Immune system is a very important and highly organized host defense system and throughout evolution, it has been developed in various ways to protect from countless pathogens in the environment. Most species in the animal kingdom rely on innate immunity, which constitutes the first line of defense against infection and attacks pathogens in non-antigen-specific manner. Recent studies have shown that innate immune systems are highly diverse among species and have uniquely developed in a species-specific manner. On the other hand, highly sophisticated adaptive immunity, which shows antigen-specific response and create immune memory, is only observed in the vertebrates; for a long time, only one type of the adaptive immune system was known in higher vertebrates, commonly referred to as jawed vertebrates (sharks to mammals). In these animals, antigen receptor genes are somatically diversified under strict regulations; genes encoding T cell receptors and immunoglobulins (Ig) are rearranged and expressed in T cells and B cells, respectively. T cells and B cells functionally interact with each other, providing the basis for highly specific and effective immune responses and the development of immune memory.

Although the most basal vertebrates, jawless vertebrates, show adaptive immune responses, they do not possess conventional antigen receptor genes. The paradox causes a question what is responsible for adaptive immune responses and whether or not they have a system to execute a highly complex immune response as jawed vertebrates. Several years ago, a study showed jawless vertebrates possess an alternative antigen receptor named variable lymphocyte receptors (VLR), which consist of multiple leucine-rich repeat (LRR) modules. In hagfish and lampreys, which are the only extant orders of jawless vertebrates, two types of the VLR gene, VLRA and VLRB, are present. Recent reports showed that in the sea lamprey (Petromyzon marinus), VLRA and VLRB are expressed each by separate lymphocyte subpopulations, and that lymphocytes expressing VLRA are T cell-like, while those expressing VLRB are B cell-like.

Diverse VLR genes are somatically generated in lymphocyte-like cells in hagfish and lampreys; the intervening sequence in the incomplete germline VLR gene is replaced by a set of LRR-coding gene segments residing around the germline VLR gene (Fig. IA). A great number of LRR segments, which come in several types, lie randomly in the genome and the size of VLRA and VLRB loci spread over 2 mega bases. The mechanism to generate diverse VLR genes was, however, largely unknown. In order to understand VLR diversification mechanism, I examined how diverse VLRB (VLRA was not reported at that time) genes were generated in the Japanese lamprey (Lethenteron japonicum). Sequence analysis of VLRB genes before, during and after somatic rearrangement revealed that short homologies were present between germline LRR segments and seemed to be used as an anchor site for LRR segment insertion.

Based on close analysis of LRR junction sequences, I have proposed that VLR assembly is mediated by a process involving copy choice/template-switching, in which DNA polymerase dissociates from one DNA region (template) to another through repeat sequences in templates. In VLR assembly, short homologies in LRR segments are used as template-switching sites and several LRR segments are inserted in order (Fig. I). For instance, the short homology in 5' constant region (5'C) of the germline VLRB gene draws a particular LRR segment, an LRRNT plus LRR (Fig. IB). An LRRNT-LRR segment is inserted into the germline VLRB gene, and an inserted sequence now contains a short homology which draws a different LRR segment, an LRR (Fig. IB). Insertion of LRR segments occurs at both 5'C and 3'C ends of the intervening sequence; several kinds of LRR segments are inserted at each end, and joined at the end to generate a VLR gene (Fig. IA). When a pair of LRR segments share homology at several sites, LRR segments were connected at different short homology sites, generating various hybrid LRRs from a single pair of LRR segments. Thus, in VLR assembly, a vast repertoire of assembled VLR genes could be generated, not only by inserting LRR segments in various combinations, but also by joining LRR segments at multiple sites.

The research of VLRB gene assembly in the Japanese lamprey also demonstrated monoallelic assembly of VLRB gene, leaving non-assembled allele intact. Since there are two types of VLR gene in the jawless vertebrates, important questions arise such as how VLRA and VLRB are assembled in a lymphocyte and how the assembly is controlled to be monoallelic. It is, in other words, whether or not jawless vertebrates also have developed a certain mechanism to ensure that a single antigen receptor is expressed in a lymphocyte, as highly strict regulations that are seen in gene rearrangement of Ig-type antigen receptors. In order to understand how VLR assembly is regulated, I analyzed the VLR gene assembly in the inshore hagfish (Eptatretus burgeri) at the single-cell level, using over 1,000 sorted lymphocytes. Single-cell PCR analysis showed that each lymphocyte assembled only one type of the VLR gene, either VLRA or VLRB, mostly in a monoallelic fashion. On the other hand, single-cell RT-PCR demonstrated that VLR transcription was detected in both alleles and not restricted to one of the two alleles. In minority of lymphocytes, VLR assembly occurred in both alleles; in many of such cases, only one allele contained a functional VLR gene, and the other allele contained a defective VLR gene with an in-frame stop codon or a frameshift.

Based on these results, I have proposed a model describing how VLR assembly proceeds, referring to feedback inhibition hypothesis that a functional VLR protein acts to prevent VLR assembly on the other allele (Fig. II). At first, lymphoid progenitors, if they exist, separate into two populations, each of which somehow activates either VLRA or VLRB locus. While transcription occurs on both alleles, VLR assembly occurs on either allele to generate a functional VLR gene. A functional VLR protein induces feedback inhibition on the other allele so that VLR assembly no longer occurs. If defective VLR is generated (or VLR protein somehow can not induce feedback inhibition), VLR assembly continues on the other allele, generating one defective and one functional VLR gene (or two apparently functional VLR genes) in a lymphocyte. When defective VLR genes are generated on both alleles, such lymphocytes might be subject to apoptosis. Only lymphocytes containing at least one functional VLR gene survive and come out into the peripheral blood.

At an early stage of vertebrate evolution, there seems to have been an overwhelming demand for adaptive immunity; two adaptive immune systems mediated by Ig-type and VLR-type antigen receptors have developed in jawed and jawless vertebrates, respectively. I have revealed that a copy-choice-involving mechanism is used for somatic assembly of the VLR gene in jawless vertebrates. Although VLR antigen receptors are very different from Ig-type antigen receptors, I have shown that various VLR genes are generated through combinatorial and junctional diversifications and that the VLR assembly is highly regulated to ensure that a single receptor is expressed by each lymphocyte, as in V(D)J recombination. Thus, although the two adaptive immune systems have evolved with distinct antigen receptors in parallel, they show common features that diverse antigen receptor genes are generated through similar diversification strategies, and each lymphocyte is regulated to express a single receptor.

Figure I. A model of VLR assembly mechanism.

(A) A model of VLR assembly. Several LRR segments are inserted in order using short homologies. Through somatic assembly, the VLR gene is greatly diversified not only by inserting LRR segments in various combinations, but also by shifting combining sites between LRR segments.

(B) A model of LRR segment insertion into incomplete VLR gene. Only the 5'C end is illustrated. Short homologies between LRR segments are used as an anchor site for LRR segment insertion. Single-stranded extension of the 5' C (recipient) hybridizes and copies the LRRNT-LRR sequence (donor). After incorporating one LRR segment, copying continues with the next segment.

Figure II. A model of VLR assembly in lymphocyte development.

Before VLR assembly takes place, lymphocytes separate into two groups, each of which undergo VLRA or VLRB assembly in a mutually exclusive manner. At each VLRA or VLRB locus, both alleles are activated in transcription. Somehow, VLR assembly occurs in a single allele and a successful assembly of a functional VLR gene would lead to feedback inhibition on the other allele. When a defective VLR gene is generated, assembly might occur on the other allele, which would result in a lymphocyte with one defective and one functional VLR. In some cases, two functional VLR genes were found in a single lymphocyte, which might be due to a failure of feedback inhibition. If a lymphocyte contains only defective VLR genes, it would result in cell death.

獲得免疫系は脊椎動物のみでみられる高度な生体防御機構だが、有顎類がimmunoglobulin (Ig)タイプの抗原受容体を用いるのに対して、現存する脊椎動物で最も下等な無顎類はleucine-rich repeat (LRR)構造をもつvariable lymphocyte receptor (VLR)抗原受容体を用いることが最近分かってきた。Igタイプの抗原受容体と同様にVLR抗原受容体はリンパ球様細胞で遺伝子再編成によって多様化するが、その仕組みについては不明であった。また、VLRタイプ抗原受容体にはVLRAとVLRBの二種類があるが、一つの細胞でVLRAとVLRB遺伝子再編成がどのように制御されているのかについても明らかになっていなかった。本研究はVLR抗原受容体の遺伝子再編成の機構とその制御について調べ、初めてその仕組みについて明らかにし獲得免疫系の起源と進化を理解する上で重要な知見をもたらした。よって当該分野において極めて重要な仕事である。

本論文は5章からなる。第1章はイントロダクションであり、獲得免疫系で主要な役割を果たす抗原受容体を中心に、既知のIg系獲得免疫系と新規のVLR系獲得免疫系に関して述べている。本研究は無顎類のVLR遺伝子再編成機構と制御を調べたので、獲得免疫系の進化、獲得免疫応答、遺伝子再編成機構を中心に当該分野における先行研究のレビューをおこない、その中に本研究を位置付けた。第2章及び第3章は、研究結果を記している。両章においてそれぞれ、研究の進め方を示しつつ実験結果を述べ、更に全体のデータを踏まえて研究で明らかになった点やその意義を考察している。第2章はVLR遺伝子の再編成機構に関する研究をまとめている。遺伝子再編成前、再編成後、及び再編成途中と思われるVLR遺伝子の配列比較から、"copy choice"と呼ばれる機構を利用して複数のLRR断片が順にコピーされることでVLR遺伝子が完成することを明らかにした。更に、VLR遺伝子はLRR断片の組み合わせのみならずその結合部位をシフトさせることによって多様化することを明らかにした。第3章はVLR遺伝子再編成の制御機構に関する研究をまとめている。単細胞レベルでVLRA、VLRBの遺伝子再編成と転写を調べることで、再編成も転写もVLRA、VLRB相互排他的に起こっていることを明らかにした。更に遺伝子再編成はほとんどの場合monoallelicに起こっており、feedback inhibitionによってallelic exclusionが成立している可能性を明らかにした。第4章は、研究全体を通した考察と結論を述べて、本研究の当該分野における寄与を示している。続けて第5章に、本研究の研究手法を述べている。ヤツメウナギとヌタウナギからのサンプルの調整、細胞の採取、PCRの条件、データ解析の方法などを詳しく述べている。

本研究では、VLR遺伝子再編成が"copy choice"と呼ばれる機構を利用しておこなわれることを明らかにし、V(D)J組み換えと同様に、LRR断片の組み合わせと結合部位をシフトさせつことにより非常に多様な抗原受容体をつくりだすことを示した。更にVLR遺伝子再編成はV(D)J組み換えと同様に、VLRAとVLRBの再編成は相互排他的に起こり、更に基本的に一方のアリールのみで再編成が起こるように制御されていることを示した。本研究により、VLR遺伝子再編成はV(D)J組み換えとは全く異なる機構で行われるにもかかわらず、多様性創出の仕組みは似ており、更に各細胞当たり一つの抗原受容体を発現するように制御する機構も似ていることが明らかにされた。有顎類と無顎類はIgタイプとVLRタイプという全く異なる分子を抗原受容体として獲得免疫系を構築してきたにもかかわらず、多様性創出の仕組みや遺伝子再編成制御機構の点において多くの共通点をもつ事が明らかになった。

なお、本論文第2章及び第3章は、松野 達哉・高橋 宜聖・高場 啓之・西住 裕文・名川 文清との共同研究であるが、論文提出者が主体となって実験及び解析を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(理学)の学位を授与できると認める。