Negative selection of self-reactive chicken B cells requires B cell receptor signaling and is independent of the bursal microenvironment.

Although the negative selection of self-reactive B cells in the bone marrow of mammals has been clearly demonstrated, it remains unclear in models of gut-associated B cell lymphopoiesis, such as that of the chicken (Gallus gallus). We have generated chicken surface IgM-related receptors in which the diversity region of the lamprey variable lymphocyte receptor (VLR) has been fused to the C region of chicken surface IgM (Tµ). Expression of a VLR:Tµ receptor with specificity for PE supported normal development of B cells, whereas a VLR:Tµ receptor specific to hen egg lysozyme (a self-antigen with respect to chicken B cells) induced, in vivo, complete deletion of VLR(HEL)Tµ-expressing B cells. In ovo i.v. injection of PE resulted in deletion of VLR(PE)Tµ-expressing Β cells in the embryo spleen, demonstrating that negative selection was independent of the bursal microenvironment. Although chickens transduced with a murine CD8α:chicken Igα fusion protein contained B cells expressing mCD8α:chIgα, cotransfection of the mCD8α:chIgα construct, together with thymus leukemia Ag (a natural ligand for mCD8α), resulted in reduced levels of mCD8α:chIgα-expressing B cells in inverse proportion to the levels of thymus leukemia Ag-expressing cells. Deletion of mCD8α:chIgα-expressing cells was specific for B cells and required active signaling downstream of the mCD8α:chIgα receptor. Ag-mediated negative selection of developing chicken B cells can therefore occur independently of the bursal microenvironment and is dependent on signaling downstream of the BCR.

Ligation of surface Ig by gut-derived antigen positively selects chicken bursal and peripheral B cells.

In many mammals and birds, B cell lymphopoiesis takes place in GALT, such as the avian bursa of Fabricius. Although BCR expression is sufficient for bursal colonization, the role of BCR ligation in the later stages of bursal B cell lymphopoiesis remains elusive. To address this directly, we introduced a surface Ig-related construct with defined Ag specificity containing the Ag-binding portion of a lamprey variable lymphocyte receptor specific for PE fused to a truncated chicken µ-chain (VLR(PE)Tµ) into developing chick embryos. VLR(PE)Tµ expression supports bursal follicle colonization, clonal expansion, and Ig V gene diversification. VLR(PE)Tµ-expressing B cells migrate to the periphery in the absence of the Ag starting from day 18 of embryogenesis. VLR(PE)Tµ-expressing B cells declined rapidly in the bursa and periphery in the absence of Ag after hatch; however, intrabursal injection of PE prolonged survival of VLR(PE)Tµ(+) bursal and peripheral B cells. Intrabursal introduction of Ag increased emigration of short-lived LT2(+) B cells. Peripheral VLR(PE)Tµ(+) B cells were maintained following intrabursal PE application and contained both short-lived LT2(+) and long-lived LT2(-) B cells. In the chicken bursa, the later stages of B cell development occur in the presence of gut-derived Ag; therefore, we conclude that Ag-mediated ligation of BCR in bursal B cells acts to positively select bursal B cells into both short-lived and long-lived peripheral B cell populations.

Recognition of the Thomsen-Friedenreich pancarcinoma carbohydrate antigen by a lamprey variable lymphocyte receptor.

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galß1-3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the ß-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the ß-strands, requiring them to bend to match the concavity of the VLR solenoid.

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.

Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galß1-3GalNAcα), aGPA, and TFα (Galß1-3GalNAcα), with affinity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affinity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.

Yeast surface display of lamprey variable lymphocyte receptors.

The variable lymphocyte receptors (VLRs) of lamprey and hagfish comprise leucine-rich repeat modules, instead of the immunoglobulin-like domain building blocks of antibodies and T-cell receptors in jawed vertebrates. Both types of vertebrate-rearranging antigen receptors are similarly diverse, with repertoires that can potentially exceed 10(14) unique receptors. In order to characterize antigen-binding properties of the VLRs, we developed a high-throughput yeast surface display platform for the isolation of monoclonal VLRs. We have isolated VLRs that specifically bind hen egg lysozyme, ß-galactosidase, cholera toxin subunit B, R-phycoerythrin, and the blood group trisaccharides A and B, with binding affinities in the mid-nanomolar to mid-picomolar range. VLRs may, thus, be excellent single-chain alternatives to Ig-based antibodies for biotechnology applications.

A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey.

Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called "variable lymphocyte receptors" (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like gammadelta T-cell receptors (TCRs) but unlike alphabeta TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus, these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and gammadelta TCRs, and the exclusive expression on the lymphocyte surface that is unique to alphabeta and gammadelta TCRs.

Structural insights into the evolution of the adaptive immune system: the variable lymphocyte receptors of jawless vertebrates.

Adaptive immunity in jawless vertebrates is mediated by antigen receptors that are fundamentally different from those of jawed vertebrates. Whereas antibodies and T cell receptors (TCRs) are composed of immunoglobulin (Ig) domains, the variable lymphocyte receptors (VLRs) of jawless fish consist of leucine-rich repeat (LRR) modules. As with antibodies and TCRs, VLRs are assembled by DNA recombination in a process that generates a vast repertoire of receptors. VLRs recognize as diverse an array of particulate and soluble antigens as Ig-based antibodies, and do so with similar affinity and specificity. X-ray crystallographic studies of VLRs in complex with protein and carbohydrate antigens have shown that these LRR-based receptors use nearly all their concave surface to bind ligands, in addition to a highly variable loop in their C-terminal LRR capping module. This structural information, combined with a comprehensive analysis of VLR sequences, has revealed an almost perfect match between antigen-contacting positions and positions with highest sequence diversity. The independent evolution approximately 500 million years ago of LRR-based and Ig-based receptors of comparable diversity and antigen-binding properties provides evidence for the survival value of adaptive immunity in vertebrates.

High-affinity lamprey VLRA and VLRB monoclonal antibodies.

Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and beta-galactosidase (beta-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, beta-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications.

Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen.

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLR, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure combined with sequence analysis revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, it is likely that we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured by 13-fold to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase.

The variable lymphocyte receptors (VLRs) of jawless vertebrates such as lamprey and hagfish are composed of highly diverse modular leucine-rich repeats. Each lymphocyte assembles a unique VLR by rearrangement of the germline gene. In the lamprey genome, we identify here about 850 distinct cassettes encoding leucine-rich repeat modules that serve as sequence templates for the hypervariable VLR repertoires. The data indicate a gene conversion-like process in VLR diversification. Genomic analysis suggested a link between the VLR and platelet glycoprotein receptors. Lamprey lymphocytes express two putative deaminases of the AID-APOBEC family that may be involved in VLR diversification, as indicated by in vitro mutagenesis and recombination assays. Vertebrate acquired immunity could have therefore originated from lymphocyte receptor diversification by an ancestral AID-like DNA cytosine deaminase.

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.

The evolution of adaptive immunity.

Approximately 500 mya two types of recombinatorial adaptive immune systems appeared in vertebrates. Jawed vertebrates generate a diverse repertoire of B and T cell antigen receptors through the rearrangement of immunoglobulin V, D, and J gene fragments, whereas jawless fish assemble their variable lymphocyte receptors through recombinatorial usage of leucine-rich repeat (LRR) modular units. Invariant germ line-encoded, LRR-containing proteins are pivotal mediators of microbial recognition throughout the plant and animal kingdoms. Whereas the genomes of plants and deuterostome and chordate invertebrates harbor large arsenals of recognition receptors primarily encoding LRR-containing proteins, relatively few innate pattern recognition receptors suffice for survival of pathogen-infected nematodes, insects, and vertebrates. The appearance of a lymphocyte-based recombinatorial system of anticipatory immunity in the vertebrates may have been driven by a need to facilitate developmental and morphological plasticity in addition to the advantage conferred by the ability to recognize a larger portion of the antigenic world.

Diversity and function of adaptive immune receptors in a jawless vertebrate.

Instead of the immunoglobulin-type antigen receptors of jawed vertebrates, jawless fish have variable lymphocyte receptors (VLRs), which consist of leucine-rich repeat (LRR) modules. Somatic diversification of the VLR gene is shown here to occur through a multistep assembly of LRR modules randomly selected from a large bank of flanking cassettes. The predicted concave surface of the VLR is lined with hypervariable positively selected residues, and computational analysis suggests a repertoire of about 10(14) unique receptors. Lamprey immunized with anthrax spores responded with the production of soluble antigen-specific VLRs. These findings reveal that two strikingly different modes of antigen recognition through rearranged lymphocyte receptors have evolved in the jawless and jawed vertebrates.

Variable lymphocyte receptors in hagfish.

A previously uncharacterized type of variable lymphocyte receptors (VLR) was identified recently in the Sea lamprey. This jawless vertebrate generates an extensive VLR repertoire through differential insertion of neighboring diverse leucine-rich repeat (LRR) cassettes into an incomplete germ-line VLR gene. We report here VLR homologs from two additional lamprey species and the presence of two types of VLR genes in hagfish, the only other order of contemporary jawless vertebrates. As in the Sea lamprey, the incomplete hagfish germ-line VLR-A and -B genes are modified in lymphocyte-like cells to generate highly diverse repertoires of VLR-A and -B proteins via a presently undetermined mechanism. This jawless-fish mode of VLR diversification starkly contrasts with the rearrangement of Ig V(D)J gene segments used by all jawed vertebrates to produce diverse repertoires of T and B lymphocyte antigen receptors. The development of two very different strategies for receptor diversification at the dawn of vertebrate evolution approximately 500 million years ago attests to the fitness value of a lymphocyte-based system of anticipatory immunity.

Variable domains and a VpreB-like molecule are present in a jawless vertebrate.

Immunoglobulins (Igs) and T cell antigen receptors (TCRs) that undergo somatic diversification have not been identified in the two extant orders of jawless vertebrates, which occupy essential positions in terms of understanding the evolution of the emergence of adaptive immunity. Using a single motif-dependent PCR-based approach coupled with a vector that allows selection of cDNAs encoding secretion signal sequences, four different genes encoding Ig V-type domains were identified in the sea lamprey (Petromyzon marinus). One of the predicted proteins encoded by these genes shares structural characteristics with mammalian VpreB molecules, including the absence of a recognizable transmembrane region, a relatively high proportion of charged amino acids in its C-terminal tail and distinctive features of its secretion signal peptide. This is the first indication of a molecule related to the B cell receptor (BCR) complex in a species that diverged prior to the jawed vertebrates in which RAG-mediated adaptive immunity is first encountered.

Prototypic T cell receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey.

All jawed vertebrates have highly diverse lymphocyte receptors, which allow discrimination between self and nonself antigens as well as the recognition of potential pathogens. Key elements of the anticipatory recombinatorial immune system in jawed vertebrates are the TCR, Ig, and MHC genes, but their ancestral genes have not been found in more basal vertebrates. In this study, we extended our analysis of the transcriptome of lymphocyte-like cells in the lamprey to identify the TCR-like and CD4-like genes. The structural features of these genes and their preferential expression in lymphocytes make them attractive candidates for ancestral TCR and CD4 genes. The TCR-like gene contains both V (variable) and J (joining) sequences in its first exon and exists as a single-copy gene that is invariant. Thus, the TCR-like gene cannot account for the receptor diversity that is required for the immune responses reported for lamprey, but it could have been easily modified to serve as an evolutionary precursor of modern TCR and Ig genes.