Phylotranscriptomics suggests the jawed vertebrate ancestor could generate diverse helper and regulatory T cell subsets

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Cartilaginous fish; Elasmobranch; Immune gene evolution; Immunity; Interleukin; Phylogenetic rooting; Shark; Site-heterogeneous models; T cell; Transcriptome.

Fig. 1

Summary of the presence/absence of major mammalian CD4+ T-cell lineages and associated genes in the jawed vertebrate ancestor. The figure and gene selection are based on Fig. 5 from Venkatesh et al. [5]. Boxed lineages were predicted to have emerged in the ancestor of jawed vertebrates by Venkatesh et al. [5] (black boxed lineages), or by Dijkstra [18] (blue boxed lineages). Crossed out genes are those thought to be absent from cartilaginous fishes and the vertebrate ancestor, while blue encircled genes are those that Dijkstra later predicted to in fact be present [18]. Dotted box/circle edges indicate uncertainty of gene or lineage presence(e.g. for IL-2R, FOXP3 [18])

Fig. 2

Posterior predictive simulations show that standard models, but not site-heterogeneous mixture models, inadequately capture site-specific biochemical diversity in all tested vertebrate immune genes

Fig. 3

Phylogenetic analysis of the IL-2 superfamily. Branches are coloured according to the taxonomic key in the figure. Statistical support is shown for key nodes as per accompanying box, wherein the analysis shown in bold is the topology shown. Root Posterior Probabilities (RPP) > 0.05 from the BEAST analysis are shown

Fig. 4

Phylogenetic analyses of the (a) IL-7/9 family and the (b) IL-4/IL-13 family. Outgroup sequences are from human. BPP and RPP values are from analyses not including the outgroups. Other details as per Fig. 3

Fig. 5

Phylogenetic analysis of the IL-6 superfamily reveals orthologues of IL-23α (p19), IL-27α (p28), and IL-11 in cartilaginous fishes. Details as per Fig. 3

Fig. 6

Phylogenetic analysis of the (a) IL-6Rα family, and the (b) IL2Rα/IL-15Rα family. Details as per Fig. 3

Fig. 7

Phylogenetic analysis of class 1 group 2 cytokine receptors reveals an IL-23R orthologue in cartilaginous fishes. Details as per Fig. 3

Fig. 8

Phylogenetic analyses of the vertebrate ROR family shows that ROR-γ existed in the jawed vertebrate ancestor and reveals a new vertebrate ROR-β paralog not found in mammals (which we name ROR-δ). Alternative rooting strategies, using (a) a relaxed clock model, (b) fruit fly HR3 as outgroup, or (c) human RARs as outgroup, show that the root of the ROR phylogeny cannot be confidently placed. Gene level clades are collapsed in (b) and (c), but contain the same taxa as a Other details as per Fig. 3

Fig. 9

Phylogenetic analyses of the vertebrate FOXP family verifies the existence of cartilaginous fish orthologues to FOXP1–4, but alternative rooting strategies, using (a) a relaxed clock model, or (b) invertebrate FOXP sequences as an outgroup, show that the root of the FOXP phylogeny cannot be confidently placed. Other details for (a) and (b) as per Figs. 3 and 6. (c) Alignment of the FOXP3 DNA-binding domain from phylogenetically representative vertebrates suggests that cartilaginous fish FOXP3 is not atypical

Fig. 10

A full set of T helper and T regulatory cell associated genes existed in the jawed vertebrate ancestor. The figure and gene selection are based on Fig. 5 from Venkatesh et al. [5], but here refer to the ancestral jawed vertebrate gene set rather than that of cartilaginous fishes. Boxed lineages were predicted to have emerged in the ancestor of jawed vertebrates by Venkatesh et al. [5] (black boxed lineages), by this study (red boxed lineages), or by this study and Dijkstra [18] (blue boxed lineages). All genes listed, except for IL-9 and IL-2Rα, have now been identified in cartilaginous fishes