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of TH-17 cells only when TGF- is abundant; otherwise, a dominant TH1 response ensues.

However, these new studies1,2 indicate that certain PRRs can induce IL-23 without IL-12. Such induction might result in a TH-17 response even in environments not tradition-ally thought to contain much TGF-. In sup-port of that idea, LeibundGut-Landmann et al. found that intravenous injection of curdlan or C. albicans supported a TH-17 response despite bypassing the TGF--rich mucosa1. In con-trast, TH-17 responses to extracellular bacteria, which would presumably induce both IL-12 and IL-23 through TLRs, have been noted after mucosal infection6. Thus, pattern recognition may have a dominant function in establishing a mucosa-homing TH-17 response. However, further research is needed to clarify the relative importance of route of exposure versus PRR agonist composition for both T helper cell fate determination and imprinting of chemokine receptor expression.

Although other studies have reported the induction of a TH-17 response after TLR liga-

tion in the presence of TGF-, LeibundGut-Landmann et al. have now shown that the fungal component curdlan preferentially induces a TH-17 response

1. This demon-strates specific induction of the differentia-tion of TH-17 cells by a pathogen-associated molecular pattern, and these findings provide a convincing demonstration that a non-TLR PRR is sufficient to activate adaptive immu-nity. In parallel, the work of Acosta-Rodriguez et al. shows that human TH-17 cells express a unique chemokine receptor repertoire that allows their identification and is indicative of homing to the skin and mucosa2.

Together these studies indicate that in addition to being involved in defense against extracellular bacteria, TH-17 responses are involved in antifungal defense. Furthermore, these studies represent an important advance in understanding of the relationship between pattern recognition and the induc-tion of tailored adaptive immune responses (Fig. 1). One implication of these findings is that innate immune recognition, perhaps along

with tissue-specific cytokine environments, can program an appropriate class of the adaptive immune response that is optimized to deal with a particular type of infection. As it is likely that additional T helper effector sub-sets will be identified, similar principles will probably apply even more broadly.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

1. LeibundGut-Landmann, S. et al. Nat. Immunol. 8, 630638 (2007).

2. Acosta-Rodriguez, E.V. et al. Nat. Immunol. 8, 639646 (2007).

3. Bettelli, E., Oukka, M. & Kuchroo, V.K. Nat. Immunol. 8, 345350 (2007).

4. Veldhoen, M. et al. Immunity 24, 179189 (2006).5. Bettelli, E. et al. Nature 441, 235238 (2006).6. Mangan, P.R. et al. Nature 441, 231234 (2006).7. Brown, G.D. & Gordon, S. Nature 413, 3637

(2001).8. Rogers, N.C. et al. Immunity 22, 507517 (2005).9. Gross, O. et al. Nature 442, 651656 (2006).10. Taylor, P.R. et al. Nat. Immunol. 8, 3138 (2007).11. Khader, S.A. et al. Nat. Immunol. 8, 369377 (2007).12. Sato, K. et al. J. Biol. Chem. 281, 3885438866

(2006).13. Cua, D.J. & Kastelein, R.A. Nat. Immunol. 7, 557559

(2006).

DNA deaminases converge on adaptive immunityDavid G Schatz

AID acts to further refine the immunoglobulin response in fish, birds and mammals. Now, evidence is uncovered to suggest AID actually arose early in evolution and can diversify antigen receptors in lampreys.

To help deal with the constantly evolving, unpredictable threat presented by infec-tious microorganisms, humans and other jawed vertebrates (gnathostomes) developed a sophisticated adaptive immune system orga-nized around B and T lymphocytes. These cells express a highly diversified repertoire of immunoglobulins and T cell receptors that is generated by somatic recombination and mutation reactions during lymphocyte devel-opment. With the discovery of somatically diversified variable lymphocyte receptors (VLRs) in lampreys and hagfish (jawless ver-tebrates, or agnathans)1,2, it became apparent that evolution had twice derived similar solu-tions to the microbial threat, albeit with two entirely different classes of immune receptors.

Now a report by Rogozin et al.3 in this issue of Nature Immunology suggests that this conver-gence of strategies runs deeper still to include key DNA cytidine deaminases involved in the somatic diversification process: activation-induced deaminase (AID) used by jawed vertebrates for immunoglobulin gene diver-sification, and newly identified genes encod-ing members of the AID-APOBEC family expressed by lamprey lymphocytes and linked to the assembly and diversification of genes encoding VLRs.

Developing lymphocytes of jawed verte-brates assemble the variable region of genes encoding immunoglobulins and T cell recep-tors from variable (V), diversity (D) and join-ing (J) gene segments by a process known as V(D)J recombination (Fig. 1, left). This reac-tion is initiated by the recombination-activat-ing proteins RAG-1 and RAG-2 and generates all of the diversity found in T cell receptors. V(D)J recombination is also the chief source of immunoglobulin diversity in humans and mice, but in many other species, such as

chickens and rabbits, it generates little or no immunoglobulin diversity4. Instead, in those species, genes encoding immunoglobulins are diversified after assembly by processes that either introduce blocks of sequence altera-tions copied from nearby pseudo-V donors (gene conversion) or generate point muta-tions (somatic hypermutation; Fig. 1, left). Gene conversion and somatic hypermutation are initiated by AID-mediated deamination of cytidine residues in V-region DNA encod-ing immunoglobulins and are completed when the resulting uracil residues are acted on by various distinct DNA-repair processes5. After B cells encounter antigen, AID is again brought into action to trigger gene conver-sion and/or somatic hypermutation to further diversify genes encoding immunoglobulins, which can facilitate the development of high-affinity antibodies by a process known as affinity maturation.

Agnathan VLRs are composed almost entirely of multiple (four to twelve) sequence-diverse leucine-rich repeat (LRR) modules,

David G. Schatz is with the Howard Hughes

Medical Institute, Department of Immunobiology,

Yale University School of Medicine, New Haven,

Connecticut 06520, USA.

e-mail: david.schatz@yale.edu

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placing them in the same far-flung family of LRR proteins as the Toll-like receptors of the innate immune system1. The two germline VLR loci (VLRA and VLRB) found in both lamprey and hagfish contain coding infor-mation for only the amino- and carboxy-terminal regions of the proteins, which are separated by noncoding sequences (Fig. 1, right). During somatic assembly of a func-tional VLR gene, the noncoding regions are replaced by a variable number of sequences encoding LRR modules contributed by large pools of such sequences thought to reside in regions of the chromosome that flank the germline loci (Fig. 1, right). By analysis of a draft version of the sea lamprey genome, Rogozin et al. have identified nearly 2,000

sequences encoding LRR modules that can contribute to either VLRA or VLRB but not both3. Because the number and identity of the module-encoding sequences inserted can vary, potential VLR diversity was estimated to be huge (about 1014), easily matching that of immunoglobulin and T cell receptor pro-teins2,6. Published studies have indicated that the lamprey and hagfish immune systems have adaptive properties1; hence, it was gratifying to find that immunization of sea lamprey lar-vae elicits a substantial increase in large VLR+ lymphocytes in blood and lymphoid tissues and a time- and dose-dependent increase in antigen-specific, soluble VLRs in the blood6. VLRs are therefore the likely mediators of adaptive immunity in agnathans.

The identification of intermediates in VLR assembly has shown that the process involves the stepwise insertion of sequences encod-ing LRR modules in-frame with the germ-line coding sequences6. That, along with the identification of short blocks of sequence homology between the ends of the mod-ules, led to the suggestion that a gene con-versionlike mechanism is involved in VLR assembly1. The study of Rogozin et al.3 as well as another study of the Japanese lam-prey7 now demonstrate that every insertion occurs at a short (6- to 50-nucleotide) region of sequence homology between the donor LRR moduleencoding sequence and the recipient VLR locus. Both studies have also shown that most domains encoding LRRs in

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Figure 1 Assembly and diversification of genes encoding antigen receptors in vertebrates. In jawed vertebrates (left), genes encoding immunoglobulins and T cell receptors are assembled by V(D)J recombination and genes encoding immunoglobulins can be further diversified by somatic hypermutation and gene conversion, which are triggered by the introduction of uracil residues (U) by AID. Igh, gene encoding immunoglobulin heavy chain; V, pseudo-V. In jawless vertebrates (right), the germline VLR locus consists of sequences encoding amino-terminal (LRRNT) and carboxy-terminal (LRRCT) LRR regions (purple rectangles) separated by a noncoding intervening sequence. Flanking those are many sequences encoding LRR modules (blue squares). Assembly is proposed to be initiated by an AID-APOBEC family deaminase that introduces uracil residues into the VLR locus, whose processing leads to single- or double-stranded DNA breaks. A short region of homology (closely spaced vertical lines) allows a 3 DNA end to prime DNA synthesis from sequence encoding an LRR module (LRRa), and subsequent short homology regions allow the extending end to jump to successive new LRR-encoding templates (LRRb, LRRc and so on) until the reaction terminates by a template jump back into the germline locus. Most LRR-encoding sequences in the final, assembled VLR locus are hybrids composed of sequences from two (or more) donor LRR moduleencoding sequences. Many elements of this model are speculative. NT and CT, sequence encoding the amino or carboxyl terminus, respectively.

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an assembled VLR gene are chimeric, with the sequence encoding the amino-terminal portion being contributed by a piece of one LRR moduleencoding sequence and the remainder being contributed by regions of one or more different LRR moduleencod-ing sequences. This ability to undergo both inter- and intramodule joining substantially increases the potential diversity of VLR genes.

How does such an assembly process occur? There is no hint of RAG1 or RAG2 homo-logs in the lamprey genome or of their rec-ognition elements in VLR loci3, nor does the precise, in-frame addition of one module after another in VLR assembly resemble the sloppy joining events that characterize V(D)J recombination. Instead, the process resembles immunoglobulin gene conversion, which also occurs at short blocks of homology between donor pseudo-V genes and the recipient immunoglobulin locus. Given the essential function of AID in immunoglobulin gene conversion, the identification by Rogozin et al. of genes encoding two cytidine deaminases of the AID-APOBEC family in the lamprey genome3 is particularly intriguing. Both are expressed in lamprey lymphocytes; one was characterized with a variety of in vitro and in vivo assays and was found to have DNA cytidine-deaminase activity similar to that of AID3.

Although the lamprey deaminases have not yet been proven to mediate VLR assem-bly, these findings support the following copy-choice model7 of VLR gene assem-bly3,7 (Fig. 1, right). The process is initiated by the deamination of cytidine residues in the germline VLR locus by an AID-like cyti-dine deaminase3. Processing of the resulting uracil residues by a DNA-repair pathway (most plausibly base-excision repair involv-ing a uracil DNA glycosylase such as UNG) creates a single- or double-stranded break in the DNA and thereby liberates a free 3 DNA end. This can invade and prime DNA syn-thesis from a donor LRR moduleencoding sequence, taking advantage of a short region of homology between the invading end and the donor. DNA synthesis extends through some or all of the donor LRR moduleencod-ing sequence and then, again taking advan-tage of a short block of homology, jumps either to the noncoding intervening DNA in the VLR locus3 or to a second (and third, and fourth and so on) LRR moduleencoding donor template7 (Fig. 1, right). After one or more rounds of deaminase-initiated recom-bination, sequence encoding a carboxy-ter-minal LRR unit is inserted with homology with the germline region of the VLR locus

encoding the carboxyl terminus, and a final template jump completes the assembly process.

Evolutionary convergence is a principal theme emerging from the findings of Rogozin et al. reported here3. Jawed and jawless verte-brates independently evolved similar anticipa-tory strategies of adaptive immunity, but the two systems make use of completely different antigen receptor proteins, and in both cases, evolution seems to have chosen DNA cyti-dine deaminases of the AID-APOBEC family as tools for receptor diversification. The abil-ity of these deaminases to trigger mutations and DNA lesions at many different positions and sequences in a target region probably helps to explain their recurrent use.

Just as the discovery of VLR genes dis-pelled the idea that jawed vertebrates have sole dominion over adaptive immunity, so do the findings of Rogozin et al. revise the understanding of the evolutionary history of the AID-APOBEC family of cytidine deami-nases8. It is now apparent that the founding member of the family must have existed early in vertebrate evolution in a common ancestor of jawed and jawless vertebrates that appeared after the emergence of deuterostomes3. That in turn has implications for consideration of the old question Which came first, RAG-1-RAG-2mediated V(D)J recombination, or AID-mediated somatic hypermutation and/or gene conversion? As V(D)J recombina-tion is confined to jawed vertebrates, it seems likely that deaminase-mediated diversifica-tion of immune receptors was first on the scene. As has been suggested9, the most plau-sible scenario is that V(D)J recombination arose from transposon insertion into a gene encoding an immunoglobulin superfamily antigen receptor that was already subject to AID-mediated diversification. Given the notably similar functions of AID-APOBEC deaminases in the assembly and diversifica-tion of genes encoding immunoglobulins and VLRs, it is possible that agnathans and gnastostomes have evolved similar mecha-nisms for specific targeting of the deaminase to loci encoding antigen receptors to avoid mutation and destabilization of other parts of the genome.

Sequence analysis of the newly identified lamprey deaminases together with that of existing deaminases led Rogozin et al. to the insight that all members of the AID-APOBEC family share several distinctive features with the Tad2p-TadA family of tRNA adeno sine deaminases3. Of note are several shared -helices and loops near the carboxyl ter-mini of the proteins that contain highly con-served amino acids involved in RNA binding

by TadA10. This includes an almost perfectly conserved phenylalanine residue noted by Rogozin et al. to be involved in base inter-actions with substrate RNA by TadA3. It is therefore possible that substrate recogni-tion by AID-APOBEC cytidine deaminases and Tad2p-TadA adenosine deaminases has conserved structural underpinnings3,11. Such considerations also support the idea that the AID-APOBEC family arose early in vertebrate evolution from an ancestral Tad2p-TadA fam-ily member3. The idea of an evolutionary link between adenosine and cytidine deaminases gains further impetus from the demonstra-tion of a Trypanosoma brucei tRNA-editing enzyme with both adenosine-deaminase activity on RNA and cytidine-deaminase activity on single-stranded DNA12. It is therefore easy to imagine that an ancestral deaminase with a flexible active site capable of accommodating adenosine and cytidine as well as RNA and DNA gave rise to many fam-ilies of enzymes, such as the AID-APOBEC family, with wide-ranging functions and active on a variety of substrates.

All of this suggests that future investiga-tions may demonstrate more unexpected findings about polynucleotide deaminases. Additional family members are sure to emerge as more genomes are sequenced, and additional functions will probably be identi-fied for those already known. Further explo-ration of these enzymes will shed light on the mechanism of VLR gene assembly and many other aspects of adaptive immunity. Given the flexibility of the deaminases, it remains possible that microbes such as trypanosomes also take advantage of deaminases in their struggle to evade the immune system12.

COMPETING INTERESTS STATEMENTThe author declares no competing financial interests.

1. Cooper, M.D. & Alder, M.N. Cell 124, 815822 (2006).

2. Pancer, Z. et al. Nature 430, 174180 (2004).3. Rogozin, I.B. et al. Nat. Immunol. 647656 (2007). 4. Flajnik, M.F. Nat. Rev. Immunol. 2, 688698

(2002).5. Di Noia, J.M. & Neuberger, M.S. Annu. Rev.

Biochem., advance online publication 28 February 2007 (doi:2010.1146/annurev.biochem.2076.061705.090740).

6. Alder, M.N. et al. Science 310, 19701973 (2005).

7. Nagawa, F. et al. Nat. Immunol. 8, 206213 (2007).

8. Conticello, S.G., Thomas, C.J., Petersen-Mahrt, S.K. & Neuberger, M.S. Mol. Biol. Evol. 22, 367377 (2005).

9. Lee, S.S., Tranchina, D., Ohta, Y., Flajnik, M.F. & Hsu, E. Immunity 16, 571582 (2002).

10. Losey, H.C., Ruthenburg, A.J. & Verdine, G.L. Nat. Struct. Mol. Biol. 13, 153159 (2006).

11. Conticello, S.G., Langlois, M.A. & Neuberger, M.S. Nat. Struct. Mol. Biol. 14, 79 (2007).

12. Rubio, M.A.T. et al. Proc. Natl. Acad. Sci. USA, advance online publication 1 May 2007 (doi: 10.1073/pnas0702394104).

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