Yoichi Sutoh

VLR-Based Adaptive Immunity

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

Identification of a third variable lymphocyte receptor in the lamprey

Jawless vertebrates such as lamprey and hagfish lack T-cell and B-cell receptors; instead, they have unique antigen receptors known as variable lymphocyte receptors (VLRs). VLRs generate diversity by recombining highly diverse leucine-rich repeat modules and are expressed clonally on lymphocyte-like cells (LLCs). Thus far, two types of receptors, VLRA and VLRB, have been identified in lampreys and hagfish. Recent evidence indicates that VLRA and VLRB are expressed on distinct populations of LLCs that resemble T cells and B cells of jawed vertebrates, respectively. Here we identified a third VLR, designated VLRC, in the lamprey. None of the ≈100 VLRC cDNA clones subjected to sequencing had an identical sequence, indicating that VLRC can generate sufficient diversity to function as antigen receptors. Notably, the C-terminal cap of VLRC exhibits only limited diversity and has important structural differences relative to VLRA and VLRB. Single-cell PCR analysis identified LLCs that rearranged VLRC but not VLRA or VLRB, suggesting the presence of a unique population of LLCs that express only VLRC.

Two forms of adaptive immunity in vertebrates: similarities and differences.

Unlike jawed vertebrates that use T-cell and B-cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors. VLRs generate diversity comparable to that of gnathostome antigen receptors by assembling variable leucine-rich repeat modules. The discovery of VLR has revolutionized our understanding of how adaptive immunity emerged and highlighted the differences between the adaptive immune systems (AISs) of jawed and jawless vertebrates. However, emerging evidence also indicates that their AISs have much in common. Particularly striking is the conservation of lymphocyte lineages. The basic architecture of the AIS including the dichotomy of lymphocytes appears to have been established in a common ancestor of jawed and jawless vertebrates. We review here the current knowledge on the AIS of jawless vertebrates, emphasizing both the similarities to and differences from the AIS of jawed vertebrates.

Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes.

The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t subunit, designated PSMB11, was identified in the cartilaginous fish, the most divergent group of jawed vertebrates compared to the other jawed vertebrates, but not in jawless vertebrates or invertebrates. Interestingly, teleost fish have two copies of apparently functional PSMB11 genes, designated PSMB11a and PSMB11b, that encode β5t subunits with distinct amino acids in the S1 pocket. BLAST searches of genome databases suggest that birds such as chickens, turkey, and zebra finch lost the PSMB11 gene, and have neither thymoproteasomes nor immunoproteasomes. In mammals, reptiles, amphibians, and teleost fishes, the PSMB11 gene (the PSMB11a gene in teleost fish) is located next to the PSMB5 gene coding for the β5 subunit of the standard 20S proteasome, indicating that the PSMB11 gene arose by tandem duplication from the evolutionarily more ancient PSMB5 gene. The general absence of introns in PSMB11 and an unusual exon–intron structure of jawed vertebrate PSMB5 suggest that PSMB5 lost introns and duplicated in tandem in a common ancestor of jawed vertebrates, with PSMB5 subsequently gaining two introns and PSMB11 remaining intronless.

Two Types of Antigen Receptor Systems in Vertebrates

Abstract Extant jawless vertebrates, represented by lampreys and hagfishes, have innate immune receptors with variable domains structurally resembling those of T/B-cell receptors. However, they appear to lack cardinal elements of adaptive immunity shared by all jawed vertebrates: major histocompatibility complex molecules and T/B-cell receptors. Thus, it was widely believed that adaptive immunity is unique to jawed vertebrates. Recently, this belief was overturned by the discovery of agnathan antigen receptors named variable lymphocyte receptors. These receptors generate diversity in their antigen-binding sites through assembling highly diverse leucine-rich repeat modules. The crystal structures of hagfish variable lymphocyte receptor monomers indicate that they adopt a horseshoe-shaped structure and likely bind antigens through the hypervariable concave surface. Secreted variable lymphocyte receptors form pentamers or tetramers of dimers and bind antigens with high specificity and avidity. The fact that variable lymphocyte receptors are structurally unrelated to T/B-cell receptors indicates that jawed and jawless vertebrates have developed antigen receptors independently.

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.

Characterization of Lamprey BAFF-like Gene: Evolutionary Implications

BAFF (TNF superfamily [TNFSF] 13B/Blys) and APRIL (TNFSF13) are important regulatory factors for lymphocyte activation and survival in mammals. A BAFF/APRIL-like relative called BAFF- and APRIL-like molecule (BALM) has also been identified in cartilaginous and bony fishes, and we report in this study a BAFF-like gene in lampreys. Our phylogenetic analysis of these genes and a related TNFSF12 gene called TNF-like weak inducer of apoptosis (TWEAK) suggest that, whereas an ancestral homolog of BAFF and APRIL was already present in a common ancestor of jawed and jawless vertebrates, TWEAK evolved early on in the jawed vertebrate lineage. Like mammalian BAFF and APRIL, the lamprey BAFF-like gene is expressed in T-like, B-like, and innate immune cells. The predicted protein encoded by this BAFF-like gene in lampreys exhibits higher sequence similarity with mammalian BAFF than APRIL. Correspondingly, we find BAFF orthologs in all of the jawed vertebrate representatives that we examined, although APRIL and/or BALM orthologs are not identifiable in certain jawed vertebrates. For example, BALM is not identifiable in tetrapods, and APRIL is not identifiable in several bony fishes or in birds, the latter of which also lack a TWEAK-like gene. Our analysis further suggests that a hybrid molecule called TWE-PRIL, which is a product of an in-genomic fusion between APRIL and TWEAK genes evolved early in mammalian evolution.

Comparative genomics of the NKG2D ligand gene family.

NKG2D ligands (NKG2DLs) are a group of stress‐inducible major histocompatibility complex (MHC) class I‐like molecules that act as a danger signal alerting the immune system to the presence of abnormal cells. In mammals, two families of NKG2DL genes have been identified: the MIC gene family encoded in the MHC region and the ULBP gene family encoded outside the MHC region in most species. Some mammals have a third family of NKG2DL‐like class I genes which we named MILL (MHC class I‐like located near the leukocyte receptor complex). Despite the fact that MILL genes are more closely related to MIC genes than ULBP genes are to MIC genes, MILL molecules do not function as NKG2DLs, and their function remains unknown. With the progress of mammalian genome projects, information on the MIC, ULBP, and MILL gene families became available in many mammalian species. Here, we summarize such information and discuss the origin and evolution of the NKG2DL gene family from the viewpoint of host–pathogen coevolution.

Evolution of two prototypic T cell lineages

Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αβ or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

The SKINT1-like gene is inactivated in hominoids but not in all primate species: implications for the origin of dendritic epidermal T cells.

Dendritic epidermal T cells, which express an invariant Vγ5Vδ1 T-cell receptor and account for 95% of all resident T cells in the mouse epidermis, play a critical role in skin immune surveillance. These γδ T cells are generated by positive selection in the fetal thymus, after which they migrate to the skin. The development of dendritic epidermal T cells is critically dependent on the Skint1 gene expressed specifically in keratinocytes and thymic epithelial cells, suggesting an indispensable role for Skint1 in the selection machinery for specific intraepithelial lymphocytes. Phylogenetically, rodents have functional SKINT1 molecules, but humans and chimpanzees have a SKINT1-like (SKINT1L) gene with multiple inactivating mutations. In the present study, we analyzed SKINT1L sequences in representative primate species and found that all hominoid species have a common inactivating mutation, but that Old World monkeys such as olive baboons, green monkeys, cynomolgus macaques and rhesus macaques have apparently functional SKINT1L sequences, indicating that SKINT1L was inactivated in a common ancestor of hominoids. Interestingly, the epidermis of cynomolgus macaques contained a population of dendritic-shaped γδ T cells expressing a semi-invariant Vγ10/Vδ1 T-cell receptor. However, this population of macaque T cells differed from rodent dendritic epidermal T cells in that their Vγ10/Vδ1 T-cell receptors displayed junctional diversity and expression of Vγ10 was not epidermis-specific. Therefore, macaques do not appear to have rodent-type dendritic epidermal T cells despite having apparently functional SKINT1L. Comprehensive bioinformatics analysis indicates that SKINT1L emerged in an ancestor of placental mammals but was inactivated or lost multiple times in mammalian evolution and that Skint1 arose by gene duplication in a rodent lineage, suggesting that authentic dendritic epidermal T cells are presumably unique to rodents.