The Evolution of Adaptive Immunity

ANRV270-IY24-16 ARI 15 February 2006 1:25

The Evolution of AdaptiveImmunityZeev Pancer1 and Max D. Cooper2

1Center of Marine Biotechnology, University of Maryland Biotechnology Institute,Baltimore, Maryland 21202; email: [email protected] Hughes Medical Institute, University of Alabama, Birmingham,Alabama 35294; email: [email protected]

Annu. Rev. Immunol.2006. 24:497–518

First published online as aReview in Advance onJanuary 16, 2006

The Annual Review ofImmunology is online atimmunol.annualreviews.org

This article’s doi:10.1146/annurev.immunol.24.021605.090542

Copyright c! 2006 byAnnual Reviews. All rightsreserved

0732-0582/06/0423-0497$20.00

Key Wordsinvertebrate, vertebrate, agnatha, gnathostome, innate immunity,variable lymphocyte receptors (VLRs), leucine-rich repeat(LRR)–containing proteins

AbstractApproximately 500 mya two types of recombinatorial adaptive im-mune systems appeared in vertebrates. Jawed vertebrates generate adiverse repertoire of B and T cell antigen receptors through the rear-rangement of immunoglobulin V, D, and J gene fragments, whereasjawless fish assemble their variable lymphocyte receptors throughrecombinatorial usage of leucine-rich repeat (LRR) modular units.Invariant germ line–encoded, LRR-containing proteins are pivotalmediators of microbial recognition throughout the plant and ani-mal kingdoms. Whereas the genomes of plants and deuterostomeand chordate invertebrates harbor large arsenals of recognition re-ceptors primarily encoding LRR-containing proteins, relatively fewinnate pattern recognition receptors suffice for survival of pathogen-infected nematodes, insects, and vertebrates. The appearance of alymphocyte-based recombinatorial system of anticipatory immunityin the vertebrates may have been driven by a need to facilitate devel-opmental and morphological plasticity in addition to the advantageconferred by the ability to recognize a larger portion of the antigenicworld.

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PAMPs:pathogen-associatedmolecular patterns

PRR: patternrecognition receptor

LRR: leucine-richrepeat

VLR: variablelymphocyte receptor

INTRODUCTIONThe earth biomass consists primarily ofmicroorganisms, many of which arepathogens capable of killing and convertingother organisms into copies of themselves.In response to this threat, eukaryotes haveconstantly evolved antipathogen devices. Inturn, microorganisms continually evolve newways to evade host defense tactics in whathas been called the host-versus-pathogenarms race. The first line of host responsesto pathogen invasion is the innate immunedefenses. Innate immunity depends on germline–encoded receptors that have evolvedto recognize highly conserved pathogen-associated molecular patterns (PAMPs).These receptors have therefore been termedpattern recognition receptors (PRRs). Inaddition to the innate defense mechanisms,jawed vertebrates (gnathostomes) haveevolved an adaptive immune system medi-ated primarily by lymphocytes. By virtueof rearrangeable immunoglobulin (Ig) V, D,and J gene segments, the jawed vertebratesgenerate a lymphocyte receptor repertoire ofsufficient diversity to recognize the antigeniccomponent of any potential pathogen ortoxin. All jawed vertebrates, beginning withcartilaginous fish, rearrange their V(D)J genesegments to assemble complete genes forthe antigen receptors expressed by T and Blymphocytes. Antigen-mediated triggering ofT and B cells initiates specific cell-mediatedand humoral immune responses (1, 2).

The Ig domains are an ancient protein su-perfamily, and in the adaptive immune sys-tem of jawed vertebrates, the IgV (variable)domains are the cardinal molecular elementsof antigen receptors. In invertebrate animals,however, evidence of a role for Ig domainsin pathogen recognition or self/nonself dis-crimination was first reported for hemolin, aunique hemolymph protein of lepidopteraninsects comprised of four Ig-like domains.Microbial challenge induces secretion of thehemolin protein, which can bind to bac-teria and yeast (3). Remarkably, a diverse

repertoire of Ig domain–containing recep-tors is generated in insects through alterna-tive splicing of the Downs syndrome adhesionmolecule gene transcripts (3a). Other immune-related, Ig-containing molecules have beenreported in invertebrates, but their directrole in immune recognition has not beendemonstrated. For instance, the freshwatersnail, Biomphalaria glabrata, has a diverse fam-ily of fibrinogen-related hemolymph proteins(FREPs) with one or two N-terminal Ig-like domains. FREPs are expressed in in-creased abundance by circulating phagocyticcells, called hemocytes, following infectionwith trematode parasites, and they can bindto soluble trematode antigens. FREP genesin the hemocytes, central nervous system tis-sue, and stomach wall muscle may undergosome type of somatic diversification (4). An-other family of genes that encode IgV region–containing chitin-binding proteins (VCBPs)was identified first in the cephalochordate am-phioxus, Branchiostoma floridae (5), and laterin the genome of the tunicate Ciona intesti-nalis (6). These amphioxus VCBP moleculesare encoded by five or more multigene fami-lies that are polymorphic within the popula-tion. VCBP gene products are secreted intothe intestine, where they may play a role inpreventing microbial invasion.

Evidence has recently been obtained thattwo very different recombinatorial systemsfor lymphocyte antigen receptor diversifica-tion appeared at the dawn of vertebrate evo-lution !500 mya (Figure 1). Lamprey andhagfish, which are the only surviving jaw-less fish (agnathans) belonging to the old-est vertebrate taxon (7, 8), have been foundto assemble diverse lymphocyte antigen re-ceptor genes through the genomic rearrange-ment of leucine-rich repeat (LRR)–encodingmodules (9, 10). These cell surface receptorsare designated variable lymphocyte receptors,or VLRs. Recombinatorial mechanisms forthe generation of anticipatory receptors thusevolved in both the jawless and jawed verte-brates, but each vertebrate group employs adifferent kind of modular protein domain.

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J CV VV J J V D J CVV D J J

Gnathostome antibody genes

Light chain Heavy chain

D J CVJ CV

Lymphocyteincomplete

germ line genes

Lymphocyte recombinatorial

genes

Cell-bound anticipatory receptors

Humoral effector molecules

RAG-mediated rearrangment

Agnathan VLR gene

StalkLRR NT

LRR CT CP

SP

SP

5`LRRCT

3`LRRCT

StalkLRR NT

LRR1

5`LRRNT

LRR V

LRR1

LRR1

LRR V

LRR V

LRR V

LRR V

LRR V

LRR V

LRR V

LRR V

Rearrangement with flanking LRR casettes

5`LRRCT

Figure 1Rearranging antigen receptors of jawless and jawed vertebrates. Agnathan variable lymphocyte receptors(VLRs) are assembled by insertion of diverse LRR modules from flanking genomic cassettes into thegerm line incomplete VLR gene. The mature VLR gene consists of a signal peptide (SP), an N-terminalLRR (LRRNT), first 18-residue LRRs (LRR1), a variable number of 24-residue LRRs (LRRV), aconnecting peptide (CP), a C-terminal LRR (LRRCT), and a threonine/proline-rich stalk. Portions ofLRRNT and LRRCT that are not encoded in the germ line VLRs are hatched. Jawed vertebratesantibody genes are assembled via random joining of Ig gene fragments consisting of variable (V), diversity(D), and joining ( J) elements as well as Ig constant (C) domains. Following somatic DNA rearrangementthese antigen receptors are expressed on the surface of lymphocytes via GPI anchorage in VLRs or via atransmembrane domain in the antibody IgM cell surface form. Upon activation VLRs can be released tothe plasma via GPI-specific phospholipase cleavage, while secreted antibodies result from isotypeswitching to the secretory forms.

The appearance of two types of recombi-natorial immune systems within a relativelyshort evolutionary period of !40 million yearsduring the Cambrian raises intriguing ques-tions. What was the selective pressure toevolve acquired immunity? Why were LRR-containing modules selected as the recom-binatorial units of antigen receptors in ag-nathans, and why were Ig domains selected bythe gnathostomes? The question of gnathos-tome Igs is not addressed here. On the otherhand, the issue of LRR-based antigen re-ceptors of jawless fish may be more easilyaddressed. Because LRR-containing proteinsare ancient mediators of antimicrobial re-sponses in both kingdoms of multicellular

organisms, it is reasonable to suggest thatthe last common ancestor of plants and ani-mals used some version(s) of LRR-containingproteins for microbial detection (11). LRR-containing proteins therefore would haveprovided natural molecular candidates forearly agnathan experimentation with somaticDNA rearrangement to achieve receptor genediversification.

We begin this review with a considerationof the emergence of lymphocytes as a novelcirculatory cell type in vertebrates and thenconsider phylogenetic aspects of the super-family of LRR-containing proteins and theirrole in immunity. We conclude with an evolu-tionary scenario that may explain the sudden

www.annualreviews.org • Evolution of Adaptive Immunity 499

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appearance of a lymphocyte-based recombi-natorial system of anticipatory immunity invertebrates.

MIGRATORY PHAGOCYTICCELLS APPEARED BEFOREIMMUNOCOMPETENTLYMPHOCYTESPhagocytic cells form the cellular arm of in-nate immune defenses in almost all animals(metazoans) that have been studied, exceptfor the nematode Caenorhabditis elegans, whichmay lack cellular immune defenses. Follow-ing pathogen invasion in C. elegans, thereis activation of an inducible defense systemmarked by an increased expression of genesencoding lectins, antimicrobial peptides, andlysozymes, but exactly how the host per-ceives infection is not yet understood (12). InDrosophila melanogaster, another protostomeinvertebrate whose genome has been se-quenced, plasmatocytes are the predominantphagocytic blood cells involved in clearanceof invading microorganisms. The Drosophilaplasmatocytes are considered the functionalequivalents of monocytes/macrophages in thevertebrates (13, 14).

Monocyte/macrophage-type cells have arelatively short life span in both invertebratesand vertebrates. Proliferation of these innateimmunocytes appears to be confined to thegenerative hematopoietic tissues. As maturecirculatory cells, these nondividing phago-cytes can be activated to become effectorcells. These innate immunocytes may expressa surprisingly large repertoire of surface re-ceptors. As one example, a multigene familyin the sea urchin Strongylocentrotus purpura-tus (an echinoderm) encodes proteins featur-ing scavenger receptor cysteine-rich repeats.The circulatory coelomocytes of individ-ual sea urchins express unique and tempo-rally varying scavenger receptor cysteine-richrepertoires that are selected from an arse-nal of hundreds of genes (15). It is thereforeconceivable that diversification mechanismsfor immune receptors evolved before the ap-

pearance of long-lived circulatory immunecells that can undergo clonal expansion fol-lowing ligand engagement of their uniquereceptors (16). Evidence suggesting somaticdiversification of the snail FREPs may supportthis view, although this diversification processmay not be limited to circulating hemocytes(4). Inevitably, self-reactivity among recep-tors generated by random somatic diversifi-cation mechanisms would present a problem.In principle, though, autoimmunity could beavoided by a developmental program includ-ing a transitory interval of immunocyte hyper-sensitivity to receptor-mediated triggering,leading to apoptosis, or another type of inacti-vation mechanism for cells with autoreactivereceptors. In this way, cells bearing nonself-reactive receptors could selectively con-tinue their maturation to become migratoryimmunocytes.

THE APPEARANCE OFLYMPHOCYTES INVERTEBRATESA new type of circulatory cell with the po-tential for self-renewal and clonal expansionappeared near the beginning of vertebrateradiation in the form of the long-lived lym-phocyte. In the jawed vertebrates, T and Blymphocytes are the acknowledged cellularpillars of adaptive immunity. T lymphocytesare primarily responsible for cell-mediatedimmunity, and B lymphocytes are responsi-ble for humoral immunity, but they work to-gether and with other types of cells to mediateeffective adaptive immunity. Along with thenatural killer cells, these specialized lymphoidcells are derived from committed progenitorsin hematopoietic tissues, which then undergounique V(D)J rearrangements of their antigenreceptors to become clonally diverse lympho-cytes. Newly formed T and B lymphocytesbearing autoreactive receptors can be elimi-nated by self-antigen contact in the thymusand bone marrow, respectively. The surviv-ing T and B cells then migrate via the blood-stream to peripheral lymphoid tissues, where,


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following antigen recognition, they may un-dergo clonal expansion and differentiationinto effector T lymphocytes or antibody-producing plasma cells or otherwise becomememory cells that await reexposure to theirspecific antigens.

Exactly when during evolution the lym-phocytes appeared as a specialized type of im-munocompetent cells is unknown, but cellscomparable to the lymphocytes in jawed ver-tebrates have never been characterized in in-vertebrates. On the other hand, increasingevidence for bona fide lymphocytes in lam-prey and hagfish suggests that lymphocytesmust have evolved in the common ancestorof the vertebrates. Most of the lymphoid cellsin lamprey and hagfish are small round cellscomposed mainly of a nucleus with condensedchromatin and a small rim of surrounding cy-toplasm that contains relatively few organelles(17). Following antigen and/or mitogen stim-ulation, agnathan lymphocytes can transforminto large lymphoblast-like cells (9). Mor-phological studies have led to the view thatagnathan lymphocytes are generated in thehematopoietic tissues, primarily the intestine-associated hematopoietic organ in lampreylarvae, called the typhlosole, and the protover-tebral arch of adult lamprey (17–19). Evidencefor a thymus-like organ in agnathans is equiv-ocal at best, however. Collections of lymphoidcells have been found in pharyngeal guttersof the lamprey gill region, but there is norecognizable capsular, stromal, or lymphoep-ithelial organization of the type that char-acterizes the lymphopoietic thymus in jawedvertebrates (17, 20–22).

The lamprey lymphocytes express ho-mologs of many genes expressed during jawedvertebrate lymphocyte differentiation, prolif-eration, migration, and intracellular signalingand perhaps also express the relatives of genesthat gnathostomes use in antigen processingand intracellular transport of antigenic pep-tides (23–27). It is important to note that pro-teins with sequence similarity to the jawed ver-tebrate rearrangeable Ig genes or MHC geneshave not been found in extensive surveys of

lamprey and hagfish leukocyte transcripts (28,29).

Until very recently, there was no cred-ible evidence for lymphocyte receptor di-versity in lamprey or hagfish. This led toconsiderable skepticism about the earlier re-ports of agnathan adaptive immunity (17, 20,30–32). This picture changed dramaticallywith the identification of VLR genes in thelamprey and hagfish (9, 10). These genes areassembled by a special recombinatorial mech-anism used to generate a diverse repertoireof anticipatory receptors. The lymphocytesof lamprey and hagfish rearrange modularLRR cassettes to create functional matureVLR genes. A VLR of unique sequence is ex-pressed by each lymphocyte in a monoallelicfashion. As in the case for the gnathostomelymphocytes, the agnathan lymphocytes mayundergo lymphoblastoid transformation fol-lowing antigen and/or mitogen stimulation.Clonal amplification appears to occur dur-ing antigen-induced proliferative responsesin lamprey and hagfish, although more ex-perimental evidence will be required to con-firm this conclusion. Lamprey lymphocytescan respond to immunization by the release oftheir antigen-specific VLRs into the plasma,thus providing the potential basis for humoralimmunity (M.N. Alder, M.D. Cooper &Z. Pancer, unpublished data). Although manyquestions about the development and func-tion of agnathan lymphocytes are still unan-swered, it is clear that the jawless vertebrateshave a lymphocyte-based recombinatorial im-mune system that differs radically from theIg-based recombinatorial immune system inthe jawed vertebrates.

IMMUNE-RELATED LRRPROTEINS OF INVERTEBRATESAND PLANTSDrosophila has two distinct families of PRRsthat are used selectively to activate one or theother NF!B-like signaling pathways, the Tollor the Immune Deficiency. Cellular activa-tion via these pathways results in induction


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Metazoans

DE

UTE

RO

STO

ME

SP

RO

TOS

TOM

ES

Gnathostomes(jawed vertebrates)

Agnathans(jawless vertebrates)

Cephalochordates(Amphioxus)

Urochordates(ascidians)

Echinoderms

Hemichordates(acorn worms)

Annelids

Molluscs

Nematodes

Arthropods

Cnidarians

Figure 2Metazoan tree oflife. A simplifiedrepresentation ofthe two majorgroups ofmulticellularanimals,protostomes anddeuterostomes,that include thechordateinvertebrates andvertebrates.

TLR: Toll-likereceptor

of antimicrobial peptide genes in the fat bodyand the secretion of antimicrobial peptidesinto the hemolymph (33). It was first real-ized in 1996 that the dorsoventral patterningreceptor Toll is also involved in protectionof flies against fungi, by way of inducing ex-pression of the antifungal peptide gene Dro-somycin (34). The Toll pathway has recentlybeen shown to be essential for protectionagainst the Drosophila X virus as well (35).

The Toll receptor has an extracellularLRR-containing domain, a transmembraneregion, and a cytoplasmic Toll/interleukin-1receptor homology domain (TIR). In additionto Toll, the Drosophila genome contains eightToll homologs, and the mosquito Anophelesgambiae genome contains ten Toll homologs.All but one of these Tolls in both insects ap-pear to be linked to developmental functionsrather than to immunity (36). Likewise, im-mune function could not be attributed to thesingle C. elegans and Caenorhabditis briggsae

Toll homolog, nor to the Toll-like receptor(TLR) reported in the horseshoe crab Tachy-pleus tridentatus (37). In striking contrast tothe TLR saga in these protostome inverte-brates, the vertebrate homologs of DrosophilaToll appear to be dedicated solely to hostdefense (38, 39).

This leads us to consider what isknown about the LRR-containing proteinsin deuterostome invertebrates, which are inthe ancestral evolutionary lineage of the ver-tebrates (Figure 2). It is known that TLRpolypeptides are frequently encoded by a con-tinuous open reading frame that is unin-terrupted by introns. Standard examples ofthis type of gene structure include Tollo,Toll 6, and Toll 7 from Drosophila and hu-man TLRs 1, 2, 4D, 5, 6, and 10 (intronlessgenes database) (40). The genome of the seaurchin S. purpuratus abounds with intronlessLRR-containing genes. A sample of 52 in-tronless TLRs, derived from those identified


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in the draft genome sequence (SequencingProject Version 0.3, Baylor College ofMedicine; http://www.hgsc.bcm.tmc.edu),is illustrated in Figure 3. These TLRs clus-ter in a very unusual branching patternconsisting of sequences with nearly identi-cal values of genetic distance among branchmembers (Figure 4), thereby indicating mul-tiple events of expansion and diversificationamong branch members. The total num-ber of sea urchin TLRs is estimated to be!340 members, or half this number if all thesegenes prove to be polymorphic (41). Cloningand expression analysis indicate that multipleTLRs may function in sea urchin immu-nity (Z. Pancer & E.H. Davidson, unpub-lished data). In addition to the TLR genes,the sea urchin genome harbors other intron-less LRR-encoding genes. One group withat least 47 members consists solely of LRRmotifs (not shown); some of these proteinshave transmembrane domains (N = 13/47).Another group also has C-terminal Ig do-mains (N = 10), and some of these are alsopredicted to have membrane anchorage do-mains (N = 6/10) (Z. Pancer & M.D. Cooper,unpublished data).

In the chordate phylum, the genome of thesolitary tunicate Ciona savignyi contains be-tween 7 and 19 TLR genes (41), only 2 ofwhich are intronless genes, rendering geneprediction uncertain. At least 22 other in-tronless LRR-containing genes were iden-tified in the C. savignyi genome (Sequenc-ing Project data, April 25, 2003, version,Whitehead Institute and MIT Center forGenome Research; http://www-genome.wi.mit.edu), and 8 of these appear to be cellsurface receptors (Z. Pancer & M.D. Cooper,unpublished data). Only three TLRs were re-ported in the genome of another solitary tu-nicate, C. intestinalis (6), and all of these areinterrupted by introns. Notably, no other in-tronless LRR genes could be identified in thisspecies.

In the amphioxus B. floridae, we identi-fied at least 42 intronless TLR genes (tracearchive, WGS Sequencing Project, DOE

Joint Genome Institute; ftp://ftp.ensembl.org/pub/traces/branchiostoma floridae/).These TLR genes cluster in equidistantbranches of the genetic distance tree, likeTLRs from the sea urchin. In addition,the B. floridae genome harbors at least 211intronless LRR-containing genes, or halfthis number if all alleles are polymorphic.Seventy-one of these LRR-containing genesare illustrated in Figure 5. Fifty-one consistonly of LRR motifs, and 12 of these includetransmembrane domains; the other 20 haveboth LRR and C-terminal Ig-like domains,and 12 of these are predicted cell surfaceproteins (Z. Pancer & M.D. Cooper, unpub-lished data). Computational analysis indicatesthat several more of the sea urchin andamphioxus intronless LRR-containing genesmay encode proteins that are tethered to thecell surface via glycosyl-phosphatidyl-inositol(GPI) anchors.

Intriguing unanswered questions abound:Why is the solitary tunicate C. intestinalis dif-ferent from other deuterostome invertebratesthat utilize multiple LRR-containing pro-teins? What may be the strategy employed bycolonial tunicates, such as Botryllus schlosseri,that are known for their highly elaborateand polymorphic self/nonself-recognitionsystems (42)?

Plant genomes harbor very large fami-lies of LRR-containing genes, and many ofthese mediate disease resistance. The mostimportant plant disease resistance genes en-code the STAND ATPase domain (43) ornucleotide-binding site (NBS)–LRR proteins,some of which include N-terminal TIR do-mains. There are also the LRR receptor–like kinases and the membrane-bound LRRreceptor–like proteins. Several of these pro-teins control resistance to a wide variety ofplant pathogens and pests, including viruses,bacteria, fungi, nematodes, and insects (44,45). In response to pathogen challenge, di-verse resistance responses in plants are ac-tivated by disease resistance proteins. Theseresponses include the production of anti-microbial peptides and a form of programmed


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Figure 3A large superfamily of sea urchin TLRs. A sample of 52 intronless TLR genes identified in the genome ofStrongylocentrotus purpuratus. Prediction of domain architecture was via the SMART server(http://smart.embl-heidelberg.de). (N-terminal LRR: light blue rectangle; LRR: green rectangle;C-terminal LRR: light blue oval; transmembrane domain: dark blue rectangles; C-terminal TIR domains:green diamond.)


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SpT

LR.1

06

SpT

LR.4

48

SpT

LR.2

60S

pTLR

.7Sp

TLR

.372

SpTL

R.19

7SpT

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77

SpTLR

.128

SpTLR.148

SpTLR.291

SpTLR.240

SpTLR.122

SpTLR.262

SpTLR.410

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SpTLR.374SpTLR.341

SpTLR.359SpTLR.233SpTLR.295SpTLR.19

SpTLR1.1

SpTLR.33

SpTLR.101

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.136

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R1.

2

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LR.67SpTLR.12SpTLR.276SpTLR.404

SpTLR.320

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SpTLR.308

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SpTLR.274

SpTLR.94

SpTLR.357

SpTLR.288

SpTLR.39

SpTLR.216SpTLR.445

SpTLR.13

SpTLR

.258

0.1

Figure 4Genetic distance among 52 sea urchin intronless TLRs. UPGMA tree constructed from the predictedamino acid sequence using the pairwise deletion option (MEGA 3 Molecular Evolutionary GeneticsAnalysis) (99).

cell death called the hypersensitive response(46). In rice, Oryza sativa, 585 predicted NBS-LRR genes account for approximately 1% ofthe genes identified in the genome; a similarfraction of the Arabidopsis thaliana genome isdedicated to disease resistance genes (44, 45).

Even with knowledge of this large arsenal ofdisease resistance genes, we still do not un-derstand how plants can detect the multitudeof infectious pathogens with a limited num-ber of PRR genes. The “guard” hypothesispostulates that resistance proteins constitute


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Figure 5A large superfamily of amphioxus intronless LRR-containing genes. A sample of 71 genes identified inthe genome of the Florida lancelet Branchiostoma floridae. Prediction of domain architecture was via theSMART server (http://smart.embl-heidelberg.de). N-terminal LRR: light blue rectangle; LRR: greenrectangle; C-terminal LRR: light blue oval; Ig superfamily domain: green oval; transmembrane domain:dark blue rectangle.


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Figure 5(Continued )


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components of larger signal perception com-plexes that are activated in response topathogen-induced perturbance of the normalfunction of host proteins. Hence, instead ofrecognition via a specific receptor for eachpathogen, detection may occur indirectly viathe damage inflicted by pathogen-derived vir-ulence proteins (45–47).

High levels of polymorphism have beennoted among members of plant resistancegenes, and the putative ligand-binding sur-faces in many families of LRR-containing re-sistance genes appear to be undergoing rapiddiversifying selection (48). Interestingly, thereis also evidence of pronounced selection forsomatic variants of disease resistance genesthat may affect the ligand specificities of par-ticular resistance proteins (49). This variationmay be generated via an unknown diversifi-cation mechanism of plant PRR genes, or itmay reflect the increased frequency in homol-ogous recombination that has been observedfor virus-infected plants (50).

Intronless LRR-containing genes are rel-atively rare in animal genomes other thandeuterostome and chordate invertebrates.Apart from the few intronless TLRs in mam-mals and insects, there are only 10 other in-tronless LRR-containing genes in the humanand mouse genomes, none in Drosophila, andonly 25 of the NBS-LRR genes in Arabidop-sis (intronless genes database) (40). Intronlessgenes most likely result from retropositionand subsequent genomic integration thoughtto occur via the reverse transcription activityof endogenous retrotransposons, such as thehuman LINE elements (51). Intronless genesmay be excellent gene templates to generaterapidly evolving arsenals of diverse germ line–encoded receptors.

IMMUNE-RELATED LRRPROTEINS OF JAWEDVERTEBRATESThe typical TLR complement for vertebratesis approximately one dozen genes. The onlyexception that has been noted thus far is fish

that have retained both copies of duplicatedTLRs resulting from a whole genome du-plication that seems to have occurred afterthe divergence of bony fish and tetrapods.For example, at least 17 predicted TLR geneswere expressed in the zebrafish, Danio rerio(52). Nearly all vertebrate TLRs belong toone of six major families (TLR1–6), and eachTLR family is capable of recognizing a gen-eral class of PAMPs. TLR2 family membersbind lipopeptide; TLR3 family members binddouble-stranded RNA; TLR4 family mem-bers bind LPS; TLR5 family members bindflagellin; members of the TLR7, TLR8, andTLR9 subfamilies bind nucleic acid and hememotifs; and TLR1 family members associatewith TLR2 members as heterodimeric recep-tors (41). Mouse TLR11 has recently beenimplicated in the response to a profilin-likeprotein of the protozoan parasite Toxoplasmagondii (53).

Soluble TLR forms, consisting of the ex-tracellular portions only, may also partici-pate in immunity. Amphibians and fish havea soluble form of the TLR5 gene that aroseby duplication of the region encoding theextracellular domain. In rainbow trout, On-chorhynchus mikiss, bacterial flagellin interactswith membrane-bound TLR5 to induce theexpression of the soluble TLR5 gene in theliver, resulting in efficient clearance of flag-ellin from the circulation (54). In chicken, al-ternatively spliced forms of TLR3 and TLR5yield soluble products (55), and human plasmaand breast milk also contain a functional solu-ble form of TLR2 that is generated by a post-translational modification (56).

The recently identified CATERPILLERfamily of LRR-containing immune-regula-tory genes encodes cytoplasmic proteins thatare structurally similar to some of the plantdisease resistance genes (43). The N termi-nus of these proteins may function as aneffector domain, mediating homotypic or het-erotypic interactions; a central NBS domainhas regulatory function, whereas the C ter-minus is composed of variable sets of LRRmotifs that may function in ligand binding.


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Heterodimers formed between different fam-ily members may in some cases increase thecombinatorial binding potential (57). Nu-cleotide oligomerization domain (NOD) 1and NOD2 members of this family are im-plicated as sensors of intracellular bacterialproducts and activators of host responsesagainst invading pathogens (57, 58). In gutregions that are rich in commensal bacteria,NOD1 functions as the detector of enteroin-vasive bacteria that have evolved the meansto prevent intestinal epithelial cell signalingthrough the TLRs. NOD2 is critical for reg-ulation of bacterial immunity within the in-testine by controlling the expression of crypt-dins, which are intestinal antimicrobial pep-tides (59). More than 20 members of thisfamily have been identified in mammaliangenomes, whereas no CATERPILLER or-thologs have been identified in Drosophila orC. elegans (57).

One strategy that is employed to main-tain balance in the arms race between in-sects and pathogens involves natural selectionof random mutations in insect PRR genes.In wild Drosophila populations, for example,!10% of the polymorphic sites in genesencoding antibacterial peptides, Toll recep-tors, signal transduction molecules, and otherpathogen recognition molecules are associ-ated with disease resistance phenotypes to asingle pathogen (60). However, the vertebrateTLRs are not rapidly evolving genes. They ap-pear to be under strong purifying selection tomaintain their PAMP recognition specificityin order to discriminate between pathogensand the host. This may be because self-reactive TLRs would be detrimental to thehost, as none of the self-tolerance mechanismsthat can purge self-reactive T and B lympho-cytes or prevent development of potentiallyharmful natural killer cells have been identi-fied for cells bearing TLRs (41). The largemultigene families of LRR-containing pro-teins in deuterostome invertebrates are espe-cially remarkable given that microbial recog-nition is served by only a handful of PRRs innematodes, insects, and vertebrates (61).

STRUCTURE AND FUNCTIONOF LRR-CONTAININGPROTEINSLRRs of 20–29 amino acids per repeat arepresent in more than 2000 proteins fromviruses, bacteria, archaea, and eukaryotes.Family members of the LRR-containing pro-teins participate in nearly all known biologicalfunctions, including plant and animal immu-nity, apoptosis, cell adhesion, signal transduc-tion, DNA repair, DNA recombination andtranscription, RNA processing, and ice nu-cleation. Nonetheless, the existence of manytypes of LRR-containing immune gene fami-lies in the genomes of both plants and animalsargues for the special role of these proteins inhost defense.

Sixteen crystallized LRR-containing pro-teins all adopt an arc or horseshoe-like shape,with the individual LRR motifs forming par-allel loops that are stacked into a coil. Mostof the LRR-containing proteins have charac-teristic N-terminal and C-terminal LRR do-mains capping the ends of the hollow tube.The concave face of the coil consists of a par-allel "-sheet, whereas there may be #-, 310-,or pII helices in the convex face (62).

Ligand-binding sites have been deter-mined for several LRR-containing receptors.The mammalian ribonuclease inhibitor, thefirst LRR structure solved, interacts with theribonuclease via multiple contact points lo-cated on the concave LRR surface of the in-hibitor (63). Glycoprotein Ib, a platelet LRR-containing receptor for the von Willebrandfactor, binds its ligand via exposed residueson the concave LRR face and via a finger-like insertion in the C-terminal LRR (64).CD14, one of the major LPS receptor units,is an LRR-containing protein expressed onmyelomonocytic cells as a GPI-linked gly-coprotein or released into the plasma as asoluble form. The crystal structure of CD14reveals a dimer in which each horseshoe-shaped monomer consists of 13 "-strands.The large hydrophobic pocket is located onthe side of the horseshoe near the N terminus


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(65). The polygalacturonase-inhibiting pro-tein of Phaseolus vulgaris is the product ofa plant disease resistance gene. It acts asan inhibitor of cell wall–degrading enzymesproduced by pathogenic fungi. A negativelycharged surface on the concave LRR faceis probably involved in binding fungal poly-galacturonases (66). The tomato, Lycoper-sicon pimpinellifolium, Cf-9 protein confersfungal resistance via residues in its N-terminalLRR; putative glycosylation sites in theouter #-helices are also essential for binding(67).

Mammalian receptors for the glycoproteinhormones (thyrotropin, lutropin, chorionicgonadotropin, and follitropin) are G protein–coupled proteins that bind their ligands viathe LRR motifs in their extracellular domains(68, 69). Internalins A and B of Listeria mono-cytogenes are LRR-containing surface proteinsthat mediate specific host-cell invasion by thebacteria. Internalin A mediates bacterial adhe-sion and initiates invasion of human intestinalepithelia through specific interaction with theE-cadherin receptor. The crystal structure ofInternalin A complexed with the N-terminaldomain of E-cadherin reveals tight interac-tion sites on the concave surface of the LRRcoil (70). The N terminus of Internalin B con-sists of an LRR domain that is C-terminallycapped by an Ig-like domain, and this portionis sufficient to induce bacterial internalizationinto host cells (71).

Promiscuity of ligand binding has beensuggested in the case of Nogo and its LRR-containing GPI-anchored receptor, whichplays a key role in inhibition of mammalianaxon regeneration. The Nogo receptor has aputative ligand-binding site within the con-cave LRR face in which multiple solvent-exposed hydrophobic and aromatic residuescreate high potential for binding cross-reactivity (72). Decorin and Opticin are smallLRR-containing extracellular matrix proteo-glycans that form antiparallel homodimersvia highly specific interactions at their con-cave LRR surfaces. It therefore seems likelythat their binding of different ligands occurs

through protein surface sites other than thoseof the concave sheet (73, 74).

The extracellular domains of TLRs con-sist of 19–25 tandem LRR motifs, most ofwhich conform to a 24-residue consensus mo-tif. Peptide insertions are present within someof the LRRs, and these have been predictedto mediate recognition of PAMPs (75). Theonly TLR crystal structure reported to date,the human TLR3 ectodomain, provides ex-perimental support for this prediction (76).The TLR3 solenoid consists of 25 LRR mo-tifs that are stacked and stabilized throughhydrogen bonds formed by conserved as-paragine residues. Glycosylation-free facesof the solenoid provide interfaces for a ho-modimeric configuration maintained via con-served surface residues and a loop formed by apeptide insertion in LRR20, whereas a secondpeptide insertion in LRR12 and two clustersof positively charged residues form the puta-tive binding site for double-stranded RNA.

Some TLRs may recognize only a lim-ited number of PAMPs. For instance, TLR9directly interacts with particular sequencesof unmethylated CpG-DNA found in bac-terial DNA (77, 78), and TLR7 on thesurface of plasmacytoid dendritic cells andB cells mediates the recognition of single-stranded RNA from vesicular stomatitis virusand influenza virus. Thus, TLR7 recognizessingle-stranded RNA viruses, whereas ei-ther TLR3 or TLR9 detects double-strandedRNA viruses (79, 80). Other TLRs have theremarkable potential to interact with struc-turally unrelated ligands. TLR2 mediates hostresponses to peptidoglycan and lipoteichoicacid from Gram-positive bacteria, lipoara-binomannan from mycobacteria, neisserialporins, bacterial tripalmitoylated and my-coplasmal diacylated lipoproteins, and yeastproducts and GPI-anchored proteins of theprotozoan Trypanosoma cruzi. Results frommutagenesis of the extracellular LRRs inTLR2 imply the existence of different bind-ing sites for different ligands (81). TLR4 canbind LPS from Gram-negative bacteria, vi-ral proteins, bacterial and host heat shock


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proteins, host oligosaccharides derived fromheperan sulfate and hyaluronic acid, hosttaxol, and fibrinogen (75).

In spite of their sequence and functionaldiversity, the horseshoe-like structure withits concave binding surface is remarkablyconserved in all the LRR-containing pro-teins. This may reflect the modular struc-ture formed by the tandem array of stackedLRR motifs. Each such structure consists ofa mosaic of conserved scaffold residues inter-spersed with highly variable residues to ac-count for the enormous diversity in ligand-binding sites.

HYPOTHESIS: EVOLUTION OFADAPTIVE IMMUNITY INVERTEBRATESAre there any fundamental differences be-tween invertebrates and vertebrates in termsof their potential pathogens? We cannot goback to the time when the earliest jawlessfish diverged from a common cephalochor-date ancestor (82), but we can speculate thatthe answer to this question is no, based onthe fossil record and studies of contempo-rary species. There is no indication for mas-sive eradication of species in the Cambrianthat could imply new types of potentiallydevastating pathogens, and it seems unlikelythat the newly evolved vertebrates becamethe favorite hosts for new kinds of pathogenssoon after their emergence. Despite 500 mil-lion years of vertebrate existence, there is adearth of evidence for significant numbersof vertebrate-specific pathogens. Conversely,many pathogens are known to infect both in-vertebrate and vertebrate hosts, including, forexample, more than 500 varieties of the ar-boviruses (83).

Germ line–encoded innate immune bar-riers protect both invertebrates and verte-brates from potential pathogens, althoughvertebrates may be better protected againstsome of the frequently recurring pathogens.Even in vertebrates, however, innate immu-nity provides the first line of defense against

pathogens because a protective level adaptiveimmune response takes at least several days tomount. Innate immune mechanisms of inver-tebrates must therefore be as efficient as thosein vertebrates for combating the rapidly evolv-ing pathogens that these animals inevitablyencounter (16).

Why then do deuterostome invertebratesneed a vastly expanded arsenal of germ line–encoded receptors when only a handful ofPRRs suffices for immunity in nematodes, in-sects, and vertebrates? The LRR-containingproteins and other multigene families ofimmune receptors of deuterostome inverte-brates may play a pivotal role in the mainte-nance and surveillance of the endosymbioticmicrobial communities that these animalsharbor. For example, it has been estimatedthat more than 60% of echinoderm speciesassociate with bacterial symbionts (84). Theintestinal floral symbionts in sea urchins maybe needed to ferment and detoxify the poorlynutrient kelp and algae on which these animalsgraze. Such a complex mode of long-termcoexistence between animals and microor-ganisms may have favored the evolution oflarge arsenals of specific microbial recogni-tion molecules, whereas the strategy of PAMPrecognition indiscriminately targets practi-cally all microorganisms as nonself. This com-plex mode of coexistence with endosymbioticmicrobes most likely was transmitted frominvertebrate ancestors to their vertebratedescendents because complex microbial com-munities exist in the intestines of all ver-tebrates; 400–1000 species are estimated tolive in the human gastrointestinal tract (85,86), and the commensal microbiota have beenshown to shape the Ig repertoire of periph-eral B lymphocytes (87). Furthermore, main-tenance of the mammalian gut flora appears torequire highly elaborate immune mechanismsand an active cross talk between the microfloraand the host mucosal immune system (88).It may be interesting to explore the mecha-nisms that invertebrates employ to distinguishbetween symbionts and potential pathogensin animals belonging to the deuterostome


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lineage as well as in protostome invertebratesthat harbor endosymbiotic microorganisms,for example, the wood-feeding termites (89)and molluscan cephalopods (octopus, squid,and cuttlefish) that harbor symbiotic bacteriain their light organs (90).

Thus far we have emphasized the similar-ities between deuterostome invertebrates andtheir vertebrate evolutionary descendents. Itmay be more relevant to consider differencesbetween these animal groups that may havefavored the development of a completely newmode of antigen recognition in vertebrates onthe basis of receptor gene rearrangement. Toaddress this enigma, we need to look back atthe Cambrian explosion !500 mya, when aunique and stunning burst of evolutionary di-versification of new vertebrate species began.In a relatively brief evolutionary period, a va-riety of free swimming jawless fish appeared inthe oceans. These fish descended from smallamphioxus-like ancestors that lived as sus-pension feeders buried in the sand in shal-low coastal waters. Early skulled vertebrates(Craniates) had a unique feature that sepa-rated them from their cephalochordate ances-tor, namely a whole genome duplication thatmost likely occurred at the beginning of ver-tebrate divergence (91, 92). This genome du-plication may have fueled the dramatic “bigleap” in vertebrate developmental, morpho-logical, and functional innovation during theCambrian period (93).

We can speculate that a large arsenal ofdiverse LRR-containing proteins was alsopart of the ancestral cephalochordate her-itage. Members of these abundant cell surfaceand soluble receptors may have engaged inserendipitous interactions with newly evolv-ing molecular determinants of early ag-nathans. If so, this interference may havebeen a rate-limiting factor in the process ofrapid vertebrate evolution. In considerationof the enormous binding versatility of LRR-containing proteins, it is conceivable that theirself-reactivity presented serious autoimmu-nity problems at a time of rapid develop-mental and morphologic innovation. Further-

more, the transition from invertebrates tovertebrates may have been associated withchanges in the endosymbiotic communities,thereby rendering many of these microbialsurveillance proteins obsolete. In any case,the rate of readjustment required to main-tain large multigene families of germ line re-ceptors as strictly nonself-reactive may havebecome overly burdensome. Consequently,early vertebrates may have been forced toabandon the invertebrate deuterostome strat-egy of large arsenals of germ line–encoded im-mune receptors. This line of reasoning leadsus to speculate that an adjustable immune sys-tem based on randomly generated receptor di-versity evolved in part to enable the burst ofvertebrate speciation in the Cambrian. Lym-phocytes bearing uniquely rearranged sur-face antigen receptors, which could undergonegative selection to purge self-reactive lym-phocytes while sparing clones expressingpotentially beneficial antigen receptors ofsufficient diversity, could have replaced thefunction of ancestral germ line arsenals.

It is also likely that the newly evolvedvertebrate lymphocytes performed innateimmune functions concomitantly with thestepwise acquisition of acquired immunefunctions. There is ample evidence that lym-phocytes have retained innate immune func-tionality. For example, B lymphocytes expressTLRs and respond to their ligands by prolifer-ation, expression of costimulatory molecules,and plasma cell differentiation (39). B cellsof the peritoneal cavity and spleen marginalzones mediate microbial destruction viasecretion of polyreactive antibodies that areessentially germ line encoded (94). Plasmacy-toid dendritic cells derived from a commonlymphoid progenitor express TLR7 and -9.These professional producers of IFN-#/" areimportant in protection against a wide rangeof viruses, bacteria, and parasites (95). TheT lymphocytes are professional producers ofIFN-$. The concerted integration betweeninnate and adaptive immune functions of lym-phocytes may explain why the outcome of ge-netic defects that prevent the development


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of T and B lymphocytes in infants withsevere combined immunodeficiency diseasesprecludes survival from viral, bacterial, andfungal infections (96).

Only hagfish, lamprey, and the jawed ver-tebrates survived from the early vertebrateradiation, and only a sparse fossil recordremains from the short period that separatesthe emergence of jawless fish and the appear-ance of jawed vertebrates (97, 98). It there-fore will be difficult to determine whether theagnathan LRR-containing VLRs were fore-runners of vertebrate immune receptors or if

the rearranging VLRs and Igs evolved as inde-pendent solutions to similar necessities. Thedevelopment of two very different modes oflymphocyte-based receptor diversification atthe dawn of vertebrate evolution neverthelessstrongly attests to the enormous fitness valueof anticipatory immunity. The benefits fromthis innovative strategy may not be limited tothe ability to recognize a nearly infinite anti-genic world. Rather, the immediate selectivepressure may instead have been facilitation ofthe developmental and morphological plastic-ity of the vertebrates.

ACKNOWLEDGMENTSWe thank Hui-Hsien Chou of Iowa State University at Ames for providing Perl scripts to ana-lyze the trace archive sequences. We also thank Matthew Alder of the University of Alabama atBirmingham; L. Aravind, Lakshminarayan Iyer, and Igor Rogozin of the National Center forBiotechnology Information, National Library of Medicine at the National Institutes of Health;and Gerardo Vasta of the Center of Marine Biotechnology, University of Maryland Biotech-nology Institute at Baltimore, for helpful discussion. We additionally thank Ann Brookshirefor her role in manuscript preparation. Z.P. was funded by National Science Foundation grantsMCB-0317460 and IBN-0321461. M.D.C. is an investigator at the Howard Hughes MedicalInstitute. This paper is contribution #05-120 from the Center of Marine Biotechnology.

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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-! 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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Documents

The Evolution of Adaptive Immunity