Germinal centers (GCs) are distinct microanatomical structures that form in the secondary lymphoid organs of endothermic vertebrates (i.e., mammals and some birds). Within GCs, B cells undergo a ...Darwinian selection process to identify clones which can respond to pathogen insult as well as affinity mature the B cell repertoire. The GC response ultimately generates memory B cells and bone marrow plasma cells which facilitate humoral immunological memory, the basis for successful vaccination programs. GCs have not been observed in the secondary lymphoid organs of ectothermic jawed vertebrates (i.e., fishes, reptiles, and amphibians). However, abundant research over the past decades has indicated these organisms can produce antigen specific B cell responses and some degree of affinity maturation. This review examines data demonstrating that the fundamentals of B cell selection may be more conserved across vertebrate phylogeny than previously anticipated. Further, research in both conventional mammalian model systems and comparative models raises the question of what evolutionary benefit GCs provide endotherms if they are seemingly unnecessary for generating the basic functional components of jawed vertebrate humoral adaptive immune responses.
Immunological memory provides long-term protection against pathogen re-infection and is the foundation for successful vaccination. We have previously shown an antigen-specific recall response in ...nurse sharks almost one year after primary exposure. Herein, we extend the time between prime and successful recall to >8 years, the longest period for which immunological memory has been shown in any non-mammalian vertebrate. We confirm that antigen binding is mediated by monomeric IgM and IgNAR, but not pentameric IgM, in both the primary and recall phases. Our inability to find target-binding clones in recombinant VNAR expression libraries suggests that, at least in this instance, antigen-specific memory cells comprise a small fraction of the IgNAR-positive B cells in epigonal and spleen. Further, that the few memory cells present can generate a robust antigen-specific IgNAR titer following re-stimulation. Our results continue to challenge the long-held, but erroneous, belief that the shark adaptive immune system is ‘primitive’ when compared to that of mammals.
•A primary antibody response was raised in a nurse shark via immunization.•The shark was rested for 8 years without further exposure to the antigen.•Re-stimulation with soluble antigen induced a robust and specific antibody recall response.•Antigen binding is mediated by monomeric IgM and IgNAR, not pentameric IgM, in the recall phase.•This is the longest memory period recorded for any non-mammalian vertebrate to date.
Interferons orchestrate host antiviral responses in jawed vertebrates. They are categorized into three classes; IFN1 and IFN3 are the primary antiviral cytokine lineages, while IFN2 responds to a ...broader variety of pathogens. The evolutionary relationships within and between these three classes have proven difficult to resolve. Here, we reassess interferon evolution, considering key phylogenetic pitfalls including taxon sampling, alignment quality, model adequacy, and outgroup choice. We reveal that cartilaginous fishes, and hence the jawed vertebrate ancestor, possess(ed) orthologs of all three interferon classes. We show that IFN3 groups sister to IFN1, resolve the origins of the human IFN3 lineages, and find that intronless IFN3s emerged at least three times. IFN2 genes are highly conserved, except for IFN-γ-rel, which we confirm resulted from a teleost-specific duplication. Our analyses show that IFN1 phylogeny is highly sensitive to phylogenetic error. By accounting for this, we describe a new backbone IFN1 phylogeny that implies several IFN1 genes existed in the jawed vertebrate ancestor. One of these is represented by the intronless IFN1s of tetrapods, including mammalian-like repertoires of reptile IFN1s and a subset of amphibian IFN1s, in addition to newly-identified intron-containing shark IFN1 genes. IFN-f, previously only found in teleosts, likely represents another ancestral jawed vertebrate IFN1 family member, suggesting the current classification of fish IFN1s into two groups based on the number of cysteines may need revision. The providence of the remaining fish IFN1s and the coelacanth IFN1s proved difficult to resolve, but they may also be ancestral jawed vertebrate IFN1 lineages. Finally, a large group of amphibian-specific IFN1s falls sister to all other IFN1s and was likely also present in the jawed vertebrate ancestor. Our results verify that intronless IFN1s have evolved multiple times in amphibians and indicate that no one-to-one orthology exists between mammal and reptile IFN1s. Our data also imply that diversification of the multiple IFN1s present in the jawed vertebrate ancestor has occurred through a rapid birth-death process, consistent with functional maintenance over a 450-million-year host-pathogen arms race. In summary, this study reveals a new model of interferon evolution important to our understanding of jawed vertebrate antiviral immunity.
Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and ...chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more "conventional" mammalian species.
Cartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the hallmark receptor of cytotoxic T ...cells and is required for the formation of T cell receptor-major histocompatibility complex (TCR-MHC) class I complexes.
RACE PCR was used to obtain gene sequences. Direct dilution was applied for the refolding of denatured recombinant CD8 protein. Hanging-drop vapor diffusion method was performed for protein crystallization.
In this study, CD8α and CD8β orthologues (termed ScCD8α and ScCD8β) were identified in small-spotted catshark (
). Both ScCD8α and ScCD8β possess an extracellular immunoglobulin superfamily (IgSF) V domain as in previously identified CD8 proteins. The genes encoding CD8α and CD8β are tandemly linked in the genomes of all jawed vertebrates studied, suggesting that they were duplicated from a common ancestral gene before the divergence of cartilaginous fishes and other vertebrates. We determined the crystal structure of the ScCD8α ectodomain homodimer at a resolution of 1.35 Å and show that it exhibits the typical topological structure of CD8α from endotherms. As in mammals, the homodimer formation of ScCD8αα relies upon interactions within a hydrophobic core although this differs in position and amino acid composition. Importantly, ScCD8αα shares the canonical cavity required for interaction with peptide-loaded MHC I in mammals. Furthermore, it was found that ScCD8α can co-immunoprecipitate with ScCD8β, indicating that it can form both homodimeric and heterodimeric complexes.
Our results expand the current knowledge of vertebrate CD8 dimerization and the interaction between CD8α with p/MHC I from an evolutionary perspective.
The cartilaginous fishes diverged from other jawed vertebrates ~ 450 million years ago (mya). Despite this key evolutionary position, the only high-quality cartilaginous fish genome available is for ...the elephant shark (Callorhinchus milii), a chimaera whose ancestors split from the elasmobranch lineage ~ 420 mya. Initial analysis of this resource led to proposals that key components of the cartilaginous fish adaptive immune system, most notably their array of T cell subsets, was primitive compared to mammals. This proposal is at odds with the robust, antigen-specific antibody responses reported in elasmobranchs following immunization. To explore this discrepancy, we generated a multi-tissue transcriptome for small-spotted catshark (Scyliorhinus canicula), a tractable elasmobranch model for functional studies. We searched this, and other newly available sequence datasets, for CD4+ T cell subset-defining genes, aiming to confirm the presence or absence of each subset in cartilaginous fishes.
We generated a new transcriptome based on a normalised, multi-tissue RNA pool, aiming to maximise representation of tissue-specific and lowly expressed genes. We utilized multiple transcriptomic datasets and assembly variants in phylogenetic reconstructions to unambiguously identify several T cell subset-specific molecules in cartilaginous fishes for the first time, including interleukins, interleukin receptors, and key transcription factors. Our results reveal the inability of standard phylogenetic reconstruction approaches to capture the site-specific evolutionary processes of fast-evolving immune genes but show that site-heterogeneous mixture models can adequately do so.
Our analyses reveal that cartilaginous fishes are capable of producing a range of CD4+ T cell subsets comparable to that of mammals. Further, that the key molecules required for the differentiation and functioning of these subsets existed in the jawed vertebrate ancestor. Additionally, we highlight the importance of considering phylogenetic diversity and, where possible, utilizing multiple datasets for individual species, to accurately infer gene presence or absence at higher taxonomic levels.
The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing that nurse sharks can produce robust antigen-specific responses and affinity mature their B cell ...repertoires. To investigate this apparent incongruity, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5high centrocyte-like B cells and a population of putative T follicular helper (Tfh) cells, surrounded by a peripheral ring of Ki67+ AID+ CXCR4+ centroblast-like B cells. Further, we reveal selection of mutations in B cell clones dissected from these follicles. We propose that the B cell sites identified here represent the evolutionary foundation of GCs, dating back to the jawed vertebrate ancestor.
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•Shark splenic follicles share functional hallmarks of mammalian germinal centers•Intact antigen accumulates in the center of splenic follicles post-immunization•AID+ CXCR4+ centroblast-like B cells form a ring at the periphery of the follicles•A population of shark T follicular helper-like cells were also identified
Matz et al. studied the spleen of nurse sharks and found that functional hallmarks of mammalian germinal centers, including centrocyte- and centroblast-like B cells, a subset of T follicular helper-like cells, and cells that can present intact antigen, are present in the oldest jawed vertebrate lineage with adaptive immunity.
Cartilaginous fishes (sharks, skates, rays, and chimeras) are the most phylogenetically distant lineage relative to mammals in which somatically rearranging immunoglobulins (Igs or antibodies) have ...also been found. Alongside their conventional (heavy-light chain) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy-chain homodimer. Naturally lacking light chains, antigen binding is mediated by two highly soluble and independently functioning variable domains, or VNARs, each having a molecular weight of approximately 12 kDa. The small size, high affinity for antigen, and extreme structural stability of single-domain VNARs make them an emerging prospect for use in therapeutic, diagnostic, and research applications. In this chapter, we detail the immunization protocol we use to raise an antigen-specific IgNAR response in the nurse shark (Ginglymostoma cirratum), the subsequent cloning of the variable domains from this isotype, and the selection of antigen-specific VNARs by phage display.
Many animals of scientific importance lack species-specific reagents (e.g., monoclonal antibodies) for in-depth studies of immune proteins. Mass spectrometry (MS)-based proteomics has emerged as a ...useful method for monitoring changes in protein abundance and modifications in non-model species. It can be used to quantify hundreds of candidate immune molecules simultaneously without the generation of new reagents. Here, we used MS-based proteomics to identify and quantify candidate immune proteins in the plasma of the nurse shark (
), a cartilaginous fish and representative of the most basal extant vertebrate lineage with an immunoglobulin-based immune system. Mass spectrometry-based LC-MS/MS was performed on the blood plasma of nurse sharks immunized with human serum albumin (n=4) or sham immunized (n=1), and sampled at days 0 (baseline control), 1, 2, 3, 5, 7, 14, 21, 28, 25, 42 and 49. An antigen-specific antibody response was experimentally confirmed post-immunization. To provide a high-quality reference to identify proteins, we assembled and annotated a multi-tissue
transcriptome integrating long- and short-read sequence data. This comprised 62,682 contigs containing open reading frames (ORFs) with a length >80 amino acids. Using this transcriptome, we reliably identified 626 plasma proteins which were broadly categorized into coagulation, immune, and metabolic functional groups. To assess the feasibility of performing LC-MS/MS proteomics in nurse shark in the absence of species-specific protein annotations, we compared the results to an alternative strategy, mapping peptides to proteins predicted in the genome assembly of a related species, the whale shark (
). This approach reliably identified 297 proteins, indicating that useful data on the plasma proteome may be obtained in many instances despite the absence of a species-specific reference protein database. Among the plasma proteins defined against the nurse shark transcriptome, fifteen showed consistent changes in abundance across the immunized shark individuals, indicating a role in the immune response. These included alpha-2-macroglobulin (A2M) and a novel protein yet to be characterized in diverse vertebrate lineages. Overall, this study enhances genetic and protein-level resources for nurse shark research and vastly improves our understanding of the elasmobranch plasma proteome, including its remodelling following immune stimulation.
Chondrichthyes (cartilaginous fishes) are fundamental for understanding vertebrate evolution, yet their genomes are understudied. We report long-read sequencing of the whale shark genome to generate ...the best gapless chondrichthyan genome assembly yet with higher contig contiguity than all other cartilaginous fish genomes, and studied vertebrate genomic evolution of ancestral gene families, immunity, and gigantism. We found a major increase in gene families at the origin of gnathostomes (jawed vertebrates) independent of their genome duplication. We studied vertebrate pathogen recognition receptors (PRRs), which are key in initiating innate immune defense, and found diverse patterns of gene family evolution, demonstrating that adaptive immunity in gnathostomes did not fully displace germline-encoded PRR innovation. We also discovered a new toll-like receptor (TLR29) and three NOD1 copies in the whale shark. We found chondrichthyan and giant vertebrate genomes had decreased substitution rates compared to other vertebrates, but gene family expansion rates varied among vertebrate giants, suggesting substitution and expansion rates of gene families are decoupled in vertebrate genomes. Finally, we found gene families that shifted in expansion rate in vertebrate giants were enriched for human cancer-related genes, consistent with gigantism requiring adaptations to suppress cancer.
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