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rather than suppress the pathogenesis of sys-temic lupus erythematosis12. Furthermore, expression of the ‘pro-tolerogenic’ enzyme indoleamine dioxygenase is found exclusively in splenic CD19+ DCs that have overlapping properties with pDCs but express distinct surface markers13. Indoleamine dioxygenase is involved in allograft tolerance induced by a soluble form of the T cell inhibitory recep-tor CTLA-4, which binds to the CD28 and CTLA-4 ligands B7-1 and B7-2. Thus, it would seem that cell surface phenotype, maturation state, immunological context and location all determine whether a DC is tolerogenic or immunogenic.

Results presented by Ochando and col-leagues indicate that pDCs follow distinct trafficking patterns in tolerant and rejecting recipient mice, suggesting that pDC location is a critical parameter determining whether a pDC will act in a tolerogenic or immunogenic way. The trafficking of pDCs to lymph nodes correlated with allograft tolerance, whereas pDC accumulation in the spleen resulted in allograft rejection (Fig. 1a). Moreover, only pDCs from lymph nodes, not those from the allografts, blood or spleens of tolerant recipi-ent mice, were able to trigger Foxp3 expres-sion in nonregulatory alloantigen-specific CD4+ T cells. The reason for the lymph node specificity of the tolerance-promoting activ-ity is unclear. It is possible that pDCs that migrate to lymph nodes represent a special-ized subset of tolerogenic pDCs. Alternatively, lymph nodes may provide additional fac-tors needed for pDCs to acquire tolerogenic

properties. Finally, pDCs may be positioned in the lymph node near the high endothelial venules to interact with alloantigen-specific T cells in a noninflammatory environment before they reach other DCs, resulting in dominant Foxp3-inducing signals; whereas a similar encounter at the site of inflamma-tion may lead to pathogenic T cell activation. The model system used by Ochando and colleagues is unique in that the vascular-ized allografts have no lymphatic drainage. Therefore, lymph node entry of other DC lineages, such as mDCs, that normally enter via afferent lymphatic vessels originating in surrounding tissues, is precluded. pDCs may thus influence immunity differentially based on the presence or absence of other antigen-presenting cells in lymph nodes. In organ transplant settings in which lymphatic drain-age develops, pDCs may not be sufficient to induce tolerance.

If there is no specific subset of DCs that uniquely forms partners with Treg cells to control the induction of immunologic tol-erance, can a consensus model emerge? One possible scenario is that immature mDCs and pDC induce adaptive Treg cells (Fig. 1b). As immature DCs are sensitive to Treg cell–mediated suppression14, they are kept immature by adaptive or natural Treg cells during steady-state conditions. Infection or other immunological insults, such as trans-plantation, ‘break’ that tolerogenic cycle and induce mDC maturation. As they mature, mDCs become resistant to Treg cell–mediated control15 and, therefore, productive immunity

ensues. Mature mDCs induce the expan-sion of antigen-specific Treg cell populations, thereby initiating a negative feedback loop to prevent further DC maturation once an immune response has been initiated. Thus, preemptive delivery of alloantigens to imma-ture DCs via a relatively immunologically quiescent process, such as donor-specific transfusion under the ‘cover’ of CD40 ligand blockade, will favor the development of antigen-specific adaptive Treg cells and ultimately promote allograft-specific tolerance. A better understanding of the rules that govern the Treg cell–DC relationship will enable scien-tists to better manage the immune response in transplant and other disease settings.

1. Ochando, J.C. et al. Nat. Immunol. 7, 652–662 (2006).

2. Bluestone, J.A. & Abbas, A.K. Nat. Rev. Immunol. 3, 253–257 (2003).

3. Hsieh, C.S., Zheng, Y., Liang, Y., Fontenot, J.D. & Rudensky, A.Y. Nat. Immunol. 7, 401–410 (2006).

4. Watanabe, N. et al. Nature 436, 1181–1185 (2005).

5. Cong, Y. et al. J. Immunol. 174, 2787–2795 (2005).

6. Jonuleit, H., Schmitt, E., Schsuler, G., Knop, J. & Enk, A.H. J. Exp. Med. 192, 1213–1222 (2000).

7. Yamazaki, S. et al. J. Exp. Med. 198, 235–247 (2003).

8. Tang, Q. et al. Nat. Immunol. 7, 83–92 (2006).9. Moseman, E.A. et al. J. Immunol. 173, 4433–4442

(2004).10. Kared, H. et al. Diabetes 54, 78–84 (2005).11. Colonna, M., Trinchieri, G. & Liu, Y.J. Nat. Immunol.

5, 1219–1226 (2004).12. Blanco, P., Palucka, A.K., Gill, M., Pascual, V. &

Banchereau, J. Science 294, 1540–1543 (2001).13. Mellor, A.L. et al. J. Immunol. 175, 5601–5605

(2005).14. Serra, P. et al. Immunity 19, 877–889 (2003).15. Pasare, C. & Medzhitov, R. Science 299, 1033–1036

(2003).

Stress management for T helper differentiationKenneth M Murphy

The integrated stress response is a complex signaling pathway that regulates myriad cell processes, including protein translation, depending on the stress conditions. Primed CD4+ T helper cells may use this response system to optimize cytokine expression.

“You can observe a lot just by watching.” Yogi Berra

In this issue of Nature Immunology, Locksley and colleagues1 describe a new, or at least

previously unappreciated, aspect of T helper

Kenneth M. Murphy is in the Department of

Pathology and Immunology, Howard Hughes

Medical Institute, Washington University School of

Medicine, St. Louis Missouri, 63110, USA.

e-mail: Murphy@immunology.wustl.edu

cell differentiation whose story provides a clas-sic example of ‘chance favoring the prepared mind’. The setup for their finding lies in the analysis of two different cytokine ‘reporter’ mice made years earlier by Locksley’s group2,3. The original purpose of the reporter mice ‘4get’ (interleukin 4–green fluorescent pro-tein–enhanced transcript) and ‘Yeti’ (yellow fluorescent protein–enhanced transcript for interferon-γ) mice was to allow in vivo track-ing of cytokine production by T cells, with the goal of elucidating T helper type 1 (TH1)–TH2 differentiation. Having helped to achieve that

goal in previous publications, the reporter mice have now provided unanticipated data indicating the involvement of a protein bio-synthetic control system, the integrated stress response (ISR), in TH1-TH2 differentiation (Fig. 1).

As usual, ‘the devil is in the details’. In this case, the demon crept in by the particular methodology used to generate the reporter mice. As is often done, these authors used an internal ribosome entry site (IRES) linked to a gene encoding either a green fluorescent or yellow fluorescent protein to simultaneously

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express Il4 or Ifng as a ‘bicistronic’ mRNA encoding a cytokine and a fluorescent protein. The IRES, it was originally assumed by Locksley and colleagues, would function simply as a structural element to enable translation of the fluorescent protein from the cytokine mRNA. Thus, transgenic mice expressing a bicistronic ‘cytokine–fluorescent protein’ message would allow in vivo identification of the ‘who, where and when’ of cytokine production. Of course, there were a few caveats to that straightforward plan, such as the possibility that the relative stability of the fluorescent proteins (versus the cytokine stability) would ‘over-report’ cells by causing them to remain fluorescent for lon-ger than the cytokine was biologically active. Nonetheless, fluorescence would at least, so it was assumed, faithfully indicate transcription of the respectively translated cytokine, which would be sufficient for the task at hand.

The 4get and Yeti reporter mice served their initial purpose2,3, but an unexpected result soon emerged that, after careful scru-tiny, could not simply be explained by dif-ferential protein stability of cytokines and fluorescent proteins. Instead, it became apparent that during the process of TH cell differentiation, which takes around 5–7 days, there was a clear-cut dissociation of the initial presence of the cytokine mRNA versus actual production of cytokine pro-tein. Thus, differentiating helper cells were fluorescent, indicating transcription of the cytokine gene, but there was no production of the cytokine protein. In fact, that discrep-ancy could have been discovered through conventional technology before the existence of the 4get or Yeti mice, but there had been no compelling reason to look for it. However, the problem now facing the authors was the pres-ence of cytokine transcription, demonstrated by the fluorescence of differentiating helper cells, but no cytokine production, a discrep-ancy difficult to ‘4get’ about. The burden now was how to explain those data.

The present paper by Locksley and col-leagues1 now clarifies that discrepancy. They find that fluorescent protein expression with-out cytokine expression can be explained by a mechanism of differential translation of cytokine and fluorescent protein genes (even though they are both present on the same mRNA) through regulation by a pathway resembling the ISR4. The ISR is conserved from yeast to mammals and regulates many cell pro-cesses, including protein biosyntheses at the level of mRNA translation. One important outcome of the ISR is adapting the translation of existing mRNA to optimize the present cel-lular requirements, mainly by ‘turning down’ the translation of many mRNA molecules, but

also by enabling continuous translation of oth-ers. To do that, the ISR integrates information from various cellular pathways that indicate specific stresses, from the ‘unfolded protein response’ in the endoplasmic reticulum5, to amino acid deprivation and oxidative stress4, to viral infection and so on. In general, diverse stress signals converge through the action of various kinases onto a critical ‘bottleneck’ in protein biosynthesis, the α-subunit of the eukaryotic initiation factor 2 (eIF2α).

To function, eIF2α must form a ternary complex by binding the methianyl-initiator tRNA and GTP, which is required for trans-lation initiation from an mRNA. However, phosphorylation of serine 51 of eIF2α by vari-ous ‘stress kinases’ prevents its being recharged from its GDP-bound state by the nucleotide-exchange factor eIF2B. In fact, phosphory-lated eIF2α can stoichiometrically bind to the cellular stores of eIF2B and prevent its acting on nonphosphorylated eIF2α, thus stopping the translation of many mRNA transcripts. It turns out that the translation-control system is even more complex than initiation require-ments through the eIF2α regulatory circuit. Some classes of mRNA, including those that have endogenous IRES elements at their 5′ end, can continue to be translated even in conditions that activate the ISR pathway6. Many such transcripts encode proteins whose function is to respond to particular cellular stresses. But ‘aye, there’s the rub’: it is pre-

cisely the IRES in the 4get and Yeti mRNA that enables translation of the cytokine genes in conditions normally present during CD4+ TH cell differentiation, which seem to resemble, according to data from Locksley’s group, the ISR (discussed below). One other important general consequence of an activated ISR is the localization of sequestrated untranslated mRNA into ‘stress granules’ that seem to be unique cellular loci geared for containing and/or degrading mRNA in stress conditions7–9. The localization of untranslated mRNA into stress granules can be mediated by cellular RNA-binding proteins such as T cell intracel-lular antigen 1 (ref. 10) and T cell intracellular antigen 1–related protein11.

What Locksley and colleagues have now shown is that the ISR pathway, or certainly aspects of the ISR pathway if not the actual pathway itself, is (are) activated during the initial period of TH cell differentiation, when antigen-activated T cells must undergo both rapid clonal expansion and complex tran-scriptional changes required for the subse-quent production of specific cytokines such as interferon-γ or interleukin 4. The authors demonstrate that despite the transcription of these effector cytokine genes, little pro-duction of cytokines occurs during this time because the ISR is activated, which effectively shuts off mRNA translation. The ISR results in increased phosphorylated eIF2α and accu-mulation of cytokine mRNA transcripts into

Figure 1 An ISR attenuates the translation of cytokines during TH cell differentiation. The initial priming of naive CD4+ T cells toward a differentiated phenotype, such as the TH2 cell presented here, requires both the rapid clonal expansion of cells and, simultaneously, the acquisition of transcriptional competence of effector cytokines, such as interleukin 4 (IL-4), through chromatin remodeling. However, the production of effector cytokines may be unnecessary until differentiated TH cells reach sites of infection. In the primed TH cell, the ISR can block translation of existing cytokine transcripts that have been produced during the process of differentiation. The block is at the point at which the initiation factor eIF2α-GTP is reconstituted to form one part of the translation initiation complex required for cap-dependent translation (left; ‘poised’ translation). After entry into sites of infection, stimulation through the T cell receptor reverses translational blockade produced by the ISR and allows rapid cytokine production (right; activated translation), as well as initiating further transcription of cytokine from their loci (not shown). 2B, eIF2B; 5, 5′ end; Met, methionine; 4E and 4G, elongation factors; 60S, 60S rRNA; 7mG, 7-methyl guanosine; 3, 3′ end; eGFP, enhanced green fluorescent protein; AAA, poly(A) tail; 40S, 40S rRNA.

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stress granules in differentiating TH cells. In a nice proof of principle, translation of the of the ‘ready but waiting’ cytokine transcripts is induced experimentally by inhibition of T cell intracellular antigen 1, further linking the process to the ISR and to stress granules. The authors suggest that this may be neces-sary during T cell differentiation to allow the T cell to ‘focus on’ clonally expanding and the remodeling of cytokine genes, which would come at the expense of cytokine production that might be unnecessary during this early period. Thus, the ISR is put to good use, letting the T cells do what they need to do when they need to do it, not before.

There may be some minor differences between the ISR in T cells and the ISR in other types of cells. For example, classical forms of stimulating the ISR, such as treat-ment with thapsigargin and tunicamycin, led to lower induction of typical targets of ISR gene expression in T cells than in other cells.

Also, in other cells, stress granules can rapidly dissipate after the removal of stress stimuli, whereas for T cells, stimulation of the TCR pathway, which delivers the signal to translate cytokine mRNA, did not cause an immedi-ate loss of stress granules. On a more gen-eral level, whereas the kinases that normally activate the ISR pathway during amino acid deprivation, endoplasmic reticulum stress, heme deficiency or viral infection are known, whether the same ones are activated by similar pathways during helper differentiation or how secondary TCR signaling reverses the ISR in helper cells is not apparent. Many other issues, such as the possible involvement of antigen-presenting cells and costimulatory pathways, must be evaluated as possible contributing factors as well.

At the very least, the discovery of an ‘ISR-like’ response during TH cell differentiation shows that T cells have multiple levels of regulation for the production of the TH1 and

TH2 effector cytokines. Now, transcriptional and translational levels of control must also be considered. More than that, the finding that the ISR pathway underlies translational control adds an important new twist to an already elaborate system of TH cell differen-tiation, one whose full contribution has yet to be appreciated.

1. Scheu, S. et al. Nat. Immunol. 7, 644–651 (2006).

2. Mohrs, M. et al. Immunity 15, 303–311 (2001).3. Stetson, D.B. et al. J. Exp. Med. 198, 1069–1076

(2003).4. Harding, H.P. et al. Mol. Cell 11, 619–633 (2003).5. Calfon, M. et al. Nature 415, 92–96 (2002).6. Komar, A.A. & Hatzoglou, M. J. Biol. Chem. 280,

23425–23428 (2005).7. Kedersha, N. & Anderson, P. Biochem. Soc. Trans.

30, 963–969 (2002).8. Brengues, M., Teixeira, D. & Parker, R. Science 310,

486–489 (2005).9. Cougot, N., Babajko, S. & Seraphin, B. J. Cell Biol.

165, 31–40 (2004).10. Tian, Q. et al. Cell 67, 629–639 (1991).11. Kawakami, A. et al. Proc. Natl. Acad. Sci. USA 89,

8681–8685 (1992).

Pin-ning down immune responses to RNA virusesNadege Goutagny, Martina Severa & Katherine A Fitzgerald

To prevent RNA virus–dependent tissue damage caused by interferon-regulatory factor 3 (IRF3)–induced type I interferons, proteasome-dependent destruction of IRF3 is orchestrated by the cytoplasmic prolyl isomerase Pin1.

Nadege Goutagny, Martina Severa and Katherine A.

Fitzgerald are in the Division of Infectious Diseases

and Immunology, Department of Medicine,

University of Massachusetts Medical School,

Worcester, Massachusetts 01605, USA.

e-mail: kate.fitzgerald@umassmed.edu

Successful host defense against virus infection relies on the rapid produc-

tion of type I interferons (IFN-αβ) and the subsequent transcription of hundreds of interferon-stimulated genes, which leads to a cellular antiviral state and prevents viral replication1. Production of IFN-αβ is one of the earliest responses induced by virus replication intermediates, such as double-stranded RNA (dsRNA), which are ‘sensed’ by germline-encoded pattern- recognition receptors. Two distinct classes of dsRNA sensors have been linked to the regu-lation of IFN-αβ. Toll-like receptor 3 (TLR3) and the RNA helicases RIG-I and Mda-5 detect dsRNA and trigger signal transduction cascades that culminate in the formation of a multiprotein complex of transcription factors, which contains ATF-2–c-Jun, transcription factor NF-κB, interferon-regulatory factor 3

(IRF3) and IRF7. Assembly of this ‘enhanceo-some’ on the IFN-β enhancer2 leads to IFN-β production. Although critical for protection of the host, the production of IFN-β must also be tightly controlled to maintain homeosta-sis and prevent tissue damage. Although considerable progress has been made in the understanding of the molecular mechanisms underlying the induction of IFN-αβ, how those mechanisms are switched off is not well understood. Lgp2, an RNA helicase related to RIG-I, has been shown to function in a nega-tive feedback loop as a postinduction inhibitor of antiviral responses3,4. In this issue of Nature Immunology, Yamaoka and colleagues indicate involvement of a different negative regula-tory mechanism, via the cytoplasmic prolyl isomerase Pin1, in control of both TLR3- and RIG-I-mediated antiviral responses5.

RIG-I and Mda-5 detect dsRNA via their RNA helicase domains and trigger ‘down-stream’ signaling via their caspase activa-tion and recruitment domains (CARDs) in collaboration with the ‘downstream’ adap-tor MAVS (also called IPS1, cardif or VISA). MAVS is localized to the outer mitochondrial membrane by its C-terminal transmembrane domain. A second pathway for sensing dsRNA

involves TLR3, a type I transmembrane recep-tor localized to endosomes. Activation of TLR3 by dsRNA is thought to occur in the lumen of endosomes, possibly after release from dying cells. After dsRNA recognition, TLR3 forms oligomers via protein-protein interactions involving Toll–interleukin 1 receptor domains present in both the receptor and the Toll–inter-leukin 1 receptor domain–containing adap-tor inducing IFN-β (TRIF). Recruitment of MAVS or TRIF creates a signaling platform to which tumor necrosis factor–associated fac-tor (TRAF) proteins and ‘downstream’ kinase complexes are recruited for signaling1.

In resting cells, NF-κB and IRF3 are both localized to the cytoplasm. NF-κB is bound to the inhibitor protein IκBα, which, after virus infection, becomes phosphorylated by the classical IκB kinase complex (IKKαβγ), ‘mark-ing’ IκBα for ubiquitination and subsequent degradation by the proteasome. Similarly, after virus infection, IRF3 becomes phosphorylated by a related kinase complex composed of IKKε (IKKi) and/or TBK1 (also called NAK or T2K)6,7, and ATF-2–c-Jun is rapidly activated by the c-Jun N-terminal kinase (Jnk) path-way. Activation of all three pathways results in nuclear translocation of NF-κB, IRF3 and

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