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The Evolution of Adaptive Immunity Zeev Pancer1 and Max D. Cooper2 1

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202; email: [email protected]

2

Howard Hughes Medical Institute, University of Alabama, Birmingham, Alabama 35294; email: [email protected]

Annu. Rev. Immunol. 2006. 24:497–518

Key Words

First published online as a Review in Advance on January 16, 2006

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

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.24.021605.090542 c 2006 by Copyright  Annual Reviews. All rights reserved 0732-0582/06/0423-0497$20.00

Abstract Approximately 500 mya two types of recombinatorial adaptive immune systems appeared in vertebrates. Jawed vertebrates generate a diverse repertoire of B and T cell antigen receptors through the rearrangement of immunoglobulin V, D, and J gene fragments, whereas jawless fish assemble their variable lymphocyte receptors through recombinatorial usage of leucine-rich repeat (LRR) modular units. Invariant germ line–encoded, LRR-containing proteins are pivotal mediators of microbial recognition throughout the plant and animal kingdoms. Whereas the genomes of plants and deuterostome and chordate invertebrates harbor large arsenals of recognition receptors primarily encoding LRR-containing proteins, relatively few innate pattern recognition receptors suffice for survival of pathogeninfected nematodes, insects, and vertebrates. The appearance of a lymphocyte-based recombinatorial system of anticipatory immunity in the vertebrates may have been driven by a need to facilitate developmental and morphological plasticity in addition to the advantage conferred by the ability to recognize a larger portion of the antigenic world.

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INTRODUCTION PAMPs: pathogen-associated molecular patterns PRR: pattern recognition receptor LRR: leucine-rich repeat VLR: variable lymphocyte receptor

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The earth biomass consists primarily of microorganisms, many of which are pathogens capable of killing and converting other organisms into copies of themselves. In response to this threat, eukaryotes have constantly evolved antipathogen devices. In turn, microorganisms continually evolve new ways to evade host defense tactics in what has been called the host-versus-pathogen arms race. The first line of host responses to pathogen invasion is the innate immune defenses. Innate immunity depends on germ line–encoded receptors that have evolved to recognize highly conserved pathogenassociated molecular patterns (PAMPs). These receptors have therefore been termed pattern recognition receptors (PRRs). In addition to the innate defense mechanisms, jawed vertebrates (gnathostomes) have evolved an adaptive immune system mediated primarily by lymphocytes. By virtue of rearrangeable immunoglobulin (Ig) V, D, and J gene segments, the jawed vertebrates generate a lymphocyte receptor repertoire of sufficient diversity to recognize the antigenic component of any potential pathogen or toxin. All jawed vertebrates, beginning with cartilaginous fish, rearrange their V(D)J gene segments to assemble complete genes for the antigen receptors expressed by T and B lymphocytes. Antigen-mediated triggering of T and B cells initiates specific cell-mediated and humoral immune responses (1, 2). The Ig domains are an ancient protein superfamily, and in the adaptive immune system of jawed vertebrates, the IgV (variable) domains are the cardinal molecular elements of antigen receptors. In invertebrate animals, however, evidence of a role for Ig domains in pathogen recognition or self/nonself discrimination was first reported for hemolin, a unique hemolymph protein of lepidopteran insects comprised of four Ig-like domains. Microbial challenge induces secretion of the hemolin protein, which can bind to bacteria and yeast (3). Remarkably, a diverse

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repertoire of Ig domain–containing receptors is generated in insects through alternative splicing of the Downs syndrome adhesion molecule gene transcripts (3a). Other immunerelated, Ig-containing molecules have been reported in invertebrates, but their direct role in immune recognition has not been demonstrated. For instance, the freshwater snail, Biomphalaria glabrata, has a diverse family of fibrinogen-related hemolymph proteins (FREPs) with one or two N-terminal Iglike domains. FREPs are expressed in increased abundance by circulating phagocytic cells, called hemocytes, following infection with trematode parasites, and they can bind to soluble trematode antigens. FREP genes in the hemocytes, central nervous system tissue, and stomach wall muscle may undergo some type of somatic diversification (4). Another family of genes that encode IgV region– containing chitin-binding proteins (VCBPs) was identified first in the cephalochordate amphioxus, Branchiostoma floridae (5), and later in the genome of the tunicate Ciona intestinalis (6). These amphioxus VCBP molecules are encoded by five or more multigene families that are polymorphic within the population. VCBP gene products are secreted into the intestine, where they may play a role in preventing microbial invasion. Evidence has recently been obtained that two very different recombinatorial systems for lymphocyte antigen receptor diversification appeared at the dawn of vertebrate evolution ∼500 mya (Figure 1). Lamprey and hagfish, which are the only surviving jawless fish (agnathans) belonging to the oldest vertebrate taxon (7, 8), have been found to assemble diverse lymphocyte antigen receptor genes through the genomic rearrangement of leucine-rich repeat (LRR)–encoding modules (9, 10). These cell surface receptors are designated variable lymphocyte receptors, or VLRs. Recombinatorial mechanisms for the generation of anticipatory receptors thus evolved in both the jawless and jawed vertebrates, but each vertebrate group employs a different kind of modular protein domain.

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Agnathan VLR gene LRR NT

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Humoral effector molecules Figure 1 Rearranging 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 the germ line incomplete VLR gene. The mature VLR gene consists of a signal peptide (SP), an N-terminal LRR (LRRNT), first 18-residue LRRs (LRR1), a variable number of 24-residue LRRs (LRRV), a connecting peptide (CP), a C-terminal LRR (LRRCT), and a threonine/proline-rich stalk. Portions of LRRNT and LRRCT that are not encoded in the germ line VLRs are hatched. Jawed vertebrates antibody 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 rearrangement these antigen receptors are expressed on the surface of lymphocytes via GPI anchorage in VLRs or via a transmembrane domain in the antibody IgM cell surface form. Upon activation VLRs can be released to the plasma via GPI-specific phospholipase cleavage, while secreted antibodies result from isotype switching to the secretory forms.

The appearance of two types of recombinatorial immune systems within a relatively short evolutionary period of ∼40 million years during the Cambrian raises intriguing questions. What was the selective pressure to evolve acquired immunity? Why were LRRcontaining modules selected as the recombinatorial units of antigen receptors in agnathans, and why were Ig domains selected by the gnathostomes? The question of gnathostome Igs is not addressed here. On the other hand, the issue of LRR-based antigen receptors of jawless fish may be more easily addressed. Because LRR-containing proteins are ancient mediators of antimicrobial responses in both kingdoms of multicellular

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organisms, it is reasonable to suggest that the last common ancestor of plants and animals used some version(s) of LRR-containing proteins for microbial detection (11). LRRcontaining proteins therefore would have provided natural molecular candidates for early agnathan experimentation with somatic DNA rearrangement to achieve receptor gene diversification. We begin this review with a consideration of the emergence of lymphocytes as a novel circulatory cell type in vertebrates and then consider phylogenetic aspects of the superfamily of LRR-containing proteins and their role in immunity. We conclude with an evolutionary scenario that may explain the sudden www.annualreviews.org • Evolution of Adaptive Immunity

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appearance of a lymphocyte-based recombinatorial system of anticipatory immunity in vertebrates.

MIGRATORY PHAGOCYTIC CELLS APPEARED BEFORE IMMUNOCOMPETENT LYMPHOCYTES Phagocytic cells form the cellular arm of innate immune defenses in almost all animals (metazoans) that have been studied, except for the nematode Caenorhabditis elegans, which may lack cellular immune defenses. Following pathogen invasion in C. elegans, there is activation of an inducible defense system marked by an increased expression of genes encoding lectins, antimicrobial peptides, and lysozymes, but exactly how the host perceives infection is not yet understood (12). In Drosophila melanogaster, another protostome invertebrate whose genome has been sequenced, plasmatocytes are the predominant phagocytic blood cells involved in clearance of invading microorganisms. The Drosophila plasmatocytes are considered the functional equivalents of monocytes/macrophages in the vertebrates (13, 14). Monocyte/macrophage-type cells have a relatively short life span in both invertebrates and vertebrates. Proliferation of these innate immunocytes appears to be confined to the generative hematopoietic tissues. As mature circulatory cells, these nondividing phagocytes can be activated to become effector cells. These innate immunocytes may express a surprisingly large repertoire of surface receptors. As one example, a multigene family in the sea urchin Strongylocentrotus purpuratus (an echinoderm) encodes proteins featuring scavenger receptor cysteine-rich repeats. The circulatory coelomocytes of individual sea urchins express unique and temporally varying scavenger receptor cysteine-rich repertoires that are selected from an arsenal of hundreds of genes (15). It is therefore conceivable that diversification mechanisms for immune receptors evolved before the ap500

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pearance of long-lived circulatory immune cells that can undergo clonal expansion following ligand engagement of their unique receptors (16). Evidence suggesting somatic diversification of the snail FREPs may support this view, although this diversification process may not be limited to circulating hemocytes (4). Inevitably, self-reactivity among receptors generated by random somatic diversification mechanisms would present a problem. In principle, though, autoimmunity could be avoided by a developmental program including a transitory interval of immunocyte hypersensitivity to receptor-mediated triggering, leading to apoptosis, or another type of inactivation mechanism for cells with autoreactive receptors. In this way, cells bearing nonselfreactive receptors could selectively continue their maturation to become migratory immunocytes.

THE APPEARANCE OF LYMPHOCYTES IN VERTEBRATES A new type of circulatory cell with the potential for self-renewal and clonal expansion appeared near the beginning of vertebrate radiation in the form of the long-lived lymphocyte. In the jawed vertebrates, T and B lymphocytes are the acknowledged cellular pillars of adaptive immunity. T lymphocytes are primarily responsible for cell-mediated immunity, and B lymphocytes are responsible for humoral immunity, but they work together and with other types of cells to mediate effective adaptive immunity. Along with the natural killer cells, these specialized lymphoid cells are derived from committed progenitors in hematopoietic tissues, which then undergo unique V(D)J rearrangements of their antigen receptors to become clonally diverse lymphocytes. Newly formed T and B lymphocytes bearing autoreactive receptors can be eliminated by self-antigen contact in the thymus and bone marrow, respectively. The surviving T and B cells then migrate via the bloodstream to peripheral lymphoid tissues, where,

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following antigen recognition, they may undergo clonal expansion and differentiation into effector T lymphocytes or antibodyproducing plasma cells or otherwise become memory cells that await reexposure to their specific antigens. Exactly when during evolution the lymphocytes appeared as a specialized type of immunocompetent cells is unknown, but cells comparable to the lymphocytes in jawed vertebrates have never been characterized in invertebrates. On the other hand, increasing evidence for bona fide lymphocytes in lamprey and hagfish suggests that lymphocytes must have evolved in the common ancestor of the vertebrates. Most of the lymphoid cells in lamprey and hagfish are small round cells composed mainly of a nucleus with condensed chromatin and a small rim of surrounding cytoplasm that contains relatively few organelles (17). Following antigen and/or mitogen stimulation, agnathan lymphocytes can transform into large lymphoblast-like cells (9). Morphological studies have led to the view that agnathan lymphocytes are generated in the hematopoietic tissues, primarily the intestineassociated hematopoietic organ in lamprey larvae, called the typhlosole, and the protovertebral arch of adult lamprey (17–19). Evidence for a thymus-like organ in agnathans is equivocal at best, however. Collections of lymphoid cells have been found in pharyngeal gutters of the lamprey gill region, but there is no recognizable capsular, stromal, or lymphoepithelial organization of the type that characterizes the lymphopoietic thymus in jawed vertebrates (17, 20–22). The lamprey lymphocytes express homologs of many genes expressed during jawed vertebrate lymphocyte differentiation, proliferation, migration, and intracellular signaling and perhaps also express the relatives of genes that gnathostomes use in antigen processing and intracellular transport of antigenic peptides (23–27). It is important to note that proteins with sequence similarity to the jawed vertebrate rearrangeable Ig genes or MHC genes have not been found in extensive surveys of

lamprey and hagfish leukocyte transcripts (28, 29). Until very recently, there was no credible evidence for lymphocyte receptor diversity in lamprey or hagfish. This led to considerable skepticism about the earlier reports of agnathan adaptive immunity (17, 20, 30–32). This picture changed dramatically with the identification of VLR genes in the lamprey and hagfish (9, 10). These genes are assembled by a special recombinatorial mechanism used to generate a diverse repertoire of anticipatory receptors. The lymphocytes of lamprey and hagfish rearrange modular LRR cassettes to create functional mature VLR genes. A VLR of unique sequence is expressed by each lymphocyte in a monoallelic fashion. As in the case for the gnathostome lymphocytes, the agnathan lymphocytes may undergo lymphoblastoid transformation following antigen and/or mitogen stimulation. Clonal amplification appears to occur during antigen-induced proliferative responses in lamprey and hagfish, although more experimental evidence will be required to confirm this conclusion. Lamprey lymphocytes can respond to immunization by the release of their antigen-specific VLRs into the plasma, thus providing the potential basis for humoral immunity (M.N. Alder, M.D. Cooper & Z. Pancer, unpublished data). Although many questions about the development and function of agnathan lymphocytes are still unanswered, it is clear that the jawless vertebrates have a lymphocyte-based recombinatorial immune system that differs radically from the Ig-based recombinatorial immune system in the jawed vertebrates.

IMMUNE-RELATED LRR PROTEINS OF INVERTEBRATES AND PLANTS Drosophila has two distinct families of PRRs that are used selectively to activate one or the other NFκB-like signaling pathways, the Toll or the Immune Deficiency. Cellular activation via these pathways results in induction www.annualreviews.org • Evolution of Adaptive Immunity

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Gnathostomes (jawed vertebrates)

Cephalochordates (Amphioxus) Urochordates (ascidians) Echinoderms

DEUTEROSTOMES

Agnathans (jawless vertebrates)

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Annelids Molluscs

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Metazoan tree of life. A simplified representation of the two major groups of multicellular animals, protostomes and deuterostomes, that include the chordate invertebrates and vertebrates.

Cnidarians

of antimicrobial peptide genes in the fat body and the secretion of antimicrobial peptides into the hemolymph (33). It was first realized in 1996 that the dorsoventral patterning receptor Toll is also involved in protection of flies against fungi, by way of inducing expression of the antifungal peptide gene Drosomycin (34). The Toll pathway has recently been shown to be essential for protection against the Drosophila X virus as well (35). The Toll receptor has an extracellular LRR-containing domain, a transmembrane region, and a cytoplasmic Toll/interleukin-1 receptor homology domain (TIR). In addition to Toll, the Drosophila genome contains eight Toll homologs, and the mosquito Anopheles gambiae genome contains ten Toll homologs. All but one of these Tolls in both insects appear to be linked to developmental functions rather than to immunity (36). Likewise, immune function could not be attributed to the single C. elegans and Caenorhabditis briggsae

TLR: Toll-like receptor

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Toll homolog, nor to the Toll-like receptor (TLR) reported in the horseshoe crab Tachypleus tridentatus (37). In striking contrast to the TLR saga in these protostome invertebrates, the vertebrate homologs of Drosophila Toll appear to be dedicated solely to host defense (38, 39). This leads us to consider what is known about the LRR-containing proteins in deuterostome invertebrates, which are in the ancestral evolutionary lineage of the vertebrates (Figure 2). It is known that TLR polypeptides are frequently encoded by a continuous open reading frame that is uninterrupted by introns. Standard examples of this type of gene structure include Tollo, Toll 6, and Toll 7 from Drosophila and human TLRs 1, 2, 4D, 5, 6, and 10 (intronless genes database) (40). The genome of the sea urchin S. purpuratus abounds with intronless LRR-containing genes. A sample of 52 intronless TLRs, derived from those identified

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in the draft genome sequence (Sequencing Project Version 0.3, Baylor College of Medicine; http://www.hgsc.bcm.tmc.edu), is illustrated in Figure 3. These TLRs cluster in a very unusual branching pattern consisting of sequences with nearly identical values of genetic distance among branch members (Figure 4), thereby indicating multiple events of expansion and diversification among branch members. The total number of sea urchin TLRs is estimated to be ∼340 members, or half this number if all these genes prove to be polymorphic (41). Cloning and expression analysis indicate that multiple TLRs may function in sea urchin immunity (Z. Pancer & E.H. Davidson, unpublished data). In addition to the TLR genes, the sea urchin genome harbors other intronless LRR-encoding genes. One group with at least 47 members consists solely of LRR motifs (not shown); some of these proteins have transmembrane domains (N = 13/47). Another group also has C-terminal Ig domains (N = 10), and some of these are also predicted to have membrane anchorage domains (N = 6/10) (Z. Pancer & M.D. Cooper, unpublished data). In the chordate phylum, the genome of the solitary tunicate Ciona savignyi contains between 7 and 19 TLR genes (41), only 2 of which are intronless genes, rendering gene prediction uncertain. At least 22 other intronless LRR-containing genes were identified in the C. savignyi genome (Sequencing Project data, April 25, 2003, version, Whitehead Institute and MIT Center for Genome Research; http://www-genome. wi.mit.edu), and 8 of these appear to be cell surface receptors (Z. Pancer & M.D. Cooper, unpublished data). Only three TLRs were reported in the genome of another solitary tunicate, C. intestinalis (6), and all of these are interrupted by introns. Notably, no other intronless LRR genes could be identified in this species. In the amphioxus B. floridae, we identified at least 42 intronless TLR genes (trace archive, WGS Sequencing Project, DOE

Joint Genome Institute; ftp://ftp.ensembl. org/pub/traces/branchiostoma floridae/). These TLR genes cluster in equidistant branches of the genetic distance tree, like TLRs from the sea urchin. In addition, the B. floridae genome harbors at least 211 intronless LRR-containing genes, or half this number if all alleles are polymorphic. Seventy-one of these LRR-containing genes are illustrated in Figure 5. Fifty-one consist only of LRR motifs, and 12 of these include transmembrane domains; the other 20 have both LRR and C-terminal Ig-like domains, and 12 of these are predicted cell surface proteins (Z. Pancer & M.D. Cooper, unpublished data). Computational analysis indicates that several more of the sea urchin and amphioxus intronless LRR-containing genes may encode proteins that are tethered to the cell surface via glycosyl-phosphatidyl-inositol (GPI) anchors. Intriguing unanswered questions abound: Why is the solitary tunicate C. intestinalis different from other deuterostome invertebrates that utilize multiple LRR-containing proteins? What may be the strategy employed by colonial tunicates, such as Botryllus schlosseri, that are known for their highly elaborate and polymorphic self/nonself-recognition systems (42)? Plant genomes harbor very large families of LRR-containing genes, and many of these mediate disease resistance. The most important plant disease resistance genes encode the STAND ATPase domain (43) or nucleotide-binding site (NBS)–LRR proteins, some of which include N-terminal TIR domains. There are also the LRR receptor– like kinases and the membrane-bound LRR receptor–like proteins. Several of these proteins control resistance to a wide variety of plant pathogens and pests, including viruses, bacteria, fungi, nematodes, and insects (44, 45). In response to pathogen challenge, diverse resistance responses in plants are activated by disease resistance proteins. These responses include the production of antimicrobial peptides and a form of programmed www.annualreviews.org • Evolution of Adaptive Immunity

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Figure 3 A large superfamily of sea urchin TLRs. A sample of 52 intronless TLR genes identified in the genome of Strongylocentrotus 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.) 504

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cell death called the hypersensitive response (46). In rice, Oryza sativa, 585 predicted NBSLRR genes account for approximately 1% of the genes identified in the genome; a similar fraction of the Arabidopsis thaliana genome is dedicated to disease resistance genes (44, 45).

Even with knowledge of this large arsenal of disease resistance genes, we still do not understand how plants can detect the multitude of infectious pathogens with a limited number of PRR genes. The “guard” hypothesis postulates that resistance proteins constitute www.annualreviews.org • Evolution of Adaptive Immunity

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Figure 5 A large superfamily of amphioxus intronless LRR-containing genes. A sample of 71 genes identified in the genome of the Florida lancelet Branchiostoma floridae. 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; Ig superfamily domain: green oval; transmembrane domain: dark blue rectangle. 506

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Figure 5 (Continued )

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components of larger signal perception complexes that are activated in response to pathogen-induced perturbance of the normal function of host proteins. Hence, instead of recognition via a specific receptor for each pathogen, detection may occur indirectly via the damage inflicted by pathogen-derived virulence proteins (45–47). High levels of polymorphism have been noted among members of plant resistance genes, and the putative ligand-binding surfaces in many families of LRR-containing resistance genes appear to be undergoing rapid diversifying selection (48). Interestingly, there is also evidence of pronounced selection for somatic variants of disease resistance genes that may affect the ligand specificities of particular resistance proteins (49). This variation may be generated via an unknown diversification mechanism of plant PRR genes, or it may reflect the increased frequency in homologous recombination that has been observed for virus-infected plants (50). Intronless LRR-containing genes are relatively rare in animal genomes other than deuterostome and chordate invertebrates. Apart from the few intronless TLRs in mammals and insects, there are only 10 other intronless LRR-containing genes in the human and mouse genomes, none in Drosophila, and only 25 of the NBS-LRR genes in Arabidopsis (intronless genes database) (40). Intronless genes most likely result from retroposition and subsequent genomic integration thought to occur via the reverse transcription activity of endogenous retrotransposons, such as the human LINE elements (51). Intronless genes may be excellent gene templates to generate rapidly evolving arsenals of diverse germ line– encoded receptors.

IMMUNE-RELATED LRR PROTEINS OF JAWED VERTEBRATES The typical TLR complement for vertebrates is approximately one dozen genes. The only exception that has been noted thus far is fish 508

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that have retained both copies of duplicated TLRs resulting from a whole genome duplication that seems to have occurred after the divergence of bony fish and tetrapods. For example, at least 17 predicted TLR genes were expressed in the zebrafish, Danio rerio (52). Nearly all vertebrate TLRs belong to one of six major families (TLR1–6), and each TLR family is capable of recognizing a general class of PAMPs. TLR2 family members bind lipopeptide; TLR3 family members bind double-stranded RNA; TLR4 family members bind LPS; TLR5 family members bind flagellin; members of the TLR7, TLR8, and TLR9 subfamilies bind nucleic acid and heme motifs; and TLR1 family members associate with TLR2 members as heterodimeric receptors (41). Mouse TLR11 has recently been implicated in the response to a profilin-like protein of the protozoan parasite Toxoplasma gondii (53). Soluble TLR forms, consisting of the extracellular portions only, may also participate in immunity. Amphibians and fish have a soluble form of the TLR5 gene that arose by duplication of the region encoding the extracellular domain. In rainbow trout, Onchorhynchus mikiss, bacterial flagellin interacts with membrane-bound TLR5 to induce the expression of the soluble TLR5 gene in the liver, resulting in efficient clearance of flagellin from the circulation (54). In chicken, alternatively spliced forms of TLR3 and TLR5 yield soluble products (55), and human plasma and breast milk also contain a functional soluble form of TLR2 that is generated by a posttranslational modification (56). The recently identified CATERPILLER family of LRR-containing immune-regulatory genes encodes cytoplasmic proteins that are structurally similar to some of the plant disease resistance genes (43). The N terminus of these proteins may function as an effector domain, mediating homotypic or heterotypic interactions; a central NBS domain has regulatory function, whereas the C terminus is composed of variable sets of LRR motifs that may function in ligand binding.

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Heterodimers formed between different family members may in some cases increase the combinatorial binding potential (57). Nucleotide oligomerization domain (NOD) 1 and NOD2 members of this family are implicated as sensors of intracellular bacterial products and activators of host responses against invading pathogens (57, 58). In gut regions that are rich in commensal bacteria, NOD1 functions as the detector of enteroinvasive bacteria that have evolved the means to prevent intestinal epithelial cell signaling through the TLRs. NOD2 is critical for regulation of bacterial immunity within the intestine by controlling the expression of cryptdins, which are intestinal antimicrobial peptides (59). More than 20 members of this family have been identified in mammalian genomes, whereas no CATERPILLER orthologs have been identified in Drosophila or C. elegans (57). One strategy that is employed to maintain balance in the arms race between insects and pathogens involves natural selection of random mutations in insect PRR genes. In wild Drosophila populations, for example, ∼10% of the polymorphic sites in genes encoding antibacterial peptides, Toll receptors, signal transduction molecules, and other pathogen recognition molecules are associated with disease resistance phenotypes to a single pathogen (60). However, the vertebrate TLRs are not rapidly evolving genes. They appear to be under strong purifying selection to maintain their PAMP recognition specificity in order to discriminate between pathogens and the host. This may be because selfreactive TLRs would be detrimental to the host, as none of the self-tolerance mechanisms that can purge self-reactive T and B lymphocytes or prevent development of potentially harmful natural killer cells have been identified for cells bearing TLRs (41). The large multigene families of LRR-containing proteins in deuterostome invertebrates are especially remarkable given that microbial recognition is served by only a handful of PRRs in nematodes, insects, and vertebrates (61).

STRUCTURE AND FUNCTION OF LRR-CONTAINING PROTEINS LRRs of 20–29 amino acids per repeat are present in more than 2000 proteins from viruses, bacteria, archaea, and eukaryotes. Family members of the LRR-containing proteins participate in nearly all known biological functions, including plant and animal immunity, apoptosis, cell adhesion, signal transduction, DNA repair, DNA recombination and transcription, RNA processing, and ice nucleation. Nonetheless, the existence of many types of LRR-containing immune gene families in the genomes of both plants and animals argues for the special role of these proteins in host defense. Sixteen crystallized LRR-containing proteins all adopt an arc or horseshoe-like shape, with the individual LRR motifs forming parallel loops that are stacked into a coil. Most of the LRR-containing proteins have characteristic N-terminal and C-terminal LRR domains capping the ends of the hollow tube. The concave face of the coil consists of a parallel β-sheet, whereas there may be α-, 310 -, or pII helices in the convex face (62). Ligand-binding sites have been determined for several LRR-containing receptors. The mammalian ribonuclease inhibitor, the first LRR structure solved, interacts with the ribonuclease via multiple contact points located on the concave LRR surface of the inhibitor (63). Glycoprotein Ib, a platelet LRRcontaining receptor for the von Willebrand factor, binds its ligand via exposed residues on the concave LRR face and via a fingerlike insertion in the C-terminal LRR (64). CD14, one of the major LPS receptor units, is an LRR-containing protein expressed on myelomonocytic cells as a GPI-linked glycoprotein or released into the plasma as a soluble form. The crystal structure of CD14 reveals a dimer in which each horseshoeshaped monomer consists of 13 β-strands. The large hydrophobic pocket is located on the side of the horseshoe near the N terminus

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(65). The polygalacturonase-inhibiting protein of Phaseolus vulgaris is the product of a plant disease resistance gene. It acts as an inhibitor of cell wall–degrading enzymes produced by pathogenic fungi. A negatively charged surface on the concave LRR face is probably involved in binding fungal polygalacturonases (66). The tomato, Lycopersicon pimpinellifolium, Cf-9 protein confers fungal resistance via residues in its N-terminal LRR; putative glycosylation sites in the outer α-helices are also essential for binding (67). Mammalian receptors for the glycoprotein hormones (thyrotropin, lutropin, chorionic gonadotropin, and follitropin) are G protein– coupled proteins that bind their ligands via the LRR motifs in their extracellular domains (68, 69). Internalins A and B of Listeria monocytogenes are LRR-containing surface proteins that mediate specific host-cell invasion by the bacteria. Internalin A mediates bacterial adhesion and initiates invasion of human intestinal epithelia through specific interaction with the E-cadherin receptor. The crystal structure of Internalin A complexed with the N-terminal domain of E-cadherin reveals tight interaction sites on the concave surface of the LRR coil (70). The N terminus of Internalin B consists of an LRR domain that is C-terminally capped by an Ig-like domain, and this portion is sufficient to induce bacterial internalization into host cells (71). Promiscuity of ligand binding has been suggested in the case of Nogo and its LRRcontaining GPI-anchored receptor, which plays a key role in inhibition of mammalian axon regeneration. The Nogo receptor has a putative ligand-binding site within the concave LRR face in which multiple solventexposed hydrophobic and aromatic residues create high potential for binding crossreactivity (72). Decorin and Opticin are small LRR-containing extracellular matrix proteoglycans that form antiparallel homodimers via highly specific interactions at their concave LRR surfaces. It therefore seems likely that their binding of different ligands occurs 510

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through protein surface sites other than those of the concave sheet (73, 74). The extracellular domains of TLRs consist of 19–25 tandem LRR motifs, most of which conform to a 24-residue consensus motif. Peptide insertions are present within some of the LRRs, and these have been predicted to mediate recognition of PAMPs (75). The only TLR crystal structure reported to date, the human TLR3 ectodomain, provides experimental support for this prediction (76). The TLR3 solenoid consists of 25 LRR motifs that are stacked and stabilized through hydrogen bonds formed by conserved asparagine residues. Glycosylation-free faces of the solenoid provide interfaces for a homodimeric configuration maintained via conserved surface residues and a loop formed by a peptide insertion in LRR20, whereas a second peptide insertion in LRR12 and two clusters of positively charged residues form the putative binding site for double-stranded RNA. Some TLRs may recognize only a limited number of PAMPs. For instance, TLR9 directly interacts with particular sequences of unmethylated CpG-DNA found in bacterial DNA (77, 78), and TLR7 on the surface of plasmacytoid dendritic cells and B cells mediates the recognition of singlestranded RNA from vesicular stomatitis virus and influenza virus. Thus, TLR7 recognizes single-stranded RNA viruses, whereas either TLR3 or TLR9 detects double-stranded RNA viruses (79, 80). Other TLRs have the remarkable potential to interact with structurally unrelated ligands. TLR2 mediates host responses to peptidoglycan and lipoteichoic acid from Gram-positive bacteria, lipoarabinomannan from mycobacteria, neisserial porins, bacterial tripalmitoylated and mycoplasmal diacylated lipoproteins, and yeast products and GPI-anchored proteins of the protozoan Trypanosoma cruzi. Results from mutagenesis of the extracellular LRRs in TLR2 imply the existence of different binding sites for different ligands (81). TLR4 can bind LPS from Gram-negative bacteria, viral proteins, bacterial and host heat shock

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proteins, host oligosaccharides derived from heperan sulfate and hyaluronic acid, host taxol, and fibrinogen (75). In spite of their sequence and functional diversity, the horseshoe-like structure with its concave binding surface is remarkably conserved in all the LRR-containing proteins. This may reflect the modular structure formed by the tandem array of stacked LRR motifs. Each such structure consists of a mosaic of conserved scaffold residues interspersed with highly variable residues to account for the enormous diversity in ligandbinding sites.

HYPOTHESIS: EVOLUTION OF ADAPTIVE IMMUNITY IN VERTEBRATES Are there any fundamental differences between invertebrates and vertebrates in terms of their potential pathogens? We cannot go back to the time when the earliest jawless fish diverged from a common cephalochordate ancestor (82), but we can speculate that the answer to this question is no, based on the fossil record and studies of contemporary species. There is no indication for massive eradication of species in the Cambrian that could imply new types of potentially devastating pathogens, and it seems unlikely that the newly evolved vertebrates became the favorite hosts for new kinds of pathogens soon after their emergence. Despite 500 million years of vertebrate existence, there is a dearth of evidence for significant numbers of vertebrate-specific pathogens. Conversely, many pathogens are known to infect both invertebrate and vertebrate hosts, including, for example, more than 500 varieties of the arboviruses (83). Germ line–encoded innate immune barriers protect both invertebrates and vertebrates from potential pathogens, although vertebrates may be better protected against some of the frequently recurring pathogens. Even in vertebrates, however, innate immunity provides the first line of defense against

pathogens because a protective level adaptive immune response takes at least several days to mount. Innate immune mechanisms of invertebrates must therefore be as efficient as those in vertebrates for combating the rapidly evolving pathogens that these animals inevitably encounter (16). Why then do deuterostome invertebrates need a vastly expanded arsenal of germ line– encoded receptors when only a handful of PRRs suffices for immunity in nematodes, insects, and vertebrates? The LRR-containing proteins and other multigene families of immune receptors of deuterostome invertebrates may play a pivotal role in the maintenance and surveillance of the endosymbiotic microbial communities that these animals harbor. For example, it has been estimated that more than 60% of echinoderm species associate with bacterial symbionts (84). The intestinal floral symbionts in sea urchins may be needed to ferment and detoxify the poorly nutrient kelp and algae on which these animals graze. Such a complex mode of long-term coexistence between animals and microorganisms may have favored the evolution of large arsenals of specific microbial recognition molecules, whereas the strategy of PAMP recognition indiscriminately targets practically all microorganisms as nonself. This complex mode of coexistence with endosymbiotic microbes most likely was transmitted from invertebrate ancestors to their vertebrate descendents because complex microbial communities exist in the intestines of all vertebrates; 400–1000 species are estimated to live in the human gastrointestinal tract (85, 86), and the commensal microbiota have been shown to shape the Ig repertoire of peripheral B lymphocytes (87). Furthermore, maintenance of the mammalian gut flora appears to require highly elaborate immune mechanisms and an active cross talk between the microflora and the host mucosal immune system (88). It may be interesting to explore the mechanisms that invertebrates employ to distinguish between symbionts and potential pathogens in animals belonging to the deuterostome www.annualreviews.org • Evolution of Adaptive Immunity

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lineage as well as in protostome invertebrates that harbor endosymbiotic microorganisms, for example, the wood-feeding termites (89) and molluscan cephalopods (octopus, squid, and cuttlefish) that harbor symbiotic bacteria in their light organs (90). Thus far we have emphasized the similarities between deuterostome invertebrates and their vertebrate evolutionary descendents. It may be more relevant to consider differences between these animal groups that may have favored the development of a completely new mode of antigen recognition in vertebrates on the basis of receptor gene rearrangement. To address this enigma, we need to look back at the Cambrian explosion ∼500 mya, when a unique and stunning burst of evolutionary diversification of new vertebrate species began. In a relatively brief evolutionary period, a variety of free swimming jawless fish appeared in the oceans. These fish descended from small amphioxus-like ancestors that lived as suspension feeders buried in the sand in shallow coastal waters. Early skulled vertebrates (Craniates) had a unique feature that separated them from their cephalochordate ancestor, namely a whole genome duplication that most likely occurred at the beginning of vertebrate divergence (91, 92). This genome duplication may have fueled the dramatic “big leap” in vertebrate developmental, morphological, and functional innovation during the Cambrian period (93). We can speculate that a large arsenal of diverse LRR-containing proteins was also part of the ancestral cephalochordate heritage. Members of these abundant cell surface and soluble receptors may have engaged in serendipitous interactions with newly evolving molecular determinants of early agnathans. If so, this interference may have been a rate-limiting factor in the process of rapid vertebrate evolution. In consideration of the enormous binding versatility of LRRcontaining proteins, it is conceivable that their self-reactivity presented serious autoimmunity problems at a time of rapid developmental and morphologic innovation. Further512

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more, the transition from invertebrates to vertebrates may have been associated with changes in the endosymbiotic communities, thereby rendering many of these microbial surveillance proteins obsolete. In any case, the rate of readjustment required to maintain large multigene families of germ line receptors as strictly nonself-reactive may have become overly burdensome. Consequently, early vertebrates may have been forced to abandon the invertebrate deuterostome strategy of large arsenals of germ line–encoded immune receptors. This line of reasoning leads us to speculate that an adjustable immune system based on randomly generated receptor diversity evolved in part to enable the burst of vertebrate speciation in the Cambrian. Lymphocytes bearing uniquely rearranged surface antigen receptors, which could undergo negative selection to purge self-reactive lymphocytes while sparing clones expressing potentially beneficial antigen receptors of sufficient diversity, could have replaced the function of ancestral germ line arsenals. It is also likely that the newly evolved vertebrate lymphocytes performed innate immune functions concomitantly with the stepwise acquisition of acquired immune functions. There is ample evidence that lymphocytes have retained innate immune functionality. For example, B lymphocytes express TLRs and respond to their ligands by proliferation, expression of costimulatory molecules, and plasma cell differentiation (39). B cells of the peritoneal cavity and spleen marginal zones mediate microbial destruction via secretion of polyreactive antibodies that are essentially germ line encoded (94). Plasmacytoid dendritic cells derived from a common lymphoid progenitor express TLR7 and -9. These professional producers of IFN-α/β are important in protection against a wide range of viruses, bacteria, and parasites (95). The T lymphocytes are professional producers of IFN-γ. The concerted integration between innate and adaptive immune functions of lymphocytes may explain why the outcome of genetic defects that prevent the development

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of T and B lymphocytes in infants with severe combined immunodeficiency diseases precludes survival from viral, bacterial, and fungal infections (96). Only hagfish, lamprey, and the jawed vertebrates survived from the early vertebrate radiation, and only a sparse fossil record remains from the short period that separates the emergence of jawless fish and the appearance of jawed vertebrates (97, 98). It therefore will be difficult to determine whether the agnathan LRR-containing VLRs were forerunners of vertebrate immune receptors or if

the rearranging VLRs and Igs evolved as independent solutions to similar necessities. The development of two very different modes of lymphocyte-based receptor diversification at the dawn of vertebrate evolution nevertheless strongly attests to the enormous fitness value of anticipatory immunity. The benefits from this innovative strategy may not be limited to the ability to recognize a nearly infinite antigenic world. Rather, the immediate selective pressure may instead have been facilitation of the developmental and morphological plasticity of the vertebrates.

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

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