Immune Related Genes Underpin the Evolution of Adaptive Immunity in Jawless Vertebrates

The study of immune related genes in lampreys and hagfish provides a unique perspective on the evolutionary genetic underpinnings of adaptive immunity and the evolution of vertebrate genomes. Separated from their jawed cousins at the stem of the vertebrate lineage, these jawless vertebrates have many of the gene families and gene regulatory networks associated with the defining morphological and physiological features of vertebrates. These include genes vital for innate immunity, inflammation, wound healing, protein degradation, and the development, signaling and trafficking of lymphocytes. Jawless vertebrates recognize antigen by using leucine-rich repeat (LRR) based variable lymphocyte receptors (VLRs), which are very different from the immunoglobulin (Ig) based T cell receptor (TCR) and B cell receptor (BCR) used for antigen recognition by jawed vertebrates. The somatically constructed VLR genes are expressed in monoallelic fashion by T-like and B-like lymphocytes. Jawless and jawed vertebrates thus share many of the genes that provide the molecular infrastructure and physiological context for adaptive immune responses, yet use entirely different genes and mechanisms of combinatorial assembly to generate diverse repertoires of antigen recognition receptors.     

无颌类脊椎动物适应性免疫系统的研究进展

在以七鳃鳗和盲鳗为代表的无颌类脊椎动物中, 虽然发现了与有颌类脊椎动物T细胞受体(T-cell receptors, TLRs)、B细胞受体 (B-cell receptors, BCRs)可变区具有相似结构的先天性免疫受体, 却从未发现有颌类脊椎动物适应性免疫系统的核心组分: TCRs、BCRs、组织相容性复合体 (Major histocompatibility complex, MHC)。因此, 长期以来, 人们一直认为适应性免疫系统只存在于有颌类脊椎动物中。但最近的一项发现彻底改变了这一传统观念, 即在无颌类脊椎动物中, 存在一种新型可变淋巴细胞受体VLRs(Variable lymphocyte receptors), VLRs通过改变亮氨酸富集序列LRRs(Leucine-rich repeats)的插入情况, 实现对特异性抗原的高效识别。晶体衍射分析发现, 盲鳗的VLRs呈现一种“马蹄”型结构, 抗原结合位点则位于“马蹄”的凹面区。分泌型的VLRs以四聚体或五聚体的形式识别、结合特异性抗原。综上所述, 无颌类和有颌类脊椎动物应用不同的抗原识别系统完成适应性免疫反应。文章对近年来无颌类脊椎动物适应性免疫系统相关分子的研究进展加以概述, 为揭示适应性免疫系统起源与进化问题提供有益参考。     

The evolution of adaptive immune systems

A clonally diverse anticipatory repertoire in which each lymphocyte bears a unique antigen receptor is the central feature of the adaptive immune system that evolved in our vertebrate ancestors. The survival advantage gained through adding this type of adaptive immune system to a pre-existing innate immune system led to the evolution of alternative ways for lymphocytes to generate diverse antigen receptors for use in recognizing and repelling pathogen invaders. All jawed vertebrates assemble their antigen-receptor genes through recombinatorial rearrangement of different immunoglobulin or T cell receptor gene segments. The surviving jawless vertebrates, lampreys and hagfish, instead solved the receptor diversification problem by the recombinatorial assembly of leucine-rich-repeat genetic modules to encode variable lymphocyte receptors. The convergent evolution of these remarkably different adaptive immune systems involved innovative genetic modification of innate-immune-system components.     

七鳃鳗鳃黏膜免疫系统类淋巴细胞免疫应答遗传基础

近年来,在无颌类脊椎动物七鳃鳗体内发现了以可变淋巴细胞受体(Variable lymphocyte receptors, VLR)为基础的抗原识别机制。为揭示七鳃鳗鳃黏膜免疫系统中类淋巴细胞适应性免疫应答的遗传基础,探索无颌类与有颌类脊椎动物在适应性免疫应答机制上的进化关系,本文构建了日本七鳃鳗(Lampetra japonica)鳃囊组织免疫前后cDNA文库并进行了高通量转录组测序及分析。通过对组装得到的88 525个独立基因(Unigene)进行功能注释,分别有21 704和9769个unigene在GO(Gene Ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库得到注释。999个unigene参与免疫系统的多个通路,其中184个与高等脊椎动物TCR(T cell receptor)和BCR(B cell receptor)信号通路的51个分子具有较高的同源关系,说明七鳃鳗体内存在高等脊椎动物适应性免疫应答信号通路的相关分子。本文还发现5个VLRA、7个VLRB和4个VLRC分子,说明七鳃鳗鳃黏膜免疫组织内至少分布3种类淋巴细胞亚群。实时荧光定量PCR结果显示,Lck、Fyn和Zap70基因在免疫激发后表达量显著上调,而Syk、Btk和Blnk基因表达没有显著变化,说明七鳃鳗鳃组织受到抗原刺激后,类似T淋巴细胞的信号转导途径被激活。本研究初步证明,尽管无颌类和有颌类脊椎动物的适应性免疫系统在抗原识别机制上存在不同,但具有共同的遗传基础。研究结果为探讨七鳃鳗VLRA⁺、VLRB⁺和VLRC⁺淋巴细胞免疫应答信号传导过程提供了有价值的线索。     

VLR Recognition of TLR5 Expands the Molecular Characterization of Protein Antigen Binding by Non-Ig-based Antibodies

Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.     

Design principles of adaptive immune systems

Both jawless vertebrates, such as lampreys and hagfish, and jawed vertebrates (encompassing species as diverse as sharks and humans) have an adaptive immune system that is based on somatically diversified and clonally expressed antigen receptors. Although the molecular nature of the antigen receptors and the mechanisms of their assembly are different, recent findings suggest that the general design principles underlying the two adaptive immune systems are surprisingly similar. The identification of such commonalities promises to further our understanding of the mammalian immune system and to inspire the development of new strategies for medical interventions targeting the consequences of faulty immune functions.     

Reconstructing immune phylogeny: new perspectives

Numerous studies of the mammalian immune system have begun to uncover profound interrelationships, as well as fundamental differences, between the adaptive and innate systems of immune recognition. Coincident with these investigations, the increasing experimental accessibility of non-mammalian jawed vertebrates, jawless vertebrates, protochordates and invertebrates has provided intriguing new information regarding the likely patterns of emergence of immune-related molecules during metazoan phylogeny, as well as the evolution of alternative mechanisms for receptor diversification. Such findings blur traditional distinctions between adaptive and innate immunity and emphasize that, throughout evolution, the immune system has used a remarkably extensive variety of solutions to meet fundamentally similar requirements for host protection.     

Structural diversity of the hagfish variable lymphocyte receptors

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.     

Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.     

The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

Genome biology of the cyclostomes and insights into the evolutionary biology of vertebrate genomes

The jawless vertebrates (lamprey and hagfish) are the closest extant outgroups to all jawed vertebrates (gnathostomes) and can therefore provide critical insight into the evolution and basic biology of vertebrate genomes. As such, it is notable that the genomes of lamprey and hagfish possess a capacity for rearrangement that is beyond anything known from the gnathostomes. Like the jawed vertebrates, lamprey and hagfish undergo rearrangement of adaptive immune receptors. However, the receptors and the mechanisms for rearrangement that are utilized by jawless vertebrates clearly evolved independently of the gnathostome system. Unlike the jawed vertebrates, lamprey and hagfish also undergo extensive programmed rearrangements of the genome during embryonic development. By considering these fascinating genome biologies in the context of proposed (albeit contentious) phylogenetic relationships among lamprey, hagfish, and gnathostomes, we can begin to understand the evolutionary history of the vertebrate genome. Specifically, the deep shared ancestry and rapid divergence of lampreys, hagfish and gnathostomes is considered evidence that the two versions of programmed rearrangement present in lamprey and hagfish (embryonic and immune receptor) were present in an ancestral lineage that existed more than 400 million years ago and perhaps included the ancestor of the jawed vertebrates. Validating this premise will require better characterization of the genome sequence and mechanisms of rearrangement in lamprey and hagfish.     

Vertebrate immunity: mutator proteins and their evolution

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.     

临床诊断与治疗的新工具——可变淋巴细胞受体

单克隆抗体(Monoclonal antibody, mAb)在癌症以及自身免疫等疾病的诊断与治疗中得到广泛应用, 并且取得了重大进展。当今应用于临床的单克隆抗体是在免疫球蛋白的基础上进行改造研发而得。然而近期发现的无颌类脊椎动物的特异性抗原受体-可变淋巴细胞受体(Variable lymphocyte receptor, VLR), 为抗体类试剂或药物的研发提供了新的视角。与免疫球蛋白(Immunoglobulins, Ig)相比, VLR与抗原结合的特异性、亲和力及稳定性都优于Ig类抗体, 并且抗原特异性单克隆VLR的制备技术日趋成熟。因此, VLR在临床诊断和治疗中具有更高的应用价值, 并可能成为新一代的抗体药物。文章就VLR的基本特征、制备方法及其应用前景进行综述, 为实现VLR在临床诊断与治疗等领域中的应用提供有益参考。     

Regulation of antigen‐receptor gene assembly in hagfish

Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T‐cell‐like, whereas those expressing VLRB are B‐cell‐like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single‐cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases—in many of which, one allele was functional and the other was defective. In fact, all VLR‐assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR‐positive lymphocytes.     

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.     

Did the molecules of adaptive immunity evolve from the innate immune system?

The antigen receptors on cells of innate immune systems recognizebroadly expressed markers on non-host cells while the receptorson lymphocytes of the adaptive immune system display a higherlevel of specificity. Adaptive immunity, with its exquisitespecificity and immunological memory, has only been found inthe jawed vertebrates, which also display innate immunity. Jawlessfishes and invertebrates only have innate immunity. In the adaptiveimmune response, T and B-lymphocytes detect foreign agents orantigens using T cell receptors (TCR) or immunoglobulins (Ig),respectively. While Ig can bind free intact antigens, TCR onlybinds processed antigenic fragments that are presented on moleculesencoded in the major histocompatibility complex (MHC). MHC moleculesdisplay variation through allelic polymorphism. A diverse repertoireof Ig and TCR molecules is generated by gene rearrangement andjunctional diversity, processes carried out by the recombinaseactivating gene (RAG) products and terminal deoxynucleotidyltransferase (TdT). Thus, the molecules that define adaptiveimmunity are TCR, Ig, MHC molecules, RAG products and TdT. Nodirect predecessors of these molecules have been found in thejawless fishes or invertebrates. In contrast, the complementcascade can be activated by either adaptive or innate immunesystems and contains examples of molecules that gradually evolvedfrom non-immune functions to being part of the innate and thenadaptive immune system. In this paper we examine the moleculesof the adaptive immune system and speculate on the existenceof direct predecessors that were part of innate immunity.     

Incarnation of classical pro- and eukaryotic mechanisms of mutagenesis in hypermutagenesis and immunity of vertebrates

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.     

Rapid evolutionary emergence of the combinatorial recognition repertoire

Although the capacity of cells to respond to environmental challengessuch as oxidative damage are ancient evolutionary developmentsthat have been carried through to modern higher vertebratesas "innate" immunity, the characteristic immune response ofvertebrates is a relatively recent evolutionary developmentthat is present only in jawed vertebrates. The vertebrate "combinatorial"response is defined by the presence of lymphocytes as specificantigen recognition cells and by the complete panel of antibodies,T cell receptors, and major histocompatibility complex moleculesall of which are members of the immunoglobulin family. Its emergencein evolution was an extremely rapid event (approximately 10million years) that was catalyzed by the horizontal transferof recombinase activator genes (RAG) from microbes to an ancestraljawed vertebrate. RAGs occur in jawed vertebrates, but havenot been found in invertebrates and other intermediate species.We propose that antigen recognition capacity contributed bythis novel combinatorial mechanism gave jawed vertebrates theability to recognize the entire range of potential antigenicmolecular structures, including self components and moleculesof infectious microbes not shared with vertebrates. The contrastwithin the vertebrates is striking because the most ancientextant jawed vertebrates, sharks and their kin, have the completepanoply of T-cell receptors, antibodies, MHC products and RAGgenes, whereas agnathans possess cells resembling lymphocytesbut ostensibly lack all of the molecules definitive of combinatorialimmunity. Another vertebrate innovation may have been the utilizationof nuclear receptor superfamily, in the regulation of lymphocytesand other cells of the immune lineage. Unlike, RAG, however,this superfamily occurs in all metazoans with the exceptionof sponges.