A polarizing situationTasha N Sims & Michael L Dustin

The physical location of key proteins may turn out to significantly influence the differentiation tack that a cell takes.If clusters of antigen receptors include interferon-γ receptors in their midst, the fate of that T cell may be tipped.

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authors did show that A20 can deubiquitinateTRAF6, which is a target of K63-ubiquitina-tion in TLR and IL-1R signaling pathways.

Although Ma and colleagues show A20dampens TLR signaling, their previous workshowed A20 deficiency does not affect IL-1β-mediated NF-κB activation. This is unex-pected, because IL-1R is homologous to theTLR in the cytoplasmic domain (the TIRdomain) and, like the TLRs, IL-1R uses theTIR domain–containing adapter MyD88 toactivate NF-κB. How then does IL-1 signalingescape A20-mediated silencing? The answermay lie in the exclusive use of MyD88 tomediate IL-1-signaling, in contrast to TLRsignaling, which requires additional adaptors.Another possible explanation for this discrep-ancy may be the two cell types analyzed,macrophages versus embryonic fibroblasts, orthe assays used, IκBα degradation versus IL-6production. Consistent with the idea that A20inhibits TLR and IL-1R signaling is the find-ing that overexpression of A20 by transienttransfection inhibits IL-1β- and TLR-inducedNF-κB activation11. More careful analyses of A20 and other A20-like proteins will be

necessary to sort out their differential effectson IL-1R, TLR and TNFR signaling.

The ability of A20 to inhibit both TLR4 andTNFR1 signaling naturally draws attention toRIP1, which is critical for TLR3-mediatedTrif-dependent NF-κB activation5. TLR4-induced NF-κB activation also requires Trif,especially at later time points, and may alsorequire RIP1. Dixit and colleagues establishedRIP1 as the target of deubiquitination andreubiquitination by A20 in the TNFR pathway.RIP1 could be similarly K63-ubiquitinated inthe TLR4 pathway and then modified again byA20 to inhibit signaling. This would explainhow A20 affects TLR4, TLR3 and TNFR, butnot IL-1R signaling. However, it does notexplain how A20 inhibits TLR2 and TLR9 sig-naling, which do not require TRIF or RIP1.

Ubiquitination and deubiquitination havemultiple functions in the control of NF-κBactivation. K48 polyubiquitination is essential‘downstream’, where IκBs are ubiquitinated bythe β-TrCP-SCF complex after IKK-mediatedphosphorylation. In contrast, K63 polyubi-quitin chains are important upstream, whereTRAF-mediated K63 ubiquitination is critical

for activation of the IKK complex. Two DUBenzymes downregulate this step in the path-way. The tumor suppressor CYLD functionsas a DUB enzyme that removes TRAF-induced K63-polyubiquitin chains andthereby inhibits NF-κB activation12. Similarly,the Ma and Dixit groups demonstrate thatA20 functions by removing the K63-polyubi-quitin chains from TRAF6 and RIP1, as well asby K48-polyubiquitinating RIP1, and therebydownregulating NF-κB activation in theTNFR and TLR pathways2,3. These new find-ings emphasize the importance of ubiquitin inthe control of NF-κB activation.

1. Lee, E.G. et al. Science 289, 2350–2354 (2000).2. Wertz, I.E. et al. Nature 430, 694–699 (2004).3. Boone, D.L. et al. Nat. Immunol. 5, 1052–1060

(2004).4. Vogel, S.N., Fitzgerald, K.A. & Fenton, M.J. Mol.

Interv. 3, 466–477 (2003).5. Meylan, E. et al. Nat. Immunol. 5, 503–507 (2004).6. Sun, L. & Chen, Z.J. Curr. Opin. Cell Biol. 16,

119–126 (2004).7. Kanayama, A. et al. Mol. Cell. 15, 535–548 (2004).8. Lee, T.H., Shank, J., Cusson, N. & Kelliher, M.A.

J. Biol. Chem. 279, 33185–33191 (2004).9. Takaesu, G. et al. J. Mol. Biol. 326, 105–115 (2003).10. Yang, J. et al. Nat. Immunol. 2, 620–624 (2001).11. Evans, P.C. et al. Biochem. J. 378, 727–734 (2004).12. Lakhani, S.R. N. Engl. J. Med. 350, 187–188 (2004).

In a recent issue of Nature, Glimcher and col-leagues forge a link between polarization of

cytokine receptors in the immunologicalsynapse and the cascade of events leading to Thelper (TH) cell polarization1. When cell biolo-gists talk about polarization, they are likely tomean that a cell has a distinct front and backthat allows directed movement or secretion. Incontrast, when immunologists talk aboutpolarization, they are most likely referring tothe differentiation of clones of proliferating THcells into either TH1 or TH2 cells, which sup-port inflammatory or allergic type reactions,respectively, during an immune response2.Polarity of T cells in the cell biological sensehas been known to be a common feature of

T cell activation since the early 1980s (ref. 3).The T cell orients its secretory apparatustoward the antigen-presenting cell (APC), andthis polarity is thought to be important for thedirected delivery of cytokines4. Glimcher andcolleagues finally connect cell biological toimmunologic polarization with the demon-stration that the interferon-γ (IFN-γ) receptoris polarized to the immunological synapse andthat being in this location shows multiplestrong correlations with a cell’s commitmentto TH1 polarization.

The immunological synapse is an organizedcellular junction between the T cell and APCthat is defined by an antigen-induced accumu-lation of a ring of the adhesion proteins LFA-1(CD11a-CD18) bound to ICAM-1 (CD54)surrounding an accumulation of T cell antigenreceptors (TCRs) bound to major histocom-patibility complex proteins5. The secretoryapparatus is positioned close to the adhesion‘bull’s eye’ and provides a mechanism fordirected secretion of cytokines. Involvement of

the immunological synapse in directedcytokine secretion has been proposed, butreports on the localization of cytokine recep-tors in the synapse had not surfaced.

Maldonado et al. consider the immunesynapse a platform for TH commitment thatmay have the decisive early function in inte-grating TCR and cytokine signals1. Theauthors demonstrate that within 30 minutes,IFN-γ receptor subunits are redistributed tothe interface between T cells and APC s, closeto the TCR-populated region. The definingsubstructures and molecules in the substruc-tures of the immune synapse (LFA-1–ICAM-1interactions) were not included in the analysis,and it will be of interest to determine ifcytokine receptors are located in one of thesubstructures of an immune synapse or areinternalized. In their subsequent experiments,the authors used antibody crosslinking as asurrogate for the APCs. These crosslinkingexperiments showed that receptors for bothIFN-γ and interleukin 2 (IL-2) localize to the

The authors are in the Department of Pathology

at the Skirball Institute of Molecular Medicine,

New York University School of Medicine, New York,

New York 10016, USA.

e-mail: dustin@saturn.med.nyu.edu

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This type of directionality for IFN-γ or IL-12release would increase the local concentrationof ligands, thus increasing likelihood that the Tcell will respond in different stages of TH1 dif-ferentiation. Assembly of the IFN-γ receptorinto a signalosome with the TCR may furtherenhance ligand-dependent, or even ligand-independent, signaling through the receptor.These interactions would thereby set up a lig-and–cytokine receptor ‘conversation’ betweenthe two cells. The immunological synapse andsubsequent conversation with the signalosomecould provide the capacitance for later full TH1development. Growing evidence suggests thatthe synapse could be a platform for cell fatedecisions of various sorts, including anergy,degree of cell proliferation or tolerance10–12, oreven T cell sequestration or imprinting.

These recent observations on the influenceof early ‘events’ like immunological synapseformation on processes that take weeks tocomplete, like T cell polarization, suggestanother unexpected function of the synapse in

T cell biology. The proof of these effects andthe underlying mechanisms will require muchmore work, but Glimcher and colleagues haveopened new avenues by linking two importantmodes of biological polarization.

1. Moldonado, R.A., Irvine, D.J., Schreiber, R. & Glimcher,L.H. Nature advance online publication, 22 September2004 (doi:10.1038/nature02916).

2. Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin,M.A. & Coffman, R.L. J. Immunol. 136, 2348–2357(1986).

3. Kupfer, A., Dennert, G. & Singer, S.J. Proc. Natl.Acad. Sci. USA 80, 7224–7228 (1983).

4. Poo, W.J., Conrad, L. & Janeway, C.A., Jr. Nature 332,378–380 (1988).

5. Dustin, M.L. & Colman, D.R. Science 298, 785–789(2002).

6. Wakil, A.E., Wang, Z.E., Ryan, J.C., Fowell, D.J. &Locksley, R.M. J. Exp. Med. 188, 1651–1656 (1998).

7. Szabo, S.J., Dighe, A.S., Gubler, U. & Murphy, K.M.J. Exp. Med. 185, 817–824 (1997).

8. Frucht, D.M. et al. Trends Immunol. 22, 556–560(2001).

9. Boes, M. et al. Nature 418, 983–988 (2002).10. Lee, K.H. et al. Science 302, 1218–1222 (2003).11. Heissmeyer, V. et al. Nat. Immunol. 5, 255–265

(2004).12. Couzin, J. Science 305, 772–773 (2004).

NATURE IMMUNOLOGY VOLUME 5 NUMBER 10 OCTOBER 2004 1013

same general region as the TCR β-chain. Theevidence that the polarization of IFN-γ recep-tors is important for TH lineage decisions isbased on a many notable correlations. First,polarization of IFN-γ receptors was strongestin the naturally TH1-leaning C57BL/6 mousestrain, compared with the TH2-biased BALB/cstrain. Second, in the presence of the TH2-promoting cytokine IL-4, the redistribution ofthe IFN-γ receptor was abolished in a STAT6-dependent way. The authors suggest that TH1may be the default phenotype, at least in theC57BL/6 strain of mice, based on constitutiveconcentration of IFN-γ receptors near theTCR. This IFN-γR polarization is antagonizedby TH2-polarizing stimuli.

These correlations provide compelling sup-port for a new model, but the mechanisms bywhich the clustering of IFN-γ receptors at theimmunological synapse would specificallypromote TH1 lineage commitment are openfor exploration. IFN-γ can have a critical earlyfunction in upregulation of the transcriptionfactor T-bet, which in turn is important for theinduction of IL-12 receptor β (IL-12Rβ)chains, which are absent on naive T cells. Thus,the immunological synapse may coordinatethe cytokine cascade leading to TH1 commit-ment, a process that would be greatly enhancedby the polarized localization of IFN-γ recep-tors. IFN-γ produced by dendritic cells can besecreted into the immunological synapse andinteract with IFN-γreceptors in a TH1 signalo-some (Fig. 1). IFN-γ could also come fromother sources, such as natural killer (NK) cells,NKT cells or memory CD8 cells, independentof an immunological synapse. The apparentconcentration of cytokine may be reduced inthis case, due to the lack of the focusing effectthat the synapse potentially generates. Earlyproduction of IFN-γby activated CD4 T cells issufficient for the control of leishmania infec-tion6. IFN-γ from these varied sources may actwith great potency in the stable synapse tomaintain expression of IL-12Rβ7. It is notknown if newly minted IL-12Rβ2 chains pro-duced in response to early TCR signals are alsodirected to the immune synapse, but a likelymodel would be that these receptors are incor-porated into the same platform, where theywould be well positioned to respond to IL-12from dendritic cells. Perhaps the relocation ofthe IFN-γ receptor in the absence of ligandallows APC s to direct IFN-γ placement overshort distances and increase the sensitivity of‘sensing’ by the T cell8 (Fig. 1). Although den-dritic cells do not polarize like T cells, a solu-tion to this problem has been proposed byPloegh and colleagues, whereby microtubulesdirect major histocompatibility complex classII proteins to the immunological synapse9.

T cell movement Polarization

Lineage commitment

Substratum

APC pMHC ICAM-1

30 min IFN-γ receptorIL-12

receptor IFN-γ

TCR

IFN-γIFN-γ

STAT1

T-bet

IL-12Rβ2IL-12

30 min

IL-4R

IL-4

TCR

STAT6

TH1 TH2

C57BL/6No IL-4

BALB/cIL-4

Figure 1 Helper T cell lineage commitment. T cells migrate on substrata until they recognizepeptide–major histocompatibility complex (pMHC) complexes and ICAM-1 on an APC. The T cell thenstops and becomes polarized toward the APC. TCRs and LFA-1 are quickly (30 s–5 min) moved to formthe immunological synapse. The TH1-favoring synapse include the IFN-γ receptor, whose distributionbecomes polarized in the absence of ligand (30 min). In the presence of IL-4 (or in TH2-prone mice),the arrangement of IFN-γ receptors does not polarize, presumably then leading to TH2 lineagecommitment. The IFN-γ receptor clustering may prime the T cell to receive IFN-γ from the APC, drivingits T cell partner toward the TH1 phenotype. The primed T cell becomes sensitive to IL-12, augmentingthe T cell’s ability to secrete IFN-γ. The autocrine IFN-γ stimulation loop commits the T cell to be a trueTH1 effector cell. In this model, a stable immunological synapse would be necessary to receive smallamounts of IFN-γ (or IL-12) directed from the APC, further driving the TH1 lineage commitment.

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