in that they are unable to activate p38 upon peptidoglycan ingestion. Interestingly, Relish mutants, in which IMD-dependent AMP tran-scription is abolished, are not impaired in their ability to activate p38. This suggests that the IMD pathway bifurcates ‘downstream’ of IMD into an inhibitor of NF-κB (IκB) kinase–Relish–dependent branch required for AMP produc-tion, and an IκB kinase–Relish–independent branch necessary for p38 activation and subse-quent Duox transcriptional activation (Fig. 1). Further experiments are needed to understand the precise molecular mechanisms through which peptidoglycan-dependent activation of IMD leads to MEKK1 activation. It is likely that the kinase Tak1, which acts ‘downstream’ of IMD in this signaling cascade, is involved in this process.

The authors then focus their attention on the peptidoglycan-independent branch. They show that cultured drosophila cells treated with dou-ble-stranded RNA directed against PLC-β fail to activate p38 after incubation with peptido-glycan-depleted microbial extracts. This finding indicates that PLC-β is involved in intracellular Ca2+ mobilization that leads to stimulation of DUOX activity and in the peptidoglycan-inde-pendent activation of the MEKK1-MKK3-p38 pathway that leads to Duox induction. Together, these results demonstrate that a peptidoglycan-independent PLC-β pathway and a peptido-glycan-dependent IMD pathway merge into MEKK1 to create a novel p38-dependent Duox expression pathway (Fig. 1). More work will be needed to understand how MEKK1 is activated by PLC-β and to identify the G protein–coupled receptor that acts ‘upstream’ of this cascade.

As indicated above, commensal bacteria–derived peptidoglycan is able to induce Relish translocation in a PGRP-LC-dependent way. Unexpectedly, however, Ha et al. observe that PGRP-LC-dependent activation of p38 does not occur in wild-type gut cells, which sug-gests that peptidoglycan-dependant expres-sion of Duox in repressed in uninfected guts. Notably, they report that in PLC-β mutants, p38 is constitutively phosphorylated in a way dependent on the presence of the commensal microbiota. Thus, in addition to its positive effect on DUOX enzymatic activity, PLC-β also prevents p38 activation induced by commen-sal microbiota. This striking result identifies a PLC-β-dependent feedback-inhibitory circuit needed to avoid excessive Duox expression in the presence of the normal gut microbiota. Through a series of in vitro and in vivo experi-ments, Ha et al. demonstrate that this inhibi-tory circuit involves sequential induction of the Ca2+-dependent phosphatase calcineurin B and the dual phosphatase MKP3, which inhibit p38 activation and dampen Duox transcription (Fig. 1). Experiments with MKP3 loss-of-function mutants clearly show that this negative regula-tion is essential in vivo for maintaining a ben-eficial commensal microbe–gut interaction. Silencing of MKP3 expression specifically in gut epithelial cells results in abnormally high ROS responses to commensal microbiota; the consequence of this is greater gut cell death and a shorter lifespan.

Together, these findings demonstrate that drosophila gut innate immune responses rely on a finely tuned balance between positive and negative inputs controlled by two regula-

tory pathways. One is initiated by an uniden-tified G protein–coupled receptor and signals through PLC-β, whereas the other is triggered by PGRP-LC and signals via IMD. These two pathways allow the host to adapt its intestinal antimicrobial response to the gut microbial load. Given the conserved function of pep-tidoglycan as elicitor of immune responses and in the establishment of beneficial recip-rocal host-microbe interactions4,11, and the conserved function of G protein–coupled receptor–PLC-β–mediated activation of p38 in the regulation of epithelial immune responses in the animal kingdom12–14, this paradigm of intestinal innate immune homeostasis provides a blueprint for host-microbe interactions that is probably conserved in other metazoans, includ-ing mammals.

1. Ley, R.E., Peterson, D.A. & Gordon, J.I. Cell 124, 837–848 (2006).

2. Ha, E.-M. et al. Nat. Immunol. 10, 949–957 (2009).

3. Lemaitre, B. & Hoffmann, J. Annu. Rev. Immunol. 25, 697–743 (2007).

4. Ryu, J.H. et al. Science 319, 777–782 (2008).5. Maillet, F., Bischoff, V., Vignal, C., Hoffmann, J. &

Royet, J. Cell Host Microbe 3, 293–303 (2008).6. Lhocine, N. et al. Cell Host Microbe 4, 147–158

(2008).7. Zaidman-Remy, A. et al. Immunity 24, 463–473

(2006).8. Bischoff, V. et al. PLoS Pathog. 2, e14 (2006).9. Ha, E.M., Oh, C.T., Bae, Y.S. & Lee, W.J. Science 310,

847–850 (2005).10. Ha, E.M. et al. Dev. Cell 16, 386–397 (2009).11. Koropatnick, T.A. et al. Science 306, 1186–1188

(2004).12. Ren, M., Feng, H., Fu, Y., Land, M. & Rubin, C.S.

Immunity 30, 521–532 (2009).13. Ziegler, K. et al. Cell Host Microbe 5, 341–352

(2009).14. Powell, J.R., Kim, D.H. & Ausubel, F.M. Proc. Natl.

Acad. Sci. USA 106, 2782–2787 (2009).

938 volume 10 number 9 september 2009 nature immunology

Regulatory T cells that become autoaggressiveDaniel Hawiger & Richard A Flavell

Foxp3 expression is not stable and may be extinguished both in vitro and in vivo in regulatory T cells that convert into proinflammatory effector T cells. The loss of Foxp3 in regulatory T cells under autoimmune conditions may result in the conversion of suppressor T cells into highly autoaggressive lymphocytes.

Daniel Hawiger is in the Department of

Immunobiology, Yale University School of Medicine,

New Haven, Connecticut, USA. Richard A. Flavell is

in the Department of Immunobiology and Howard

Hughes Medical Institute, Yale University School of

Medicine, New Haven, Connecticut, USA.

e-mail: richard.flavell@yale.edu

Foxp3 is a transcription factor that serves a critical role in the homeostasis of the

immune system through its crucial function in

maintaining the suppressor phenotype of natu-ral regulatory T cells (Treg cells)1,2. Foxp3 is also expressed in some activated peripheral CD4+ T cells3,4, and although some such adaptive or induced Treg cells have true suppressor activity, new expression of Foxp3 does not always con-fer a regulatory phenotype on a T cell5. Foxp3 has been proposed to amplify and maintain the genetic ‘signature’ of Treg cells induced by various developmental and environmental cues rather than acting as a ‘master switch’

that turns on the suppressor phenotype6,7. Moreover, Foxp3 expression in Treg cells is not stable and may be extinguished both in vitro and in vivo in Treg cells that divert from their original phenotype and become, for example, interleukin 17–producing helper T cells8,9 or follicular helper T cells10. However, the impli-cations of such unstable Foxp3 expression on the immune response have remained uncertain. In this issue of Nature Immunology, Bluestone and colleagues11 reveal that the loss of Foxp3

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in Treg cells under autoimmune conditions may result in the conversion of suppressor T cells into highly autoaggressive lymphocytes.

The authors have developed an elegant double-transgenic reporter experimental sys-tem to monitor the fates of T cells that either continue to express or have ceased to express Foxp3 in vivo. This genetic fate-mapping sys-tem is based on two components: a bacterial artificial chromosome transgene expressing a fusion protein of green fluorescent protein (GFP) and Cre recombinase from the Foxp3 promoter, and a Rosa26-YFP reporter in which expression of yellow fluorescent protein (YFP) is dependent on Cre-mediated excision of a loxP-flanked stop cassette. Therefore, acti-vation of the Foxp3 promoter induces expres-sion of GFP and Cre, and the latter leads to irreversible and permanent expression of YFP from a constitutively active Rosa26 promoter. The authors confirm faithful expression of the GFP-Cre fusion protein in Foxp3+ Treg cells and ascertain that such T cells also express YFP, as the expression of YFP is induced by Cre. Consequently, the majority of mature Foxp3+ T cells express both GFP and YFP, but only negligible numbers of Foxp3– cells express GFP-Cre in this mouse model.

Surprisingly, the authors also observe 10–20% of GFP–YFP+ cells among all YFP+ T cells in thymus and periphery in the double- transgenic reporter mice. Although such ‘exFoxp3’ cells are Foxp3–, their Foxp3 promot-ers must have been active at some point to allow expression of the GFP-Cre that is required for the induction of YFP expression. The exFoxp3 cells exhibit a phenotype distinct from Foxp3+ cells in that they show uniformly low expres-sion of CD25 and the immunomodulatory receptor GITR and heterogeneous expression of other Treg cell markers such as CTLA-4 or CD103. In addition to the loss of the phe-notypic characteristics of Treg cells, exFoxp3 cells acquire some new features, including the ability to produce effector cytokines such as interferon-γ and interleukin 17. Importantly, the exFoxp3 cells do not seem to represent some default ‘end-stage’ state in the develop-ment of Treg cells. Instead, the authors present compelling evidence for the active induction of exFoxp3 cell formation during an ongoing autoimmune process.

In their experiments, the authors cross double-transgenic reporter mice with non-obese diabetic mice that develop spontaneous insulitis and diabetes. They find that such mice have a higher percentage of exFoxp3 cells and a lower percentage of GFP+YFP+ Treg cells in the pancreas than in pancreatic or inguinal lymph nodes. This enrichment of exFoxp3 cells seems to be driven at least in part by recognition of specific autoantigens, as higher percentages of exFoxp3 cells are found in the pancreatic lymph nodes of mice transgenic for the BDC2.5 T antigen cell receptor (TCR), in which T cells bear a TCR specific for a pancreatic islet self antigen. Perhaps most remarkably, these exFoxp3 cells are not just ‘innocent bystanders’ generated by the ongoing autoimmune process. Instead, these cells are themselves autoaggres-sive and may contribute to autoimmune dis-ease. Indeed, BDC2.5 TCR–transgenic exFoxp3 cells are diabetogenic and are able to induce insulitis and diabetes when transferred into T cell–deficient nonobese diabetic mice.

One very interesting issue raised by this study is the origin of the exFoxp3 cells in vivo. By performing direct transfer experiments, the authors establish that natural Treg cells can become exFoxp3 cells. At the same time, analy-sis of the TCR repertoire in double-transgenic reporter mice suggests that some exFoxp3 cells might be derived from adaptive or induced Treg cells that initially developed from conventional T cells in the periphery. Therefore, exFoxp3 cells probably represent a heterogeneous population, a possibility reflected by the strik-ing diversity of effector cytokines produced by exFoxp3 cells. Interestingly, like conven-tional CD4+ T cells, a majority of exFoxp3 cells exhibit CpG methylation of the Foxp3 locus. This observation raises the question of whether exFoxp3 cells can undergo a process of remethylation at their Foxp3 loci as they cease to express Foxp3.

On the basis of their findings, Bluestone and colleagues put forth the provocative and highly interesting hypothesis that at least in some cases, autoimmunity does not simply overcome the regulatory component of the immune system but instead develops as a direct consequence of suppressor T cells’ becoming autoaggressive effector T cells (Fig. 1). Such an idea underscores the need for effective Treg cell–

based immunotherapy as a potential substitute for the Treg cells that may have been lost during autoimmunity. But at the same time, caution should be taken in plans to introduce Treg cells that could potentially downregulate Foxp3 expression and become harmful in situ. Given the growing number of reports describing the induction of antigen-specific Treg cells in the periphery and the developmental instability of some Treg cells3,8,9, we might further speculate that immune homeostasis may be maintained by switching of specific T cells among various activated, regulatory and effector or memory functional compartments. Therefore, more research on the functional state of antigen-specific T cells under different conditions is needed to understand how they contribute to the collective actions of other T cell clones in the same functional subset.

1. Fontenot, J.D., Gavin, M.A. & Rudensky, A.Y. Nat. Immunol. 4, 330–336 (2003).

2. Sakaguchi, S. Annu. Rev. Immunol. 22, 531–562 (2004).

3. Apostolou, I. et al. J. Clin. Immunol. 28, 619–624 (2008).

4. Curotto de Lafaille, M.A. & Lafaille, J.J. Immunity 30, 626–635 (2009).

5. Shevach, E.M., Tran, D.Q., Davidson, T.S. & Andersson, J. Eur. J. Immunol. 38, 915–917 (2008).

6. Gavin, M.A. et al. Nature 445, 771–775 (2007).7. Williams, L.M. & Rudensky, A.Y. Nat. Immunol. 8, 277–

284 (2007).8. Zhou, L., Chong, M.M. & Littman, D.R. Immunity 30,

646–655 (2009).9. Feuerer, M., Hill, J.A., Mathis, D. & Benoist, C. Nat.

Immunol. 10, 689–695 (2009).10. Tsuji, M. et al. Science 323, 1488–1492 (2009).11. Zhou, X. et al. Nat. Immunol. 10, 1000–1007 (2009).

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Foxp3+ Treg

Induces Autoimmunity

Foxp3– exFoxp3

Inhibits

Promotes

Figure 1 Loss of Foxp3 in some Treg cells under autoimmune conditions may result in the conversion of former suppressor T cells into highly autoaggressive lymphocytes. Such exFoxp3 cells lose their original suppressor capacity and become effector or memory cells that can promote autoimmune disease.

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