Shohei Hori

Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species

Bacteria of the genus Clostridium promote the induction of suppressor T cells in the colons of mice. CD4+ T regulatory cells (Tregs), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor–β and affected Foxp3+ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4+ CD45RBhi T cells in Rag1−/− mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host–microbe interactions establish immunological homeostasis in the gut.

Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.

Summary:  Naturally arising CD25+CD4+ regulatory T (Treg) cells, most of which are produced by the normal thymus as a functionally mature T‐cell subpopulation, play key roles in the maintenance of immunologic self‐tolerance and negative control of a variety of physiological and pathological immune responses. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3‐expressing natural Tregs, whether they are CD25+ or CD25–, activates even weak or rare self‐reactive T‐cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)‐2 for their survival in the periphery. In addition to Foxp3 and IL‐2/IL‐2 receptor, deficiency or functional alteration of other molecules, expressed by T cells or non‐T cells, may affect the development/function of Tregs or self‐reactive T cells, or both, and consequently tip the peripheral balance between the two populations toward autoimmunity. Elucidation of the molecular and cellular basis of this Treg‐mediated active maintenance of self‐tolerance will facilitate both our understanding of the pathogenetic mechanism of autoimmune disease and the development of novel methods of autoimmune disease prevention and treatment via enhancing and re‐establishing Treg‐mediated dominant control over self‐reactive T cells.

Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization

Interleukin (IL)-2 plays a crucial role in the maintenance of natural immunologic self-tolerance. Neutralization of circulating IL-2 by anti–IL-2 monoclonal antibody for a limited period elicits autoimmune gastritis in BALB/c mice. Similar treatment of diabetes-prone nonobese diabetic mice triggers early onset of diabetes and produces a wide spectrum of T cell–mediated autoimmune diseases, including gastritis, thyroiditis, sialadenitis, and notably, severe neuropathy. Such treatment selectively reduces the number of Foxp3-expressing CD25+ CD4+ T cells, but not CD25− CD4+ T cells, in the thymus and periphery of normal and thymectomized mice. IL-2 neutralization inhibits physiological proliferation of peripheral CD25+ CD4+ T cells that are presumably responding to normal self-antigens, whereas it is unable to inhibit their lymphopenia-induced homeostatic expansion in a T cell–deficient environment. In normal naive mice, CD25low CD4+ nonregulatory T cells actively transcribe the IL-2 gene and secrete IL-2 protein in the physiological state. IL-2 is thus indispensable for the peripheral maintenance of natural CD25+ CD4+ regulatory T cells (T reg cells). The principal physiological source of IL-2 for the maintenance of T reg cells appears to be other T cells, especially CD25low CD4+ activated T cells, which include self-reactive T cells. Furthermore, impairment of this negative feedback loop via IL-2 can be a cause and a predisposing factor for autoimmune disease.

Preferential Generation of Follicular B Helper T Cells from Foxp3+ T Cells in Gut Peyer's Patches

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyers patches through a process that requires the presence of CD4+ follicular B helper T(TFH) cells. The nature of these TFH cells in Peyers patches has been elusive. Here, we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyers patches. The conversion of Foxp3+ T cells into TFH cells requires the loss of Foxp3 expression and subsequent interaction with B cells. Thus, environmental cues present in gut Peyers patches promote the selective differentiation of distinct helper T cell subsets, such as TFH cells.

Heterogeneity of natural Foxp3+ T cells: A committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity

Natural regulatory T cells (Treg) represent a distinct lineage of T lymphocytes committed to suppressive functions, and expression of the transcription factor Foxp3 is thought to identify this lineage specifically. Here we report that, whereas the majority of natural CD4+Foxp3+ T cells maintain stable Foxp3 expression after adoptive transfer to lymphopenic or lymphoreplete recipients, a minor fraction enriched within the CD25− subset actually lose it. Some of those Foxp3− T cells adopt effector helper T cell (Th) functions, whereas some retain “memory” of previous Foxp3 expression, reacquiring Foxp3 upon activation. This minority “unstable” population exhibits flexible responses to cytokine signals, relying on transforming growth factor-β to maintain Foxp3 expression and responding to other cytokines by differentiating into effector Th in vitro. In contrast, CD4+Foxp3+CD25high T cells are resistant to such conversion to effector Th even after many rounds of cell division. These results demonstrate that natural Foxp3+ T cells are a heterogeneous population consisting of a committed Treg lineage and an uncommitted subpopulation with developmental plasticity.

Foxp3 Inhibits RORγt-mediated IL-17A mRNA Transcription through Direct Interaction with RORγt

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.

Regulatory T cells: Recommendations to simplify the nomenclature

c o rr e s p o n d e n c e npg

Control of Autoimmunity by Naturally Arising Regulatory CD4+ T Cells

Naturally acquired immunological self-tolerance is not entirely accounted for by clonal deletion, anergy, and ignorance. It is now well established that the T cell-repertoire of healthy individuals harbors self-reactive lymphocytes with a potential to cause autoimmune disease and these lymphocytes are under dominant control by a unique subpopulation of CD4+ T cells now called regulatory T cells. Efforts to delineate these Treg cells naturally present in normal individuals have revealed that they are enriched in the CD25+ CD4+ population. The identification of the CD25 molecule as a useful marker for naturally arising CD4+ regulatory T cells has made it possible to investigate many key aspects of their immunobiology, including their antigen specificities and the cellular/molecular pathways involved in their development and their mechanisms of action. Furthermore, reduction or dysfunction of the CD25+ CD4+ regulatory T cell population can be responsible for certain autoimmune diseases in humans.

Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice

CD25+4+ regulatory T cells (Treg) play an indispensable role in preventing autoimmunity. Little is known, however, about the antigen specificities required for their development and effector functions. Mice transgenic for an anti-myelin basic protein (MBP) T cell antigen receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE) when deficient for the RAG-1 gene (T/R−), whereas RAG-1-competent transgenic animals (T/R+) remain healthy, protected by CD4+ Treg-expressing endogenous TCRs. We have now investigated the role and specificity of CD25+4+ Treg in this system. The results show that T/R+ animals contain MBP-specific suppressive CD25+4+ cells, whereas T/R− do not. Adoptive transfer of CD25+4+ cells from nontransgenic or T/R+ donors into T/R− mice prevented the development of EAE. Surprisingly, transfer of nontransgenic CD25+4+ cells purified from T/R+ donors conferred only a limited protection, possibly because of their restricted repertoire diversity that we demonstrate here. Absence of transgenic CD25+4+ cells in animals deficient for endogenous TCRα chains and analyses of endogenous TCR gene expression in subsets of CD4+ cells from T/R+ mice demonstrate that development of transgenic MBP-specific CD25+4+ Treg depends on the coexpression of endogenous TCRα chains. Taken together, these results indicate that specificity to MBP is required for effector functions but is not sufficient for thymic selection/commitment of CD25+4+ Treg preventing EAE.