells express Toll‐like receptors (TLRs) and respond directly to TLR ligands, suggests that signals from commensal bacteria may directly affect T‐cell responses in the gut. In this review, we focus on evidence that defects in Treg cells may underlie IBD in humans. In addition, we discuss evidence that direct signaling via TLRs to T cells can affect IBD and that T‐cell‐dependent responses to bacterial proteins, such as flagellin, are central to the aetiology of this disease.

Regulatory T‐cell therapy for inflammatory bowel disease: more questions than answers

T regulatory (Treg) cells are critical for maintaining immune homeostasis and establishing tolerance to foreign, non‐pathogenic antigens including those found in commensal bacteria and food. Because of their multiple suppressive mechanisms, Tregs represent a promising strategy for engineering tolerance to self and non‐self antigens in chronic inflammatory diseases. Already in clinical trials in the transplantation setting, the question remains whether this therapy would be effective for the treatment of mucosal inflammatory diseases that do not pose an immediate threat to life. In this review we will discuss evidence from both animal models and patients suggesting that Treg therapy would be beneficial in the context of inflammatory bowel disease (IBD). We will examine the role of T‐cell versus Treg dysfunction in IBD and discuss the putative antigens that could be potential targets of antigen‐directed Treg therapy. Finally, the challenges of using Treg therapy in IBD will be discussed, with a specific emphasis on the role that the microbiota may play in the outcome of this treatment. As Treg therapy becomes a bedside reality in the field of transplantation, there is great hope that it will soon also be deployed in the setting of IBD and ultimately prove more effective than the current non‐specific immunosuppressive therapies.

Human CD4+FOXP3+ regulatory T cells produce CXCL8 and recruit neutrophils

One of the defining features of the majority of FOXP3+ Tregs is their inability to produce typical T‐cell‐derived cytokines. Little is known, however, about their capacity to produce chemokines. As Tregs are constitutively present in, and rapidly traffic to, non‐lympoid tissues, we hypothesized that they may produce chemokines to direct the composition of cells that infiltrate inflamed tissues. Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL‐8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2‐5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8‐dependent mechanism. These data provide the first evidence that although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells.

TLR5 is not required for flagellin-mediated exacerbation of DSS colitis

Background: The two forms of human inflammatory bowel disease, Crohns disease (CD) and ulcerative colitis (UC), are both associated with loss of tolerance to gut microbial antigens. The dominant antigen recognized by antibody and T‐cell responses in patients with CD is bacterial flagellin. Flagellin is also the only known ligand for Toll‐like receptor 5 (TLR5), a key protein in innate immunity. Although flagellin activates TLR5 to produce inflammatory responses in many cell types in the gut, there is conflicting evidence as to whether TLR5 is harmful or protective in CD and murine colitis models. A recent study found that administration of flagellin enemas to mice along with dextran sodium sulfate (DSS) made their colitis worse. Methods: We sought to determine whether this exacerbation was due to TLR5 ligation, or to TLR5‐independent adaptive immune responses to flagellin as an antigen, by using a transposon insertional mutant of the Escherichia coli H18 flagellin, 2H3, which lacks TLR5 stimulatory activity. Results: We found that flagellin enemas produced only a mild exacerbation of DSS colitis, and that 2H3 was equivalent to or worse than wildtype flagellin. Moreover, we found that DSS colitis was more severe in TLR5−/− mice than wildtype C57BL/6 mice. Conclusions: Together, these results suggest that flagellin‐mediated exacerbation of colitis is independent of TLR5. (Inflamm Bowel Dis 2010;)

The Stress signal extracellular ATP modulates antiflagellin immune responses in intestinal epithelial cells

Background: Although intestinal epithelial cells (IECs) are continually exposed to commensal microbes, under healthy conditions they contribute to intestinal homeostasis while keeping inflammatory responses in check. In response to invading pathogens, however, IECs respond vigorously by producing inflammatory mediators. To better understand the signals that regulate the inflammatory responses of IECs, we investigated whether the danger signal ATP (which is released from injured cells) could alter responses to bacterial products. Methods: We measured chemokine production from Caco‐2 cells stimulated with the Toll‐like receptor 5 agonist flagellin with or without ATP. ATP increased flagellin‐induced IL‐8 secretion but reduced CCL20 secretion via distinct signaling pathways. Results: ATP‐enhanced IL‐8 production was only partly blocked by the P2 receptor antagonist suramin and required activation of NF‐&kgr;B while ATP‐mediated reduction of CCL20 was completely blocked by suramin and required activation of ERK1/2. The effects of ATP on both chemokines required extracellular calcium but not phospholipase C, implicating P2X receptor involvement. To investigate how ATP alters IEC responses to bacterial products in vivo, mice receiving dextran sodium sulfate were given intrarectal flagellin with or without ATP. Addition of ATP to flagellin caused greater weight loss and increased antiflagellin antibody titers, as well as decreased colonic interferon gamma (IFN‐&ggr;) and higher antiflagellin IgG1/IgG2 ratios, which indicate decreased Th1 polarization. Conclusions: Together, these data indicate that stress, in the form of extracellular ATP, reshapes both the inflammatory response of flagellin‐stimulated IECs and downstream adaptive immunity, representing a possible strategy by which these cells differentiate between commensal and pathogenic bacteria. (Inflamm Bowel Dis 2011;)

Response to Comment on “Helios+ and Helios− Cells Coexist within the Natural FOXP3+ T Regulatory Cell Subset in Humans”

Ayyoub et al. raise two long-standing questions in their comment addressing the publication by Himmel et al. ([1][1]): what is the best way to define T regulatory cells (Tregs) from different origins in humans; and which population of Tregs is optimal for cell therapy applications? A consistent