genetic analysis of host resistance: toll-like receptor signaling and immunity at large

ANRV270-IY24-12 ARI 15 February 2006 4:15

Genetic Analysis of HostResistance: Toll-LikeReceptor Signaling andImmunity at LargeBruce Beutler, Zhengfan Jiang, Philippe Georgel,Karine Crozat, Ben Croker, Sophie Rutschmann,Xin Du, and Kasper HoebeDepartment of Immunology, Scripps Research Institute, La Jolla, California 92037;email: [email protected]

Annu. Rev. Immunol.2006. 24:353–89

The Annual Review ofImmunology is online atimmunol.annualreviews.org

This article’s doi:10.1146/annurev.immunol.24.021605.090552

Copyright c© 2006 byAnnual Reviews. All rightsreserved

0732-0582/06/0423-0353$20.00

Key Words

mutagenesis, Mendelian genetics, infection, innate immunity

AbstractClassical genetic methods, driven by phenotype rather than hy-potheses, generally permit the identification of all proteins that servenonredundant functions in a defined biological process. Long beforethis goal is achieved, and sometimes at the very outset, genetics maycut to the heart of a biological puzzle. So it was in the field of mam-malian innate immunity. The positional cloning of a spontaneousmutation that caused lipopolysaccharide resistance and susceptibil-ity to Gram-negative infection led directly to the understanding thatToll-like receptors (TLRs) are essential sensors of microbial infec-tion. Other mutations, induced by the random germ line mutagenENU (N-ethyl-N-nitrosourea), have disclosed key molecules in theTLR signaling pathways and helped us to construct a reasonably so-phisticated portrait of the afferent innate immune response. A stillbroader genetic screen—one that detects all mutations that compro-mise survival during infection—is permitting fresh insight into thenumber and types of proteins that mammals use to defend themselvesagainst microbes.

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Forward genetics:the genetic approachthat begins withphenotype ofunknown origin andends with theidentification ofculpable mutation(s)

INTRODUCTION

Phenomena and Phenotypes: TheGeneticist’s View of Resistance toInfectious Disease

Few immunologists are content to forsake hy-potheses and follow a classical genetic strat-egy. But perhaps more of them should. Theforward genetic approach to biological prob-lems is an unbiased one. It does not beginwith broad speculation about how a biolog-ical system might operate, or even with morerestricted hypotheses, such as guesses aboutthe function of a particular protein or howcells interact. On the contrary, genetic ex-ploration begins with no preconceptions atall. Hypotheses are the outcome of geneticinvestigation rather than the starting point(Figure 1).

In genetics, as in biology at large, phenom-ena come first. They are the ultimate sourceof all curiosity and all inquiry. Some phenom-

Figure 1Summary of the classical (“forward”) genetic approach. Hypotheses (andexperiments to test them) are the result of genetic inquiry rather than thestarting point.

ena (the shapes of leaves, the presence of spotson the wings of moths, and the fact that dogsbeget dogs whereas humans beget humans)are either so subtle or so commonplace thatmost people hardly think about them at all,although their importance may belie their ba-nality. Other phenomena (aging, conscious-ness, and cancer) are uppermost in the popu-lar mind and have impelled investigation on abroad scale, using all the tools technology canoffer.

However prosaic or glamorous the phe-nomena might seem, their analysis by classi-cal genetic methods entails the identificationof phenotypes: alternative forms of phenom-ena. Where such phenotypes do not exist, theymust be created. Once a monogenic pheno-type is at hand, existing technology invariablypermits identification of the DNA sequencedifference that is behind it. Ultimately, all pro-teins with nonredundant functions in a partic-ular biological phenomenon may be identifiedin this manner. Even if it falls short of this ide-alized goal, classical genetic analysis createsa substrate upon which other methods mayoperate.

The central phenomenon at issue in im-munology is resistance to infection: the factthat we do not passively succumb to mi-crobes when inoculated with them. It is aphenomenon that declares itself every day,and it would certainly fall into the “common-place” category if not for the impressive con-sequences of its failure. Resistance to infec-tion usually goes unnoticed, but it is essentialto the survival of individuals and species.

Genetic reasoning immediately tells usthat the preponderance of resistance to in-fection is inherited rather than acquired,despite the quintessentially environmentalstatus of microbes themselves. To argue inthe most general way, different mammalianspecies show great variation in susceptibilityto specific infectious agents. In fact, it is safeto say that there are no universal pathogens,i.e., microbes to which all multicellular or-ganisms (or even all vertebrates) are suscep-tible. Never yet has a human died of mouse

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cytomegalovirus (MCMV) infection, at leastas far as we know. Never yet has a mouse diedof human immunodeficiency virus (HIV) in-fection. Admittedly, a vast number of muta-tional differences have set humans and miceforever apart. But a discrete and ultimately de-finable collection of mutations must specifyability to resist infection by these organismsand all others.

Although far fewer mutational events dis-tinguish individuals belonging to a singlespecies, formal measurement of heritabilityreveals that here, too, susceptibility to infec-tion is largely determined by genetic makeup.Premature death from infection in a biolog-ical parent confers a high probability of pre-mature death in a child separated from theparent by adoption (1). Moreover, variationin susceptibility to a given infectious agent isfrequently demonstrable among individuals ofa single species and usually has a genetic ba-sis. Among mice, polymorphisms at specificloci influence susceptibility to MCMV (dis-cussed further below) (2–5), and among hu-mans, polymorphisms at specific loci influ-ence susceptibility to HIV (6, 7).

What types of immunity are there, andwhich type confers the heritability of resis-tance to infection? The term “adaptive” isused to describe a particular type of immu-nity, represented only in vertebrates as far aswe know and markedly influenced by the en-vironment, i.e., by previous exposure to an in-fectious agent or antigen, which impels the ex-pansion of lymphoid clones that are directedtoward specific recognition of the same tar-get. The term “innate” is used to describeall other forms of immunity. There is a ten-dency to regard innate immunity as heritableand adaptive immunity, with its remarkableplasticity, as acquired. But, of course, the factthat adaptive immunity can be eliminated by awell-placed mutation reveals that it, too, is in-herited. To the geneticist, all heritable effectson immunity are of interest, and the distinc-tion between innate and adaptive systems is ofsecondary importance, although often easy tomake.

MCMV: mousecytomegalovirus

Resistome: the setof all genes encodingproteins withnonredundantfunctions in hostresistance

Adaptive immunity evolved at least twice(8). The exact selective pressures that drovethe development of our own adaptive immunesystem are unknown and can only be imag-ined. Perhaps an anticipatory immune sys-tem offered species a means of coping withfrequent and devastating plagues caused bymicrobes that escaped containment by the rel-atively static innate immune response. What-ever the reason, it may be assumed that theadaptive immune system arose in the contextof an advanced and highly functional innateimmune system, and it remains largely depen-dent on the innate system today. Subsequentto the rise of adaptive immunity, certain innateimmune functions must have become redun-dant and as a consequence were lost; hence,innate immunity alone offers suboptimal pro-tection to present-day vertebrates. The innatesystem has come to require certain moleculesof adaptive immune origin (for example, IFN-γ) to function properly. For these reasons,many of the mutations that affect innate im-mune function also affect adaptive immunefunction and vice versa.

The Concept of the Resistome

The resistome is defined as the set of genesencoding proteins with nonredundant func-tion in resistance to infection (9). It is possi-ble to speak of a universal resistome, as wellas of the resistome for specific organisms.Many components of the resistome are en-tirely conditional. If infectious organisms arenot present, there is no need for these genesto exist. In effect, they are fully dedicated toimmune function. Other components of theresistome fulfill separate and essential biolog-ical functions, having recently been appro-priated by evolution to create resistance, orconversely, to serve a function unrelated toresistance.

In some instances, it is difficult to decide onthe primary function of a protein that is knownto participate in resistance. Toll, a transmem-brane receptor with a dual function in hostresistance (10) and in embryonic development

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TIR: Toll/IL-1R/Resistance

TLR: Toll-likereceptor

in Drosophila (11), probably was inherited as amediator of immunity and co-opted to servea developmental function, based on the useof the TIR (Toll/IL-1R/Resistance) domainfor immunity in most species (12). But thepremise is debatable, and some believe thatthe primordial function of Toll was develop-mental. On occasion, a protein that clearlyevolved to serve a function that has nothingto do with immunity may offer strong resis-tance to infection in one allelic isoform, butit may fail to do so in another form (e.g.,sickle hemoglobin, which confers resistanceto Plasmodium falciparum, versus hemoglobinA, which does not). Examples of this sort maybe viewed as evolutionary works in progress,and sufficient selective pressure might drivethe mutant isoform to homozygosity, where-upon, like Toll, the hemoglobin protein wouldnecessarily be viewed as bifunctional, witha role in both oxygen transport and inimmunity.

Some resistome components (certainlymost of them) confer resistance to a broadrange of pathogens. In this sense, the resis-tome is degenerate. Where the innate im-mune system is concerned, low specificity isthe general rule, to allow for effective resis-tance to many microbes within the constraintsimposed by limitations in genomic size. Somecomponents of the resistome, however, arehighly specific in their effects, and althoughthey serve to recognize or overcome a veryrestricted repertoire of microbes, they may beessential to survival of the species.

Irrespective of functional category, a con-certed and determined genetic approach willultimately reveal every protein that plays anonredundant role in resistance and, in prin-ciple, will even identify those with latent re-sistance functions, given that such functionsdo not yet exist. Such a genetic approach willdo so even if such functions of the proteinsin question are beyond the imagination, arewithout precedent, and are unpredictable byany exercise of logic. Therein lies the appealof the genetic approach and the source of itspower.

The classical genetic approach is beholdento keen observation and is empowered by ex-perimental designs that reveal phenotypes in-apparent in the absence of special conditions.But the classical genetic approach does notdepend on guesswork and may ultimately suc-ceed where hypotheses fail. The classical ge-netic approach led to the identification of theToll-like receptors (TLRs) as the key proteinsthat allow us to recognize infection and, inlarge part, to the decipherment of signalingpathways that are activated by the TLRs. Ge-netic reasoning has also pointed to the exis-tence of alternative pathways leading to acti-vation of both the innate and adaptive immunesystems.

THE PHENOMENON: HOSTRESISTANCE

Cell-Autonomous Immunity and theEvolution of Specialized Systems forHost Resistance

Resistance to microbes must be measuredwith reference to a control rather than in ab-solute terms because defining a state in whichno resistance exists is presently impossible.For example, in the absence of the type I IFNreceptor, mice are markedly compromised intheir ability to resist MCMV infection (13).If the type II IFN receptor is also elimi-nated, susceptibility is still greater (13). Giventhat IFN-independent resistance mechanismsalso may exist, susceptibility might be furtherenhanced by additional mutations. For thepresent, resistance must be assayed by measur-ing mean lethal inoculum, mean cytolytic in-oculum, or the proliferation of the agent thatis tested.

Even in the absence of a functioning im-mune system, cultured cells offer resistanceto the growth of microbes. For example,many cells, including fibroblasts, are capableof mounting a type I IFN response. Cell-autonomous immunity presumably operatesin vivo as well as in vitro. However, ad-vanced multicellular organisms have evolved

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additional defensive strategies that make useof specialized cells designed to sense mi-crobes, alert the host, and destroy microbes.The innate immune system depends heavilyon macrophages, several classes of dendriticcells (DCs), natural killer (NK) cells, NKTcells, and granulocytes for host defense. In-terestingly, although lymphoid cells providethe cellular basis for adaptive immunity, theyhave also been used for purely innate immunefunctions. The responses of macrophages andmyeloid DCs have been studied in greatest de-tail; NK cells have also received considerableattention.

Sensing, Response, andSelf-Tolerance: The Strategy of theInnate Immune System

Immune responses are potentially injuriousto the host and are also energy intensive.Although antimicrobial peptides are consti-tutively produced in some compartments,most responses are induced by infection. Thisrequires that a system for recognition ofmicrobes must exist, and in almost all biolog-ical systems recognition implies the presenceof specific receptors. These receptors and thesignals they elicit are part of the sensing ap-paratus of the host.

In adaptive immune responses, recombi-nation and clonal selection permit the gen-eration of highly diverse, clonally expressedreceptors that recognize microbial determi-nants, but not determinants of the host. How-ever, this system is not called into play untilseveral days after inoculation has occurred,and the host must initially rely on innate im-mune responses to contain an infection.

The mammalian innate immune sensingapparatus has evolved to recognize broadlyconserved molecules that are not found inthe host but are represented in many micro-bial taxa. These include molecules such aslipopolysaccharide (LPS), bacterial lipopep-tides, double-stranded RNA (dsRNA), andDNA bearing unmethylated CpG motifs. Butoccasionally, rather narrowly expressed deter-

VSV: vesicularstomatitis virus

LPS:lipopolysaccharide

minants are sensed as well. As discussed be-low, the G-glycoprotein of VSV is detectedvia the CD14/TLR4 apparatus of the host, asensor that is principally devoted to LPS de-tection in mammals. Moreover, some verte-brates (fish, amphibians, reptiles) do not ex-hibit any response at all to LPS, and in somevertebrates (adult birds) the response is min-imal. Yet representative birds and fish retainTLR4 orthologs. The simple model of recep-tors that are highly degenerate in their speci-ficity may therefore need to be modified withtime.

An immune system must also create anenvironment that is inhospitable or lethal tomicrobes. This implies an effector arm of im-mune function. Processes that we associatewith inflammation, including the generationof toxic radicals within phagocytic cells, theelaboration of hydrolytic enzymes, and mech-anisms for containment of microbes (e.g., co-agulation, granuloma formation), are com-ponents of the effector arm. It is reasonableto think that inflammation evolved chiefly tocounter infection. Yet sterile inflammation,when it occurs, is a major cause of morbid-ity. In all likelihood, it depends on the samebiochemical pathways as those used by the in-nate immune system.

The final requirement of the immune sys-tem is self-tolerance. A part of self-toleranceis self-/nonself-discrimination. This is accom-plished by the innate immune system throughan evolutionary process: The principal recep-tors of the innate immune system fail to rec-ognize molecules of the host, at least undernormal circumstances. There may be excep-tions in that TLR3, -7, -8, and -9 recog-nize nucleic acids and, at least under someconditions, are able to recognize mammaliannucleic acids (14). Hence, one may speakof innate autoimmunity and adaptive au-toimmunity (15) rather than autoimmunityper se.

In addition to self-/nonself-discrimi-nation, self-tolerance assumes that bystandereffects should be minimized. As already men-tioned, inflammation often damages healthy


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tissues. When infections are widespread, theprocess may be lethal. This appears to be anecessary imperfection in a system designedto enforce containment of small infections be-fore they grow out of control. Small inoc-ula occur far more commonly than inoculathat are so large as to provoke life-threateningresponses; hence, an aggressive system fordealing with microscale infections would befavored by evolution, even if this system isharmful when infections involve a substantialvolume of tissue.

Figure 2The Lps mutation held the key to understanding innate immune sensing.All LPS-induced phenomena were abolished by the mutation, which waswidely assumed to affect the LPS receptor.

Microbial Sensing, LPS, and the LinkBetween Pathology and Protection

Molecules detected by the innate immune sys-tem began to be identified in the early part ofthe twentieth century, and structural analysisof some such molecules was completed morethan 30 years ago (16), leaving open the ques-tion of which receptors must be responsiblefor their recognition.

The prototypic microbial inducer, sensedstrongly by mammals although not by all ver-tebrates (17), is LPS. The toxicity of LPSalso depends on mononuclear phagocytic cells(18); most other cells of the host are essen-tially unresponsive to it. Ultimately, toxic-ity depends on specific cytokines elaboratedby these cells, notably tumor necrosis factor(TNF) (19) and type I IFN (20). Widely usedto induce local and systemic inflammationand known to reproduce most of the patho-logic features of sepsis, LPS elicits responsesat picomolar concentrations, a fact that spokestrongly in favor of the existence of a specificreceptor.

The dependence of LPS signaling on asingle protein was revealed by the identifi-cation, in 1965, of a remarkable phenotypein mice (21) seemingly caused by a sponta-neous mutation and later traced to a single lo-cus, termed Lps (22–24). The phenotype wasfirst observed in mice of the C3H/HeJ strainand was marked by a very specific and pro-found insensitivity to LPS. Neither the lethaleffect of LPS nor any of the cellular effectsof LPS occurred in these mice. This includedthe well-known adjuvant effect of LPS (25),i.e., its ability to provoke an adaptive immuneresponse to coadministered protein antigens(26). Later, it was shown that mice of theC57BL/10ScCr strain had an allelic defect(27). The Lps mutation was widely believed toaffect the LPS receptor or an essential compo-nent of the receptor, but formal proof awaitedpositional cloning data (Figure 2).

Long in advance of the positional cloningof Lps, researchers showed that failure tosense LPS lowered the mean lethal inoculum

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of Salmonella typhimurium organisms by ap-proximately four orders of magnitude (28,29). Similar observations were later made forother Gram-negative microbes administeredby other routes (30, 31). These studies pro-vided solid proof that sensing LPS was impor-tant to an effective innate immune response toGram-negative microbes or, conversely, thatLPS was one of the key molecules recognizedby the innate immune system. Finding the Lpslocus assumed great practical importance be-cause it seemed to offer the key to understand-ing innate immune sensing.

Hidden in these studies was an importantprinciple already mentioned above: The in-nate response evolved to be highly aggressiveon a microscopic scale. The same response,when disseminated, can be lethal to the host,as in the example of endotoxin-induced shock.The innate response has evolved to balancethe need for containment of microscale mi-crobial infections, which are very common,against the risk of sepsis. Sepsis occurs as theresult of a large infusion of microbes (an un-common event) or because early containmentof an infection has been unsuccessful. Sepsismay be seen, in most cases, as the result ofinnate immune failure.

The Basis of Innate Immune Sensingas Revealed by a Phenotype

The positional cloning of the Lps locus wasachieved by following a phenotype (failure ofmacrophages to produce TNF in response toLPS). This was deemed a biologically rele-vant phenotype because TNF had earlier beenshown to be one of the central executors ofthe toxicity of LPS (19). TNF production waswell known to depend on nuclear transloca-tion of NF-κB (32). However, the primarysignals elicited by LPS were unknown at thattime.

The mutation responsible for LPS un-responsiveness was found to be a missenseerror (P712H) altering the cytoplasmic do-main of TLR4 (33). TLR4 was then knownonly as one of five mammalian paralogs of

Toll (34–37), a Drosophila protein with a dualrole in development and in innate immunity.Specifically, flies with mutations affecting Tollor components of the Toll signaling path-way failed to mount an adequate antimicro-bial peptide response to fungi (10) [and, as waslater shown, to Gram-positive bacteria as well(38)].

The Toll superfamily also included theIL-1 receptor (IL-1R) and IL-18R, whichbear cytoplasmic domain homology to Toll,provoke inflammatory responses, and acti-vate NF-κB (39–41). Because IL-1R and IL-18R served immune functions, investigatorsspeculated that TLR proteins in mammalsmight have either developmental functions(35) or immune function (36), or perhapsboth. In fact, investigators had shown that lig-ation of TLR4, enforced by the creation ofa chimeric protein in which a sequence en-coding CD4 was substituted for the nativeTLR4 ectodomain-encoding sequence, couldactivate NF-κB in transfected cells (36), anobservation consistent with the fact that Tolland IL-1R could activate NF-κB. However,this observation in itself did not actually ad-dress the function of the TLRs because NF-κB activation has many consequences, somerelated to immunity and some not. Moreover,the experiment did not give any insight intothe natural ligand for TLR4, nor did it re-veal whether the ligand was endogenous (asin the case of Toll, IL-1R, and IL-18R) orexogenous.

The fact that a point mutation in TLR4entirely prevented mammalian responses toLPS and to Gram-negative bacteria, yet hadno developmental consequences, strongly fa-vored the interpretation that the TLRs hadan immunologic function and specifically sug-gested that the TLRs acted as the long-soughtsensors of molecules made by microbes. Al-though LPS was sensed specifically by TLR4,investigators immediately postulated that theother TLRs might sense other inflamma-tory molecules (e.g., dsRNA, unmethylatedDNA, glucans, and lipopeptides), and thatcollectively the mammalian TLR paralogs


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might alert the host to infection, triggering animmune response.

The Structure of the TLRs

All TLRs are single-spanning transmembraneproteins with ectodomains largely composedof leucine-rich repeats (LRRs), and with a cy-toplasmic domain largely composed of a TIRdomain (Figure 3).

Figure 3Schematic rendering of TLRs, based on the structure of the TLR3ectodomain and the TLR2 TIR motif. TLR ectodomains are all likely tobe dimerized, horseshoe-shaped, curved solenoids composed of numerousrepeating LRRs. The cytoplasmic domains are compact and globular,consisting mostly of a TIR motif. In some cases (e.g., TLR2, TLR4),multiple accessory molecules may be required for ligand engagement.Adapter proteins, four of which are known to carry TLR signals, also haveTIR domain structures. It is believed that ligands elicit a conformationalchange, allowing recruitment of specific adapter(s). The critical proline inthe BB loop and the critical valine at the Pococurante site are rendered atthe atomic level and colored red and yellow, respectively. These residuesare essential for most, but not all, TIR motif interactions, as discussed intext. Ligands are presumed to engage TLRs near the point of interfacebetween LRR subunits. (Rendering of figure was performed withPyMOL, 2005 DeLano Scientific LLC.)

The structure of a representative TLR(TLR3) has recently been solved through X-ray crystallography, revealing that TLR3 is adimeric protein composed of two horseshoe-shaped subunits that stack together side byside. A large quantity of carbohydrate projectsfrom each horseshoe in one direction, so thatonly the carbohydrate-free surfaces can re-main in contact with one another. At the pointof interaction between subunits, a positivelycharged cluster of residues marks the point ofpresumed interaction between TLR3 and itsligand, dsRNA (42).

The cytoplasmic domains of TLR1 andTLR2 have also been crystallized and theirstructures solved (43). Each is compact andglobular, and there is no evidence of inter-action between subunits in the unit cell. Asurface structure known as the BB loop ispresent in each TIR domain and contains theresidue corresponding to residue that is al-tered in the TLR4 protein of C3H/HeJ mice(P712H for TLR4; P681H for TLR2). Themutation does not prevent normal folding ofthe TIR domain, as shown by crystallizationstudies (43). However, it does abolish signaltransduction from most of the TLRs (withsome key exceptions, noted below), indicatingthat the residue in question is, in those cases,part of the signaling interface for interactionwith adapter proteins, as discussed below. Thesimilarity between TIR domains is such thatthreading programs can be used to model eachTIR domain on those that have been directlyanalyzed by X-ray crystallography (44).

It is widely believed that all the TLRsare dimeric, with some homodimeric andothers (TLR2/TLR6 and TLR2/TLR1)heterodimeric. Dimeric structure is as-sumed because enforced dimerization trig-gers a response (36) and because membrane-proximal modifications of cysteine residuesin Toll cause dorsalization of the embryo inDrosophila (45). Moreover, IL-1R and IL-18Rare heterodimers and signal via TIR domains,although the extracellular domain of each re-ceptor contains immunoglobulin superfam-ily repeats. Dimeric structure must be taken

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into account in considering precisely how theTLRs signal.

Some TLRs are clearly intracellular, resid-ing predominantly or entirely within the en-dosomes. These include TLR3, -7, -8, and -9,which sense nucleic acids. TLR1, -2, -4, and -6are at least largely expressed on the surface, al-though their presence within the phagosomeand later components of the endocytic path-way is not excluded.

Other Components of the LPSReceptor

At least some TLRs depend on other pro-teins to signal effectively (Figure 4). In thecase of the TLR2/TLR6 heterodimer, CD36,a double-spanning plasma membrane proteinof the class B scavenger receptor family, isknown to participate in signaling events, asshown by the phenotype of mutant mice iden-tified in a TLR signaling screen (46), de-scribed in detail below. The TLR2/TLR6heterodimer also depends in part on CD14to signal effectively, and mutations that elim-inate CD14 partly (∼50%) impair sensing ofall TLR2/TLR6 ligands.

The LPS receptor TLR4 depends evenmore strongly on CD14 than does theTLR2/TLR6 heterodimer, and withoutCD14, TLR4 cannot mobilize all of theadapter proteins that it requires for fullsignaling activity. This dependence suggeststhat TLR4 is organized into a supramolecularcomplex through interaction with CD14 andwith its ligand, LPS. In fact, CD14 was firstshown to enhance LPS signals in 1990 andwas the first component of the LPS receptorcomplex to be identified (47). Only morerecently was it shown, through a forwardgenetic approach, that CD14 is specifically re-quired for the detection of smooth LPS (LPSwith abundant O-glycosylation) rather thanrough LPS or lipid A, and for signaling viathe TRIF/TRAM pathway. CD14 recognizesother TLR4 ligands as well, such as glycopro-tein G of VSV (P. Georgel, Z. Jiang, S. Kunz,K. Hoebe, E. Janssen, M. Oldstone, and B.Beutler, manuscript submitted). In addition,a small protein known as MD-2 associateswith the TLR4 ectodomain and is requiredfor LPS signal transduction. Mutations inMD-2 entirely prevent LPS signaling (48),matching the effect of mutations in TLR4.

Figure 4Shared and unique components of TLR complexes. Germ line mutations have proven the participationof CD14, CD36, and MD-2 in signaling by TLR2 and TLR4 complexes. Coreceptors broaden thespecificity of the receptor complexes and in some cases influence the choice of adapters that are recruited.MALP-2 and LTA require CD36 for full signaling efficacy; zymosan and PAM2CSK4 do not. CD14 ispartly required by all TLR2/TLRX and TLR2/TLR6 agonists, is fully required by smooth LPS, and ispartly required by rough LPS. TLRX indicates uncertainty concerning the partner for TLR2 in thePAM2CSK4 receptor complex; it is most likely a second TLR2 subunit. (Abbreviations: VSV-G, vesicularstomatitis virus glycoprotein G; MMTV-G, mouse mammary tumor virus surface glycoprotein.)


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TRIF: Toll/IL-1Rdomain-containingadapter inducingIFN-β

TRAM:TRIF-relatedadapter molecule

PAM3CSK4:triacylatedlipopeptide(N-Palmitoyl-S[2,3-bis(palmitoyloxy)-(2RS)-propy1]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x3 HCI)

MALP-2:macrophageactivatinglipopeptide 2

LTA: lipoteichoicacid

The Sensing Function of OtherTLRs

Targeted gene deletion has revealed thatTLR2/TLR1 heterodimers are required fordetection of triacylated bacterial lipopeptidesor synthetic peptides such as PAM3CSK4

(49). Some diacylated lipopeptides, such asthe Mycoplasma-derived macrophage activat-ing lipopeptide 2 (MALP-2), signal via theTLR2/TLR6 heterodimer, and they do so ina stereospecific manner: Only the R enan-tiomer uses this signaling complex (50). TheTLR2/TLR6 complex reportedly also recog-nizes lipoteichoic acid (LTA) and β-glucanssuch as zymosan, although some cautionis needed in interpreting cellular signalingevents induced by microbial fractions suchas these, given that they are not always en-tirely pure. Other diacylated peptides (e.g.,PAM2CSK4), although TLR2 dependent, re-quire neither TLR1 nor TLR6 (51). Either aTLR2 homodimer or TLR2 associated withTLRX (candidates include TLR11, -12, and-13) might serve recognition of these molec-ular species.

TLR5 recognizes flagellin, a protein rep-resented in both Gram-positive and Gram-

negative microbes (52). TLR7 (in mice andhumans) and TLR8 (in humans only) recog-nize nucleoside analogs such as resiquimodor imiquimod (53), drugs with antineoplas-tic and antiviral potential that are now be-lieved to mimic the natural ligand ssRNA (54–56). TLR9 senses DNA bearing unmethylatedCpG-containing motifs (57) and does so in aspecies-specific fashion (58).

Humans (but not mice) express TLR10.Mice (but not humans) express TLR11, -12,and -13. There is some confusion of nomen-clature in that TLR11 has been called TLR12by some authors, and vice versa (59, 60).The molecular specificity of TLR10 remainsunknown, although because of its structuralsimilarity to TLR1 and TLR6, TLR10likely senses lipopeptides. Its absence in micehas prevented examination of the knockoutphenotype. In mice, TLR11 recognizes aprofilin-like component of Toxoplasma gondii(61) (evidently distinguishable from the hostmolecule). The functions of mouse TLR12and TLR13 have not yet been determined(Table 1).

Particularly where nucleotide ligands areconcerned, specificity is not absolute, and

Table 1 TLR ligand specificities, adapters used either alone or in combination withone another, and species representationa

Ligands TLR Adapters SpeciesPAM3CSK4 1,2 MyD88, MAL Human, MousePAM2CSK4 2,X MyD88, MAL Human, MouseMALP-2, LTA, Zym 2,6 MyD88, MAL Human, MousedsRNA 3 TRIF Human, MouseLPS, VSV-G, MMTV-G 4 MyD88, MAL, TRIF, TRAM Human, MouseFlagellin 5 MyD88 Human, MousessRNA, IAQ 7 MyD88 Human, MousessRNA, IAQ 8 MyD88 Humanb

CpG-ODN 9 MyD88 Human, MouseUnknown 10 Unknown HumanProfilin 11 MyD88 MouseUnknown 12 Unknown MouseUnknown 13 Unknown Mouse

aIAQ, imidazoquinolines, including resiquimod and imiquimod; MAL, MyD88 adapter–like; CpG-ODN,synthetic oligodeoxynucleotides containing CpG motifs. Other abbreviations as described in text.bTLR8 is encoded in the mouse genome, but activating ligands, if any, are unknown.

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host molecules can also trigger inflamma-tory responses, although perhaps less intenseresponses than those initiated by microbes.TLR3, -7, -8, and -9 are largely (although notnecessarily entirely) dedicated to the detec-tion of viral infection. However, other TLRs,most notably TLR4, also play an importantrole in viral sensing.

The Net Importance of TLRs inResistance to Microbes

TLRs signal via four adapter proteins(MyD88, Mal, TRIF, and TRAM), discussedin greater detail below. Without the adapterprotein MyD88, which is required for all sig-nal transduction by many of the TLRs andfor full signaling by TLR4, mice frequentlysuccumb to infections derived from theirown oral flora [mixed infections of the sub-mandibular nodes with α-hemolytic strepto-cocci and Pasteurella pneumotropica have beenobserved (Z. Jiang & P. Georgel, unpublishedobservations)]. When both MyD88 and theadapter TRIF are absent, mice are even moresusceptible to infection, and very few surviveto weaning age (B. Beutler & K. Hoebe, un-published observation). Individual TLR mu-tations are also compromising. For example,TLR4 mutations cause enhanced susceptibil-ity to Salmonella typhimurium (28) and Es-cherichia coli (29) infections; TLR2 deletioncauses enhanced susceptibility to Staphylococ-cus aureus (62) and other organisms. TLR3deficiency (59) and to an even greater extentTLR9 deficiency (59, 63) cause susceptibil-ity to MCMV infection, a highly prevalentpathogen in wild mice. By implication, thesame mutations may enhance susceptibility toβ-herpesviruses in other species. These ob-servations suggest that the TLRs make an es-sential contribution to the recognition of in-fectious organisms.

The Special Case of Viruses

Viruses were the last pathogens found to berecognized by TLRs (59, 63–68), and at the

MyD88: myeloiddifferentiation factor88

MMTV: mousemammary tumorvirus

time, it came as something of a surprise tosee that they were so recognized. Viruses areprimarily, although not entirely, sensed byTLRs dedicated to the detection of unmethy-lated DNA, dsRNA, and ssRNA. As such,the challenge of self-/nonself-discriminationis greater because host nucleic acids are onlyslightly different than virally encoded nu-cleic acids. ssRNA is, of course, very abun-dant in host cells, and most RNA species haveat least some dsRNA regions. Some CpGdinucleotides within the mammalian genomeare unmethylated and should therefore berecognized by TLR9. These facts have ledto the suggestion that autoimmune diseasesare fueled in part by TLR signaling, stimu-lated by host nucleoprotein complexes (14).At the very least, viruses test the limits ofself-/nonself-discrimination, and why TLRsare so effective in recognizing them is still notentirely clear. TLRs 3, 7, 8, and 9 not onlydetect structural differences between viral nu-cleic acids and host nucleic acids, but also mustengage the nucleic acids within endosomes, acompartment from which host nucleic acidsare normally excluded (69).

There are numerous examples of viral pro-tein detection by TLRs as well. The enve-lope glycoprotein of mouse mammary tumorvirus (MMTV) (65, 66) and glycoprotein Gof VSV (P. Georgel, manuscript in prepara-tion) are both recognized via TLR4. It hasalso been reported that the F-glycoproteinof respiratory syncitial virus activates TLR4(64), although this assertion has recently beenchallenged (70).

The ability of TLR4 to detect viruses isinteresting for several reasons. Like TLR3,TLR4 uses the adapter protein TRIF and iscapable of stimulating type I IFN responses.But TLR4 is normally thought of as theLPS receptor, and it is surprising to find thatentirely different molecules are engaged byit. The fact that they are raises structuraland evolutionary questions. The ability ofreceptors to recognize structurally disparatemolecules is not without precedent. For ex-ample, opiates are plant alkaloid agonists that


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TIR-DEPENDENT AND TIR-INDEPENDENTADAPTIVE IMMUNE ACTIVATION

TLRs mediate adjuvant effects induced by microbes, knownsince the time of Freund. The adjuvant signals initiated bythe TLRs are largely directed toward a CD4 response. Butthey are not required for adaptive immune activation per se.In the absence of TLR adapter proteins MyD88 and TRIF,TLR signaling is at least greatly diminished. However, normallymphoid development, IgG production, and allograft rejec-tion are observed. These findings point to the existence ofTIR-independent pathways for adaptive immune activation.Certain types of cell death, including death induced by UV-or γ-irradiation or by Fas ligation, are capable of triggeringa strong immunoadjuvant pathway that is chiefly (althoughnot exclusively) directed toward CTL activation, and entirelyTLR independent. This pathway is known to depend on theintegrity of the type I IFN receptor and UNC-93B (the 12-spanning ER protein affected by the 3d mutation). To theextent that the pathway can (weakly) activate CD4 cells, itis additionally dependent on CD36 (CD4 activation is selec-tively impaired by the oblivious mutation). However, CD36plays no part in the internalization of antigen molecules. TheTIR-independent immunoadjuvant pathway, which presum-ably serves the recognition of cell death triggered by cer-tain viral infections, is currently being analyzed using ENUmutagenesis.

IRF: IFN regulatoryfactor

IRAK:IL-1R-associatedkinase

trigger a response from endorphin receptors.However, it is a different task for receptorsto maintain contact with multiple targets thatare driven to evade detection. In the case ofVSV glycoprotein G, mutations might nul-lify recognition, permitting the virus to evadedetection. In the case of the MMTV enve-lope glycoprotein, investigators believe thatthe virus uses TLR4 for a subversive purpose,stimulating IL-10 production rather than thefull spectrum of TLR signals, and minimizingthe immune response (66).

The Connection Between Innateand Adaptive Immunity

As described so far, the function of TLRs inmammals was originally established by find-ing the receptor for LPS, a molecular en-

tity long known as something made by mi-crobes that was sensed by the host (33). TLRsare the key sensors of microbial infections ofmany and perhaps all types. It follows that theyare ultimately responsible for most (althoughnot necessarily all) infection-related phenom-ena, no matter how complex those phenom-ena may be (15).

One of these infection-related phenom-ena is the adjuvant effect of microbes. Thegold standard of adjuvants is Freund’s com-plete adjuvant, which is made using mycobac-teria suspended in an oil-in-water emulsion.Molecules of microbial origin must have adju-vant properties. In 1955, investigators showedthat LPS is endowed with adjuvant properties(26), and in subsequent years other moleculesof microbial origin were also shown to haveadjuvant effects (71–76).

Among the key molecular events leading toan adaptive immune response is upregulationof costimulatory molecules (including but notlimited to CD80 and CD86), which must oc-cur on the surface of antigen-presenting cells(APCs) to activate T cell mitogenesis (77).LPS elicits upregulation of these moleculeson APCs. Medzhitov and colleagues (36) oncespeculated that this occurs through the abil-ity of TLRs to activate NF-κB. However, it isnow clear that the key event in upregulation ofcostimulatory molecules is the activation of atype I IFN response (78, 79). This, in turn,depends on LPS activation of the MyD88-independent pathway and on activation of theadapters TRIF (67, 80), TRAM (81), and IRF-3, discussed in more detail below. Other mi-crobial adjuvants activate type I IFN synthesisthrough activation of the adapter MyD88, viaIRAK-1 and IRF-7 (82).

TIR-Dependent andTIR-Independent Pathways:A Death-Driven Pathway forCTL Activation

The fact that TLRs mediate microbial ad-juvanticity led to early suggestions that theymight mediate adaptive immune activation at

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Figure 5An immunoadjuvant pathway driven by programmed cell death. The pathway does not depend on TLRsignaling, and the triggering ligands induced by programmed cell death are unknown. The 3d mutationprevents the APC from presenting exogenous antigen, so that both priming and cross-priming areinhibited. The oblivious mutation prevents priming, but not cross-priming. The process also depends onthe production of type I IFN and on its signaling via IFNAR on the APC.

large. However, in mice that lack two of theadapters required for TLR signaling (MyD88and TRIF), there is little or no response tomost microbial ligands. Although we have al-ready noted that such mice are drastically im-munocompromised (K. Hoebe & B. Beutler,unpublished observation), they exhibit nor-mal concentrations of circulating antibodies,have normal lymphoid architecture, and canreject allografts. Although Freund’s adjuvantdoes not promote an immune response in suchmice, alternative pathways for adaptive im-mune activation clearly exist and are presum-ably activated by nonmicrobial stimuli.

The danger model of costimulation holdsthat cell death or injury may provide theessential signal for an adaptive immune re-sponse (83). It appears that some, but notall, forms of cell death trigger a strong andTIR-independent signal that leads to the in-duction of type I IFN synthesis, as well asto priming and cross-priming of T cells withspecificity for antigens expressed by the dy-

ing cell (Figure 5). Programmed cell deathinduced by γ- or UV-irradiation or by Fas lig-ation will trigger such a response (E. Janssen,K. Tabeta, M. Barnes, S. McBride, S. Schoen-berger, A. Theofilopoulos, B. Beutler, andK. Hoebe, manuscript submitted).

The cells that are capable of sensing pro-grammed cell death are B220− and CD8low

lymphoid elements, and they can be differ-entiated from bone marrow precursors us-ing Flt-3 ligand. They acquire antigen bynibbling. Notably, the system is not repre-sented in myeloid DCs or in macrophages;the macrophages probably dispose of manyor most of the cells that die through senes-cence or other processes of attrition in vivo.The system for adaptive immune activationmay be called into service when normalroutes for removal of apoptotic cells are over-whelmed. Moreover, cell death that is inducedby pathologic stimuli (viruses, irradiation) islikely qualitatively different than that occur-ring as a result of constitutive apoptosis, and it


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ENU: N-ethyl-N-nitrosourea

leads to the expression of recognizable surfacemolecules that are as yet unknown.

The death-driven pathway evokes a farmore powerful cytolytic T lymphocyte (CTL)response than Freund’s adjuvant. Antigens areusually detected by the system when injectedinto mice in picogram to nanogram quanti-ties, expressed by syngeneic cells that havebeen induced to undergo apoptosis. The sys-tem presumably evolved to detect cell deathinduced by viral infections and comprises aroute to rapid adaptive immune activation inresponse to this stimulus. Because it is TIRindependent, it would be effective when TLRsensing is not. This system of recognitionconceivably participates in diverse immuno-logical events, including allograft rejectionand autoimmunity.

THE INTENTIONAL CREATIONOF PHENOTYPE

Although a spontaneous mutation led tothe concept that TLRs detect molecules ofmicrobial origin, investigators have had to in-duce mutations, either deliberately or at ran-dom, to dissect the TLR signaling pathwaysand to identify participating molecules thatare structurally dissimilar from those that areknown. The induction of mutations that causephenotypes of interest, and subsequent iden-tification of those mutations, is the essenceof the classical or forward genetic approach.The germ line mutagen used for this purposeis N-ethyl-N-nitrosourea (ENU).

ENU and Its Efficiency as a Mutagen

In using a germ line mutagen, one would ide-ally like to create as many mutations as possi-ble, consistent with the production of mono-genic (as opposed to polygenic) phenotype,because only monogenic phenotype is readilymapped and positionally cloned. Practicallyspeaking, ENU dosing is limited by the factthat male mice become sterile if administeredtoo high a dose. ENU has been tested in manydifferent strains of mice, and the standard pro-

tocol that has emerged, in C57BL/6 mice, isadministration of 100 mg/kg three times atweekly intervals. A 12- to 15-week period ofinfertility follows. During this interval, thetestis is repopulated by spermatogonia, aris-ing from approximately 10 to 100 precursors.After this, mice may be bred to transmit germline mutations to the G1 population.

For studies of immunity, C57BL/6 femalesare best used in breeding to maintain a puregenetic background. Among G1 mice, domi-nant phenotype may be identified. To producerecessive phenotype, G1 mice are crossedagain to C57BL/6 females, and then to theirown daughters. Among G3 mice, recessivephenotype may be identified.

The potency of a germ line mutagen is ul-timately limited by the mutational load com-patible with life. Even if ENU did not inducesterility, it clearly induces nearly the maxi-mum number of mutations tolerated in theG3 population. Kile et al. (84) have analyzedthe frequency of lethal hits induced by ENUwithin a restricted area of the genome using abalancer chromosome, a dominantly marked,inverted chromosome that carries a homozy-gous lethal mutation. Balancer chromosomescan be used to capture lethal mutations be-cause only carriers of the balancer are evi-dent in the G3 population, whereas homozy-gotes for the mutagenized chromosome andhomozygotes for the balancer itself are absent.Approximately 1/13 G1 mice born to mutag-enized sires carried lethal hits within the bal-ancer region, with lethality defined as a failureto survive to the age of weaning.

Because the inverted region encompassedonly about 5% of all genes in the genome, andbecause lethal hits occur elsewhere as well,it may be surmised that each G1 mouse is,on average, heterozygous for about 1.5 reces-sive lethal hits genome-wide. If a G1 mousebears a lethal hit, the probability of its trans-mission to homozygosity in each G3 mouseis 1/8 (0.125), and the likelihood of nontrans-mission to homozygosity in each G3 mouseis 1–0.125, or 0.875. Hence, 1–0.8751.5, orabout 18%, of the G3 population is lost as

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a result of homozygosity for ENU-inducedmutation.

If the efficiency of ENU were tenfoldhigher (15 lethal hits per G1 mouse), 86% ofthe G3 population would be lost, an amountof attrition that might be considered unde-sirable because multiple litters would be re-quired to produce a very small number of micefor screening. In effect, ENU provides muta-genic power approaching the maximum thatcan be tolerated.

The Concept of a Genomic Footprint

For any phenotype that the investigator de-fines, a certain and very exact set of nucleotidechanges across the genome are capable ofproducing that phenotype. These nucleotidescomprise the genomic footprint of the phe-notype in question.

Estimates from direct sequencing of sen-tinel regions of G1 genomic DNA reveal thatENU changes about 1 base pair (bp) per mil-lion bp in the haploid genome (85), and 1/8of these changes are transmitted to homozy-gosity in each G3 mouse. If we assume thatall bp within a genomic footprint are equallyat risk from ENU, it is possible to calculatethe size of the genomic footprint based on thefrequency of observed phenotypic change. If100 bp are at risk, and if pedigree construc-tion is such that 50% of G1 mutations arecaptured in the homozygous state (e.g., twoG2 mice per G1 sire and three G3 mice perG2 dam), one would expect to see the pheno-type once, on average, for every 120,000 G3mice examined. If 100,000 bp are at risk, thephenotype should be apparent once in every120 G3 mice examined. Probably the pheno-type with the largest genomic footprint of all islethality, which, as noted earlier, is realized inabout 18% of G3 mice, suggesting a genomicfootprint of about 1.6 million bp.

The genomic footprint of a phenotype maybe small because it is masked by other pheno-types (typically lethality). For example, a par-ticular immunodeficiency phenotype mightrarely be observed because mice do not survive

Genomic footprint(of a phenotype):the set of allnucleotides that cancause the phenotypein question whenmutated

to the age required by the assay. Alternatively,the genes that support a particular immunefunction might also be required for develop-ment in utero, and only the exceptional nu-cleotide change will produce the phenotypeyet be compatible with viability to term. Al-though viable hypomorphic alleles probablyexist for all genes, the nucleotide substitu-tions that produce them are sometimes scarce.Rather, it is more common that a mutationalchange has no measurable effect on the func-tion of a protein at all, or that it utterly de-stroys the protein.

On other occasions, the genomic foot-print of a phenotype may be small becausethe phenomenon under analysis is served byredundant pathways. Few genes are truly re-dundant because locus duplication most com-monly leads to the formation of a degeneratepseudogene. Most commonly, the claim ofredundancy belies a screen that is not suffi-ciently powerful to resolve genes with over-lapping function, although the functions ofthe two genes are actually distinguishable.But some recently duplicated genes do existin the genome, and their functions, althoughimportant, might not be revealed by ENUmutagenesis.

How the Footprint Is Parceled intoGenes

The genomic footprint of a phenotype is scat-tered among genes that support the phe-nomenon under study. The mathematicaldistribution of the target nucleotide popu-lation among genes has not been describedand is not known from experiment becausegenome-wide saturation of a phenotype hasnot been approached. Gene size presumablyinfluences the distribution; perhaps more in-fluential still is variation in the resilience ofencoded proteins to mutagenic change. In ad-dition, it is possible (though uncertain) thatnucleotides differ regionally in their suscepti-bility to ENU.

The distribution of the genomic footprintamong genes is a matter of importance in


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considering the approach to saturation. Start-ing with the assumption that all nucleotidesare equally vulnerable to change induced byENU [which is assuredly untrue because thegreat majority of base changes are known tobe A → T transversions or A → G transitions(86)], and with the fact that 1/8 of 1 millionthof the genome is altered in homozygous formin each G3 mouse, we might imagine that5.9% of all nucleotides would be struck if onemillion G3 mice were examined, following thesame pedigree parameters as those outlinedabove.

One might thus assume that all genes rele-vant to the phenotype would be revealed. Thisassumption, however, depends entirely on thedistribution of the target nucleotide popu-lation. A given phenotypic footprint mighthave 10,000 nucleotides (a typical number)parceled into 50 genes (perhaps also a typi-cal number). The average gene would have200 target nucleotides and would indeed berevealed. But a gene with 10 vulnerable nu-cleotides would have only a 46% chance ofbeing revealed. And the footprint might in-clude many such genes.

At any rate, no ENU screen has yet ap-proached a depth of one million mice. Accord-ing to our best estimate, using a protocol inwhich half of all mutations are transmitted tohomozygosity, ∼10% phenotypic saturationis achieved with the analysis of ∼10,000 G3mice.

Some Summary Statistics

Almost all ENU-induced phenotypes resultfrom coding change (missense errors, non-sense errors, splicing errors, or, more rarely,single base pair deletions), although excep-tions have been reported (87, 88). The cod-ing region of the mouse genome encompassesapproximately 42 Mb of DNA (1.5% of thegenome as a whole). It may therefore be calcu-lated that if ENU creates 1 bp change per mil-lion bp, about 42 mutations fall within partsof the genome where a phenotypic effect canbe exerted. About 76% of nucleotide changes

that fall within a coding sequence create cod-ing change. Therefore, 32 coding changes oc-cur in each G1 mouse born to a mutagenizedsire. About four of these changes are transmit-ted to homozygosity in each G3 mouse.

About 18% of mice die as a result of fourrandom homozygous changes in coding se-quence, which suggests that each individualchange has approximately a 4.8% chance ofdelivering the lethal blow. The number ofgenes that are targets in this process (i.e., thenumber of genes with recessive lethal alle-les) remains unknown. But if we assume that1/3 of all genes have lethal alleles (a low-endestimate), we could conclude that, on aver-age, about 16% of random coding changeswithin genes that can cause lethality do causelethality. If 2/3 of all genes have lethal alle-les (a high-end estimate), the figure wouldbe 8%. This reveals the average resilience ofproteins with respect to a defined qualitativephenotype.

The Design of Screens

A good screen reflects the designer’s appreci-ation of a biological phenomenon and curios-ity about its cause. It avoids phenomena thatare well understood, avoids redundancy, and,where possible, probes a large genomic foot-print. The screening assay must be sufficientlyrobust to avoid even rare false positives. It isbest if the assay is qualitative or, better still, bi-nomial. But whatever the screen, phenotypesselected for analysis must be strong enoughand penetrant enough to permit meiotic map-ping, without which no progress can bemade.

THE GENETIC DISSECTION OFTLR SIGNALING PATHWAYS

What Was Known from the Start

Analysis of TLR signaling pathways began be-fore the mammalian TLRs were recognized,with the discovery that IL-1R signaled byway of the adapter protein MyD88 (89) and

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Figure 6Proteins known prior to beginning ENU mutagenesis to participate in signaling from seven of the TLRsto the level of TNF activity.

with recognition that the serine kinase IRAKtransduces IL-1 signals (90, 91), dependingon TRAF-6 (92) and leading to the activationof NF-κB. Similarities between the IL-1 sig-naling pathway and the Toll developmentalpathway were noted early on (93), as was thesimilarity of the IL-1R cytoplasmic domainto the cytoplasmic domain of Toll (94). Earlyand rapid progress in understanding TLR sig-nal transduction rested on this similarity. Ho-mology searches led to the identification of allthe TLRs that are presently known, and genetargeting established the specificity of mostof them once the LPS sensing function ofTLR4 was determined by positional cloning(33). Homology searches have also disclosedthe existence of at least four cytoplasmic TIRadapter proteins. Whereas MyD88 and Mal(also known as Tirap) were easily detectedby using the Basic Linear Alignment SearchTool (BLAST), TRIF and TRAM were farmore distant and were found belatedly usingthe Hidden Markov Model search algorithm(HMMER).

TRAF-6: TNFreceptor–associatedfactor 6

However, the participation of proteinsstructurally unrelated to the TLRs was muchmore difficult to ascertain, and in that re-search a pure genetic approach has also playeda valuable role. By 2000, a total of 22 pro-teins were known to participate in TLR sig-naling from the level of seven of the TLRs tothe level of TNF bioactivity, and these pro-teins could be designated as potential targets(Figure 6).

A genetic screen was applied to identifyadditional components of the pathway. Thescreen was performed by stimulating peri-toneal macrophages with TLR agonists andthen measuring TNF production by biolog-ical assay (killing of L-929 cells). To date, atotal of 11 mutations have been identified byscreening approximately 20,000 mice. Thesemutations alter a total of 10 genes. Five ofthe genes were known at the outset of screen-ing. The other five, however, were unknowncomponents of the TLR signaling apparatus.Three of these have been positionally clonedto date.


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Calculation of Pathway Complexity

A forward genetic approach permits unbi-ased estimation of the total number of pro-tein components that are required for signal-ing from TLRs to TNF because each serves anonredundant function. Given that 5 of thegenes identified fell within the category ofknown genes (22 of which exist in all) whereas5 did not, it might be guessed that 44 targetsexist in total.

Taking an independent approach to esti-mate the number of nonredundant proteins,23% (5/22) of the known protein populationwas identified in the screen, corresponding tothe degree of saturation achieved in a study of20,000 mice. If 23% saturation nets 10 genes,43 targets may be estimated to exist in all.

Figure 7The position of Lps2 on the signaling map. The phenotype imparted bythe mutation was one in which TLR3 and TLR4 were both unable toactivate IRF-3. Production of TNF was also minimal, presumablybecause the normal protein also contributes to the activation of NF-κB.

Independently again, if one assumes thatall targets have uniform size (each subsumesan equal fraction of the genomic footprint),the identification of one instance of allelismamong 11 mutations would suggest the pres-ence of approximately 53 targets. Althoughthese estimates are tentative and deeper satu-ration must be pursued to determine the num-ber of targets with confidence, one may con-clude that a relatively small number of targetsexist and that by this time most of them areprobably known.

Lps2 and the Identification of TRIF

MyD88-deficient mice showed evidence of aMyD88-independent pathway, whereby LPS,acting via TLR4, could partially stimulateNF-κB translocation after a brief delay andactivate type I IFN production without im-pediment (95). With the targeted deletionof both MyD88 and Mal genes, the samephenomenon was observed (96). TLR3 sig-naling also had no requirement for eitheradapter. The molecular basis of the MyD88-independent pathway remained unclear. Thehallmark of MyD88-independent signalingwas not NF-κB activation, but rather IRF-3activation and subsequent type I IFN synthe-sis. However, the MyD88-independent path-way is also capable of activating NF-κB andpresumably does so by activating a compo-nent of the cascade that is distal to MyD88 it-self, thereby bypassing the lesion imposed bymutations in MyD88 and/or Mal (Figure 7).Researchers now believe that the point of in-tersection between MyD88-independent andMyD88-dependent pathways is TRAF-6 (97,98), a protein that coordinates the activationof numerous kinases, including TAK-1 [trans-forming growth factor (TGF)-β-activated ki-nase 1], which is required for the phosphory-lation of IKKβ (99, 100), leading to NF-κBactivation.

A mutation termed Lps2 first revealed themolecular basis of MyD88-independent sig-naling. The Lps2 mutation prevented IRF-3phosphodimer formation and IFN synthesis

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in response to either LPS or dsRNA, indi-cating that it affected a protein required forsignaling from both TLR4 and TLR3. More-over, Lps2 rendered mice susceptible to infec-tion by MCMV (101).

Positional cloning of the Lps2 mutationrevealed a frameshift error in a new adapterprotein, independently identified by homol-ogy searching (102) and by two-hybrid sys-tem analysis (103), and respectively knownas TRIF or TICAM (TIR-containing adaptermolecule)-1. On the basis of transfection stud-ies, investigators concluded that this proteinstimulates type I IFN production; however,investigators initially disagreed as to whetherthe protein served all TLRs (102) or onlyTLR3 (103). The phenotype of the mutationrevealed a specific role in signaling via TLR4and TLR3 (104), a conclusion confirmed bygene targeting (80).

Compound mutants lacking MyD88 andhomozygous for Lps2 show no detectable re-sponses to LPS and, indeed, show no re-sponses at all to most TLR ligands (104).However, researchers have recently observedthat TLR4 can indeed signal by way ofTRAM, even in the absence of both MyD88and TRIF, when activated by the VSV gly-coprotein G (P. Georgel, Z. Jiang, S. Kunz,K. Hoebe, E. Janssen, M. Oldstone, andB. Beutler, manuscript submitted).

Inferences Concerning the Functionof TRAM

BLAST analysis using the TRIF protein se-quence as a query disclosed a fourth adapter,termed adapter X (104) and known elsewhereas TICAM-2 (105) or TRAM (81). A spe-cific role for TRAM in TLR4 signaling wassuggested by the finding that in cells ho-mozygous for the TrifLps2 allele, a fractionof macrophages remained capable of produc-ing TNF in response to LPS. This TRIF-independent population of cells must de-pend on another adapter, and that adaptercould not be MyD88 because all cells (andnot merely some of them) were MyD88-

dependent; hence, MyD88 must be expressedand used in all cells (67). Hoebe et al. pro-posed that adapter X was TRAM, and thatTRAM must serve MyD88-independent LPSsignaling alongside TRIF, but not dsRNA sig-naling (67). Gene targeting, performed in-dependently (81), substantiated this proposal(Figures 8 and 9).

It is now clear that TRAM (but not TRIF)mediates signals initiated by the VSV gly-coprotein G, which depends on CD14 andTLR4. Hence, TRAM can function entirelyby itself and is not necessarily codependenton TRIF (P. Georgel, Z. Jiang, S. Kunz,K. Hoebe, E. Janssen, M. Oldstone, and B.Beutler, manuscript submitted).

Heedless Gives Fresh Insight into theNature of the TLR4 SignalingComplex

The Heedless mutation was identified becauseit prevented TNF production in response tosmooth LPS, that is, LPS with abundant O-glycosylation. Oddly, however, unlike TLR4mutants (which show no responses to LPSof any kind), Heedless mutants were able tomake almost normal quantities of TNF inresponse to rough LPS or synthetic lipid A(106). Remarkably, lipid A could not acti-vate IRF-3 phosphodimerization, nor couldit trigger type I IFN production, suggest-ing a defect of MyD88-independent signal-ing (106). Macrophages from Heedless micealso showed diminished responses to allTLR2/TLR6 ligands and were less capableof coping with infection by VSV, as weremice with the P712H mutation of TLR4(106).

The positional cloning of Heedless revealeda premature stop codon in CD14, previouslyregarded as a coreceptor for LPS signalingin general. The new interpretation of CD14function is therefore as follows:

1. Rough LPS or lipid A can directlyengage TLR4/MD-2 complexes, caus-ing recruitment and activation of


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MyD88, whereas smooth LPS cannot(Figure 10a, left);

2. CD14 nullifies the distinction betweenrough and smooth isoforms, permittingboth types of LPS to activate TLR4(Figure 10a, right);

3. CD14 is absolutely required for LPS-induced recruitment of TRIF andTRAM, which leads to the activation ofIRF-3;

4. CD14 is additionally required for fullactivation of the TLR2/TLR6 complex(which, as noted below, also requiresCD36 for sensing of some ligands)(Figures 8, 9).

5. CD14 and TLR4 mediate sensing ofVSV (Figures 8, 9).

Because CD14 is a glycosylphospho-inositol-tethered protein that projects fromthe surface of the cell, it would seem incapableof directly interacting with the cytoplasmicdomains of TLRs and must influence signal-ing through interaction with the ectodomains.We may conclude that CD14 coordinates theinteraction of ectodomains so as to permit therecruitment of adapter molecules that wouldotherwise be excluded from that activatedcomplex (Figure 10b). These observations es-tablish that TLR4 is a switch with several pos-sible “on” positions. It can signal in differentmodes depending on the inducing ligand andon the profile of coreceptors and/or adapterspresent in the responding cell.

Macrophage production of IFN-β, in-duced by UV-inactivated VSV, is abolished bythe Heedless mutation, as well as by mutation ofTLR4 or TRAM (or, to a much lesser extent,TRIF), and by the Feckless mutation (described

below). The inducing molecule present in thevirion is glycoprotein G rather than a nu-cleic acid (P. Georgel, Z. Jiang, S. Kunz, K.Hoebe, E. Janssen, M. Oldstone, and B. Beut-ler, manuscript submitted). Glycoprotein Gof VSV is a conserved molecule, broadly rep-resented in Rhabdoviridae, encompassing ver-tebrate pathogens such as VSV, rabies, andhemorrhagic viral septicemia virus of fish. Asalready noted, LPS is chiefly a stimulus tomammalian cells. Yet TLR4 is represented infish, which do not respond to LPS. We mayspeculate that its primordial function entailedthe detection of viruses, which would explainits connection to the type I IFN inductionpathway. This function has been retained inmammals as well, but only in mammals hasthe ability to sense LPS also been acquired.

CpG1 and the Role of TLRs inResistance to Viral Infection

TLR3, -7, -8, and -9 are at least mostly in-tracellular molecules, synthesized in the ER,and are eventually transferred to the endo-somes, possibly via transit over the cell sur-face and endocytosis (107). Several mutationsaffected signaling via these TLRs. The firstof these to be identified was one that blockedCpG sensing (CpG1). It was traced to a mis-sense error in the ectodomain of TLR9. TheLps2 mutation had earlier suggested the im-portance of the TLR3 → TRIF axis in sensingMCMV infection and responding to it (67).The CpG1 mutation was associated with evenmore severe susceptibility to MCMV (59), aswas a MyD88 mutation (59), indicating thatthe TLR9 → MyD88 and TLR3 → TRIF

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 8An overview of the pathways discussed in text. All TLRs, whether located at the cell surface (TLR1, -2,-4, -5, and -6) or within endosomes [TLR3, -7 (or -8), and -9], converge on four adapter proteins, whichact in discrete combinations with one another. These adapters lead to activation of protein kinases,including IRAK, IRAK4, TBK1, and IKKi. These kinases ultimately lead to activation of transcriptionfactors, including NF-κB, IRF-3, and IRF-7, which mediate many of the inflammatory effects ofmicrobial inducer molecules. Among the thousands of genes modulated by TLR signaling, TNF andIFN-β may be taken as landmarks of the response and are themselves known to be of key importance insubsequent activation of innate and adaptive immune responses.

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axes played nonredundant, additive roles inMCMV resistance. Although both axes con-tribute to type I IFN production, they mayhave type I IFN–independent effects as well,some of which may be nonoverlapping andmay contribute to resistance (e.g., the acti-vation of IL-12 production, which leads toIFN-γ production).

The importance of TLR9 and MyD88 inMCMV resistance was independently ana-lyzed using knockout mice, with similar con-clusions (63). Moreover, TLR9 was shown tocontribute to the response to herpes simplexvirus I (HSV-I) (68).

Pococurante and Lackadaisical, MyD88Alleles with Receptor-SelectiveFunction, and Their Interpretation

The Pococurante (Poc) phenotype is one inwhich there is an absence of MyD88 signal-ing, with the exception of signals that em-anate from the TLR2/TLR6 heterodimer (Z.Jiang, P. Georgel, C. Li, J. Choe, K. Crozat,S. Rutschmann, X. Du, T. Bigby, S. Mudd, S.Sovath, I. Wilson, A. Olson, and B. Beutler,manuscript in preparation). The Lackadaisi-cal (Lkd) phenotype shows normal MyD88signaling, except for signals from TLR7 andTLR9, which are markedly diminished. Bothmutations are missense errors in MyD88,each distinguishable from the knockout allele(Figure 11). The Poc mutation (I179N) affectsa surface residue within the TIR domain ofMyD88, whereas the Lkd mutation (Y116C)affects a portion of the polypeptide chain be-tween the death domain and the TIR domain.Because the modified adapter protein is, ineach case, capable of transmitting signals fromsome of the TLRs, it may be inferred that themutations do not destroy the protein. Rather,they must affect the signaling interfaces thatunite the adapters and receptors.

The Poc mutation causes pronounced sus-ceptibility to MCMV. However, mice arespared the severe immunodeficiency diseasecaused by a MyD88-knockout mutation andare demonstrably more resistant to at leastsome Gram-positive infections, suggestingthat limited signaling through a single TLRcomplex (TLR2/TLR6) is sufficient to offerfairly strong protection against a wide varietyof microbes. The Lkd mutation has little or noeffect on susceptibility to MCMV, probablybecause some signaling via TLR9 is retainedin Lkd homozygotes, compared with the situ-ation in CpG1 homozygotes.

Pococurante and Insouciant RevealSomething Special about MyD88Signaling from the TLR2/TLR6Complex

Poc is a particularly interesting mutationbecause it does disrupt signaling initiatedby some TLR2/TLR6 ligands, notably zy-mosan and LTA. However, it does not dis-rupt signaling by MALP-2 or PAM2CSK4,both diacylated lipopeptides that signal viathe TLR2/TLR6 heterodimer and via TLR2(alone or in conjunction with a still un-known TLR), respectively. Insouciant (Int),a point mutation in the ectodomain ofTLR6 (V327A), abolishes zymosan, LTA,and MALP-2 signaling but not PAM2CSK4

signaling.When engrafted onto TLR4 or TLR9, the

Poc mutation abolishes MyD88-dependentsignaling. However, when engrafted ontoTLR2 or TLR6, the mutation does not affectsignaling by MALP-2 or PAM2CSK4. More-over, the BB loop mutation, if engrafted ontoMyD88 (P200H), does not prevent down-stream signal transduction.

These observations suggest that cer-tain TLR2/TLR6 and TLR2/TLRX ligands

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 9An overview of the pathways discussed in text, with mutants shown with red X. Mutations shown wereinduced with ENU, and all but two (Spacey and Feckless) have been identified positionally.

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Figure 10(a) The Heedless mutation reveals that TLR4 can signal in two modes.Without CD14, Lipid A activates TLR4, causing recruitment of MyD88and MAL (MyD88 adapter–like) only, whereas smooth LPS (longpolysaccharide chain) cannot activate TLR4 at all. In the presence ofCD14 (bent solenoid), both smooth and rough LPS can recruit bothMyD88/MAL and TRIF/TRAM pathways. (b) Surface view of TLR4dimer, with or without MyD88. A supramolecular effect on TLR4aggregation is envisioned, so that with CD14, the geometry of interactionbetween subunits is correct for recruitment of all adapters. WithoutCD14, smooth LPS is excluded from the activation complex and Lipid Ais only able to stimulate MyD88/MAL recruitment.

(MALP-2 and PAM2CSK4) cause a confor-mational change in the receptor that favors aunique mode of MyD88 association with thereceptor complex (Figure 12). Although mostTLRs (and for some ligands, TLR2/TLR6 aswell) associate with MyD88 in a manner thatcauses both the BB loop and the Poc site tobe included in the signaling interface, diacy-lated lipopeptides trigger a different form ofreceptor activation, requiring the participa-tion of neither residue. The TIR domains ofMyD88 and the TLR2/TLR6 receptor com-plex likely engage one another in a recipro-cal manner in both cases. However, MyD88clearly has two different ways of propagating asignal.

Oblivious: A Co-Factor forTLR2/TLR6 Signaling

Still another point of complexity concerningTLR2/TLR6 signaling is that the diacylatedlipopeptide MALP-2 (but not PAM2CSK4)and LTA are largely dependent on CD36for signaling, a fact revealed by a prematurestop codon in CD36 that caused a phenotypecalled oblivious (46). Other TLR2/TLR6 lig-ands (zymosan, for example) are not. Hence,the TLR2/TLR6 complex uses CD36 as a co-receptor for some of its ligands. Perhapsfor this reason, oblivious mice show exag-gerated susceptibility to Gram-positive in-fections (46). The oblivious mutation alsoprevents CD4 priming by cells exposed toantigens in the context of apoptosis. Thus,oblivious impairs one endpoint of the death-driven immunoadjuvant pathway describedearlier in this review (E. Janssen, K. Tabeta,M. Barnes, S. McBride, S. Schoenberger, A.Theofilopoulos, B. Beutler, and K. Hoebe,manuscript submitted). This impairment sug-gests that CD36 has other immunologicalfunctions yet to be found.

CD36 is a double-spanning plasma mem-brane protein and is one of three membersof the class B scavenger receptor family (46,108). It is known as a receptor for throm-bospondin (109), for a role in the translocation

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Figure 11The Pococurante and Lackadaisical phenotypes. MyD88 is required for signaling via most of the TLRs, butnot TLR3, and is only partly required for signaling via TLR4. Pococurante mimics a null allele exceptinsofar as it permits full signaling by some of the ligands that use the TLR2/TLR6 and TLR2/TLRXcomplexes. Lackadaisical is similar to wild-type MyD88, except for diminished signaling via TLR7 andTLR9.

of fatty acids (110, 111), and for its abilityto bind oxidized LDL (112). Some endoge-nous molecules with inflammatory effects maystimulate TLR2/TLR6 signaling by way ofCD36, although specific examples of this havenot yet been demonstrated.

PanR1: A Point Mutation in TNFon a Pure C57BL/6 Background

The codominant PanR1 phenotype was sonamed for pan resistance to all TLR lig-

ands, where TNF activity was the end-point of response. The mutation was mappedto the TNF locus itself, and PanR1 wasfound to be a missense allele (P138T) withdominant inhibitory effects, resulting fromthe fact that a single mutant subunit pre-vents engagement of the TNF trimer bythe p55 TNF receptor (Figure 13). Littleif any TNF activity is evident in homozy-gotes, which consequently show exagger-ated susceptibility to Listeria monocytogenes (S.Rutschmann, K. Hoebe, J. Zalevsky, X. Du,

Figure 12The Pococurante mutation (Poc) reveals that MyD88 can interact with TLR2 complexes in twoconformationally distinct ways. Poc site and the BB loop site mutations inactivate most TLRs, and alsoinactivate MyD88, preventing interactions when engrafted onto either molecule. The TLR2/TLR6heterodimer and the TLR2/TLRX heterodimer are unique in that they are capable of interacting withMyD88 in two ways: one that is disrupted by Poc or BB loop mutations in either molecule, and one that isnot (special signaling mode). The latter mode of interaction is induced only by diacylated lipopeptides.


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Figure 13The location of the PanR1 mutation, which affects a surface residue(P138T) on the TNF homotrimer (yellow). Subunits are each shown indifferent colors.

N. Mann, P. Steed, and B. Beutler, manuscriptsubmitted).

Mice with the mutation, unlike mice witha knockout allele of TNF, have entirely nor-mal lymphoid development, including Peyer’spatches and marginal zone B cells in thespleen and lymph nodes (S. Rutschmann,K. Hoebe, J. Zalevsky, X. Du, N. Mann, P.Steed, and B. Beutler, manuscript submit-ted). This may suggest that the Tnf geneknockout affects expression of the neighbor-ing lymphotoxin (LTa) gene, which is knownto be required for lymphoid development,and that TNF itself has no developmentalrole. Alternatively, an exceedingly low levelof TNF activity—present in the homozygousPanR1 mutants but not in homozygous knock-out mice—may be sufficient for lymphoiddevelopment.

3d: A Defect of Signaling via TLR3,-7, and -9, Coupled with DefectiveExogenous Antigen Presentation

A combined defect of nucleic acid sensingvia TLR3, -7, and -9 was designated TripleD (3d) to denote a triple defect of signaling(113). Probably because nucleic acid sensingvia both TLR3 and TLR9 is impaired, themice are highly susceptible to MCMV infec-tion. However, they also show susceptibility toother organisms, such as Staphylococcus aureus,which is not effectively cleared and sometimescauses metastatic infections when inoculatedintradermally.

The 3d mutation abolishes exogenousantigen presentation, preventing both prim-ing and cross-priming of T cells in re-sponse to ovalbumin, administered eitheras a particulate antigen (expressed by cellsthat were induced to undergo apoptosis) oras a soluble protein. However, normal lev-els of class I and class II MHC proteinsare expressed on the surface of 3d APCs(113).

The phenotype was shown to result froma missense error affecting the 12-spanning,ER-resident membrane protein UNC-93B,which had no previously recognized func-tion in mammals. The effect of the muta-tion implies communication between the ERand the endosomal/lysosomal pathway. Wespeculate that 3d is required for the traffick-ing of certain membrane proteins within thecell, including proteins required for signal-ing via TLR3, -7, and -9 and other pro-teins required for exogenous antigen pre-sentation (Figure 14). A named homologof UNC-93B (UNC-93A) is also encodedin the mouse genome, and still more dis-tant homologs, as yet unnamed, are identi-fiable as well. These homologs are relatedto a Caenorhabditis elegans protein, known tobe a component of a two-pore potassiumchannel (114). However, UNC-93B appar-ently does not fulfill this function in mammals(113).

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Figure 14Possible mode of action of UNC-93B. The traffic of specific membrane proteins required for signalingby TLR3, -7, -8, and -9 is postulated to depend on the ER-resident protein UNC-93B. These co-factorsfor TLR activity, and probably other proteins required for presentation and cross-presentation ofexogenous antigens, cannot traverse the normal route from ER to endosome. Note that theTLR-independent cell death–induced activation pathway is also endocytic and, like the TLR pathway,leads to the induction of IFN-β. However, IFN-β induction does not depend on UNC-93B.

Feckless: A Protein Required forTLR3 to Activate NF-κB and forthe Response to VSV

As previously noted, TRIF activates IRF-3,yielding activation of the IFN-β gene, butalso activates NF-κB via an interaction withTRAF-6 (97, 98). The Feckless phenotype wascharacterized by failure to activate NF-κB inresponse to dsRNA, although dsRNA couldinitiate IFN-β production (Z. Jiang & B.Beutler, unpublished data).

Macrophages from Feckless mice were,moreover, found to be highly susceptible toVSV, and UV-inactivated VSV was incapableof stimulating a type I IFN response in Fecklesscells. The G-glycoprotein of VSV is knownto activate a type I IFN response via CD14,TLR4, TRAM, and IRF-7 (P. Georgel, Z.Jiang, S. Kunz, K. Hoebe, E. Janssen, M. Old-stone, and B. Beutler, manuscript submitted),but does not require TRIF to do so. Clearlyrequired to carry a signal from TLR3 andTRIF to activate NF-κB, Feckless may also in-

Reverse genetics:the genetic approachthat begins with agene of unknownfunction and endswith a phenotype

TBK1:TANK-bindingkinase 1

IKKi: IκB kinase i

teract with TRAM in an entirely independentpathway, leading to activation of IRF-7.

Spacey

Spacey homozygotes are runted animals withlow fertility and were identified because of di-minished ability to respond to TLR9. Themutation has been mapped to chromosome10 and is unlinked to the TLR9 locus itself.No genes known to be required for TLR sig-naling reside within the Spacey critical region.However, the mutation remains to be found.

REVERSE GENETICINVESTIGATIONS

TBK1 and IKKi, IRF-7 and IRF-5

Intercurrent investigations using reverse ge-netic methods revealed that TBK1 and IKKiare the protein kinases responsible for activa-tion of IRF-3 (98, 116–118), which is chiefly


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responsible for induction of IFN-β gene ex-pression caused by LPS or dsRNA. TBK1 ap-pears to be the more important of the two(119). IRF-7, which exists at minimal con-centrations in resting cells, is induced (pre-sumably autoinduced) to high concentrationsin response to TLR7 or TLR9 stimulation.These TLRs do not signal by way of TRIF orTRAM, and they activate type I IFN produc-tion via IRF-7 rather than IRF-3 (120). IRF-5is required for MyD88-dependent signalinginitiated by diverse MyD88-dependent TLRs(121). Investigators have suggested, althoughnot formally proved, that IRF-5 is activatedby IRAK-4. Alternatively, IRF-5 may be re-quired for the transcription of genes encodingcertain components of the pathway itself.

Inhibitors of Signaling

IRAK-M (122) and SOCS (suppressor ofcytokine signaling)-1 (123) function as in-hibitors of signaling via TLRs, the formerprobably inhibiting IRAK/IRAK-2/IRAK-4signaling and the latter preventing STAT (sig-nal transducer and activator of transcription)-1 signaling. The orphan TIR domainreceptors SIGIRR (single Ig repeat–relatedprotein) (124) and ST2 (125) exert inhibitoryinfluences as well, probably by virtue ofdirect interactions with TLRs. Upregula-tion of the phosphatase SHIP (Src homol-ogy inducible phosphatase)-1, which dependson autocrine stimulation by TGF-β, alsoinhibits LPS signal transduction, possiblyblocking both MyD88-dependent and TRIF-dependent pathways (126). The phenomenonof endotoxin tolerance is thus partially ex-plained (127) and appears to operate at severaldifferent levels.

Saturation: How Many Genes Servethe Pathways?

We can now point to 30 genes that makepositive (as opposed to inhibitory) contribu-tions to TLR signal transduction. As outlinedabove, roughly 40 to 50 genes are expected

to make nonredundant contributions to sig-naling, subject to fairly broad limits of er-ror. These figures might be taken to indicatethat 10 to 20 genes remain to be found. Weshould emphasize that these figures includeonly those genes that are required for the pro-duction of TNF activity in response to ligandsthat depend on 7 of the 12 mouse TLRs. If thescreen for responses were less focused, and ifall the TLRs were stimulated, more targetsmight be detected.

A BROADER LOOK: THE MCMVRESISTOME AND ITS SIZE

Because 11 transmissible mutations wereidentified by screening about 20,000 G3 mice,an authentic phenotype is picked up in about1 of every 1800 mice examined. A sub-stantially higher hit rate is observed in ascreen that tests the integrity of all essentialsystems for the containment of MCMV infec-tion. Approximately 1 in 400 G3 mice scorepositive in this screen, in which 105 PFU ofvirus (an inoculum insufficient to affect nor-mal C57BL/6 animals) is administered by anintraperitoneal route, and sickness or deathis recorded within the first seven days to as-sure that only innate immunodeficiency phe-notypes are detected. This corresponds to1 recessive mutation in every 33 pedigrees ex-amined, and to a genomic footprint that en-compasses about 45,000 nucleotides, parceledamong about 300 genes. The innate resistomefor MCMV thus corresponds to about 1% ofthe genome.

Some of the mutations identified in ascreen for MCMV susceptibility cause cosus-ceptibility to other viral pathogens. For exam-ple, macrophage susceptibility to VSV is com-monly observed. But some mutations have aspecific effect on NK cell function, or ratherbroad effects on vesicle transport, leading tohypopigmentation coupled with immunode-ficiency.

As of this writing, approximately 30MCMV mutations have been identified inscreening, and 10 mutations are displayed in

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Table 2 Nine MCMV susceptibility mutations and their phenotypic characteristics

NameOutcome of MCMV

infectionVSV

Susceptible?aIFN-α/βRescue?b

NK celldefect? Cloned?

Domino (recessive) death on day 4 Necrotic spleen Yes No Yes Yes(STAT-1)

Goodnight(recessive)

death on day 2–3 Necroticliver

n.d. n.a. n.d. No

Solitaire (recessive) death on day 2–3 n.d. n.a. n.d. NoSlumber (recessive) death on day 2–3 n.d. n.a. n.d. NoJinx (recessive) Elevated viral load in spleen Yes Yes Yes NoWarmflash(co-dominant)

Elevated viral load in spleen Yes Yes No No

May Day (recessive) death on day 3 n.d. n.a. n.d. NoParis (recessive) Exanthem n.d. n.a. n.d. NoMoneypenny(recessive)

death on day 6 Yes Yes n.d. No

Havelock (recessive) Elevated viral load in spleen No n.a. No No

aRefers to the susceptibility of macrophages ex vivo.bRefers to rescue of the VSV phenotype.n.a., not applicable; n.d., not determined.

Table 2. One among these has been posi-tionally cloned. This mutation, Domino, isa missense allele of STAT-1, modifying theDNA-binding domain of the protein and alsopreventing normal phosphorylation and ex-pression of the protein in cells (K. Crozat, P.Georgel, S. Rutschmann, N. Mann, X. Du,K. Hoebe, and B. Beutler, manuscript sub-mitted). Remarkably, many of the mutationsidentified to date cause far greater suscepti-bility to MCMV than the Domino mutation,which effectively disrupts both type I and typeII IFN signaling, as well as IL-27 signaling.We must therefore conclude that some pro-teins are far more important to the host thanthe IFNs, given the circumstance of MCMVinfection. Clearly, much remains to be learnedabout antiviral defense.

CONCLUSIONS

A classical genetic inquiry led directly to ourpresent understanding of how the immunesystem detects infection. But beyond point-ing to the function of the TLRs and helping

to elucidate their signaling pathways, the ge-netic approach has done much more as well.Intracellular sensors of microbes also exist.The nucleotide-binding oligomerization do-main (NOD)-1 and NOD2 proteins, for ex-ample, are believed to function as cytoplasmicsensors of peptidoglycans (128–130). Theirgeneral role in immunity was originally dis-covered by a pure genetic investigation, un-dertaken in humans rather than in mice (131,132). The NK receptor Ly49H, which detectsthe MCMV-encoded protein m157 (133), wasidentified by positional cloning as well (2,3). So, too, was the key transcription fac-tor required for the development of regu-latory T cells (134). Mutagenesis is beingdirectly applied to the problem of autoimmu-nity and has already yielded impressive results(135, 136).

What other large-scale phenomena inter-est us in immunology? This is for the reader toponder. Sometimes, asking the question—orin this case, seeing the phenomenon, cleverlyconcealed as it is—may be the most difficultpart of the process.


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APPENDIX: GLOSSARY OF MUTATIONS

Domino (Dom): STAT-1 (missense; null)

Feckless (fks): unknown (failure to activate NF-κB with preserved IFN production inresponse to polyI:C)

Heedless (hdl): CD14 (nonsense; null)

Insouciant (Int): TLR6 (missense; null)

Lackadaisical (Lkd): MyD88 (missense; diminished signaling via TLRs 7 and 9)

Lps: TLR4 (missense; null)

Lps2: TRIF (frameshift; null; codominant or haploinsufficient)

Oblivious (Obl): CD36 (nonsense; null)

Pococurante (Poc): MyD88 (missense; retains signaling only via TLR2/TLR6 andTLR2/TLRX)

PanR1: TNF (missense; extreme hypomorph; dominant)

Triple D (3d): unc-93b (missense; null)

Spacey (Spy): unknown (diminished TLR9 signaling)

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Contents ARI 7 February 2006 11:41

Annual Reviewof Immunology

Volume 24, 2006Contents

FrontispieceJack L. Strominger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � x

The Tortuous Journey of a Biochemist to Immunoland and What HeFound ThereJack L. Strominger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Osteoimmunology: Interplay Between the Immune System and BoneMetabolismMatthew C. Walsh, Nacksung Kim, Yuho Kadono, Jaerang Rho, Soo Young Lee,

Joseph Lorenzo, and Yongwon Choi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �33

A Molecular Perspective of CTLA-4 FunctionWendy A. Teft, Mark G. Kirchhof, and Joaquín Madrenas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Transforming Growth Factor-β Regulation of Immune ResponsesMing O. Li, Yisong Y. Wan, Shomyseh Sanjabi, Anna-Karin L. Robertson,

and Richard A. Flavell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �99

The EosinophilMarc E. Rothenberg and Simon P. Hogan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 147

Human T Cell Responses Against MelanomaThierry Boon, Pierre G. Coulie, Benoît J. Van den Eynde,

and Pierre van der Bruggen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 175

FOXP3: Of Mice and MenSteven F. Ziegler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 209

HIV VaccinesAndrew J. McMichael � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

Natural Killer Cell Developmental Pathways: A Question of BalanceJames P. Di Santo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 257

Development of Human Lymphoid CellsBianca Blom and Hergen Spits � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 287

Genetic Disorders of Programmed Cell Death in the Immune SystemNicolas Bidère, Helen C. Su, and Michael J. Lenardo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 321

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Contents ARI 7 February 2006 11:41

Genetic Analysis of Host Resistance: Toll-Like Receptor Signaling andImmunity at LargeBruce Beutler, Zhengfan Jiang, Philippe Georgel, Karine Crozat, Ben Croker,

Sophie Rutschmann, Xin Du, and Kasper Hoebe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 353

Multiplexed Protein Array Platforms for Analysis of AutoimmuneDiseasesImelda Balboni, Steven M. Chan, Michael Kattah, Jessica D. Tenenbaum,

Atul J. Butte, and Paul J. Utz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 391

How TCRs Bind MHCs, Peptides, and CoreceptorsMarkus G. Rudolph, Robyn L. Stanfield, and Ian A. Wilson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 419

B Cell Immunobiology in Disease: Evolving Concepts from the ClinicFlavius Martin and Andrew C. Chan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 467

The Evolution of Adaptive ImmunityZeev Pancer and Max D. Cooper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 497

Cooperation Between CD4+ and CD8+ T Cells: When, Where,and HowFlora Castellino and Ronald N. Germain � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 519

Mechanism and Control of V(D)J Recombination at theImmunoglobulin Heavy Chain LocusDavid Jung, Cosmas Giallourakis, Raul Mostoslavsky,

and Frederick W. Alt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 541

A Central Role for Central ToleranceBruno Kyewski and Ludger Klein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 571

Regulation of Th2 Differentiation and Il4 Locus AccessibilityK. Mark Ansel, Ivana Djuretic, Bogdan Tanasa, and Anjana Rao � � � � � � � � � � � � � � � � � � � � � � � 607

Diverse Functions of IL-2, IL-15, and IL-7 in Lymphoid HomeostasisAveril Ma, Rima Koka, and Patrick Burkett � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 657

Intestinal and Pulmonary Mucosal T Cells: Local Heroes Fight toMaintain the Status QuoLeo Lefrançois and Lynn Puddington � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 681

Determinants of Lymphoid-Myeloid Lineage DiversificationCatherine V. Laiosa, Matthias Stadtfeld, and Thomas Graf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 705

GP120: Target for Neutralizing HIV-1 AntibodiesRalph Pantophlet and Dennis R. Burton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 739

Compartmentalized Ras/MAPK SignalingAdam Mor and Mark R. Philips � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 771

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genetic analysis of host resistance: toll-like receptor signaling and immunity at large

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