Ancient Use of Ig Variable Domains Contributes Significantly to the TCRδ Repertoire.

The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCRδ rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCRδ repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCRδ rearrangements. Furthermore, the Ig-TCRδ rearrangements are expressed with TCRγ, canonically found in the TCRδ heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCRδC), and canonical (TCRδV-TCRδC) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCRαδ locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCRδ locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCRδ translocus, grouped with IgHV-like rearrangements we found expressed with TCRδ (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCRδ-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCRδ repertoire employing TCRδVs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCRδ-associated Ig-like V segments.

Genomic organization of the zebrafish (Danio rerio) T cell receptor alpha/delta locus and analysis of expressed products.

In testing the hypothesis that all jawed vertebrate classes employ immunoglobulin heavy chain V (IgHV) gene segments in their T cell receptor (TCR)δ encoding loci, we found that some basic characterization was required of zebrafish TCRδ. We began by annotating and characterizing the TCRα/δ locus of Danio rerio based on the most recent genome assembly, GRCz10. We identified a total of 141 theoretically functional V segments which we grouped into 41 families based upon 70 % nucleotide identity. This number represents the second greatest count of apparently functional V genes thus far described in an antigen receptor locus with the exception of cattle TCRα/δ. Cloning, relative quantitative PCR, and deep sequencing results corroborate that zebrafish do express TCRδ, but these data suggest only at extremely low levels and in limited diversity in the spleens of the adult fish. While we found no evidence for IgH-TCRδ rearrangements in this fish, by determining the locus organization we were able to suggest how the evolution of the teleost α/δ locus could have lost IgHVs that exist in sharks and frogs. We also found evidence of surprisingly low TCRδ expression and repertoire diversity in this species.

Mutation rate and spectrum in obligately outcrossing Caenorhabditis elegans mutation accumulation lines subjected to RNAi-induced knockdown of the mismatch repair gene msh-2.

DNA mismatch repair (MMR), an evolutionarily conserved repair pathway shared by prokaryotic and eukaryotic species alike, influences molecular evolution by detecting and correcting mismatches, thereby protecting genetic fidelity, reducing the mutational load, and preventing lethality. Herein we conduct the first genome-wide evaluation of the alterations to the mutation rate and spectrum under impaired activity of the MutSα homolog, msh-2, in Caenorhabditis elegans male-female fog-2(lf) lines. We performed mutation accumulation (MA) under RNAi-induced knockdown of msh-2 for up to 50 generations, followed by next-generation sequencing of 19 MA lines and the ancestral control. msh-2 impairment in the male-female background substantially increased the frequency of nuclear base substitutions (∼23×) and small indels (∼328×) relative to wildtype hermaphrodites. However, we observed no increase in the mutation rates of mtDNA, and copy-number changes of single-copy genes. There was a marked increase in copy-number variation of rDNA genes under MMR impairment. In C. elegans, msh-2 repairs transitions more efficiently than transversions and increases the AT mutational bias relative to wildtype. The local sequence context, including sequence complexity, G + C-content, and flanking bases influenced the mutation rate. The X chromosome exhibited lower substitution and higher indel rates than autosomes, which can either result from sex-specific mutation rates or a nonrandom distribution of mutable sites between chromosomes. Provided the observed difference in mutational pattern is mostly due to MMR impairment, our results indicate that the specificity of MMR varies between taxa, and is more efficient in detecting and repairing small indels in eukaryotes relative to prokaryotes.

Bergerac strains of Caenorhabditis elegans revisited: expansion of Tc1 elements imposes a significant genomic and fitness cost.

The DNA transposon Tc1 was the first transposable element to be characterized in Caenorhabditis elegans and to date, remains the best-studied transposable element in Caenorhabditis worms. While Tc1 copy-number is regulated at approximately 30 copies in the laboratory Bristol N2 and the vast majority of C. elegans strains, the Bergerac strain and its derivatives have experienced a marked Tc1 proliferation. Given the historical importance of the Bergerac strain in the development of the C. elegans model, we implemented a modern genomic analysis of three Bergerac strains (CB4851, RW6999, and RW7000) in conjunction with multiple phenotypic assays to better elucidate the (1) genomic distribution of Tc1 and (2) phenotypic consequences of transposable element deregulation for the host organism. The median estimates of Tc1 copy-number in the Bergerac strains ranged from 451 to 748, which is both (1) greater than previously estimated and (2) likely to be an underestimate of the actual copy-numbers since coverage-based estimates and digital droplet polymerase chain reaction results both suggest higher Tc1 numbers. All three Bergerac strains had significantly reduced trait means compared with the N2 control for each of four fitness-related traits, with specific traits displaying significant differences between Bergerac strains. Tc1 proliferation was genome-wide, specific to Tc1, and particularly high on chromosomes V and X. There were fewer Tc1 insertions in highly expressed chromatin environments than expected by chance. Furthermore, Tc1 integration motifs were also less frequent in exon than noncoding sequences. The source of the proliferation of Tc1 in the Bergerac strains is specific to Tc1 and independent of other transposable elements. The Bergerac strains contain none of the alleles that have previously been found to derepress transposable element activity in C. elegans. However, the Bergerac strains had several Tc1 insertions near or within highly germline-transcribed genes which could account for the recent germline proliferation.

Using PacBio SMRT data for identification of class I MHC alleles in a wildlife species, Zalophus californianus (California sea lion).

High allelic polymorphism and association with disease susceptibility has made the genes encoding major histocompatibility complex (MHC) antigen presentation molecules in humans, domesticated animals, and wildlife species of wide interest to ecologists, evolutionary biologists, and health specialists. The often multifaceted polygenism and extreme polymorphism of this immunogenetic system have made it especially difficult to characterize in non-model species. Here we compare and contrast the workflows of traditional Sanger sequencing of plasmid-cloned amplicons to Pacific Biosciences SMRT circular consensus sequencing (CCS) in their ability to capture alleles of MHC class I in a wildlife species where characterization of these genes was absent. We assessed two California sea lions (Zalophus californianus), a species suffering from a high prevalence of an aggressive cancer associated with a sexually transmitted gamma herpesvirus. In this pilot study, SMRT CCS proved superior in identifying more alleles from each animal than the more laborious plasmid cloning/Sanger workflow (12:7, 10:7), and no alleles were identified with the cloning/Sanger approach that were not identified by SMRT CCS. We discuss the advantages and disadvantages of each approach including cost, allele rarefaction, and sequence fidelity.

A Broad Role for Cysteines in Bovine Antibody Diversity.

Ab diversity in most vertebrates results from the assortment of amino acid side chains on CDR loops formed through V(D)J recombination. Cows (Bos taurus) have a low combinatorial diversity potential because of a small number of highly homologous V, D, and J gene segments. Despite this, a subset of the Ab repertoire (∼10%) contains exceptionally long CDR H chain (HC) 3 (H3) regions with a rich diversity of cysteines and disulfide-bonded loops that diversify through a single V-D-J recombination event followed by massive somatic hypermutation. However, the much larger portion of the repertoire, encoding shorter CDR H3s, has not been examined in detail. Analysis of germline gene segments reveals noncanonical cysteines in the HC V regions and significant cysteine content in the HC D regions. Deep sequencing analysis of naturally occurring shorter CDR H3 (<40 aa) Ab genes shows that HC V and HC D regions preferentially combine to form a functional gene with an even number of total cysteines in the final V region, suggesting that disulfide bonds contribute to diversity not only in ultralong CDR H3 bovine Abs but in shorter CDR H3 bovine Abs as well. In addition to germline "hard-coded" cysteines, the bovine Ab repertoire can produce additional cysteine codons through somatic hypermutation, further diversifying the repertoire. Given the limited combinatorial diversity at the bovine Ig loci, this helps to explain how diversity is created in shorter CDR H3 Abs and potentially provides novel structural paratopes in bovine Ab combining sites.

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique ß-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F ß-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

The Unusual Genetics and Biochemistry of Bovine Immunoglobulins.

Antibodies are the key circulating molecules that have evolved to fight infection by the adaptive immune system of vertebrates. Typical antibodies of most species contain six complementarity-determining regions (CDRs), where the third CDR of the heavy chain (CDR H3) has the greatest diversity and often makes the most significant contact with antigen. Generally, the process of V(D)J recombination produces a vast repertoire of antibodies; multiple V, D, and J gene segments recombine with additional junctional diversity at the V-D and D-J joints, and additional combinatorial possibilities occur through heavy- and light-chain pairing. Despite these processes, the overall structure of the resulting antibody is largely conserved, and binding to antigen occurs predominantly through the CDR loops of the immunoglobulin V domains. Bovines have deviated from this general paradigm by having few VH regions and thus little germline combinatorial diversity, but their antibodies contain long CDR H3 regions, with substantial diversity generated through somatic hypermutation. A subset of the repertoire comprises antibodies with ultralong CDR H3s, which can reach over 70 amino acids in length. Structurally, these unusual antibodies form a ß-ribbon "stalk" and disulfide-bonded "knob" that protrude far from the antibody surface. These long CDR H3s allow cows to mount a particularly robust immune response when immunized with viral antigens, particularly to broadly neutralizing epitopes on a stabilized HIV gp140 trimer, which has been a challenge for other species. The unusual genetics and structural biology of cows provide for a unique paradigm for creation of immune diversity and could enable generation of antibodies against especially challenging targets and epitopes.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αß T cell repertoire in sharks, the first reported use in vertebrates.

The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity.

Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity.

DNP-KLH Yields Changes in Leukocyte Populations and Immunoglobulin Isotype Use with Different Immunization Routes in Zebrafish.

Distinct methods are required for inducing mucosal versus systemic immunity in mammals for vaccine protection at the tissues most commonly breached by pathogens. Understanding of mucosal immunization in teleost fish is needed to combat aquaculture disease, understand emerging ecological threats, and know how vertebrate adaptive immunity evolved. Here, we quantitatively measured expression levels of IgM as well as the teleost mucosal immunoglobulin, IgZ/IgT, in zebrafish given an antigen systemically via intraperitoneal (i.p.) injection or mucosally via bath immersion. Both immunoglobulin isotypes and the B cell activating factor gene transcription was induced in fish injected with antigen as compared to saline injected or antigen immersed fish, though these failed to reach statistical significance. Here we provide additional reference hematology for this model species. Differential blood counts revealed a greater lymphocyte percentage in both i.p. and immersed fish, with increase in large lymphocyte counts and decrease in neutrophils. These humoral adaptive gene transcription and cytological data should provide a foundation for more studies connecting immunology in this dominant developmental and genetic fish model to other species where mucosal immunization is of greater commercial importance.

Expressed IgH µ and τ transcripts share diversity segment in ranched Thunnus orientalis.

It is now appreciated that in addition to the immunoglobulin (Ig)M and D isotypes fish also make the mucosal IgT. In this study we sequenced the full length of Ig τ as well as µ in the commercially important Thunnus orientalis (Pacific bluefin tuna), the first molecular analysis of these two Ig isotypes in a member of the order Perciformes. Tuna IgM and IgT are each composed of four constant (CH) domains. We cloned and sequenced 48 different variable (VH) domain gene rearrangements of tuna immunoglobulins and grouped the VH gene sequences to four VH gene segment families based on 70% nucleotide identity. Three VH gene families were used by both IgM and IgT but one group was only found to be used by IgM. Most interestingly, both µ and τ clones appear to use the same diversity (DH) segment, unlike what has been described in other species, although they have dedicated IgT and IgM joining (JH) gene segments. We complemented this repertoire study with phylogenetic and tissue expression analysis. In addition to supporting the development of humoral vaccines in this important aquaculture species, these data suggest that the DH-JH recombination rather than the VH-DH recombination may be instructive for IgT versus IgM/D bearing lymphocyte lineages in some fish.