Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

The immunogenetics of sexual parasitism.

Sexual parasitism has evolved as a distinctive mode of reproduction among deep-sea anglerfishes. The permanent attachment of males to host females observed in these species represents a form of anatomical joining, which is otherwise unknown in nature. Pronounced modifications to immune facilities are associated with this reproductive trait. The genomes of species with temporarily attaching males lack functional aicda genes that underpin affinity maturation of antibodies. Permanent attachment is associated with additional alterations, culminating in the loss of functional rag genes in some species, abolishing somatic diversification of antigen receptor genes, the hallmark of canonical adaptive immunity. In anglerfishes, coevolution of innate and adaptive immunity has been disentangled, implying that an alternative form of immunity supported the emergence of this evolutionarily successful group of vertebrates.

Cytidine deaminase 2 is required for VLRB antibody gene assembly in lampreys.

The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage-associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage-specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.

Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity.

AID/APOBEC deaminases (AADs) convert cytidine to uridine in single-stranded nucleic acids. They are involved in numerous mutagenic processes, including those underpinning vertebrate innate and adaptive immunity. Using a multipronged sequence analysis strategy, we uncover several AADs across metazoa, dictyosteliida, and algae, including multiple previously unreported vertebrate clades, and versions from urochordates, nematodes, echinoderms, arthropods, lophotrochozoans, cnidarians, and porifera. Evolutionary analysis suggests a fundamental division of AADs early in metazoan evolution into secreted deaminases (SNADs) and classical AADs, followed by diversification into several clades driven by rapid-sequence evolution, gene loss, lineage-specific expansions, and lateral transfer to various algae. Most vertebrate AADs, including AID and APOBECs1-3, diversified in the vertebrates, whereas the APOBEC4-like clade has a deeper origin in metazoa. Positional entropy analysis suggests that several AAD clades are diversifying rapidly, especially in the positions predicted to interact with the nucleic acid target motif, and with potential viral inhibitors. Further, several AADs have evolved neomorphic metal-binding inserts, especially within loops predicted to interact with the target nucleic acid. We also observe polymorphisms, driven by alternative splicing, gene loss, and possibly intergenic recombination between paralogs. We propose that biological conflicts of AADs with viruses and genomic retroelements are drivers of rapid AAD evolution, suggesting a widespread presence of mutagenesis-based immune-defense systems. Deaminases like AID represent versions "institutionalized" from the broader array of AADs pitted in such arms races for mutagenesis of self-DNA, and similar recruitment might have independently occurred elsewhere in metazoa.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

Evolution of Alternative Adaptive Immune Systems in Vertebrates.

Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.

Characterization of Lamprey IL-17 Family Members and Their Receptors.

IL-17 is an ancient cytokine implicated in a variety of immune defense reactions. We identified five members of the sea lamprey IL-17 family (IL-17D.1, IL-17D.2, IL-17E, IL-17B, and IL-17C) and six IL-17R genes (IL-17RA.1, IL-17RA.2, IL-17RA.3, IL-17RF, IL-17RE/RC, and IL-17RD), determined their relationship with mammalian orthologs, and examined their expression patterns and potential interactions to explore their roles in innate and adaptive immunity. The most highly expressed IL-17 family member is IL-17D.1 (mammalian IL-17D like), which was found to be preferentially expressed by epithelial cells of skin, intestine, and gills and by the two types of lamprey T-like cells. IL-17D.1 binding to rIL-17RA.1 and to the surface of IL-17RA.1-expressing B-like cells and monocytes of lamprey larvae was demonstrated, and treatment of lamprey blood cells with rIL-17D.1 protein enhanced transcription of genes expressed by the B-like cells. These findings suggest a potential role for IL-17 in coordinating the interactions between T-like cells and other cells of the adaptive and innate immune systems in jawless vertebrates.

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αß and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

Selection of the lamprey VLRC antigen receptor repertoire.

The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

The T-cell receptor is not hardwired to engage MHC ligands.

The bias of αß T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

T cell receptor CDR3 loops influence alphabeta pairing.

T cell receptor transfer is an attractive strategy for the generation of antigen specific T cells to target infection and malignancy. Cross pairing of the transduced and endogenous TCR chains produces new and potentially auto-reactive specificities and dilutes the therapeutic TCR. This is further complicated as the efficiency of pairing for each alphabeta pair is unpredictable and the factors which influence it are not well characterized. Complementarity determining region 3 (CDR3) loops are the main sources of TCR alpha and beta diversity due to nucleotide insertion and deletion at V(D)J junctions. Given the variability in composition and length of these non-germ line encoded structures, it is likely that structural strain may occur during formation of some TCR hetero-dimers contributing to the observed pairing restrictions. The beta chain of the HY specific T cell receptor C6 is such an example. Despite pairing efficiently with the C6 alpha chain, it pairs poorly with many other alpha chains. To investigate whether the long, C6 beta CDR3 region underlies this effect, it was replaced with a short, artificial CDR3 region that restored efficient pairing with the endogenous alpha chain repertoire. Molecular modelling is consistent with the beta chain CDR3 region causing steric incompatibility. Despite poor pairing and low surface expression, the WT C6 beta chain mediates positive selection in retrogenic mice.

Peptide-specific, TCR-alpha-driven, coreceptor-independent negative selection in TCR alpha-chain transgenic mice.

As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCR signaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.

Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator.

We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

Discovery of potent & selective inhibitors of activated thrombin-activatable fibrinolysis inhibitor for the treatment of thrombosis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) has emerged as a key link between the coagulation and fibrinolysis cascades and represents a promising new target for the treatment of thrombosis. A novel series of imidazolepropionic acids has been designed that exhibit high potency against activated TAFI (TAFIa) and excellent selectivity over plasma carboxypeptidase N (CPN). Structure activity relationships suggest that the imidazole moiety plays a key role in binding to the catalytic zinc of TAFIa, and this has been supported by crystallographic studies using porcine pancreatic carboxypeptidase B as a surrogate for TAFIa. The SAR program led to the identification of 21 (TAFIa Ki = 10 nM, selectivity TAFIa/CPN > 1000) as a candidate for clinical development. Compound 21 exhibited antithrombotic efficacy in a rabbit model of venous thrombosis, yet had no effect on surgical bleeding in the rabbit. In addition, 21 exhibited an excellent preclinical and clinical pharmacokinetic profile, characterized by paracellular absorption, low clearance, and a low volume of distribution, fully consistent with its physicochemical properties of low molecular weight (MW = 239) and high hydrophilicity (log D = -2.8). These data indicate 21 (UK-396,082) has potential as a novel TAFIa inhibitor for the treatment of thrombosis and other fibrin-dependent diseases in humans.

Design of selective thrombin inhibitors based on the (R)-Phe-Pro-Arg sequence.

Potent and selective inhibitors of thrombin were sought based on the (R)-Phe-Pro-Arg sequence. The objective was to generate similar binding interactions to those achieved by potent competitive inhibitors of the argatroban type, so eliminating the need for covalent interaction with the catalytic serine function, as utilized by aldehyde and boronic acid type inhibitors. Improving the S(1) subsite interaction by substitution of arginine with a 4-alkoxybenzamidine residue provided potent lead 2 (K(i) = 0.37 nM). Though an amide bond, which H-bonds to the active site, is lost, modeling indicated that a new H-bond is generated between the alkoxy oxygen atom and the catalytic Ser-195 hydroxyl group. Substitution of the benzamidine system by 1-amidinopiperidine then gave compound 4, which provided a further gain in selectivity over trypsin. However, previous work had shown that these compounds were likely to be too lipophilic (Log D +0.4 and +0.2, respectively) and to suffer rapid hepatic extraction, presumably via biliary elimination. Accordingly, both proved short-acting when administered intravenously to rats and showed poor activity when given intraduodenally. The aim was then to reduce lipophilicity below a log D of -1.2, which in a previously reported series had been effective in preventing rapid clearance. It was anticipated that compounds of this type would rely on the cation selective paracellular route of absorption from the gastrointestinal tract. Potent polar analogues with selectivity >1000 over trypsin were obtained. The best in vivo activity was shown by compound 12. However, in the final analysis, its oral bioavilability proved poor, relative to analogues with similar physicochemical properties derived from argatroban, consistent with the hypothesis that molecular shape is an additional important determinant of paracellular absorption.