Evolution of innate and adaptiveimmunity: can we draw a line?Martin F. Flajnik1,* and Louis Du Pasquier2

1Department of Microbiology and Immunology, University of Maryland at Baltimore School of Medicine, Baltimore,

MD 21201-1559, USA2Institute of Zoology, University of Basel, Rheinsprung 9, CH-4051, Basel, Switzerland

Several recent findings in the field of comparative

immunology have reinforced the importance of examin-

ing the molecular and functional features of immune

systems in a variety of organisms. Particularly exciting

are the discoveries of a new gene rearrangement

mechanism in lampreys and a somatic diversification

of mollusk immune genes. These immune features

being found in animals previously believed only to

have innate immunity, as well as the flood of infor-

mation on immune genes, molecules and mechanisms

in many different creatures, have prompted us to revisit

the artificial dichotomy between adaptive and innate

immune systems. Although we draw no startling

conclusions, we hope to encourage different thought

patterns when viewing immune systems.

With the remarkable discovery in the late 1990s thatmammalian Toll-like receptors (TLRs), homologues ofinsect Toll receptors, are important for both innate andadaptive immunity [1,2], the field of comparative immu-nology has gained new respect. This was the first time thatan immune pathway discovered outside mammals couldbe superimposed onto the textbook human immunesystem. Now, with the sequencing of entire genomesfrom several invertebrates (e.g. the fruitfly Drosophila,the nematode Caenorhabditis elegans, the tunicate Ciona,the mosquito Anopheles) and structure–function analysesof defense genes in a variety of animals, there has been arush to uncover new paradigms that might be transposedonto our general knowledge of immune system regulationand function [3]. For comparative immunologists, thesenew sequencing data have turned us into kids in a candyshop and have spawned new insights into the integrationof different immune mechanisms.

Origins of adaptive immunity in jawed vertebrate

The adaptive immune system, with its randomly gener-ated and vast antigen–receptor diversity, gives us thepotential to evade any invader. The human adaptiveimmune system is defined by lymphocytes, antigenreceptors [Ig and T-cell receptor (TCR)], MHC I and II,genes involved in rearrangement [e.g. recombination

Corresponding authors: Martin F. Flajnik (MFlajnik@som.umaryland.edu),Louis Du Pasquier (dupasquier@dial.eunet.ch).

* Martin F. Flajnik has been a member of the Trends in Immunology EditorialBoard since 1999.

Available online 14 October 2004

www.sciencedirect.com 1471-4906/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved

activating gene (RAG)], somatic hypermutation, geneconversion and specialized primary and secondary lym-phoid tissues. Over the last ten years, we have come to theconclusion that: (i) this system does not exist in livinganimals derived from ancestors older than the cartilagi-nous fish (e.g. sharks); and (ii) the sharks (and all jawedvertebrates) have every single one of the defining hall-marks described here (Figure 1). The only adaptive systemproperties lacking in sharks (and all bony fish) aregerminal centers and the Ig class switch [4]. The differentadaptive features were selected as distinct lineages ofgenes or mechanisms, some of which are obvious (e.g. thepirating and duplication of proteasome subunits andtransporters for class I antigen processing), and othersthat we can only speculate on.

The birth of the adaptive immune system is believed tohave occurred when an Ig superfamily (Igsf) gene of thevariable (V) type was invaded by a transposable elementcontaining RAG1 and RAG2 genes [5,6]. This innovativeevent, which greatly augmented receptor diversity (andheightened the risk of autoimmunity), must have beencrucial for the adaptive immune system, even if it were nottruly the initiating event [7] because all of the antigenreceptor loci rearrange in a similar fashion (see later). Thereare several invertebrate Igsf family members havingfeatures of Ig or TCR V genes, which could be related tothe ancestral gene invaded by the transposon [8–10].

An Igsf putative ancestral receptor, identified in theprotochordate Ciona (Figure 1), is a member of the CTX(cortical thymocyte marker for Xenopus) [junctionaladhesion molecule (JAM)] family and is structurally lesssimilar to an ideal Igsf ancestor (V-C2 rather than theV-C1; canonical C1 domains are rare, found in Ig, TCR andMHC proteins) but which is perhaps associated with viralresponses. Strikingly, all human homologs of the Ciona(and Amphioxus, another protochordate) Igsf members,with receptor features including an external Igsf domainand transmembrane or cytoplasmic regions (nectins,CD47, CTX/JAM, CD166L), segregate on paralogousregions (human chromosomes 3, 1, 11, 21), where theycluster with Igsf MHC-restricted T-cell associated mol-ecule (CRTAM, of which a homologue, Beat, exists inDrosophila) and with CD80 and CD86, co-stimulatoryreceptors with a V-C1-like architecture. The duplication ofthis ancient ‘minigene complex’ might have donated V-C1domains to antigen receptors and the MHC-linked

Opinion TRENDS in Immunology Vol.25 No.12 December 2004

. doi:10.1016/j.it.2004.10.001

TRENDS in Immunology

UROCHORDATES

HEMICHORDATES Acorn worms

ECHINODERMS

MOLLUSKS

ANNELIDS Earthworms hemolysins, lectins, NK-L, PPO

SIPUNCULIDS Peanut worms NK-L

NEMERTEANS Ribbon worms

ARTHROPODS

NEMATODES C. elegans (Igsf V-I-C2), lectins, PPO, AMP (Alt. Splice), PCD, PGRP?

CNIDARIA Corals C’/TEP, PPO, PCD

PORIFERA Sponges (Igsf V-I-C2), lectins, PPO

PROTOZOA

CEPHALOCHORDATES

Sea squirts LRR, lectins, C’/TEP,TNF, PPO, (Igsf V-I-C2-C1-L), NK-L, MHC synteny

Amphioxus C’/TEP, lectins, PPO, (Igsf V) Igsf VCBP?, MHC synteny

FliesMosquitoesShrimpButterflies

Sea urchins LRR, TLRs, lectins, C’/TEP, PPO, ([SCR, Alt. Splice]), MHC synteny, Rel, TIR

Snails PPO, lectins, AMP, Igsf V-I-C2, MDM, Igsf FREPs, som. DNA, Alt. Splice, Rel

JAWLESS VERTEBRATES Lamprey/hagfish

JAWED VERTEBRATES Sharks to mammals

LRR, lectins, C’/TEP, (IgsfVC), VLRs, som. DNA

PLATHYHELMINTHS Flatworms

LRR, Toll, Lectins, PPO, Igsf Hemolin, (Igsf V-I-C2), AMP, C‘/TEP,[PGRP, penaedins, Alt. Splice], MHC synteny, Rel, TIR

PLANTS LRR, TIR

LRR, TLR, lectins, C’, SCR, TNF, AMP, Igsf, NK,Igsf V-C1 Ig /TCR, RAG1-2, MHC I-II som. DNA, PCD, Rel, TIR

Deu

tero

stom

es

Bila

teria

Pro

tost

omes

Figure 1. Immunemolecules and mechanisms found throughout (primarily) the animal kingdom. Red indicates molecules or mechanisms involved in innate immunity. Blue

indicatesmolecules whose genes diversify somatically and are involved (or believed to be involved) in adaptive immunity. Orange indicates somaticmechanisms at the DNA

or RNA level to generate or maximize immune diversity (underlined taxa indicate that representatives in these groups have diversification mechanisms of immune genes).

Green indicates conserved signaling pathways. Black indicates that genes have been found in linkage groups similar to those in the jawed vertebrateMHC. Abbreviations and

short descriptions of each category are all found in Table 1. Details for each of thesemolecules andmechanisms can be found in Refs [2–4,7,9,10,14,19,21,23,24], which are all

recent reviews on the subject. Abbreviation: C. elegans, Caenorhabditis elegans.

Opinion TRENDS in Immunology Vol.25 No.12 December 2004 641

tapasin, as well as the C1 to MHC I and II, as has beenspeculated [9]. Interestingly, V domains of most membersof this group act as virus-binding receptors: the binding ofvirus to V-JAM triggers apoptosis, a form of local immun-ity. In primitive animals, such as corals, ‘induced suicide’with sacrifice of part of the colony occurs after allorecogni-tion [11]. We know little about antiviral responses outsideof mammals, especially in animals lacking the jawed-vertebrate adaptive immune system. However, viruses areindeed abundant in seawater [12] and probably imposed astrong selection on the immune system, perhaps promot-ing the development of adaptive immunity.

Regarding the origins of the MHC, there has been nosign of MHC I or II genes in animals older than thecartilaginous fish. This is rather unexpected because wehad hoped to find such molecules in the genome sequenc-ing projects from Ciona [8] or other related deuterostomes(Figure 1). However, genes in the jawed-vertebrateMHC III region, including C3/4, factor B/C2 and tumornecrosis factor (TNF) family members, were detected in the

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Ciona and Amphioxus genomes, some of them geneticallylinked [13]. In Amphioxus, a forerunner of the MHC wasfound in a single copy (pre-duplication),whereas in all of thejawed vertebrates examined so far there are severalduplicated ‘MHC’ regions, consistent with large-scale(even whole genome) duplications at vertebrate origins[14]. One speculative question is whether there was a‘pre-MHC’ before the emergence of class I and class IIgenes and before the early en bloc duplications, whichmight have accommodated immune response genesinvolved in innate immunity.

Questions regarding the origins of the adaptive immunesystem will be further addressed by ongoing expressedsequence tag (EST) andgenomic sequencingprojects innon-jawed vertebrates and pre-vertebrates (lamprey, hagfish,tunicates, echinoderms). Already, other V genes withfeatures similar to Ig or the TCR and potentially related tothe ancestral Igsf gene invaded by the transposonhave beenisolated, although we know nothing of their functions orgenetic linkage groups [10,15].

Opinion TRENDS in Immunology Vol.25 No.12 December 2004642

Another big surprise: an invertebrate adaptive

immunity?

Comparative immunologists tried for many years to findIg, TCR, MHC and RAG in the oldest vertebrates, thejawless agnathans (lamprey and hagfish), to no avail.Even EST projects from lamprey lymphocyte-like cellsrevealed no member of this adaptive family, althoughseveral genes found are homologues of lymphocyte-specifichuman genes [16,17]. Expressed genes from immune-stimulated ‘lymphocytes’ were enriched for cDNAs withleucine-rich repeats (LRRs), which are found in many cell-surface molecules, including the famous TLRs [18]. Notonly were there many of these specific LRRs but they wereextremely diverse [prompting their revised name, variablelymphocyte receptors (VLRs)], which is seemingly a resultof a gene rearrangement mechanism independent of RAG.A single-copy locus is comprised of an incomplete VLRgene encoding the N- and C-terminal LRR domains.Functional VLR genes are presumably generated by theinsertion of LRR gene cassettes found 5 0 and 3 0 of theincomplete gene. Furthermore, similar to Ig and the TCRin jawed vertebrates, preliminary data suggest monotypicexpression of VLRs in each lymphocyte. The mechanism ofrearrangement, existence of the system in other speciesand potential role(s) in adaptive immunity are alluntouched areas, however, this is a spectacular result inthe annals of comparative immunology; this work sug-gests an independent acquisition of somatic diversity in amolecular defense module (LLR) involved in immunity inplants and animals [19].

Even if these VLRs turn out to be related to the jawed-vertebrate adaptive system, for example, the monotypicVLR expression in lamprey lymphocytes is somehow aforerunner to the mechanism of allelic exclusion in Ig orthe TCR. It is nevertheless likely that the advent of thejawed-vertebrate adaptive system resulted in an enor-mous immune system complexity over a short timespan.MHC processing and presenting genes, thymus andspleen, large numbers of cytokines, chemokines andhematopoietic cell-specific transcription factors and sixdifferent types of rearranging gene families that use thesame rearrangement mechanism seem to all emerge afterthe appearance of the jawed vertebrates. This immuneexplosion was, in part, probably due to the large-scalegene duplications mentioned earlier [14], although thenew RAG-mediated rearrangement mechanism to gener-ate diversity might indeed have been the innovative eventthat was generative, resulting in our current view ofadaptive immunity. By analogy, the innovative inventionof the car generated paved roads, oil refineries, rapidinnovations in the steel industry, rubber plantations andmany other industries [7]; we speculate that RAG droveour placoderm (primal, extinct jawed vertebrates) ances-tors into full-blown adaptive immunity.

The complexity of innate immunity

The seminal work by Janeway and Medzhitov [1] andFearon [20] in the late 1990s propelled a reawakeningof interest in the innate immune system. This work onthe TLR system and the role of complement in stimu-lating adaptive immunity were the wake-up calls that

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transformed mainstream immunology. These days, alongwith regulatory T cells, innate immunity, classically thegenetically pre-selected immune system, which providesour first line of defense, is all the rage. Thus, there is now aclear justification and it is politically correct for anyimmunologist to be interested in how fruitflies defendthemselves against pathogens!

Recent studies of innate immunity in various inverte-brates have provided much information on the types ofgene families involved in innate immunity (Figure 1 andTable 1; all of the molecules are reviewed in Ref. [3]). Onemajor message from these analyses, which is not at allsurprising in retrospect, is that invertebrate immunesystems are not simple and different organisms varygreatly in the levels of complexity in their forms of defense[21]. Recognition and effector molecules show diversityand multiplicity not only in their structure but also intheir mode of usage. However, as much as these have beenanalyzed, recognition molecules are often associated withconserved signaling cascades throughout phylogeny [2]. Asecond message is that, although genes from multiplefamilies are present throughout the animal kingdom(Table 1), similar to the jawed vertebrate immune genes,they evolve rapidly [22,23], both in their sequences and ingene expansion or contractions; this is apparent whencomparing the defense molecules of Drosophila and themosquito, both of which are insects [3]. A third message isthat, although we are waiting for the smoke to clear, itseems that the major gene families involved in defensearose early and thus the ‘raw material’ for selection to acton is found in all of the major animal lineages [3,10,23].Besides the TLRs, one fascinating area of study has beenthe thioester-containing proteins, members of the com-plement pathway in jawed vertebrates [24]. Recentevidence suggests that such molecules are found in allanimals and, although direct lineages to C3/4/5 have beendifficult to trace from the proteostome lineage in manycases, nevertheless the molecules [thioester-containingproteins (TEPs)] are involved in defense through at leastone of the textbook defense functions of complement,opsonization [25]. The capacity to make a covalent bondwith a pathogen is unique to members of this family; thisfeature has been well served and is clearly evolutionarilyconserved. There have been independent expansions ofthis gene family in different animals (or animal groups),reinforcing the relative immune importance of TEPs [24].

As more genomes are sequenced, we will be able toobserve how the major gene families have been exploitedfor innate immunity, and we think it will be especiallyexciting to examine the immune systems of invertebratesthat are long-lived or complex. We would expect suchcreatures to have exploited the ‘raw material’ in interest-ing ways, either quantitatively or qualitatively.

A big surprise in invertebrate adaptive immunity

In mollusks, secreted fibrinogen-related proteins (FREPs)bind to parasites [21]. These molecules, encoded by alarge number of genes falling into O10 families, haveN-terminal Igsf domains connected to fibrinogen domains.In a recent issue of Science [26], it was reported that theFREP genes somatically diversify, apparently through

Table 1. Immune defense molecules and functions found throughout the animal kingdoma

Molecule/mechanism Function (or example)

Leucine-rich repeat (LRR) Various (e.g. TLR, VLR)

Toll-like receptor (TLR) Signal innate and alert adaptive immunity

Variable lymphocyte receptor (VLR) Lamprey (potential adaptive) defense molecules

Pattern recognition receptor (PRR) Defense molecule recognizing conserved molecules on pathogens, or

pathogen-associated molecular patterns (PAMPs)

Lectins (Galectin, C-type or S-type lectin) Various (e.g. NK receptors, selectins)

Complement (C 0) Opsonization, lysis, inflammation

C3-Factor B/Thioester-containing protein (C 0/TEP) Opsonization

Scavenger receptor (SCR) Various (e.g. PRR)

Tumor necrosis factor (TNF) Proinflammatory cytokine

Antimicrobial peptides (AMP) Various (e.g. Defensins)

Immunoglobulin superfamily (Igsf) Various [e.g. Ig, fibrinogen-related protein (FREP) – see below]

V region chitin-binding protein (VCBP) Amphioxus Igsf member related to Ig/TCR

Natural Killer (NK) cell; NK-L (NK-like) Kill virally infected cells (Igsf and Lectin)

Prophenoloxidase (PPO) Invertebrate defense molecule

Fibrinogen-related proteins (FREPs) Mollusk (potential adaptive) defense molecules

Hemolysin Cell lysis

Penaedin Defense molecule

Mollusk defense molecule (MDM); Hemolin Igsf defense molecules

Variable (V); Constant-1 (C-1); Constant-2 (C2); Intermediate (I) Igsf domain types

Immunoglobulin (Ig) Humoral adaptive defense

T cell receptor (TCR) Adaptive defense

Recombination-activating gene (RAG 1-2) Enzyme required for VDJ rearrangement

MHC l or I/II Antigen (peptide) presentation

Somatic DNA modification (Som. DNA) VDJ gene rearrangement, hypermutation, or conversion

Alternative splicing (Alt. splice) Generation of multiple transcripts for one defense molecule

Programmed cell death (PCD) Induction of suicide or apoptosis in a target cell

Rel Conserved signaling pathway (e.g. NFkB)

Toll-interleukin 1 receptor domain (TIR) Conserved signaling pathwayaNote that not all of these features are found in all organisms.

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gene conversion and/or somatic hypermutation (as well asby alternative splicing). It is not clear whether the systemis indeed adaptive, however, we have come to associatesomatic mutations with acquired immunity. As with thelamprey work, many more questions were raised thanwere answered, although being shocked at this result wasprobably unjustified. We are so inured to the RAG[activation-induced cytidine deaminase (AID)]-diversifica-tion processes in the jawed vertebrates that perhaps wehave overlooked or given short shrift to other mechanismsof diversification, such as alternative splicing, expressionof constellations of genes at the single cell level anddiversity achieved by combinatorial association of proteinsor other mechanisms. In other words, perhaps we havebeen too draconian in how we categorize ‘adaptive’ and‘innate,’ and what we are learning from the invertebrates,as well as in the jawed vertebrates, might make us softenour fundamentalist views.

How do we decide what is innate and what is adaptive?

Every immunology student is taught that the jawed-vertebrate adaptive immune system was grafted onto theinnate system, co-opting pre-existing effector mechanismsof defense [27]. For example, antibodies slipped into theentrenched alternative or lectin-initiated complementpathway as well as the ancient opsonic pathways.‘Adaptive’ cytokines, such as interferon-g, stimulatephagocytes to upregulate very old intracellular pathwaysof defense against parasites. It has not been shown yet,however, the killing mechanisms used by cytotoxicT lymphocytes (CTLs) and natural killer (NK) cells areprobably ancient pathways associated with cells havingdifferent recognition systems [28,29]. Of course it works

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the other way round as well. Even before Janeway andFearon, we knew that the innate immune system alertedthe adaptive to invaders. Anaphylatoxins (C5a) generatedby the alternative complement pathway and proinflam-matory cytokines produced by many ‘innate’ cells arepotent stimulators of an inflammatory response thatpaves the way for adaptive immunity. Opsonic receptorson macrophages [now called pattern recognition receptors(PRRs)] have long been known to signal to macrophages toupregulate molecules that stimulate T cells.

Perhaps the innate–adaptive distinction should not bethought of in black-and-white terms [23]. Again, theschoolchild notion of adaptive immunity includes thefollowing features: great diversity of somatically gener-ated antigen receptors; capacity to generate a memoryresponse to a previously encountered antigen throughmemory lymphocytes, which have been clonally expanded;monotypic expression of receptors on cells, resulting inclonal selection and thus a delay in the response; andhighly evolved self-tolerance-induction mechanisms aris-ing as a penalty for generating such a diverse and randomantigen-receptor repertoire. This is a fine framework forviewing jawed-vertebrate adaptive immunity, however,there are gray areas that are not so well defined.

In mammals, clones of NK cells, which bear combin-ations of receptors from a pool of genes in the leukocyteIg-like receptor complex (LRC) and natural killer genecomplex (NKC), and which recognize self-MHC as wellas molecules of pathogens, do not easily fit into thePRR–pattern-associated molecular pattern (PAMP)innate paradigm [23]. Recent work in the invertebrateAmphioxus has suggested a high diversity of Igsf genes,combinations of which seem to be specific to each

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individual [30]. Throw in the molluscan FREPs for goodmeasure, as well as incredibly diverse (putative) defensemolecules in sea urchins (L.C. Smith, pers. commun.) andin bony fish [10], and we come to the conclusion that theview of the two major immune categories should bebroadened. What is not well understood, even for thewell-studied NK cells, is how tolerance is achieved in thesesystems. With such diverse receptor repertoires, whethersomatically generated or not, old-school adaptive immun-ologists like us would argue that there must be tolerancemechanisms at the cellular level to account for the greatdiversity. The other hallmarks of the jawed vertebrateadaptive responses – delay due to clonal selection andmemory – will probably depend on the system examined.Even some jawed vertebrates with the capacity to haveefficient immune systems do not optimally exploit themechanisms [4].

Concluding remarks

Independent pathways of immunity functioning on differ-ent principles of recognition were originally developed inparallel, we assume, each being challenged to diversifybecause of adaptation to the changing environment.Examination of receptor–effector diversification in eachof these pathways has revealed independent acquisitionsof new innate and adaptive features, including conver-gences, mimicry, duplications, dead ends, complementa-tions or even perhaps competitions, with a trend towardsindividualization of responses to the point where thedistinctions between adaptive and innate immunity areuncertain. Whether a species is predominantly under K orr selection might influence the type of balance betweeninnate and adaptive immunity within the particularspecies and the choice of future models for comparisonshould take this aspect into account.

Now that we have a broader picture, analogies becomemore frequent and reveal more about selection pressuresexerted on the different systems as well as the specificfunctional properties of these systems. It will always beimportant to track homologies, to understand the historyof different defense lineages, although the jump intocommitment is expected when there are different contextsand contingencies in different taxa, for example, the use ofthe binding properties of Igsf members in new evolution-ary immune pathways. We should adopt a different andintegrated view of how the different mechanisms feed offone another; the individual defense mechanisms do notwork in isolation, rather they are part of a coherent whole.

AcknowledgementsWe thank Michael Criscitiello for critical reading of the manuscript.M.F.F. is supported by NIH grants RR06603 and AI27877.

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