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Prospects & Overviews Evolution of adaptive immunity: Implications of a third lymphocyte lineage in lampreys Natsuko Kishishita1)2) and Fumikiyo Nagawa3)
An alternative antigen receptor, named the variable lymphocyte receptor (VLR), was first identified in lampreys in 2004. Since then, the mechanism of VLR diversification via somatic gene assembly and the function of VLRexpressing lymphocytes have been the subject of much research. VLRs comprise leucine-rich repeat (LRR) motifs and are found only in the most phylogenetically distant vertebrates from mammals, lampreys, and hagfish. Previous reports showed that VLRA and VLRB are reciprocally expressed by lymphocytes that resemble T- and B-cells; however, more recent reports show that another VLR, VLRC, is expressed on a third lymphocyte lineage, which may be equivalent to gd T cells. The existence of three major lymphocyte lineages one B-cell-like and two T-celllike and their development in lampreys, parallels the mammalian adaptive immune system. This suggests that these three cell lineages were present in the common vertebrate ancestor approximately 500 million years ago.
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Keywords: antigen receptor; jawless vertebrate; leucine-rich repeat; lymphocyte lineage; somatic diversification; variable lymphocyte receptor
Introduction The adaptive immune system is a highly interconnected selfdefense system that effectively attacks and eliminates a wide variety of pathogens and shows evidence of conservation from humans to sharks (jawed vertebrates). The main players in this system are immunoglobulin superfamily proteins, B-cell and T-cell receptors (TCR), and major histocompatibility complex (MHC) molecules. While B cells produce antibodies that bind to pathogens for elimination, T cells express TCRs, which bind processed antigens that are displayed on MHC molecules, and exert cellular immunity via cytokines and chemokines. The jawless vertebrates (lampreys and hagfish), the most phylogenetically distant vertebrates from mammals, possess an alternative form of adaptive immune system that is mediated by antigen receptors that contain a leucine-rich repeat (LRR) motif; these receptors are called variable lymphocyte receptors (VLRs) [1–4]. Three VLR genes (VLRA, VLRB, and VLRC) have been identified in lampreys and hagfish [1, 4–7], and all undergo somatic gene assembly through the insertion of several germline LRR cassettes into the incomplete germline VLR gene to create a high level of diversity [1, 5, 8, 9]. The assembled VLRs show considerable diversity similar to that of the immunoglobulin repertoire, and VLRs bind antigen on the concave surface where the sequence is most variable [10–14].
DOI 10.1002/bies.201300145 1)
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Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan Laboratory of Vaccine Science, Immunology Frontier Research Center, World Premier International Research Center, Osaka University, Suita, Osaka, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Abbreviations: AID, activation-induced cytosine deaminase; APOBEC, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like; CDA, cytosine deaminase; CP, connecting peptide; iNkT, invariant natural killer T; LRR, leucine-rich repeat; LRRCT, C-terminal LRR; LRRNT, N-terminal LRR; MHC, major histocompatibility complex; TCR, T-cell receptor; TLR, Toll-like receptor; VLR, variable lymphocyte receptor.
*Corresponding author: Fumikiyo Nagawa E-mail: [email protected]
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Lampreys possess one B-cell-like and two T-cell-like lymphocytes Kasamatsu et al. first identified VLRC in a unique population of lymphocytes from lampreys [6]. Recently, Hirano et al. characterized VLRCþ cells by analyzing the sites at which they localized, their role in the immune response, and their gene expression profile. Flow cytometry analysis using monoclonal antibodies specific for VLRC confirmed that VLRCþ cells in lampreys do not express VLRA or VLRB [17]. Thus, there appear to be three separate lymphocyte lineages that express VLRA, VLRB, or VLRC in a mutually exclusive manner. Immunofluorescence analysis revealed that VLRAþ and VLRCþ cells showed a similar distribution in the typhlosole, kidneys, and gills. On the other hand, digitate-shaped VLRCþ cells were also found in some areas, and they were numerically predominant in the epidermal region of the skin. In addition, the repertoire of VLRC genes within VLRCþ cells in the skin was very limited. The digitate-shape and conservative repertoire of epidermal VLRCþ cells are interesting because of the similarity with mouse gd T cells [19]. The responses of VLRCþ cells to antigenic stimulation are similar to those of VLRAþ cells in that they respond to antigens and mitogens with similar morphological changes,
but do not appear to differentiate into VLRC-secreting plasma cells [17]. In terms of gene expression profiles, VLRAþ and VLRBþ cells showed a clear distinction; however, VLRCþ cells were, in some respects, shown to be similar to VLRAþ cells. Interestingly, VLRAþ cells show unique expression of molecules such as T-cell factor 1 [20] and CTLA4 [21], both of which are seemingly important for the development and function of ab T cells. By contrast, VLRCþ cells uniquely express molecules such as the SRY-box containing gene 13, which is necessary for the development of gd T cells [22], and the Toll-like receptor (TLR) 3, which possibly helps VLRCþ cells to respond to RNA viral infections. This is consistent with reports showing that TLR ligands modulate the functions of gd T cells [23]. Thus, the presence of VLRAþ, VLRBþ, and VLRCþ cells in lampreys could suggest a potential correspondence to ab T cells, B cells, and gd T cells in mammals, respectively.
The genomic organization and assembly of the VLRC locus is similar to that of VLRA and VLRB The structure of the assembled VLRC gene is basically the same as that of VLRA and VLRB [1, 4–7, 18]. It comprises an N-terminal LRR (LRRNT), LRR1, multiple other LRRs, a connecting peptide (CP), a C-terminal LRR (LRRCT), and an invariant stalk region (Fig. 1A). The genomic organization of the VLRC locus, its assembly, and the mechanisms underlying its diversification are also fairly similar to those of the VLRA and VLRB loci, albeit with some unique features [1, 4–9, 18] (Fig. 1A, B). The germline VLRC gene contains short stretches of sequence that encode mainly invariant regions of N- and Ctermini and a portion of the LRR cassettes, with non-coding sequences in the middle; on the other hand, the short LRRencoding sequences, encoding several kinds of LRR cassettes, are randomly scattered throughout the locus. There are >500 LRR cassettes in the VLRA locus, >800 LRR cassettes in the VLRB locus, and 200 LRR cassettes in the VLRC locus [5, 18]. The difference in the number of germline LRR cassettes could
Figure 1. Organization of VLR gene locus and its assembly. A: The structure of assembled VLR gene is basically the same for all VLRs. It " comprises a 50 UTR (gray), which is interrupted by an intron, a signal peptide (dark blue), an LRRNT (blue), LRR1 (light blue), multiple other LRRs (green), a CP (red), an LRRCT (orange), an invariant C-terminal domain (brown), and the 30 UTR (gray). The organization of the gene loci of three lamprey VLRs and three hagfish VLRs is similar in that the germline VLR gene only encodes a fraction of the LRR sequence and the invariant constant region at the 50 and 30 termini, flanking a non-coding intervening sequence. The 50 UTR, an intron, a signal peptide, and an LRRNT are encoded at the 50 side of the germline VLR. A fragment of LRRCT, a C-terminal domain, and the 30 UTR are encoded at the 30 side. Within the locus, LRR cassettes are randomly scattered around the germline VLR (an example of the lamprey VLRC gene locus is shown here). Germline LRR cassettes encode “incomplete” (shown as “round”) sequences as they contain sequences from the middle of one LRR cassette to the middle of another. Many of the fragments contain more than one LRR cassette (not shown here). B: During VLR assembly, a cleavage is probably introduced, and LRR cassettes are inserted into the germline VLR in a specific order: at the 50 side, the LRRNT cassette is inserted first, followed by LRR1 and LRR. At the 30 side, LRRCT is inserted first, followed by CP and LRR. Non-coding intervening sequences are ultimately replaced by LRR cassettes. The lengths of the assembled VLRs differ because of different numbers of LRR cassettes (green) inserted in the middle. Lamprey and hagfish VLRAs and VLRCs usually possess four LRR cassettes (green), whereas lamprey VLRB usually contains two and hagfish VLRB usually contains three. C: A schematic model showing how an LRR cassette is inserted at the 50 side of a germline VLR. Although it is not experimentally demonstrated yet, VLR assembly will be initiated when a cleavage is introduced somewhere within the germline VLR, which could generate a single-stranded extension of the sequence encoded in the germline VLR. The single-stranded extension binds to the corresponding part of the germline LRRNT cassette via a short homologous sequence, then copies the LRRNT-LRR1 sequence. Next, the insertion of the LRR1-LRR cassettes occurs via another short homologous sequence, which is generated during the first insertion. In this way, VLR assembly can insert different types of LRR cassette in the correct order (and in various combinations) to generate highly diverse VLR genes.
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In lampreys, VLRAþ cells develop in a “thymus-like” region (the so-called “thymoid” at the tip of the gill filaments in the gill basket) [15]. These cells do not secrete VLRA molecules upon antigenic stimulation, and show T-cell-like characteristics [16]. VLRBþ cells develop in hematopoietic sites (typhlosole, an internal fold of the inner wall of the intestine) and differentiate into plasma cells that secrete soluble VLRB upon antigenic stimulation; thus, their function is essentially equivalent to that of B cells [1, 10, 16]. A third gene, VLRC, has also been identified, and the organization of the VLRC gene locus, its assembly mechanism, and the characteristics of VLRCþ cells have been reported [6, 17, 18]. These studies provide valuable evidence for the evolutionary development of the adaptive immune system.
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the LRR-encoding sequences identified thus far are all short, and do not encode an entire module [18]. In addition, only two LRRCT sequences have been identified [6, 18]. Sequence analysis of the VLRC locus suggested that there are several duplication events within the germline LRR cassettes. These events affect 1 to 15 LRR cassettes, either in tandem or separately, and one or more duplications [18].
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VLRCþ lymphocytes represent a distinct lineage with some similarity to VLRAþ lymphocytes VLRAþ and VLRBþ represent different cell lineages in both lampreys and hagfish [16, 25] and, as mentioned above, Hirano et al. identified VLRCþ cells as a third lineage in lampreys [17]. So how do these cells develop into different lineages? Since VLRAþ and VLRCþ cells do not assemble or transcribe the VLRB gene, they are clearly distinct from VLRBþ cells. On the other hand, the relationship between VLRCþ and VLRAþ cells is more complicated. Hirano et al. reported that the gene expression profiles of VLRAþ and VLRCþ cells overlap to a certain extent, and that the VLRA and VLRC genes are both transcribed in VLRAþ and VLRCþ cells, indicating that they are transcriptionally regulated in the same way. Kasamatsu et al. used single cell PCR analysis to show mono-allelic assembly in the majority of VLRAþ and VLRCþ cells, although in one case both the VLRA and VLRC genes were assembled in the same cell [6]. Taken together, these results suggest that VLRAþ and VLRCþ cells might go through the same stages of lymphocyte development.
VLRC assembly is prone to generating non-functional genes Hirano et al. further examined gene assembly in sorted VLRAþ, VLRBþ, and VLRCþ cells [17]. VLRAþ cells contained equal proportions of assembled and germline VLRA genes (50/50), suggesting that only one allele is assembled per cell; however, VLRCþ cells contained more assembled VLRC genes than germline VLRC genes (65/35), although 15% of the assembled VLRCs were non-productive. If one assumes feedback regulation, wherein the generation of a functional VLR prevents further gene assembly, then the failure to assemble one allele would precede the assembly of the other. This would result in diallelic assembly and only one productively assembled VLR allele [25]. The fact that VLRCþ cells appear to contain many diallelically assembled VLRC genes, many of which are non-productive, suggests that the VLRC assembly process is somehow prone to generating nonfunctional genes. The reason for this is not yet clear, although it may be due to the structures of LRR cassettes; they can be short, and quite often contain an internal stop codon or show highly diverse sequence at the 50 or 30 regions [17]. Bioessays 36: 244–250, ß 2013 WILEY Periodicals, Inc.
VLR assembly status suggests the existence of bi-potent progenitors that differentiate into either VLRCþ or VLRAþ lymphocytes Hirano et al. also reported that some VLRAþ cells contain a fully assembled VLRC gene (indeed, the majority is nonfunctional), whereas VLRCþ cells rarely contain an assembled VLRA gene (those that are assembled are non-functional). The authors went on to propose a model for the assembly of VLRA and VLRC genes, and suggested that if both VLRC alleles within a cell fail to assemble, then VLRA assembly may occur (Fig. 2A). At this point, it is possible to assume that VLRA and VLRC assembly is not mutually exclusive (Fig. 2B), as in the case of V(D)J recombination at TCR b, g, and d loci [19]. The progenitors can begin assembly on either the VLRA or the VLRC gene and, even when the first assembly generates a nonfunctional VLRC (or VLRA) gene, the next can occur on either VLRA or VLRC (Fig. 2B). This would generate VLRAþ or VLRCþ cells that harbor a non-productively assembled VLRC (or VLRA) gene. As mentioned above, the generation of many VLRAþ cells harboring a non-productively assembled VLRC gene may be due to the fact that VLRC assembly is somehow prone to generating non-functional genes. Further detailed analyses of gene assembly in individual cells are needed to clarify these issues. Taking the above into account, the following scenario is possible: VLRA/Cþ and VLRBþ cells are derived from the same lymphoid progenitor, although there is no evidence for this at present. The VLRA/VLRC progenitors move to the thymoid, which is located in the gill, whereas VLRB progenitors move to the typhlosole in the gut. The cells then undergo gene assembly. Those that successfully assemble and express functional VLRs are selected and move to the periphery, whereas those that generate a non-functional VLR gene are subjected to another round of assembly. This may continue until a functional gene is assembled. If gene assembly fails to produce a functional VLR gene, cells will probably die through apoptosis. Before cells enter the periphery, they would need to undergo some form of selection to ensure that they express a functional non-self-reactive antigen receptor.
Did three types of lymphocyte exist before the development of an adaptive immune system? Since three different lymphocyte lineages, VLRBþ, VLRAþ, and VLRCþ (possibly equivalent to mammalian B, ab T, gd T cells) exist in lampreys, Hirano et al. suggested that three primordial lymphocyte lineages may have existed and served functions in a primordial “non-adaptive” immune system before the rearrangement of antigen receptor genes evolved [17]. This is quite important if we are to fully understand the evolution of adaptive immunity. The hagfish is interesting from an evolutionary perspective because it belongs to a group of jawless vertebrates that is both anatomically and developmentally different from the lamprey; indeed, their evolutionary relationship with the
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VLR assembly occurs in a step-wise manner via short homologous sequences [5, 8, 9, 18]. The intervening sequence is replaced by inserting LRR cassettes in various combinations; short homologous sequences guide the copying of flanking LRR sequences (Fig. 1B and C). The insertion of an LRR cassette starts either 50 or 30 of the germline VLR (Fig. 1B and C), although the exact points (areas) at which assembly is initiated have yet to be identified. Interestingly, although the germline C-terminal region containing LRR sequences is often retained in mature VLRs, it is sometimes replaced with other germline LRR cassettes [18]. At this point, the insertion start point can be either within an intervening sequence or within the LRR-coding germline region.
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Figure 2. A schematic model of lymphocyte development in lampreys. A: In this model, presumptive lymphocyte progenitor cells will first be committed to either the VLRB or VLRA/C lineage. The factors that determine lymphocyte lineage are not yet clear. VLRB assembly occurs in lymphocytes at the typhlosole, and successful assembly and expression leads to the inhibition of further VLRB assembly. Unsuccessful assembly leads to VLRB assembly in the other allele. VLRA and VLRC assembly occurs in lymphocytes in the “thymoid”, in which both the VLRA and VLRC loci become transcriptionally active. Successful assembly and expression leads to the inhibition of further VLRA and VLRC assembly, while unsuccessful assembly leads to assembly in the other allele. VLRC assembly is more prone to failure and may be unsuccessful in both alleles. In such cases, VLRA assembly might then occur to generate a productive VLRA gene. Eventually, three lymphocyte lineages are generated (VLRAþ, VLRBþ, and VLRCþ cells), each of which expresses only a single functional VLR. B: Another model where VLRA and VLRC assembly is not mutually exclusive. Only the VLRA/C lineage is shown. The bi-potent progenitors can begin assembly on either the VLRA or the VLRC gene and, when the first assembly generates a non-functional gene, the next assembly can occur on either VLRA or VLRC. This would generate VLRAþ or VLRCþ cells that harbor a non-productively assembled VLRC or VLRA gene.
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What is the origin of VLR gene assembly? It is widely accepted that V(D)J recombination originated from the accidental insertion of a transposon into a primordial receptor gene [30]. V(D)J recombination is the mechanism that ensures the diversity of immunoglobulins and TCRs in jawed vertebrates. So what are the origins of VLR gene assembly? During VLR assembly, only the short homologous sequences are used to incorporate the LRR cassettes [5, 9, 18]. VLR assembly differs from ordinary gene conversion in which overall sequence similarity is required along the entire converted region [31]. Therefore, we have proposed a mechanism that involves a process called “copy choice” or “template switching” [9], in which DNA polymerase switches templates using short homologous sequences (Fig. 1C). The activity of activation-induced cytosine deaminase (AID), which belongs to the AID-apolipoprotein B mRNAediting, enzyme-catalytic, polypeptide-like (APOBEC) family of cytosine deaminases (CDAs) [32], is required for gene conversion of antibody genes in birds, rabbits, and cattle [33, 34]. Two genes, CDA1 and CDA2, which are similar to one of the AID-APOBEC CDAs, APOBEC3 [32], have been detected in the lamprey [5], and it has been suggested that CDA1 and CDA2 are involved in the assembly of VLRA/C and VLRB, respectively [15, 16], although their actual functions have not been demonstrated. To initiate VLR assembly, the germline VLR gene needs to be cleaved at or near the constant region. Other factor(s), in addition to CDA1 and CDA2, may be involved in this process. To understand the origins of VLR assembly, we need to identify the underlying mechanism(s). For example, which enzymes are responsible and how does the reaction proceed during lymphocyte development?
gene rearrangement. Both V(D)J recombination and VLR gene assembly are capable of producing over 1014 different antigen receptors, which are not directly encoded by the genome [8]. This creates a new risk: the accidental production of receptors that recognize self-antigens. Therefore, a system that eliminates or inactivates such potentially harmful antigen receptors is required. Importantly, lampreys and hagfish do not appear to possess MHC genes. It would be interesting to examine how VLR-based adaptive immune systems eliminate self-reactive receptors. As for the hagfish VLRB, self-reactive receptors are likely to be eliminated, although the mechanism by which this is achieved has yet to be identified [35]. In the mammalian immune system, if a rearranged antigen receptor is reactive to a self-antigen, the variable region can be replaced with another sequence [36]. What happens in jawless vertebrates? It is conceptually possible that a fully assembled VLR gene undergoes gene conversion using other LRR cassettes. Even if this reaction happens, it will be difficult to identify because the initial assembly process and secondary editing via gene conversion would yield indistinguishable VLR sequences.
Conclusions
How do jawless vertebrates discriminate self from non-self?
The identification and analysis of VLRC antigen receptor in the lamprey has made a significant contribution to our understanding of the evolution of adaptive immunity. Hirano et al. analyzed a third lymphocyte lineage, VLRCþ cells, and suggested that they could be equivalent to mammalian gd T cells. The existence of three major lymphocyte lineages and their development patterns suggest that these three cell lineages were present in the common vertebrate ancestor before the advent of antigen receptor gene rearrangement. Although both the antigen receptors and the mechanisms of gene rearrangement are completely different between jawed and jawless vertebrates, they share many of the basic features, including the diversification of antigen receptor genes via gene rearrangement, the expression of only one type of antigen receptor per lymphocyte, and three different lineages of lymphocytes expressing three different antigen receptors. And yet, many questions remain. For instance, what are the functions of the VLRs and how do VLRA and VLRC recognize antigens? Do VLRAþ and VLRCþ cells provide cellular immunity and interact with VLRBþ cells? Does the lamprey possess other types of immune cell, such as B1-like cells [37] or invariant natural killer T (iNKT)-like cells [38]? Do lampreys and hagfish eliminate self-reactive antigen receptors, and if so, how? Do hagfish possess three types of lymphocyte? If so, are they functionally equivalent to those in lampreys? What is the mechanism underlying VLR gene assembly and what are the functions of CDA1 and CDA2? Further study of the convergent evolution of adaptive immunity will improve our fundamental understanding of this elegant system.
Before the evolution of antigen receptor gene rearrangement, the genes encoding receptors that were reactive with “self” molecules were probably lost from the genome over time. The need to eliminate self-reactive receptors emerged when antigen receptor genes gained the ability to diversify through
Acknowledgements We thank A. Otsuka (The University of Hawaii) and Y. Takahashi (National Institute of Infectious Diseases) for critical reading of the paper. This work was supported by the
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lamprey is still controversial, although recent molecular analysis has shown that hagfishes and lampreys are monophyletic with respect to vertebrates [26–29]. Until recently, only VLRA and VLRB were reported in hagfish; however, Li et al. have now identified the third VLR gene [7]. By comparing the structures of the three hagfish VLRs with those of the lamprey, they concluded that the third hagfish VLR is the counterpart of lamprey VLRA, and that the previously identified hagfish VLRA is actually the counterpart of lamprey VLRC (Fig. 1A). The diversity of the mature third hagfish VLR was comparable with that of the other VLRs. Further study of the hagfish lymphocytes that express the third VLR will advance our understanding of the evolution of VLR-based adaptive immunity.
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grants (24659218) from the Japan Society for the Promotion of Science. The authors have declared no conflict of interest.
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18. Das S, Hirano M, Aghaallaei N, Bajoghli B, et al. 2013. Organization of lamprey variable lymphocyte receptor C locus and repertoire development. Proc Natl Acad Sci USA 110: 6043–8. 19. Vantourout P, Hayday A. 2013. Six-of-the-best: unique contributions of gd T cells to immunology. Nat Rev Immunol 13: 88–100. 20. Yu Q, Sharma A, Sen JM. 2010. TCF1 and beta-catenin regulate T cell development and function. Immunol Res 47: 45–55. 21. Rudd CE, Taylor A, Schneider H. 2009. CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol Rev 229: 12–26. 22. Melichar HJ, Narayan K, Der SD, Hiraoka Y, et al. 2007. Regulation of gd versus ab T lymphocyte differentiation by the transcription factor SOX13. Science 315: 230–3. 23. Wesch D, Peters C, Oberg HH, Pietschmann K, et al. 2011. Modulation of gd T cell responses by TLR ligands. Cell Mol Life Sci 68: 2357–70. 24. Chien YH, Konigshofer Y. 2007. Antigen recognition by gammadelta T cells. Immunol Rev 215: 46–58. 25. Kishishita N, Matsuno T, Takahashi Y, Takaba H, et al. 2010. Regulation of antigen-receptor gene assembly in hagfish. EMBO Rep 11: 126–32. 26. Caputo Barucchi V, Giovannotti M, Nisi Cerioni P, Splendiani A. 2013. Genome duplication in early vertebrates: Insights from agnathan cytogenetics. Cytogenet Genome Res 141: 80–9. 27. Sugahara F, Murakami Y, Adachi N, Kuratani S. 2013. Evolution of the regionalization and patterning of the vertebrate telencephalon: what can we learn from cyclostomes? Curr Opin Genet Dev 23: 475–83. 28. Oisi Y, Ota KG, Kuraku S, Fujimoto S, et al. 2013. Craniofacial development of hagfishes and the evolution of vertebrates. Nature 493: 175–80. 29. Janvier P. 2013. Developmental biology: led by the nose. Nature 493: 169–70. 30. Schatz DG. 2004. Antigen receptor genes and the evolution of a recombinase. Semin Immunol 16: 245–56. 31. Chen JM, Cooper DN, Chuzhanova N, Fe´rec C, et al. 2007. Gene conversion: mechanisms, evolution and human disease. Nat Rev Genet 8: 762–75. 32. Conticello SG, Langlois M-A, Yang Z, Neuberger MS. 2007. DNA deamination in immunity: AID in the context of its APOBEC relatives. Adv Immunol 94: 37–73. 33. Arakawa H, Buerstedde J-M. 2004. Immunoglobulin gene conversion: insights from bursal B cells and the DT40 cell line. Dev Dyn 229: 458–64. 34. Di Noia JM, Neuberger MS. 2007. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem 76: 1–22. 35. Takaba H, Imai T, Miki S, Morishita Y, et al. 2013. A major allogenic leukocyte antigen in the agnathan hagfish. Sci Rep 3: 1716. 36. Verkoczy LK, Ma˚rtensson AS, Nemazee D. 2004. The scope of receptor editing and its association with autoimmunity. Curr Opin Immunol 16: 808–14. 37. Zhang X. 2013. Regulatory functions of innate-like B cells. Cell Mol Immunol 10: 113–21. 38. Van Kaer L, Parekh VV, Wu L. 2011. Invariant natural killer T cells: bridging innate and adaptive immunity. Cell Tissue Res 343: 43–55.
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Prospects & Overviews Evolution of adaptive immunity: Implications of a third lymphocyte lineage in lampreys Natsuko Kishishita1)2) and Fumikiyo Nagawa3)
An alternative antigen receptor, named the variable lymphocyte receptor (VLR), was first identified in lampreys in 2004. Since then, the mechanism of VLR diversification via somatic gene assembly and the function of VLRexpressing lymphocytes have been the subject of much research. VLRs comprise leucine-rich repeat (LRR) motifs and are found only in the most phylogenetically distant vertebrates from mammals, lampreys, and hagfish. Previous reports showed that VLRA and VLRB are reciprocally expressed by lymphocytes that resemble T- and B-cells; however, more recent reports show that another VLR, VLRC, is expressed on a third lymphocyte lineage, which may be equivalent to gd T cells. The existence of three major lymphocyte lineages one B-cell-like and two T-celllike and their development in lampreys, parallels the mammalian adaptive immune system. This suggests that these three cell lineages were present in the common vertebrate ancestor approximately 500 million years ago.
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Keywords: antigen receptor; jawless vertebrate; leucine-rich repeat; lymphocyte lineage; somatic diversification; variable lymphocyte receptor
Introduction The adaptive immune system is a highly interconnected selfdefense system that effectively attacks and eliminates a wide variety of pathogens and shows evidence of conservation from humans to sharks (jawed vertebrates). The main players in this system are immunoglobulin superfamily proteins, B-cell and T-cell receptors (TCR), and major histocompatibility complex (MHC) molecules. While B cells produce antibodies that bind to pathogens for elimination, T cells express TCRs, which bind processed antigens that are displayed on MHC molecules, and exert cellular immunity via cytokines and chemokines. The jawless vertebrates (lampreys and hagfish), the most phylogenetically distant vertebrates from mammals, possess an alternative form of adaptive immune system that is mediated by antigen receptors that contain a leucine-rich repeat (LRR) motif; these receptors are called variable lymphocyte receptors (VLRs) [1–4]. Three VLR genes (VLRA, VLRB, and VLRC) have been identified in lampreys and hagfish [1, 4–7], and all undergo somatic gene assembly through the insertion of several germline LRR cassettes into the incomplete germline VLR gene to create a high level of diversity [1, 5, 8, 9]. The assembled VLRs show considerable diversity similar to that of the immunoglobulin repertoire, and VLRs bind antigen on the concave surface where the sequence is most variable [10–14].
DOI 10.1002/bies.201300145 1)
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Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan Laboratory of Vaccine Science, Immunology Frontier Research Center, World Premier International Research Center, Osaka University, Suita, Osaka, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Abbreviations: AID, activation-induced cytosine deaminase; APOBEC, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like; CDA, cytosine deaminase; CP, connecting peptide; iNkT, invariant natural killer T; LRR, leucine-rich repeat; LRRCT, C-terminal LRR; LRRNT, N-terminal LRR; MHC, major histocompatibility complex; TCR, T-cell receptor; TLR, Toll-like receptor; VLR, variable lymphocyte receptor.
*Corresponding author: Fumikiyo Nagawa E-mail: [email protected]
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Lampreys possess one B-cell-like and two T-cell-like lymphocytes Kasamatsu et al. first identified VLRC in a unique population of lymphocytes from lampreys [6]. Recently, Hirano et al. characterized VLRCþ cells by analyzing the sites at which they localized, their role in the immune response, and their gene expression profile. Flow cytometry analysis using monoclonal antibodies specific for VLRC confirmed that VLRCþ cells in lampreys do not express VLRA or VLRB [17]. Thus, there appear to be three separate lymphocyte lineages that express VLRA, VLRB, or VLRC in a mutually exclusive manner. Immunofluorescence analysis revealed that VLRAþ and VLRCþ cells showed a similar distribution in the typhlosole, kidneys, and gills. On the other hand, digitate-shaped VLRCþ cells were also found in some areas, and they were numerically predominant in the epidermal region of the skin. In addition, the repertoire of VLRC genes within VLRCþ cells in the skin was very limited. The digitate-shape and conservative repertoire of epidermal VLRCþ cells are interesting because of the similarity with mouse gd T cells [19]. The responses of VLRCþ cells to antigenic stimulation are similar to those of VLRAþ cells in that they respond to antigens and mitogens with similar morphological changes,
but do not appear to differentiate into VLRC-secreting plasma cells [17]. In terms of gene expression profiles, VLRAþ and VLRBþ cells showed a clear distinction; however, VLRCþ cells were, in some respects, shown to be similar to VLRAþ cells. Interestingly, VLRAþ cells show unique expression of molecules such as T-cell factor 1 [20] and CTLA4 [21], both of which are seemingly important for the development and function of ab T cells. By contrast, VLRCþ cells uniquely express molecules such as the SRY-box containing gene 13, which is necessary for the development of gd T cells [22], and the Toll-like receptor (TLR) 3, which possibly helps VLRCþ cells to respond to RNA viral infections. This is consistent with reports showing that TLR ligands modulate the functions of gd T cells [23]. Thus, the presence of VLRAþ, VLRBþ, and VLRCþ cells in lampreys could suggest a potential correspondence to ab T cells, B cells, and gd T cells in mammals, respectively.
The genomic organization and assembly of the VLRC locus is similar to that of VLRA and VLRB The structure of the assembled VLRC gene is basically the same as that of VLRA and VLRB [1, 4–7, 18]. It comprises an N-terminal LRR (LRRNT), LRR1, multiple other LRRs, a connecting peptide (CP), a C-terminal LRR (LRRCT), and an invariant stalk region (Fig. 1A). The genomic organization of the VLRC locus, its assembly, and the mechanisms underlying its diversification are also fairly similar to those of the VLRA and VLRB loci, albeit with some unique features [1, 4–9, 18] (Fig. 1A, B). The germline VLRC gene contains short stretches of sequence that encode mainly invariant regions of N- and Ctermini and a portion of the LRR cassettes, with non-coding sequences in the middle; on the other hand, the short LRRencoding sequences, encoding several kinds of LRR cassettes, are randomly scattered throughout the locus. There are >500 LRR cassettes in the VLRA locus, >800 LRR cassettes in the VLRB locus, and 200 LRR cassettes in the VLRC locus [5, 18]. The difference in the number of germline LRR cassettes could
Figure 1. Organization of VLR gene locus and its assembly. A: The structure of assembled VLR gene is basically the same for all VLRs. It " comprises a 50 UTR (gray), which is interrupted by an intron, a signal peptide (dark blue), an LRRNT (blue), LRR1 (light blue), multiple other LRRs (green), a CP (red), an LRRCT (orange), an invariant C-terminal domain (brown), and the 30 UTR (gray). The organization of the gene loci of three lamprey VLRs and three hagfish VLRs is similar in that the germline VLR gene only encodes a fraction of the LRR sequence and the invariant constant region at the 50 and 30 termini, flanking a non-coding intervening sequence. The 50 UTR, an intron, a signal peptide, and an LRRNT are encoded at the 50 side of the germline VLR. A fragment of LRRCT, a C-terminal domain, and the 30 UTR are encoded at the 30 side. Within the locus, LRR cassettes are randomly scattered around the germline VLR (an example of the lamprey VLRC gene locus is shown here). Germline LRR cassettes encode “incomplete” (shown as “round”) sequences as they contain sequences from the middle of one LRR cassette to the middle of another. Many of the fragments contain more than one LRR cassette (not shown here). B: During VLR assembly, a cleavage is probably introduced, and LRR cassettes are inserted into the germline VLR in a specific order: at the 50 side, the LRRNT cassette is inserted first, followed by LRR1 and LRR. At the 30 side, LRRCT is inserted first, followed by CP and LRR. Non-coding intervening sequences are ultimately replaced by LRR cassettes. The lengths of the assembled VLRs differ because of different numbers of LRR cassettes (green) inserted in the middle. Lamprey and hagfish VLRAs and VLRCs usually possess four LRR cassettes (green), whereas lamprey VLRB usually contains two and hagfish VLRB usually contains three. C: A schematic model showing how an LRR cassette is inserted at the 50 side of a germline VLR. Although it is not experimentally demonstrated yet, VLR assembly will be initiated when a cleavage is introduced somewhere within the germline VLR, which could generate a single-stranded extension of the sequence encoded in the germline VLR. The single-stranded extension binds to the corresponding part of the germline LRRNT cassette via a short homologous sequence, then copies the LRRNT-LRR1 sequence. Next, the insertion of the LRR1-LRR cassettes occurs via another short homologous sequence, which is generated during the first insertion. In this way, VLR assembly can insert different types of LRR cassette in the correct order (and in various combinations) to generate highly diverse VLR genes.
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In lampreys, VLRAþ cells develop in a “thymus-like” region (the so-called “thymoid” at the tip of the gill filaments in the gill basket) [15]. These cells do not secrete VLRA molecules upon antigenic stimulation, and show T-cell-like characteristics [16]. VLRBþ cells develop in hematopoietic sites (typhlosole, an internal fold of the inner wall of the intestine) and differentiate into plasma cells that secrete soluble VLRB upon antigenic stimulation; thus, their function is essentially equivalent to that of B cells [1, 10, 16]. A third gene, VLRC, has also been identified, and the organization of the VLRC gene locus, its assembly mechanism, and the characteristics of VLRCþ cells have been reported [6, 17, 18]. These studies provide valuable evidence for the evolutionary development of the adaptive immune system.
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the LRR-encoding sequences identified thus far are all short, and do not encode an entire module [18]. In addition, only two LRRCT sequences have been identified [6, 18]. Sequence analysis of the VLRC locus suggested that there are several duplication events within the germline LRR cassettes. These events affect 1 to 15 LRR cassettes, either in tandem or separately, and one or more duplications [18].
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VLRCþ lymphocytes represent a distinct lineage with some similarity to VLRAþ lymphocytes VLRAþ and VLRBþ represent different cell lineages in both lampreys and hagfish [16, 25] and, as mentioned above, Hirano et al. identified VLRCþ cells as a third lineage in lampreys [17]. So how do these cells develop into different lineages? Since VLRAþ and VLRCþ cells do not assemble or transcribe the VLRB gene, they are clearly distinct from VLRBþ cells. On the other hand, the relationship between VLRCþ and VLRAþ cells is more complicated. Hirano et al. reported that the gene expression profiles of VLRAþ and VLRCþ cells overlap to a certain extent, and that the VLRA and VLRC genes are both transcribed in VLRAþ and VLRCþ cells, indicating that they are transcriptionally regulated in the same way. Kasamatsu et al. used single cell PCR analysis to show mono-allelic assembly in the majority of VLRAþ and VLRCþ cells, although in one case both the VLRA and VLRC genes were assembled in the same cell [6]. Taken together, these results suggest that VLRAþ and VLRCþ cells might go through the same stages of lymphocyte development.
VLRC assembly is prone to generating non-functional genes Hirano et al. further examined gene assembly in sorted VLRAþ, VLRBþ, and VLRCþ cells [17]. VLRAþ cells contained equal proportions of assembled and germline VLRA genes (50/50), suggesting that only one allele is assembled per cell; however, VLRCþ cells contained more assembled VLRC genes than germline VLRC genes (65/35), although 15% of the assembled VLRCs were non-productive. If one assumes feedback regulation, wherein the generation of a functional VLR prevents further gene assembly, then the failure to assemble one allele would precede the assembly of the other. This would result in diallelic assembly and only one productively assembled VLR allele [25]. The fact that VLRCþ cells appear to contain many diallelically assembled VLRC genes, many of which are non-productive, suggests that the VLRC assembly process is somehow prone to generating nonfunctional genes. The reason for this is not yet clear, although it may be due to the structures of LRR cassettes; they can be short, and quite often contain an internal stop codon or show highly diverse sequence at the 50 or 30 regions [17]. Bioessays 36: 244–250, ß 2013 WILEY Periodicals, Inc.
VLR assembly status suggests the existence of bi-potent progenitors that differentiate into either VLRCþ or VLRAþ lymphocytes Hirano et al. also reported that some VLRAþ cells contain a fully assembled VLRC gene (indeed, the majority is nonfunctional), whereas VLRCþ cells rarely contain an assembled VLRA gene (those that are assembled are non-functional). The authors went on to propose a model for the assembly of VLRA and VLRC genes, and suggested that if both VLRC alleles within a cell fail to assemble, then VLRA assembly may occur (Fig. 2A). At this point, it is possible to assume that VLRA and VLRC assembly is not mutually exclusive (Fig. 2B), as in the case of V(D)J recombination at TCR b, g, and d loci [19]. The progenitors can begin assembly on either the VLRA or the VLRC gene and, even when the first assembly generates a nonfunctional VLRC (or VLRA) gene, the next can occur on either VLRA or VLRC (Fig. 2B). This would generate VLRAþ or VLRCþ cells that harbor a non-productively assembled VLRC (or VLRA) gene. As mentioned above, the generation of many VLRAþ cells harboring a non-productively assembled VLRC gene may be due to the fact that VLRC assembly is somehow prone to generating non-functional genes. Further detailed analyses of gene assembly in individual cells are needed to clarify these issues. Taking the above into account, the following scenario is possible: VLRA/Cþ and VLRBþ cells are derived from the same lymphoid progenitor, although there is no evidence for this at present. The VLRA/VLRC progenitors move to the thymoid, which is located in the gill, whereas VLRB progenitors move to the typhlosole in the gut. The cells then undergo gene assembly. Those that successfully assemble and express functional VLRs are selected and move to the periphery, whereas those that generate a non-functional VLR gene are subjected to another round of assembly. This may continue until a functional gene is assembled. If gene assembly fails to produce a functional VLR gene, cells will probably die through apoptosis. Before cells enter the periphery, they would need to undergo some form of selection to ensure that they express a functional non-self-reactive antigen receptor.
Did three types of lymphocyte exist before the development of an adaptive immune system? Since three different lymphocyte lineages, VLRBþ, VLRAþ, and VLRCþ (possibly equivalent to mammalian B, ab T, gd T cells) exist in lampreys, Hirano et al. suggested that three primordial lymphocyte lineages may have existed and served functions in a primordial “non-adaptive” immune system before the rearrangement of antigen receptor genes evolved [17]. This is quite important if we are to fully understand the evolution of adaptive immunity. The hagfish is interesting from an evolutionary perspective because it belongs to a group of jawless vertebrates that is both anatomically and developmentally different from the lamprey; indeed, their evolutionary relationship with the
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VLR assembly occurs in a step-wise manner via short homologous sequences [5, 8, 9, 18]. The intervening sequence is replaced by inserting LRR cassettes in various combinations; short homologous sequences guide the copying of flanking LRR sequences (Fig. 1B and C). The insertion of an LRR cassette starts either 50 or 30 of the germline VLR (Fig. 1B and C), although the exact points (areas) at which assembly is initiated have yet to be identified. Interestingly, although the germline C-terminal region containing LRR sequences is often retained in mature VLRs, it is sometimes replaced with other germline LRR cassettes [18]. At this point, the insertion start point can be either within an intervening sequence or within the LRR-coding germline region.
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Figure 2. A schematic model of lymphocyte development in lampreys. A: In this model, presumptive lymphocyte progenitor cells will first be committed to either the VLRB or VLRA/C lineage. The factors that determine lymphocyte lineage are not yet clear. VLRB assembly occurs in lymphocytes at the typhlosole, and successful assembly and expression leads to the inhibition of further VLRB assembly. Unsuccessful assembly leads to VLRB assembly in the other allele. VLRA and VLRC assembly occurs in lymphocytes in the “thymoid”, in which both the VLRA and VLRC loci become transcriptionally active. Successful assembly and expression leads to the inhibition of further VLRA and VLRC assembly, while unsuccessful assembly leads to assembly in the other allele. VLRC assembly is more prone to failure and may be unsuccessful in both alleles. In such cases, VLRA assembly might then occur to generate a productive VLRA gene. Eventually, three lymphocyte lineages are generated (VLRAþ, VLRBþ, and VLRCþ cells), each of which expresses only a single functional VLR. B: Another model where VLRA and VLRC assembly is not mutually exclusive. Only the VLRA/C lineage is shown. The bi-potent progenitors can begin assembly on either the VLRA or the VLRC gene and, when the first assembly generates a non-functional gene, the next assembly can occur on either VLRA or VLRC. This would generate VLRAþ or VLRCþ cells that harbor a non-productively assembled VLRC or VLRA gene.
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What is the origin of VLR gene assembly? It is widely accepted that V(D)J recombination originated from the accidental insertion of a transposon into a primordial receptor gene [30]. V(D)J recombination is the mechanism that ensures the diversity of immunoglobulins and TCRs in jawed vertebrates. So what are the origins of VLR gene assembly? During VLR assembly, only the short homologous sequences are used to incorporate the LRR cassettes [5, 9, 18]. VLR assembly differs from ordinary gene conversion in which overall sequence similarity is required along the entire converted region [31]. Therefore, we have proposed a mechanism that involves a process called “copy choice” or “template switching” [9], in which DNA polymerase switches templates using short homologous sequences (Fig. 1C). The activity of activation-induced cytosine deaminase (AID), which belongs to the AID-apolipoprotein B mRNAediting, enzyme-catalytic, polypeptide-like (APOBEC) family of cytosine deaminases (CDAs) [32], is required for gene conversion of antibody genes in birds, rabbits, and cattle [33, 34]. Two genes, CDA1 and CDA2, which are similar to one of the AID-APOBEC CDAs, APOBEC3 [32], have been detected in the lamprey [5], and it has been suggested that CDA1 and CDA2 are involved in the assembly of VLRA/C and VLRB, respectively [15, 16], although their actual functions have not been demonstrated. To initiate VLR assembly, the germline VLR gene needs to be cleaved at or near the constant region. Other factor(s), in addition to CDA1 and CDA2, may be involved in this process. To understand the origins of VLR assembly, we need to identify the underlying mechanism(s). For example, which enzymes are responsible and how does the reaction proceed during lymphocyte development?
gene rearrangement. Both V(D)J recombination and VLR gene assembly are capable of producing over 1014 different antigen receptors, which are not directly encoded by the genome [8]. This creates a new risk: the accidental production of receptors that recognize self-antigens. Therefore, a system that eliminates or inactivates such potentially harmful antigen receptors is required. Importantly, lampreys and hagfish do not appear to possess MHC genes. It would be interesting to examine how VLR-based adaptive immune systems eliminate self-reactive receptors. As for the hagfish VLRB, self-reactive receptors are likely to be eliminated, although the mechanism by which this is achieved has yet to be identified [35]. In the mammalian immune system, if a rearranged antigen receptor is reactive to a self-antigen, the variable region can be replaced with another sequence [36]. What happens in jawless vertebrates? It is conceptually possible that a fully assembled VLR gene undergoes gene conversion using other LRR cassettes. Even if this reaction happens, it will be difficult to identify because the initial assembly process and secondary editing via gene conversion would yield indistinguishable VLR sequences.
Conclusions
How do jawless vertebrates discriminate self from non-self?
The identification and analysis of VLRC antigen receptor in the lamprey has made a significant contribution to our understanding of the evolution of adaptive immunity. Hirano et al. analyzed a third lymphocyte lineage, VLRCþ cells, and suggested that they could be equivalent to mammalian gd T cells. The existence of three major lymphocyte lineages and their development patterns suggest that these three cell lineages were present in the common vertebrate ancestor before the advent of antigen receptor gene rearrangement. Although both the antigen receptors and the mechanisms of gene rearrangement are completely different between jawed and jawless vertebrates, they share many of the basic features, including the diversification of antigen receptor genes via gene rearrangement, the expression of only one type of antigen receptor per lymphocyte, and three different lineages of lymphocytes expressing three different antigen receptors. And yet, many questions remain. For instance, what are the functions of the VLRs and how do VLRA and VLRC recognize antigens? Do VLRAþ and VLRCþ cells provide cellular immunity and interact with VLRBþ cells? Does the lamprey possess other types of immune cell, such as B1-like cells [37] or invariant natural killer T (iNKT)-like cells [38]? Do lampreys and hagfish eliminate self-reactive antigen receptors, and if so, how? Do hagfish possess three types of lymphocyte? If so, are they functionally equivalent to those in lampreys? What is the mechanism underlying VLR gene assembly and what are the functions of CDA1 and CDA2? Further study of the convergent evolution of adaptive immunity will improve our fundamental understanding of this elegant system.
Before the evolution of antigen receptor gene rearrangement, the genes encoding receptors that were reactive with “self” molecules were probably lost from the genome over time. The need to eliminate self-reactive receptors emerged when antigen receptor genes gained the ability to diversify through
Acknowledgements We thank A. Otsuka (The University of Hawaii) and Y. Takahashi (National Institute of Infectious Diseases) for critical reading of the paper. This work was supported by the
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lamprey is still controversial, although recent molecular analysis has shown that hagfishes and lampreys are monophyletic with respect to vertebrates [26–29]. Until recently, only VLRA and VLRB were reported in hagfish; however, Li et al. have now identified the third VLR gene [7]. By comparing the structures of the three hagfish VLRs with those of the lamprey, they concluded that the third hagfish VLR is the counterpart of lamprey VLRA, and that the previously identified hagfish VLRA is actually the counterpart of lamprey VLRC (Fig. 1A). The diversity of the mature third hagfish VLR was comparable with that of the other VLRs. Further study of the hagfish lymphocytes that express the third VLR will advance our understanding of the evolution of VLR-based adaptive immunity.
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grants (24659218) from the Japan Society for the Promotion of Science. The authors have declared no conflict of interest.
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