All jawed vertebrates share fundamental components of the adaptive immune system. The cartilaginous fish (including sharks) are the most divergent jawed vertebrate group relative to mammals and use a polymorphic major histocompatibility complex (MHC) (Kasahara et al., 1992), multiple isotypes of immunoglobulin (Ig) heavy and light chains (Flajnik, 2002; Criscitiello and Flajnik, 2007), and the typical four T-cell receptor (TcR) chains (Rast et al., 1997). Shark lymphocyte antigen receptors are diversified by RAG-mediated V(D)J somatic rearrangement (Bernstein et al., 1994). After antigen exposure, B cells also use the enzyme activation-induced cytidine deaminase (AID) for receptor modification via somatic hypermutation (SHM) (Conticello et al., 2005), allowing activated B cells to extensively alter their rearranged Ig variable region genes (Muramatsu et al., 2000). Some of the variants produced by this process bind antigen with higher affinity, enhancing humoral immunity through affinity maturation. In addition to SHM in all jawed vertebrate Igs, AID catalyzes the processes of heavy chain class switch recombination (CSR) in tetrapods (and is implicated in shark CSR [Zhu et al., 2012]) and Ig gene conversion (in birds and some mammals) (Barreto and Magor, 2011). AID is a member of the APOBEC family of nucleic acid mutators, two of which likely diversify the variable lymphocyte receptor (VLR) system in the more ancient vertebrate lineages of lamprey and hagfish (Alder et al., 2005; Guo et al., 2009).

Although in general immunologists think TcR loci do not undergo somatic hypermutation, a few reports do exist of AID-mediated SHM in T cells. However, non-productive TcRα rearrangements in hybridomas (Marshall et al., 1999), TcRβ sequences from HIV-positive individuals (Cheynier et al., 1998), and reports of SHM in TcRα murine germinal center T cells (Zheng et al., 1994) are not thought to describe any normal physiology (Bachl and Wabl, 1995). More recent studies indicate that TcR δ and γ in the dromedary camel and TcR γ in the sandbar shark somatically hypermutate (see below) (Antonacci et al., 2011; Chen et al., 2012). Despite these findings, the general consensus has remained that AID does not target TcR loci (Pavri and Nussenzweig, 2011; Choudhary et al., 2018). In over 30 years of studies of TcR repertoires, it has been clear that SHM is not functioning to either generate or further enhance the TcR repertoire of mouse and human.

Recent studies in the sandbar shark (Carcharhinus plumbeus) revived the notion of SHM at TcR loci. Sequencing of the entire TcRγ translocon in C. plumbeus showed definitively that SHM is occurring at that locus (Chen et al., 2009). Shark TcRγ SHM occurs in two distinct patterns: point mutations and tandem mutations characteristic of B cell SHM in cartilaginous fish (Anderson et al., 1995; Lee et al., 2002; Rumfelt et al., 2002; Zhu et al., 2012), possibly suggesting two different cellular mechanisms for generating mutations (Chen et al., 2012). The sandbar shark analysis found targeted nucleotide motifs of AID activity at the TcRγ locus. Chen et al. (2012) examined ratios of replacement (R) and silent (S) mutations between CDR and framework regions to determine if mutation altered affinity of receptors, a method commonly used to study B cell affinity maturation by SHM. Finding no difference between R/S ratios in CDR versus framework regions, they concluded that TcRγ uses SHM to generate a more diverse repertoire rather than for affinity maturation. SHM-induced changes to TcRδ in camels showed similar results.

Early work in our lab also suggested that SHM occurs in the less restricted γδ T cells in nurse shark (Ginglymostoma cirratum) and perhaps in the alpha chain of MHC-restricted αβ T cells (Criscitiello et al., 2010), encouraging us to examine this phenomenon further. Thus, we performed a systematic analysis of this process in shark primary and secondary lymphoid tissues using thymocyte clones containing the same unique third complementarity-determining region (CDR3). Our data suggest that SHM of TcRα is involved in primary T cell repertoire diversification and the enhancement of positive or negative selection in the thymic cortex. This finding is consistent with a model put forward by Niels Jerne over 40 years ago to explain antigen receptor positive selection (Jerne, 1971).

The role and diversifying mechanisms of SHM in B cells are well known (Li et al., 2004), as are the consequences of off-target AID activity (Álvarez-Prado et al., 2018). In B cells, AID mediates SHM within germinal centers of lymph nodes and spleen in mammals (Crouch et al., 2007), and we predict this is similarly occurring in the B cells zones identified in the shark splenic white pulp (Rumfelt et al., 2002). Somatic mutations occur in rearranged variable regions of B cells responding to antigen at rates of 10⁻³ mutations per base pair per cell division (Odegard and Schatz, 2006). These changes are dominated by point mutations (and in shark, tandem mutations), biased toward transitions (G:A and C:T), and preferentially targeted to the AID motifs DGYW/WRCH (and less to WA/TW) (Li et al., 2004; Malecek et al., 2005; Odegard and Schatz, 2006; Rogozin and Diaz, 2004). The sequences of B cell V genes have evolved to maximize mutational effects, targeting the accumulation of replacement mutation within the antigen-binding CDRs and limiting mutation within the more structural FRs. In humans, this focused mutation correlates with the long-term survival of B cell receptor repertoires (Saini and Hershberg, 2015). The ability of B cells to use SHM for receptor diversification and improved antigen affinity is the basis of adaptive immunity (Saini and Hershberg, 2015). Despite having similar developmental machinery as B cells (Gellert, 2002), the assumption has long been held that αβ T cells do not undergo SHM because mutation could have deleterious effects on the binding of TcRs to MHC:Ag complexes (MHC:Ag) (Wagner et al., 1995; Mantovani et al., 2002). Despite some suggestion that SHM was occurring in T cells, studies designed to either quantify or characterize mutation in mouse or human TcRs did not gain traction and the textbook definition of SHM still defines it as an exclusively B cell mechanism (Murphy and Weaver, 2017).

Recent studies reported the incidence of SHM in the γ chain of γδ T cells of sandbar shark (Chen et al., 2012; Chen et al., 2009) and in both γ and δ chains of dromedary camel (Antonacci et al., 2011; Ciccarese et al., 2014; Vaccarelli et al., 2012). In each study, SHM mirrored the mutational patterns observed in B cells during affinity maturation. However, in both sandbar shark γ and dromedary camel γ and δ chains, the authors hypothesized that T cells employ mutation as a means to generate a more diverse receptor repertoire rather than to improve receptor affinity to Ag (Chen et al., 2012; Antonacci et al., 2011; Vaccarelli et al., 2012). In contrast to αβ T cells, γδ T cells that interact with non-classical MHC often recombine tissue-specific, restricted sets of genes that have limited junctional diversity (Allison et al., 2001; Adams et al., 2005). Thus, it is reasonable to consider that SHM could be used as a receptor-diversifying mechanism to fine-tune ligand recognition within a prospective tissue or to allow changes within the loci that allow receptors to evolve more rapidly to changing ligand environments (Adams et al., 2005; Kazen and Adams, 2011). Further, many γδ T cells typically bind Ag in a manner more similar to that of Ig than to αβ T cells, recognizing and directly binding to small molecules and intact proteins without presentation by classical MHC:Ag complexes (Adams et al., 2005; Allison and Garboczi, 2002; Allison et al., 2001). Inflammation stimulates activation of γδ T cells earlier in an immune response, releasing pro-inflammatory cytokines and killing infected macrophages. Thus, γδ T cells combine an innate-like response with an adaptive recognition strategy, providing both an immediate response to pathogen invasion and an ongoing, adaptive response to inflammation (Adams et al., 2005; Allison and Garboczi, 2002; Allison et al., 2001). It is evident that how SHM presents a useful solution for accomplishing these tasks by creating a more diverse repertoire of these antibody-like γδ TcRs. Taken together, these studies clearly demonstrate that we can no longer regard SHM as a uniquely B cell mechanism. Considering the diversity of TcRs and TcR diversification mechanisms being found even in mammals (Hansen and Miller, 2015; Miller, 2010), perhaps we should prepare for more surprises in TCR antigen recognition.

In the present study, we verified SHM occurring within the γ and δ chains of γδ T cells in both thymic and peripheral immune tissue of nurse shark. Remarkably, we also detected SHM occurring in the α chain of αβ T cells. We observed mutational characteristics within α chain of nurse sharks similar to those found in B cell SHM. We observed an overall mutation frequency of 0.0226 substitutions per nucleotide (S/N) and a bias toward transition mutations. Further, we detected both single and tandem mutations, a pattern unique to sharks that also occurs in shark B cells. Changes to G and C nucleotides comprise 66.1% of all mutations. Mutation was twice as frequent in CDRs as in FRs (0.0352 versus 0.0188 S/N, respectively), and substitutions in CDRs were significantly more likely to result in amino acid changes. Further, mutations were strongly associated with AID hotspots, and substitutions to G and C nucleotides occurred nearly 1.4x as often within CDR hotspots than FR hotspots. Out of curiosity, we compared counts of AID hotspot motifs within CDR and FR regions between our 11 nurse shark TcR αV consensus sequences and 6 human TcR αV segments (V1.1, V1.2, V2, V3, V4 and V5). We found that shark TcR V segments exhibit far more WRCH/DGYW motifs per sequence than do human V segments (p=0.02). Further, motifs in CDRs of shark occurred 2-3x as often as in humans [human: average of 2.27 motifs per FR (range 1.97–2.65), 2.28 per CDR (range 1.97–2.59); shark: average of 3.25 motifs per FR (range 2.85–3.88), 5.09 per CDR (range 4.02–6.16); data not shown]. The bias we found for nonsynonymous and non-conservative mutations in TcRα CDRs in the shark thymus are consistent with more than simple repertoire diversification; it suggests selection for changes in paratope.

We identified SHM from identical cDNA clones originating from both thymus and spiral valve tissues (see Table 6), suggesting that T cells with SHM-modified receptors must have originated within the thymus and then traveled to peripheral gut-associated lymphoid tissue. Unsurprisingly, we detected the most AID expression within the inner cortex, medulla, and CMJ of shark thymus, where rearrangement and testing of TcRα takes place in mammals. Positive selection on self-MHC/self peptide for mature thymocytes begins with the CD4/CD8 double positive (DP) stage of development while differentiation into CD4/CD8 single positive (SP) cells requires that the TcR interact with MHC (Huesmann et al., 1991). If there is no TcR: MHC/peptide match found, T cell differentiation stalls with failure to be positively selected (Reinherz et al., 1999). However, the unusual nature of the TcRα locus, with up to 100 J segments depending on species, permits multiple successive rearrangements within a single cell, rescuing non-productive or self-selectable receptors with further gene rearrangements, a process called receptor editing (Bedel et al., 2012; Borgulya et al., 1992; Guo et al., 2002; Petrie et al., 1993). In mice, unlike the situation in developing B cells, receptor editing does not seem to rescue T cells from negative selection (Kreslavsky et al., 2013) and thus provides several opportunities for positive selection of DP thymocytes. Thymic nurse cells may help optimize these opportunities for selection by providing microenvironments favorable to secondary alpha chain rearrangement (Nakagawa et al., 2012).

In developing shark thymocytes, SHM in TcRα loci in conjunction with receptor editing (note that sharks, like all other gnathostomes, have a large number of TcRα J segments) could be involved in salvaging cells for positive selection or rescuing cells from death by negative selection. If AID-induced SHM occurs in conjunction with receptor editing and positive selection, AID should be upregulated in cells undergoing RAG-mediated alpha rearrangement (and thus in cells also expressing RAG). However, if SHM occurs after rearrangement of TcR α and thus used for rescuing cells during negative selection, the same T cell would not express both AID and RAG. While we cannot determine conclusively without RAG expression data, the patterns of AID and TcRα expression (Figure 9) suggest that AID is upregulated after cells proliferate and diversify following alpha rearrangement (within the ‘ring’ of cells). Thus, it is likely that AID is used primarily to rescue cells from negative selection, providing a ‘mini-expanded self-referential repertoire’ (Figure 10) and reducing the ‘profligate waste of thymocytes’ (Murphy and Weaver, 2017). However, based on the works above by Kreslavsky et al. and Nakagawa et al. in mice, we cannot discount the possibility that TcRs use SHM in conjunction with receptor editing for positive selection since developing shark T cells could still undergo negative selection after SHM (Figure 10). We cannot completely rule out AID use in mature shark T cells, although our sequence data show no greater mutation frequency in the periphery, and abatement of AID expression in the thymic medulla are consistent with AID being a mechanism used only in T cell development. Further studies examining expression data from single cells could elucidate the timing of AID-catalyzed SHM in relation to T cell development.

Figure 10

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These results are not without precedent. Qin et al. (2011) reported endogenous AID expression by peripheral CD4⁺ T cells and immature B cells in mice. T cells that expressed AID also produce a distinctive cytokine profile, are associated with cell activation, and increase in abundance with age, suggesting these cells have distinctive long-term functions in aging cells. In immature B cells, AID expression may help negatively select autoreactive B cells and contribute to primary repertoire diversity. Together these results may indicate that AID expression in mice is a relic of a more extended expression in earlier species (Qin et al., 2011). Some mammals (e.g. rabbit, sheep, and cattle) use SHM in B cell primary repertoire diversification in the gut associated lymphoid tissues (Alitheen et al., 2010; Archer et al., 1963; Becker and Knight, 1990; Butler et al., 2011; Lanning et al., 2000; Reynaud et al., 1995; Reynaud et al., 1991; Reynolds and Morris, 1983). Even more recently, research implicated AID in central B cell tolerance in mammalian pre-B cells (Cantaert et al., 2015; Kuraoka et al., 2011), and there are examples of AID-mediated SHM being used alongside RAG-mediated V(D)J recombination in primary B cell repertoire generation in many mammals and in AID-mediated primary B cell diversification during gene conversion in the bird bursa of Fabricius (Reynaud et al., 1987; Thompson and Neiman, 1987). We suggest that these B-cell-specific features are much later mechanisms of AID-driven primary lymphocyte repertoire diversification and honing, perhaps mechanisms convergent with the thymic process we describe here. However, we predict that studies of this type in other vertebrate species will reveal the use of SHM as a T cell diversifying mechanism in a much broader collection of species.

Although recent studies report B cells present in the thymus of mice (Perera and Huang, 2015; Perera et al., 2013), Miracle et al. examined B cell expression levels (IgM and IgX) in various tissues (including thymus) at different ages in the clearnose skate. While skate thymus expressed both IgM and IgX in early life stages, adult skates no longer express Ig in the thymus (Miracle et al., 2001). Criscitiello and Flajnik, 2007 also found little evidence that Ig light chain is expressed in adult nurse shark thymus (see Fig S3 of paper), corroborating older northern blot studies with heavy chain in the species (Rumfelt et al., 2001; Rumfelt et al., 2004). A concurrent study within our lab used RT-qPCR to analyze IgM expression within shark thymus and found that, while young sharks do express IgM in the thymus, adult nurse sharks (which we used in the current study) do not express IgM (data not shown). Further, preliminary results using FISH probes to IgM constant region also indicate that adult nurse sharks do not express IgM in the thymus. What little Ig expression we found in the thymus did not co-localize with AID expression.

Shark TcRs are capable of a wide variety of diversification mechanisms. In addition to RAG-mediated combinatorial and junctional diversity from complex loci, TcRδ (and possibly α) rearrange Ig-TcR chimera using IgM and IgW V exons with TcR D-J-C (Criscitiello et al., 2010). The TcR δ locus also encodes the doubly rearranging NAR-TcR, a δ chain with two diverse V domains (Criscitiello et al., 2006). Interestingly, our existing data do not suggest that SHM targets the IgHV or the TcR δV of the NAR-TcR δV chains for diversification. This suggests great control over the (nearly synchronous if not concomitant) expression of the potentially genotoxic AID and RAG. Thus, SHM at the γ, δ, and α loci adds to the battery of extraordinary diversification mechanisms used by shark lymphocytes in antigen recognition, although we do not yet understand the full effects of SHM on the animal’s immunity to infectious disease. As for the dangers of any aberrant mutational activity genome-wide, sharks could be more resilient than other taxa due to their inherent slow rate of mutation (Martin, 1999), possibly linked to the exceptional longevity of some individuals (Nielsen et al., 2016).

In the broader scope of lymphocyte evolution, we must consider whether the ancestral vertebrate lymphocyte employed APOBEC-family-mediated diversification before the ‘big bang’ of RAG (reviewed in [Hirano, 2015]). Lamprey lymphocytes express at least two lymphocyte-specific cytidine deaminases (CDA1/CDA2) in the AID/APOBEC family. These deaminases emerged phylogenetically as the closest sister group in the AID/APOBEC family to the AID used by gnathostomes for Ig class-switch recombination, somatic hypermutation, and Ig gene conversion (Rogozin et al., 2007). CDA-mediated gene rearrangement in lampreys occurs in a manner similar to AID-induced immunoglobulin gene conversion in some birds and mammals (Rogozin et al., 2007; Zheng et al., 1994). One study suggested that VLRA (the analog to αβ TcR in jawed vertebrates) also might use CDA to affinity mature its receptors, indicating that CDA contributes both to repertoire generation and to somatic mutation after antigen exposure (Deng et al., 2010; Flajnik, 2014). If this is true, it may be possible that the ancestor to modern vertebrates also used an AID-like enzyme to assist with lymphocyte receptor development in a thymus-like organ. The expression of AID in the thymus of primitive sharks may be a remnant of this ancestral process, a mechanism lost in later vertebrates because of its potential for breaking down self-tolerance in mature lymphocytes. Perhaps, agnathans evolved specific APOBEC molecules for diversification of their B and T like VLRs, while gnathostomes evolved AID for T cell primary repertoire diversification (Neils Jerne’s ‘mutant breeding organ’ [Jerne, 1971]) and B cell affinity maturation, eventually co-opting AID for use in class switch recombination at IGH translocons, and later still, gene conversion and SHM for primary B cell repertoires.

From this trend of comparative TcR studies, we conclude with two hypotheses that we will test with further immunogenetic and functional studies in shark and other vertebrate models. First, the division between B and T cell repertoire diversification components and mechanisms was not as clear-cut in ancestral lymphocytes as in modern humans and mice. The second is that different vertebrate groups have not only evolved myriad diversifications for Ig repertoires and function, but TcR biology may be just as varied. This premise is already accumulating ample supporting evidence as IgHV domains (Parra et al., 2010; Parra et al., 2012), high allelic polymorphism (Criscitiello et al., 2004a; Criscitiello et al., 2004b), germline joined V exons (Wang and Miller, 2012; Wang et al., 2011), and now mechanisms such as SHM, all once considered the immune privilege of Ig or MHC genes, are also employed for TcRs.

T cell receptor sequences have been deposited in Genbank of NCBI. Alpha KY189332-KY189354 and KY366469-KY355487; Beta KY351708-KY366487; Gamma KY351639-KY351707; Delta KY346705-KY346816.

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Author details

1. Jeannine A Ott

   Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3537-8631

2. Caitlin D Castro

   Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Thaddeus C Deiss

   Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, United States

   Contribution

   Software, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Yuko Ohta

   Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Martin F Flajnik

   Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, United States

   Contribution

   Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

6. Michael F Criscitiello

   1. Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, United States
   2. Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, Texas A&M University, Texas, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration, Writing—review and editing

   For correspondence

   mcriscitiello@cvm.tamu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4262-7832

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