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Page 1: Structural Insights into the Evolution of the Adaptive Immune System

BB42CH08-Mariuzza ARI 15 February 2013 18:45

RE V I E W

S

IN

AD V A

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Structural Insights into theEvolution of the AdaptiveImmune SystemLu Deng,1 Ming Luo,2,3 Alejandro Velikovsky,2,4

and Roy A. Mariuzza2,4

1Division of Hematology, Center for Biologics Evaluation and Research, Food and DrugAdministration, Bethesda, Maryland 208922University of Maryland Institute for Bioscience and Biotechnology Research, W.M. KeckLaboratory for Structural Biology, Rockville, Maryland 20850; email: [email protected] National Laboratory for Physical Sciences at Microscale and School of Life Sciences,University of Science and Technology of China, Hefei, Anhui 230027, China4Department of Cell Biology and Molecular Genetics, University of Maryland, College Park,Maryland 20742

Annu. Rev. Biophys. 2013. 42:8.1–8.25

The Annual Review of Biophysics is online atbiophys.annualreviews.org

This article’s doi:10.1146/annurev-biophys-083012-130422

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

antibody, T cell receptor, variable lymphocyte receptor, jawless vertebrate,immunoglobulin fold, leucine-rich repeat

Abstract

The adaptive immune system, which is based on highly diverse antigen re-ceptors that are generated by somatic recombination, arose approximately500 mya at the dawn of vertebrate evolution. In jawed vertebrates, adaptiveimmunity is mediated by antibodies and T cell receptors (TCRs), which arecomposed of immunoglobulin (Ig) domains containing hypervariable loopsthat bind antigen. In striking contrast, the adaptive immune receptors ofjawless vertebrates, termed variable lymphocyte receptors (VLRs), are con-structed from leucine-rich repeat (LRR) modules. Structural studies of VLRshave shown that these LRR-based receptors bind antigens though their con-cave surface, in addition to a unique hypervariable loop in the C-terminalLRR capping module. These studies have revealed a remarkable exampleof convergent evolution in which jawless vertebrates adopted the LRR scaf-fold to recognize as broad a spectrum of antigens as the Ig-based antibodiesand TCRs of jawed vertebrates, with altogether comparable affinity andspecificity.

8.1

Review in Advance first posted online on February 28, 2013. (Changes may still occur before final publication online and in print.)

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TLR: Toll-likereceptor

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2ANTIGEN RECOGNITION BY ANTIBODIES

AND T CELL RECEPTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3ANTIGEN RECEPTOR DIVERSIFICATION IN JAWED VERTEBRATES . . . . . 8.5EVOLUTIONARY ORIGIN OF THE ADAPTIVE IMMUNE

SYSTEM OF JAWED VERTEBRATES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6ADAPTIVE IMMUNE RECEPTORS OF JAWLESS VERTEBRATES. . . . . . . . . . . . 8.8ASSEMBLY AND DIVERSIFICATION OF VLR GENES . . . . . . . . . . . . . . . . . . . . . . . . 8.10VLRA AND VLRB LYMPHOCYTES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10ANTIGEN RECOGNITION BY VLRBs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11ANTIGEN RECOGNITION BY VLRAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12STRUCTURAL BASIS FOR ANTIGEN RECOGNITION

BY VLRs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12Structure of VLRB–Antigen Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13Structure of a VLRA–Antigen Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14Architecture of the VLR Antigen-Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.16Sequence Variability of VLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.16

COMPARISON OF ANTIGEN RECOGNITION BY VLRS,ANTIBODIES, AND TCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.19

FUTURE ISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.20

INTRODUCTION

Immune responses involve recognition of pathogens or other foreign materials followed by reac-tions to eliminate them. The responses fall into two broad categories, termed innate and adaptive.Before they can cause perceptible disease, most infectious microorganisms are detected and de-stroyed by the innate immune system, which is evolutionarily conserved from insects to mammals.By contrast, adaptive immunity is restricted to vertebrates and comes into play when innate de-fenses are evaded or overwhelmed by the invading pathogen. Adaptive immunity is believed tohave arisen approximately 500 mya at the beginning of vertebrate evolution (62). Whereas innateimmune responses are mediated by recognition molecules that are of restricted diversity, adaptiveimmunity is mediated by highly diverse receptors responsible for specific antigen recognition.Because they do not require clonal expansion, innate responses occur rapidly, often within hoursof infection. In contrast, adaptive responses are characterized by a latent period of clonal expan-sion lasting several days before the proliferating lymphocytes mature into effector cells capable ofeliminating an infection. Thus, innate responses can repel a pathogen at an early stage, or at leasthold it in check until an adaptive response can be mounted. Recent discoveries have highlightedimportant functional links between innate and adaptive immune responses (40).

Innate immunity is carried out by germline-encoded receptors that have evolved to recognizeunique products of microbial metabolism not produced by the host. These microbial ligands areknown as pathogen-associated molecular patterns (PAMPs), and the innate immune receptorsthat bind them are termed pattern recognition receptors (PRRs) (40). Examples of PAMPs rec-ognized by PRRs such as Toll-like receptors (TLRs) (12), peptidoglycan recognition proteins(PGRPs) (71), and NOD-like receptors (NLRs) (79) include lipopolysaccharide of gram-negative

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Gnathostomes:vertebrates with jaws,including bony fish,reptiles, and mammals

TCR: T cell receptor

Immunoglobulin (Ig)fold: protein domaincomposed of twoβ-pleated sheets, eachcomprising three tofive antiparallelβ-strands, that arelinked by a disulfidebond

Agnathans:jawless vertebrates,comprising lampreysand hagfish

Leucine-rich repeat(LRR):β-strand–turn–α-helixstructural motif of20–30 residues that isthe building block forLRR proteins,including variablelymphocyte receptors

VLR: variablelymphocyte receptor

bacteria, lipoteichoic acid of gram-positive bacteria, nonmethylated CpG sequences, flagellin, andpeptidoglycan of gram-negative and gram-positive bacteria. Similarly, RIG-I and MDA-5 detectRNA viruses by binding double-stranded RNA (66). Cellular activation by PRRs results in acuteinflammatory responses involving chemokine and cytokine production, direct attack against thepotential pathogen, and in the case of vertebrates, activation of the adaptive component of theimmune system (40).

In addition to innate immunity, all jawed vertebrates (gnathostomes), from sharks to mammals,have evolved an adaptive immune system that is mediated by highly diverse antigen receptorsrepresented by antibodies and T cell receptors (TCRs), which are clonally expressed on B andT lymphocytes, respectively (24, 53). These receptors, which are composed of immunoglobulin(Ig) domains, are generated by somatic recombination of variable (V), diversity (D), and joining( J) gene segments (9). The resulting repertoires are sufficiently vast to recognize any invadingmicroorganism or toxin. Among extant jawed vertebrates, the cartilaginous fish (sharks) are themost ancient organisms to possess an adaptive immune system (24). Following specific activationby antigen, B and T lymphocytes initiate humoral and cellular immune responses.

Like jawed vertebrates, jawless vertebrates (agnathans), of which the only extant examples arelampreys and hagfish, were long known to mount specific immune responses to a variety of bacteria,viruses, and xenogenic cells, as manifested by the appearance of antigen-specific agglutinins in theserum (10, 23, 25, 51). Furthermore, lampreys and hagfish were found to accept skin autograftsbut to reject allografts, which is indicative of cellular immunity (23, 64). However, transcriptomeanalyses of leukocytes from jawless fish and database searches for Ig domain–containing immune-type molecules failed to detect genes encoding antibodies or TCRs (55, 77, 81).

This mystery was solved by the remarkable discovery in lampreys and hagfish of an alternativeadaptive immune system that evolved independently of antibodies and TCRs. This non-Ig systemutilizes proteins called variable lymphocyte receptors (VLRs), which consist of leucine-rich repeat(LRR) modules that are assembled into functional receptors by DNA recombination (4, 61, 63).The LRR motif is also found in many innate immune receptors, including TLRs, NLRs, andplant disease resistance proteins, highlighting its enormous capacity for microbial recognition.The independent development of two very different antigen recognition systems in jawed andjawless vertebrates strongly attests to the fitness value of adaptive immunity (9, 10).

In this review, we first describe the basic features of antigen recognition by the antibodies andTCRs of jawed vertebrates, with an emphasis on structural insights into the evolutionary origin ofthese Ig-based adaptive immune receptors. For detailed discussions of genetic and cellular aspectsof immune system evolution, we refer the reader to several excellent reviews (9, 24, 53). We thenfocus on recent studies of the diversification and antigen-binding properties of VLRs and of thestructure of VLR–antigen complexes. Collectively, these studies have revealed a striking exampleof convergent evolution in which lampreys and hagfish adopted the LRR scaffold to recognizeas diverse an array of antigens as the Ig-based antibodies and TCRs of jawed vertebrates, withcomparable specificity and affinity.

ANTIGEN RECOGNITION BY ANTIBODIESAND T CELL RECEPTORS

Antibody molecules of jawed vertebrates are typically composed of two identical light (L) chainscovalently linked to two identical heavy (H) chains. The L and H chains are divided into N-terminal variable (V) and C-terminal constant (C) portions. Each L chain consists of two domains(VL and CL) of two antiparallel β-sheets, whereas each H chain comprises up to five such do-mains (VH, CH1, CH2, CH3, and CH4 in the case of IgM), depending on antibody isotype. These

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1

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PeptidePeptide

CH

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T22

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Peptide

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MHC class II T22HEL

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Figure 1Antigen recognition by adaptive immune receptors of jawed vertebrates. (a) Upper panel: Side view of an antibody–antigen complexformed by mouse antibody HyHEL-63 binding to HEL (1DQJ) (50). Lower panel: Top view of the complex showing positions of theVL and VH CDR loops (numbered 1–3) on the HEL surface. (b) Upper panel: Side view of an αβ TCR–peptide–MHC class IIcomplex formed by human αβ TCR HA1.7 binding to an influenza virus hemagglutinin peptide and HLA-DR1 (1FYT). Lower panel:Top view of the complex showing positions of the Vα and Vβ CDR loops on the peptide–HLA-DR1 surface. Vα CDR3 and Vβ

CDR3 straddle the MHC-bound peptide. (c) Upper panel: Side view of a γδ TCR–ligand complex formed by mouse γδ TCR G8binding to the nonclassical MHC molecule T22 (1YPZ) (2). Lower panel: Top view of the complex showing positions of the Vγ andVδ CDR loops on the T22 surface. Vγ CDR1 and Vγ CDR2 do not contact T22. Abbreviations: CDR, complementarity-determiningregion; HEL, hen egg white lysozyme.

CDR:complementarity-determiningregion

structurally very similar β-sheet domains are termed Ig domains (76). Each VL and VH domaincontains three loops that connect the β-strands and are highly variable in length and sequenceamong different antibodies. These so-called complementarity-determining regions (CDRs) lie inclose proximity on the surface of the V domains and determine the conformation of the bind-ing site. The central paradigm of antigen–antibody recognition is that the three-dimensionalstructure formed by the six CDRs recognizes a complementary surface (epitope) on the antigen(76) (Figure 1a). Antibodies are expressed on the surface of B cells as membrane-bound antigenreceptors or are secreted into the plasma following B cell stimulation by antigen and cognateinteractions with T cells. Antibody molecules composed of only H chains are found in camelidsand cartilaginous fish (18, 28), but these molecules are most likely derived evolutionary features(24, 53).

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MHC: majorhistocompatibilitycomplex

Similar to antibodies, TCRs are composed of two chains, α and β (or γ and δ), each of whichcomprises an N-terminal V region and a C-terminal C region. The Vα and Vβ (or Vγ and Vδ)regions fold into Ig domains that closely resemble the VL and VH domains of antibody molecules(72). Loops corresponding to the CDRs of antibodies are located at the membrane-distal ends ofVα and Vβ, where they collectively form the antigen-binding site. Whereas antibodies recognizeantigens in native form, αβ TCRs recognize peptide fragments bound to major histocompatibilitycomplex (MHC) molecules on the surface of antigen-presenting cells in the form of peptide–MHCcomplexes (Figure 1b). These peptides are generated by proteolytic degradation of self or foreignantigens within cells expressing MHC class I or class II molecules (antigen processing). MHCmolecules consist of two polypeptide chains (α and β2-microglobulin for MHC class I; α and β

for MHC class II). The MHC α and β chains compose the peptide-binding site, which is a longgroove formed by two α-helices with a β-sheet floor. The αβ TCR interacts with the compoundsurface created by the MHC α-helices and the MHC-bound peptide (72) (Figure 1b). In contrastto αβ TCRs, which require antigen processing, γδ TCRs recognize antigens directly (47). Forexample, the mouse γδ TCR G8 binds directly to the nonclassical MHC molecule T22 in theabsence of antigen processing (2) (Figure 1c). Unlike antibodies, TCRs are expressed exclusivelyas transmembrane proteins and are never secreted by T cells.

Phylogenetic analysis of TCR sequences has provided evidence that γδ TCRs are evolutionarilymore ancient than αβ TCRs and that the primordial antigen receptors of jawed vertebrates wereγδ-like (68). According to this analysis, γδ T cells exhibit the primitive condition of direct antigenbinding, whereas indirect, MHC-restricted antigen recognition is a derived characteristic of αβ

T cells. However, an alternative hypothesis maintains that γδ TCRs (and possibly antibodies)evolved from primordial αβ-like receptors that lost the requirement for antigen presentation byMHC-like molecules (9).

ANTIGEN RECEPTOR DIVERSIFICATION IN JAWED VERTEBRATES

Antibodies and TCRs are assembled in B and T cell progenitors through the recombination ofV, D, and J gene segments (80) (Figure 2a). The V(D)J recombination process is mediated byrecombination-activating gene (RAG) proteins, which are absent from jawless vertebrates (26). Itis believed that antibody and TCR genes evolved from uninterrupted predecessor genes and thatthe first antigen receptor genes consisted of split variable regions that were spliced together byRAG proteins to generate complete genes encoding functional antigen receptors (15, 73).

Whereas CDR1 and CDR2 are encoded within V gene segments, CDR3 is formed by thejuxtaposition of VL and JL segments for antibody L chains (Vα and Jα for TCR α chains) andof VH, D, and JH segments for H chains (Vβ, D, and Jβ for TCR β chains). As a result, theCDR3 loops account for most of the variability of antibody- and TCR-binding sites, whereasvariability from CDR1 and CDR2 is restricted to that already existing in the germline. Furtherdiversification of CDR3 sequences is carried out by terminal deoxynucleotidyl transferase(TdT), a template-independent DNA polymerase that introduces random nucleotides at thejunctions of V, D, and J segments (53). The somatically generated CDR3 loops are located atthe geometrical center of the antigen-binding site and typically dominate interactions with theligand (Figure 1a–c), both in terms of number of contacts and energetic contribution to affinity(11, 76). In TCR–peptide–MHC complexes, the highly variable CDR3 loops are generallypositioned over the antigenic peptide, whereas the less diverse CDR1 and CDR2 loops mediategermline-encoded interactions with MHC (54) (Figure 1b).

In addition, antibodies (but not TCRs) undergo somatic hypermutation, by which base changesare introduced throughout the sequences encoding L and H chains (20). Selection of high-affinity

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CPCP

a bGermline heavy-chain gene

Germlinelight-chain gene

Germline VLR gene

Rearranged genes

Rearranged gene

Membrane-boundantibodies

Secretedantibodies

Membrane-bound VLRs

Secreted VLRs

V V V D D D J J J C

V V V

V

J J J C

CCV D J J

LRRNT

LRR1

LRR1

LRRV

LRRV

LRRV

SP 5'LRRNT

5'LRRCT

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Stalk 5'LRRCT

SPLRRNT

LRR1

LRRV

LRRV

LRRV

LRRV

CP LRRCT

Stalk

Figure 2Rearranging antigen receptors of jawed and jawless vertebrates. (a) Jawed vertebrate antibody genes are assembled via RAG-mediatedjoining of antibody gene fragments consisting of V, D, and J elements, as well as constant (C) exons. D gene segments are found only inH chain genes. Antibodies are expressed on the surfaces of lymphocytes or secreted into the plasma. TCRs generate diversity in asimilar manner, but TCRs occur exclusively in membrane-bound form. (b) VLR genes of jawless vertebrates are assembled, via geneconversion, by insertion of LRR cassettes from flanking arrays into the incomplete germline gene. A VLR comprises a set of highlydiverse LRR modules capped by N- and C-terminal LRRNT and LRRCT modules. LRR1 is followed by 1–8 LRRVs and a connectingpeptide (CP). The invariant portions of VLRs include a signal peptide (SP) and stalk region, which attaches the VLR to the lymphocytesurface. VLRBs are expressed on the surfaces of lymphocytes or secreted into the plasma as disulfide-linked multimers. VLRAs andVLRCs, like TCRs, exist only in membrane-bound form. Abbreviations: RAG, recombination-activating gene; LRRV, variableleucine-rich repeat; VLR, variable lymphocyte receptor.

AID:activation-inducedcytosine deaminase

receptors is then achieved by expansion of B cell clones on the basis of improved antigen binding,as a result of greater shape and chemical complementarity at the antigen–antibody interface (50,85). Through this rapid evolutionary process of mutation and selection, antibody affinity typicallyincreases up to 100-fold over the course of an immune response, enhancing host defense. Antibodysomatic hypermutation is mediated by activation-induced cytosine deaminase (AID), a memberof the AID-APOBEC family of cytosine deaminases (20).

EVOLUTIONARY ORIGIN OF THE ADAPTIVE IMMUNESYSTEM OF JAWED VERTEBRATES

Although the origin of genes encoding antibodies and TCRs remains obscure, the evolutionarypredecessors of these rearranging antigen receptors were probably nonrearranging Ig-typedomains (48, 52, 53). Tantalizing insights into the phylogeny of adaptive immunity have comefrom studies of the cephalochordate amphioxus (Branchiostoma floridae and B. belcheri ) (13, 22,37, 38), an invertebrate that is thought to represent the most basal extant chordate lineage (65).The amphioxus genome contains some basic components of vertebrate-type adaptive immunesystems, such as the proto-MHC region (1), RAG-like genes (42), TdT-like genes, and other

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VCBP: variableregion–containingchitin-binding protein

FGFG

BCFG

B A

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GF

C

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C''''

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VCBP3 V1 TCR Vδ

CDR3CDR1

CDR2

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C'C''

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BC

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A

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C’D E

FG

C'A

A'

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C

C’D E

FG

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Figure 3Structure of VCBP from amphioxus. (a) Ribbon diagram of the V1 domain of VCBP3 (1XT5) (32), whichadopts a V-type immunoglobulin fold. The CDR-analogous loops are labeled: BC loop (CDR1), C′C′ ′ loop(CDR2), and FG loop (CDR3). (b) Structure of a human TCR Vδ domain (1TVD) (49). (c) Structure of theV1 and V2 domains of VCBP3 (2FBO) (32). The CDR-analogous loops are on opposite ends of thestructure. (d ) Surface representation of VCBP3 V1V2, showing the location of polymorphic residues (red ) inVCBP sequences. Abbreviations: CDR, complementarity-determining region; VCBP, variableregion-containing chitin-binding protein.

enzymes involved in DNA rearrangement and repair (35). Although the amphioxus genomelacks definitive orthologs of antibody or TCR genes, it encodes multiple families of diversifiedimmune-type molecules known as variable region–containing chitin-binding proteins (VCBPs).VCBPs are soluble proteins comprising two tandem N-terminal Ig V domains (V1 and V2)and a C-terminal chitin-binding domain. VCBPs exhibit exceptionally high levels of germlinepolymorphism that is concentrated in the N-terminal portions of the V1 and V2 domains(13, 22). Such extensive germline polymorphism may compensate for the absence of somaticdiversification in maintaining receptor diversity. The high sequence variation in VCBPs andtheir chimeric Ig–lectin structure suggest a role for these putatively bifunctional proteins inpathogen recognition. Indeed, VCBPs have been shown to bind to bacterial surfaces throughtheir V domains and to enhance bacterial phagocytosis (21).

Structural studies of VCBPs have revealed features that may reflect the evolutionary transitionfrom nonrearranging innate pattern recognition molecules to the rearranging adaptive immune re-ceptors of modern-day jawed vertebrates (32). Ig superfamily domains are classified as V-, interme-diate (I)-, or C-type, depending on β-strand topology (31). The crystal structure of VCBP3 showsthat both Ig domains are V-type, in which a front β-sheet comprising strands A′GFCC′C′ ′ packsagainst a back β-sheet comprising strands ABED (32) (Figure 3a). Among V-type domains, the

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greatest similarity is to a TCR Vδ domain (49) (Figure 3b). The classification of VCBP Ig domainsas V-type implies that the V-type domain had already been adopted for use as diversified antigenrecognition molecules by the time vertebrates separated from invertebrates in evolution (32, 53).

The V domains of antibodies and TCRs pack in a head-to-head orientation, which juxtaposesthe CDR loops on one end of a VLVH or VαVβ heterodimer to form the antigen-binding site(Figure 1a,b). By contrast, the V1 and V2 domains of VCBP3 are oriented head-to-tail, such thatVCBP3 regions corresponding to CDRs (CDR1: BC loop; CDR2: C′C′ ′ loop; CDR3: FG loop)appear on opposite poles of the V1V2 structure (Figure 3c). In this head-to-tail configuration,the CDR-analogous loops of VCBP3 cannot form a contiguous binding site. Nevertheless, mostresidues at the V1V2 interface are equivalent by homology to those involved in VLVH and VαVβ

dimerization. Sequence variability in VCBPs is not localized in the CDR-analogous loops butrather in the N-terminal portions of the V1 and V2 domains (A, A′, and B strands and theirconnecting loops) (32) (Figure 3d ). These hypervariable residues may be involved in pathogenrecognition (21).

Importantly, VCBPs are not the only example of polymorphic, nonrearranging immune-typeproteins that may retain structural characteristics of ancestral forms of antibodies and TCRs.Amphioxus also expresses a family of Ig-based transmembrane receptors termed V and C domain-bearing proteins (VCPs), whose ectodomains consist of one predicted V-type and one predictedC-type domain (52, 87). Both VCP domains display significant polymorphism at the populationlevel. In mollusks, fibrinogen-related proteins (FREPs) encode extraordinarily diversified Ig super-family domains that may mediate immune recognition (88). Drosophila can potentially express morethan 18,000 isoforms of the receptor Dscam (Down syndrome cell adhesion molecule) (84), whichcomprises multiple I-type Ig domains (57) that have been implicated in binding to bacteria (84). Foreach of these innate immune systems (VCBP, FREP, VCP, Dscam), extensive germline polymor-phism may compensate for the absence of somatic diversification in maintaining receptor diversity.

ADAPTIVE IMMUNE RECEPTORS OF JAWLESS VERTEBRATES

Adaptive immunity in jawless vertebrates is mediated by antigen receptors that are fundamentallydifferent from those of jawed vertebrates. Whereas antibodies and TCRs are composed of Igdomains, the VLRs of jawless fish consist of LRR modules (4, 61, 63). Indeed, VLRs are theonly known adaptive immune receptors to utilize a non-Ig scaffold. Of note, the LRR motif isalso present in many innate immune receptors, such as TLRs, NLRs, and plant disease resistanceproteins, which demonstrates its remarkable versatility in recognizing pathogens (60).

The use of two structurally unrelated scaffolds for the same purpose is not without precedentin immunology. For example, recognition of MHC class I molecules by natural killer (NK) cellsin primates is mediated by killer Ig-like receptors that belong to the Ig superfamily (59). Bycontrast, the functionally equivalent NK receptors in rodents are C-type lectin-like moleculesknown as Ly49s. Genetic evidence suggests that a common ancestor of primates and rodentspossessed precursor genes for both Ig-like and C-type lectin-like NK receptors but that thesespecies adopted different structural solutions for MHC class I recognition (34, 86). An analogousscenario may be envisaged for the evolution of Ig- and LRR-based adaptive immune receptors inthe ancestors of jawed and jawless vertebrates (10, 24).

Like other LRR proteins, notably TLRs (4), VLRs are constructed of tandem 24-residue LRRmodules with the consensus sequence XLXXLXXLXLXXNXLXXLPXXXFX (44). Each LRRmodule contains a β-strand–turn–α-helix motif, and the assembled VLR adopts a solenoid shapewith an interior parallel β-sheet on the concave surface and an exterior array of α-helices onthe convex surface (17, 30, 44, 46, 83) (Figure 4a–d ). One face of the β-sheet and one side of

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a b

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Figure 4Structures of VLR–antigen complexes. (a) Ribbon diagram of the VLRB.2D–HEL complex (3G3A) (83) showing the concaveantigen-binding surface of the VLR solenoid: LRRNT; LRR1, LRRV1, and LRRVe; CP; LRRCT; HEL. (b) VLRB.RBC36–H-trisaccharide complex (3E6J) (30). (c) VLR4–BclA complex (3TWI) (46). (d ) VLRA.R21–HEL complex (3M18) (17). (e) TLR4–MD-2complex (2Z65) (45). MD-2 makes no contacts with LRRCT of TLR4. Abbreviations: CP, connecting peptide; HEL, hen egg whitelysozyme; LRR, leucine-rich repeat; TLR, T cell receptor; VLR, variable lymphocyte receptor.

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the α-helix array are exposed to solvent and are dominated by hydrophilic residues. The regionbetween the helices and sheets constitutes the hydrophobic core of the VLR and is tightly packedwith leucine residues. The hydrophobic core is capped by disulfide-containing N- and C-terminalLRR modules. As discussed below, VLRs engage antigens through the concave surface formed bythe parallel β-sheet that contains most of the variable residues.

There are three types of VLR, denoted A, B, and C, which are clonally expressed by dis-tinct lymphocyte lineages as membrane-bound proteins (10). All VLRs contain an N-terminalLRR capping module (LRRNT), a variable number of LRR modules, and a C-terminal LRRcapping module (LRRCT) (4, 43, 61, 63) (Figure 2b). The LRR modules are subdivided intothe first LRR (LRR1), up to nine variable LRRs (LRRVs), a variable end LRR (LRRVe), anda truncated LRR designated the connecting peptide (CP) (Figure 4a–d ). VLRs also contain aC-terminal stalk region rich in threonine and proline residues (Figure 2b). The stalk includesa glycosylphosphatidylinositol (GPI) membrane-anchorage motif for tethering VLRBs to thelymphocyte membrane via a cleavable GPI linkage. Secreted VLRBs are assembled into disulfide-linked multimers that functionally resemble antibodies (33). By contrast, VLRAs and VLRCs, likeTCRs, are transmembrane proteins that are not secreted (10, 29).

ASSEMBLY AND DIVERSIFICATION OF VLR GENES

Although VLRs are structurally unrelated to antibodies and TCRs, they too are generated inlymphocytes from incomplete genomic elements by DNA recombination (Figure 2b). However,VLRs use different mechanisms to achieve somatic diversification. Unlike RAG-mediated re-combination of antibody and TCR genes, VLR genes are assembled by a gene conversion–likemechanism that appears to be mediated by cytosine deaminases of the AID-APOBEC family (58,70). Two lineage-specific cytosine deaminases have been implicated in VLR gene assembly (70):CDA1 for VLRA (and possibly VLRC) assembly and CDA2 for VLRB assembly (6, 29).

Germline VLRA, -B, and -C genes do not encode functional proteins but only portions ofthe N- and C-termini of the mature VLRs. Flanking each germline gene are several hundredcassettes encoding LRRNT, LRRV, LRRVe, CP, and LRRCT modules (Figure 2b). Duringlymphocyte development, multiple LRR cassettes are sequentially incorporated into the germlinegene framework to form a mature VLR. Because VLR assembly occurs monoallelically, eachlymphocyte expresses a single functional VLR. Similar to antibody and TCR genes, VLR genesare believed to have evolved from an uninterrupted predecessor gene, with parts of this geneexcised and translocated to positions near the original gene, such that gene conversion couldassemble these parts into complete VLR genes (9, 70).

The combinatorial process of VLR assembly and diversification can generate a vast repertoireof receptors, estimated at 1014–1017 unique VLRs, which is entirely comparable to the potentialdiversity of antibodies and TCRs (70). Hence, VLRs should be sufficiently diverse to recognizemost, if not all, potential pathogens.

VLRA AND VLRB LYMPHOCYTES

In lampreys, VLRAs and VLRBs are expressed by mutually exclusive lymphocyte populations(4, 61, 63). Whereas VLRB lymphocytes resemble the B cells of jawed vertebrates, VLRA lym-phocytes are surprisingly similar to T cells (29). Thus, VLRB lymphocytes respond to antigensby proliferating and differentiating into plasmacytes that secrete multimeric VLRBs specific forprotein, carbohydrate, or other epitopes. By contrast, VLRA lymphocytes do not secrete theirreceptors following antigen activation. Like TCRs, VLRAs are expressed exclusively as trans-membrane proteins (29). As observed for B and T cells, VLRB and VLRA lymphocytes develop

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HEL: hen egg whitelysozyme

at anatomically distinct sites in the lamprey (6). VLRB gene assembly occurs in a hematopoietictissue known as the typhlosole, which may be equivalent to the bone marrow of jawed vertebrates,where B cells develop. By contrast, VLRA gene assembly is restricted to lymphoepithelial struc-tures termed thymoids located at the tips of the gill filaments, which may fulfill the role of thethymus in T cell development (6).

Furthermore, a number of genes selectively expressed by VLRA lymphocytes are orthologs ofgenes typically expressed by T cells (29). Remarkably, VLRA lymphocytes express IL-8 receptorand IL-17, whereas VLRB lymphocytes express IL-8 and IL-17 receptor. This suggests that VLRAcells secreting IL-17 may attract VLRB cells bearing IL-17 receptor in a manner resembling theirT cell counterparts. Conversely, VLRB cells may utilize IL-8 to attract and engage VLRA cellsexpressing IL-8 receptor. The discovery of T- and B-like lymphocytes in the lamprey suggeststhat adaptive immunity is compartmentalized into cellular and humoral responses in both jawedand jawless vertebrates (6, 29). In addition, because both jawed and jawless vertebrates possess B-and T-like cells, it has been proposed that the extinct common ancestor of these sister vertebrategroups also had these two lymphocyte lineages (9).

ANTIGEN RECOGNITION BY VLRBs

Lampreys have been shown to respond vigorously to immunization with bacteria and bacterialantigens (4, 29, 33). Several weeks following injection of Bacillus anthracis spores, a particulateantigen, lamprey plasma accumulated VLRBs that reacted specifically with the spores and theirBclA glycoprotein component (4, 33). BclA-binding VLRB cells underwent proliferation anddifferentiation into plasmacytes. Other experiments demonstrated proliferation of specific VLRBlymphocytes and secretion of multimeric VLRBs after immunization with human erythrocytes,Salmonella typhimurium, Escherichia coli, and Streptococcus pneumoniae (29). Surprisingly, no responsewas detected after inoculation with soluble antigens, which suggests that the lamprey immunesystem is biased toward repetitive epitopes displayed by particulate antigens (3). In another study,however, high-titer plasma VLRB responses were reported in lampreys immunized with henegg white lysozyme (HEL) and β-galactosidase, demonstrating adaptive immune responses tosoluble antigens (78). Moreover, VLRBs are highly specific, as demonstrated by their ability todiscriminate between very closely related carbohydrate antigens (36).

Although affinity measurements have been carried out for only a few VLRBs, it appears thatthese adaptive immune receptors bind antigens with relatively low (micromolar) affinities. Thus,an anti-HEL VLRB (VLRB.2D) bound its antigen with KD = 0.43 μM (83) and an antianthraxVLRB (VLR4) bound BclA with KD = 2.6 μM (46), as measured using recombinant monomericforms of the proteins. These affinities resemble those of IgM antibodies and are considerablyweaker than the nanomolar affinities of IgG antibodies that have undergone affinity maturation.However, similar to IgMs, VLRBs exist only as multimers in lamprey serum. These oligomersare composed of four or five disulfide-linked VLRB dimers, reminiscent of IgM pentamers (33).Notably, the natural multivalent form of VLR4 was found to bind BclA with high avidity. Hence,VLRBs, like IgMs, appear to overcome their weak monomeric affinities for antigen by forminghigh-avidity multimers.

Whether VLRBs undergo somatic hypermutation to generate high-affinity binders is at presentunknown. In jawed vertebrates, somatic hypermutation is mediated by the AID-APOBEC cyto-sine deaminase AID (20). AID is typically expressed in B cells following antigen encounter, andits mutagenic activity is restricted to Ig loci. Intriguingly, VLRB lymphocytes express CDA2, anAID-APOBEC cytosine deaminase that could theoretically contribute to VLRB diversification(70).

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ANTIGEN RECOGNITION BY VLRAs

In sharp contrast to VLRBs, VLRAs are expressed solely as cell-surface proteins and are notsecreted or released following antigenic stimulation (29). Although both VLRB and VLRAlymphocytes responded to immunization with B. anthracis spores with comparable levels ofproliferation, VLRA lymphocytes, unlike VLRB lymphocytes, were unable to bind anthraxspores or to secrete VLRA proteins that bound this antigen. Similar results were obtained afterimmunizing lampreys with S. typhimurium, E. coli, and Streptococcus pneumonia (29). A possibleexplanation for the inability of VLRA lymphocytes to bind immunogens that induced theirproliferation is that VLRA lymphocytes only recognize antigens in processed form, in a manneranalogous to the T cells of jawed vertebrates. Although lampreys lack genes that encode bonafide MHC molecules (55, 77, 81), alternative mechanisms for self/nonself discrimination hasbeen described (56), including a highly polymorphic histocompatibility gene locus ( fuhc) inthe basal chordate Botryllus schlosseri that encodes transmembrane proteins comprising multipleextracellular Ig and EGF domains (19). Indeed, given the complete structural dissimilarity ofVLRAs and TCRs (see below), there is no a priori reason to expect the antigen-presentingmolecules (if any) of lampreys to bear any structural resemblance to conventional MHCmolecules.

In another study, yeast surface display was used to access the VLRA repertoire of an HEL-immunized lamprey (78). Thirteen closely related clones that appeared to derive from a singlelymphocyte precursor were isolated. However, it is unknown whether these VLRA variants aroseas a result of antigen-driven affinity maturation, as in all jawed vertebrates from shark to man(24), or as part of the primary repertoire, as in rabbits and sheep (8, 67). Time-resolved analysis ofVLRA sequences during an immune response will be required to settle this question. Remarkably,these anti-HEL VLRAs exhibited KDs in the nano- to picomolar range (26 nM–270 pM) (78). Suchaffinities approach the 100-pM affinity ceiling of IgG antibodies produced during mammalianimmune responses (7) and far exceed the micromolar affinities of αβ TCRs for peptide–MHCligands (82). Thus, at least some VLRAs can recognize antigens directly, without a requirementfor processing or assistance by specialized antigen-presenting molecules. In this respect, suchVLRs resemble antibodies and γδ TCRs, which, unlike αβ TCRs, recognize antigens directly(47).

Resolving the question of direct or indirect antigen recognition by VLRA lymphocytes willprobably require the development of conditions for culturing these cells in vitro in order to definetheir specific requirements for antigen stimulation. It should also be emphasized that these twopossibilities are not mutually exclusive, as exemplified by αβ and γδ TCRs. No information ispresently available on antigen recognition by VLRCs. However, the finding that VLRCs arephylogenetically more closely related to VLRAs than to VLRBs suggests that VLRC lymphocytesmay functionally resemble T cells more than B cells (43).

STRUCTURAL BASIS FOR ANTIGEN RECOGNITIONBY VLRs

Several structures of VLRs in unbound form or bound to carbohydrate or protein antigens havenow been reported: (a) three unbound hagfish VLRs (44); (b) the complex between a lampreyVLRB (VLRB.RBC36) and H-antigen trisaccharide (30); (c) the complex between a lampreyVLRB (VLRB.2D) and HEL (83); (d ) the complex between a lamprey VLRB (VLR4) and BclA,the immunodominant glycoprotein of B. anthracis spores (64); and (e) the complex between a lam-prey VLRA (VLRA.R2.1) and HEL (17). VLRB.RBC36 and VLR4 were isolated from lampreys

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GpIbα: plateletreceptor glycoproteinIbα

immunized with human O erythrocytes and B. anthracis spores, respectively, using a mammalianexpression system in which HEK293 cells were transfected with VLR cDNAs from the lympho-cytes of immunized lampreys (30, 33). VLRB.2D and VLRA.R21 were derived from lampreysimmunized with HEL using yeast display technology, in which VLR libraries generated fromlymphocyte cDNA of immunized lampreys were displayed on the yeast surface and screened byflow cytometry for HEL binders (78).

Collectively, these studies have begun to reveal the structural features that endow VLRs withspecificity and affinity for diverse antigens, with the important caveat that the structural database forVLRs is far smaller than that for antibodies or TCRs, for which hundreds of structures have beendetermined. We first discuss antigen recognition by VLRBs, which are expressed on lymphocytesthat resemble the B cells of jawed vertebrates, followed by a discussion of VLRAs, which are foundon the T cell–like lymphocytes of hagfish and lampreys.

Structure of VLRB–Antigen Complexes

In the VLRB.2D–HEL complex, the lamprey VLRB binds HEL through its concave surface,which is composed of six parallel β-strands (two from LRRNT, three from LRRVs, and one fromCP) (83) (Figure 4a). The convex surface, which does not contact antigen, is formed by four 310

helices, one α-helix (in LRRCT), and several connecting loops. This binding mode resemblesthat between other LRR family members and protein ligands, such as the interaction of TLR4with MD-2 (45) (Figure 4e).

The LRRCT module contains a distinctive, and highly variable, insert following its α-helixthat is uniquely shared with the platelet glycoprotein receptor Ibα (GpIbα) but is absent from theLRRCTs of other LRR-containing proteins, including TLRs (70). On this basis, it was proposedthat VLRs and the pan-vertebrate GpIbα originated from an ancestral platelet glycoprotein re-ceptor that was adapted for hemostatic function. A duplication of GpIbα in agnathans may havegenerated the ancestral VLR, which was recruited as an antigen receptor. Indeed, the VLRB.2D–HEL and VLR4–BclA complexes (46, 83) are reminiscent of the interaction of GpIbα with itsprotein ligand, von Willebrand factor (VWF) (39) (Figure 5). However, HEL and BclA are shiftedtoward LRRCT and do not contact LRRNT, whereas VWF engages both the N- and C-terminalcapping modules of GpIbα.

The VLRB.2D–HEL and VLR4–BclA complexes bury total surface areas of ∼1,500 A2, similarto the surfaces buried in complexes between Ig-based antibodies and protein antigens (1,400–2,300 A2) (76). The VLRB.RBC36–H-trisaccharide complex buries considerably less surface areathan does either VLRB–protein complex (∼700 A2), due to the smaller size of the trisaccharideligand. In all three complexes, the antigen interacts with LRR1, the LRRVs, CP, and LRRCT(Figure 4a–c). Overall, however, antigen recognition by VLRs is focused on the C-terminalLRRs, which probably reflects the importance of the LRRCT module, with its distinctive insert,for binding and specificity.

In the VLRB.RBC36–H-trisaccharide complex, the oligosaccharide is lodged in a cleft betweenthe concave surface of the VLR and the variable insert in LRRCT (30) (Figure 4b). This pocketappears to be functionally equivalent to the cleft between the CDR3 loops of the L and H chains ofVLVH antibodies, where small ligands typically bind (76). VLRB.2B binds directly over the catalyticsite of HEL, with the LRRCT insert loop penetrating deep into the carbohydrate-binding cleft(83) (Figure 6a). In the VLR4–BclA complex, the LRRCT insert lies across a shallow groove onthe antigen (46). The different interaction modes exhibited by anticarbohydrate and antiproteinVLRBs underscore the versatility of LRRCT inserts in mediating specific contacts with structurallyunrelated ligands.

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Gplbα VLRB.2D

VWF-A1

HEL

Figure 5Comparison of ligand recognition by VLRs and platelet glycoprotein receptor Ibα. Superposition of theVLRB.2D–HEL and GpIbα–VWF (1M10) (39) complexes is shown. VLRB.2D, GpIbα, and the VWF-A1domain are depicted as α-carbon traces, and HEL is shown as a surface representation in gray. Abbreviations:HEL, hen egg white lysozyme; VLR, variable lymphocyte receptor; VWF, von Willebrand factor.

Remarkably, the HEL epitope targeted by VLRB.2B almost totally overlaps those recognizedby camel antibody cAb-Lys3 (16) and shark antibody PBLA8 IgNAR (75), which contain only asingle VH domain (Figure 6a–c). In the cAb-Lys3–HEL and PBLA8 IgNAR–HEL complexes,VH CDR3 projects directly into the active site cleft in a manner analogous to the LRRCT insert.However, the VLRB.2D epitope is completely distinct from those recognized by mouse VLVH

antibodies, which bind to flatter surfaces on HEL (14) (Figure 7a).

Structure of a VLRA–Antigen Complex

In the VLRA.R21–HEL complex (17), the VLRA binds antigen through its concave surface,composed of nine parallel β-strands, with additional contacts mediated by a protruding loop ofLRRCT (Figure 4d ), as in the case of VLRBs. The 2,400-fold higher affinity of VLRA.R21 thanVLRB.2D for HEL (KD = 0.43 μM versus 180 pM) is at least in part explained by the considerablygreater shape complementarity of the VLRA.R21–HEL than of the VLRB.2D–HEL interface,based on shape correlation statistics (Sc) of 0.76 and 0.67, respectively (Sc = 1.0 for interfaceswith geometrically perfect fits) (17). Indeed, an Sc of 0.76 is at the upper end of the range forprotein–protein, including antigen–antibody, complexes (76).

VLRA.R21 recognizes a flat epitope on HEL, with the highly variable LRRCT insert packingagainst one side of the antigen (Figure 4d ) in a manner resembling the VLR4–BclA interaction(Figure 4c). By contrast, VLRB.2D targets a concave epitope on HEL, with its LRRCT insertprojecting into this cleft (Figure 6b). These two epitopes are effectively distinct, despite someoverlap (Figure 7b). The VLRA.R21 epitope is also distinct from those recognized by all anti-HEL

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VLRB.2D

HEL

cAb-Lys3

HEL HEL

PBLA8 IgNAR8 IgNARBLA8 IP

a b c

LRRNTCP

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LRRV1 LRRVe

Figure 6Comparison of HEL recognition by VLRB.2D and antibodies. (a) VLRB.2D–HEL complex oriented to highlight the interactionbetween the LRRCT insert and the active site cleft of HEL. (b) Structure of the complex between the camel single-domain VHantibody cAb-Lys3 and HEL (1MEL) (16). (c) Structure of the complex between the shark single-domain VH antibody PBLA8 IgNARand HEL (1T6V) (75). Abbreviations: HEL, hen egg white lysozyme; LRR, leucine-rich repeat; VLR, variable lymphocyte receptor.

ccAb-Ly3Ab-Ly3

VLRA

VLRB

D1.3

HyHEL-10

HyHEL-5

cAb-Ly3

a b

VLRA andVLRA andVLRBVLRB

VLRA andVLRB

Figure 7Antigenic sites on hen egg white lysozyme (HEL) recognized by antibodies and variable lymphocyte receptors (VLRs). (a) Antibodyepitopes on HEL. Epitopes for mouse antibodies D1.3, HyHEL-5, and HyHEL-10 (14) are depicted. The epitope for camel antibodycAb-Ly3 (16), which coincides with the epitope recognized by VLRB.2D (Figure 6a,b), is depicted in the center. (b) Comparison ofVLRA and VLRB epitopes on HEL. The orientation of HEL is the same as in panel a. Residues that interact exclusively withVLRA.R2.1 in the VLRA.R2.1–HEL complex are depicted along with residues that interact exclusively with VLRB.2D in theVLRB.2D–HEL complex, and residues forming contacts with HEL in both complexes.

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antibodies characterized to date from mouse, camel, and shark (14, 16, 75) (Figure 7a), whichimplies significant differences in the antigenic structure of proteins in jawed and jawless vertebrates.It is unknown at present whether the direct antigen binding manifested by VLRA.R21 is a generalfeature of VLRAs or whether VLRAs more typically recognize antigens only in processed form,as implied by the failure of VLRA lymphocytes to bind anthrax spores following immunization oflamprey larvae (29) (see above).

Architecture of the VLR Antigen-Binding Site

The VLR–antigen complexes described above have enabled us to understand the enormoussequence diversity of VLRs in terms of the molecular architecture of their binding sites, aswas achieved for antibodies and TCRs beginning in the 1970s. Both VLRBs and VLRAs(and most likely VLRCs) engage ligands through their concave face and distinctive LRRCTinsert (Figure 4a–d ). The antigen-binding concave face is composed of three parallel ridges(designated R1, R2, and R3) associated with the beginning, middle, and end, respectively, ofthe β-strand of the LRR modules (Figure 8a–d ). Each β-strand contains a six-residue motif,X1L(I)X3LX5X6, in which leucine or isoleucine at positions 2 and 4 compose the hydrophobiccore of the VLR solenoid and the remaining four residues form the three ridges on its concavesurface that engage the antigen: R1 (position 1), R2 (position 3), and R3 (positions 5 and 6).Importantly, all antigen-contacting positions within these ridges are characterized by highsequence variability, as discussed below. Moreover, the distribution of contacting residues onthe ridges themselves is highly variable, depending on the nature and positioning of the ligand(Figure 8a–d ). Involvement can range from nearly full engagement, as for R2 of VLRB.2Dbound to HEL (Figure 8a), to no contacts at all, as for R1 of VLRB.RBC36 bound toH-antigen trisaccharide (Figure 8b). Such differential engagement is reminiscent of antibodiesand TCRs, which bind ligands using all six CDR loops or only subsets of these loops (76)(Figure 1).

Sequence Variability of VLRs

For both VLRBs and VLRAs, sequence comparisons combined with structural information haverevealed a nearly perfect match between antigen-contacting positions and positions with the high-est sequence diversity (17, 83). In these analyses, diversity was measured by Shannon entropy peraligned position for LRR modules from 588 lamprey VLRB sequences and 208 lamprey VLRAsequences (Figure 9). For both types of VLR, the most variable positions in the LRRs (relativeentropy > 2.0) are distributed almost exclusively along the three parallel ridges on the concave faceof the VLRs (Figure 9a–j) or are located in the LRRCT inset (Figure 9k,l). In general, the fourhighest-entropy positions in the LRR1, LRRV, LRRVe, and CP modules correspond to residuesforming the three ridges: position 1 in the β-strand segment X1L(I)X3LX5X6 (R1), position 3(R2), and positions 5 and 6 (R3) (Figure 8a). These highest-entropy positions also account for themajority of ligand-contacting residues in the four available VLR–antigen structures. The remain-ing ligand-contacting residues mostly coincide with high-entropy positions in LRRCT inserts(Figure 9k,l). This striking correlation between high-entropy and antigen-contacting positionsstrongly suggests that these positions represent the generalized binding interface utilized by allVLRs, including VLRCs.

At the same time, structure-based sequence comparisons of VLRAs, VLRBs, and VLRCs haverevealed clear differences among these three types of VLR that suggest possible specializationin terms of antigen recognition. The average number of LRRV modules is greater in VLAs

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RR1

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Figure 8Molecular architecture of the antigen-binding site of variable lymphocyte receptors (VLRs). (a) Surface representation of hen egg whitelysozyme (HEL)-contacting residues in the R1, R2, and R3 ridges of VLRB.2D (3G3A) (83). The side chains of additional residues inR1–3 that could potentially contact antigen are shown in stick representation. (b) Surface representation of H-trisaccharide–contactingresidues in R2 and R3 of VLRB.RBC36 (3E6J) (30). The trisaccharide does not interact with R1. (c) Surface representation ofBclA-contacting residues in R2 and R3 of VLR4 (3TWI) (46). (d ) Surface representation of HEL-contacting residues in the R1, R2,and R3 ridges of VLRA.R2.1 (3M18) (17).

and VLRCs than in VLRBs, indicating a potentially larger antigen-binding surface. In bothlampreys and hagfish, VLRAs display much less variation in the length of the LRRCT insertthan do VLRBs (83). This difference recalls the constrained length distribution of the CDR3sof αβ TCRs compared with those of antibody TCRs (69). The average length of the LRRCTinsert in VLRCs is much less than in VLRAs or VLRBs, and sequence variation in the LRRCTmodule of VLRCs is substantially lower than in LRRCT of the other two VLR types (43).As shown in Figure 9, the first two LRRs of VLRAs (LRRNT and LRR1, but especiallyLRRNT) are characterized by a relative paucity of high-entropy positions: a total of three inVLRAs compared with eight in VLRBs. The far lower sequence diversity of LRRNT in VLRAsthan in VLRBs could imply the existence of conserved interactions between LRRNT andaccessory molecules that may be uniquely required for activating T cell–like VLRA lymphocytes.Such molecules could include (but are not limited to) functional equivalents of the CD4 andCD8 coreceptors on T cells or convergent analogs of MHC molecules on antigen-presentingcells.

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A C P S Q C S C S G T T V D C S G K S L A S V P T G0

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COMPARISON OF ANTIGEN RECOGNITION BY VLRS,ANTIBODIES, AND TCRs

It is now apparent that VLRs bind their cognate antigens with affinities and specificities alto-gether comparable to those of Ig-based antibodies and TCRs from jawed vertebrates. Althoughthe structural features that endow VLRs with these properties are less well understood than forantibodies or TCRs, several salient characteristics of antigen recognition by LRR-based adaptiveimmune receptors have been brought to light.

X-ray crystallographic studies of numerous antigen–antibody complexes have shown that cleftson protein surfaces are generally avoided by VLVH antibodies (16, 76). By contrast, antibodiesfrom camelids and sharks, which contain only a single VH domain, preferentially target clefts(16, 75). The striking proclivity of VLVH antibodies for planar epitopes may be explained by theirrelatively planar binding sites, which are also characteristic of αβ TCRs that bind peptide–MHCcomplexes, whereas the convex shape of the binding sites of VH antibodies, with their protrudingCDR3s, favors recognition of concave epitopes. In this respect, the interaction of VLRB.2D withHEL is highly reminiscent of the way camel and shark VH antibodies bind this same antigen(Figure 6).

However, VLRs are clearly not restricted to targeting clefts, as demonstrated by the VLR4–BclA and VLRA.R21–HEL complexes (17, 46), in which the LRRCT inserts are oriented soas to flank the ligand, rather than point directly toward it, as in the VLRB.2D–HEL complex.Moreover, LRRCT inserts display a broad length distribution, ranging from 0–13 residues (83),such that VLRs with short LRRCT inserts, in particular VLRCs (43), are unlikely to contain longprotruding loops that could hinder recognition of planar epitopes. It should also be noted that theantigen-binding site of VLR solenoids comprises a large concave surface composed of as many as 12parallel β-strands, in addition to the LRRCT insert. This surface could potentially engage antigensindependently of LRRCT, as suggested by other complexes involving LRR family members,such as TLR4–MD-2 (Figure 4e) and Listeria internalin–E-cadherin (45, 74). These LRR-basedreceptors recognize planar or convex surfaces on proteins using only their concave faces, with noparticipation by the C-terminal capping modules. Thus, the VLRs of jawless vertebrates appearto have evolved to bind as topologically diverse an array of epitopes as VLVH and VH antibodiescombined but do so using only one basic structure, a monomeric LRR solenoid.

Conformational changes in the CDR loops of antibodies and TCRs have been shown to con-tribute to antigen recognition (5, 41, 76). Due to its structural rigidity, the concave surface ofVLRs probably does not undergo conformational rearrangements other than side chain move-ments. However, the LRRCT insert of VLRA.R21, which was well defined in the VLRA.R21–HEL structure, was almost completely disordered in the structure of the unbound VLR, suggestingconformational flexibility (17). The apparent mobility of the LRRCT insert suggests a mechanism

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 9Sequence variability and antigen-contacting positions of VLRBs and VLRAs. (a–e, k) Sequence diversity plots for VLRBs. Shannonentropy per aligned position is shown separately for LRRNT, LRR1, LRRVs, LRRVe, CP, and LRRCT. Entropy was calculated from588 lamprey VLRB sequences using the Shannon entropy formula, H = −∑M

i=1 Pi log2 Pi , where Pi is the fraction of a given aminoacid i and M the total number of different amino acids (83). Entropy bars corresponding to ridges R1, R2, and R3 are depicted as inFigure 8. All other bars are depicted in gray. Dots above the entropy bars mark the antigen-contacting positions of VLRB.2D,VLRB.RBC36, and VLR4. The VLRB.2D sequence is shown below the plots as a reference. ( f–j,l ) Sequence diversity plots forVLRAs. Shannon entropy per aligned position is shown for LRRNT, LRR1, LRRVs, LRRVe, CP, and LRRCT. Entropy wascalculated from 208 lamprey VLRA sequences. HEL-contacting positions of VLRA.R2.1 are marked by dots above the entropy bars.The sequence shown below is that of VLRA.R21. Abbreviations: CP, connecting peptide; HEL, hen egg white lysozyme;LRR, leucine-rich repeat; LRRV, variable LRR; LRRVe, variable end LRR; VLR, variable lymphocyte receptor.

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for VLR binding based on conformational selection from a dynamic equilibrium of preexistingisomers, as proposed for antibodies and TCRs (5, 41). According to this mechanism, the intrin-sically mobile LRRCT insert samples multiple conformations, only one of which is competentto bind a specific ligand. Such conformational diversity may enable a single receptor to engagemultiple ligands, thereby expanding the effective size of the VLR repertoire.

FUTURE ISSUES

The genetic and structural characterization of immune receptors from phylogenetically disparatespecies, such as amphioxus and lamprey, has provided unique insights into the evolution of adaptiveimmunity that could not possibly have been gained by studying mice and humans alone, whichare the traditional vertebrate models of immunologists. At the same time, studies of alternativemodels have raised fundamental questions regarding self-reactivity and antigen presentation invertebrates.

Somatic diversification associated with adaptive immunity carries the serious risk of generatingself-reactive antigen receptors that may induce deleterious autoimmune reactions. However, thesurvival advantage of a diverse antigen receptor repertoire must have outweighed the risk of self-reactivity as somatic diversification is maintained throughout vertebrate evolution. To deal with theproblem of self-reactivity, jawed vertebrates have evolved elaborate mechanisms for distinguishingbetween self and nonself. For example, in mammals and presumably other jawed vertebrates, Tcells recognizing self-peptides presented by MHC molecules in the thymus are eliminated duringdevelopment if they interact strongly with self-peptide–MHC complexes (negative selection) (27).Since some VLRs are expected to be self-reactive, jawless fish presumably also evolved mechanismsfor self/nonself discrimination, although these mechanisms, as well as the molecules that mediatethem, remain to be discovered.

In jawed vertebrates, the protective function of T cells depends on their ability to recog-nize host cells harboring viruses or bacteria. T cells accomplish this by using αβ TCRs to rec-ognize pathogen-derived peptides bound to MHC molecules on the surface of infected cells(Figure 1b). These peptides are generated by proteolytic degradation, or processing, of nativeproteins from intracellular microbes for presentation to T cells. Whether the T cell–like VLRAand/or VLRC lymphocytes of lampreys and hagfish detect intracellular microbes through ananalogous antigen presentation mechanism is at present unknown, as so far only direct binding ofVLRAs to unprocessed antigen has been demonstrated (17, 78). Although lampreys lack MHC orMHC-like molecules (55, 77, 81), alternative mechanisms for antigen presentation, mediated byconvergent analogs of MHC molecules, are entirely possible. The identification of these putativepresenting elements would further underscore the amazing parallels that are now being discoveredin the independently evolved adaptive immune systems of jawed and jawless vertebrates.

SUMMARY POINTS

1. Adaptive immunity in jawless vertebrates (lampreys and hagfish) is mediated by highlydiverse rearranging VLRs that are fundamentally different from the antigen receptors ofjawed vertebrates.

2. Whereas the antibodies and TCRs of jawed vertebrates are composed of Ig domains,VLRs consist of multiple tandem LRR modules that form a horseshoe-shaped solenoidstructure characteristic of LRR proteins.

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3. The Ig-based VCBPs of the cephalochordate amphioxus display structural features thatmay reflect the evolutionary transition from nonrearranging innate pattern recognitionmolecules to the rearranging antibodies and TCRs of jawed vertebrates.

4. Lampreys possess three types of VLRs: VLRBs (similar to antibodies) are expressed onB cell–like lymphocytes and are secreted, whereas VLRAs and VLRCs (similar to TCRs)are expressed on T cell–like lymphocytes exclusively as transmembrane proteins.

5. VLRs bind antigens though their concave surface, in addition to a distinctive hypervari-able loop in the C-terminal LRR capping module.

6. For both VLRBs and VLRAs, there is a nearly perfect match between antigen-contactingpositions and positions with the highest sequence diversity.

7. VLRs bind as structurally diverse an array of antigens as antibodies or TCRs, withcomparable affinity and specificity.

8. The independent development of two antigen recognition systems in vertebrates based onentirely different protein scaffolds represents a striking example of convergent evolutionthat attests to the survival value of adaptive immunity.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Work in the laboratory of R.A.M. is supported by National Institutes of Health grants AI036900and AI073654. M.L. is supported by the Joint Supervision Ph.D. Project of the China ScholarshipCouncil.

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