Developmental and Comparative Immunology 115 (2021) 103873

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Developmental and Comparative Immunology

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Review

The immunoglobulins of cartilaginous fishes

Hanover Matz a, b, Danish Munir c, James Logue a, Helen Dooley a, b, *
a
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA
b
Institute of Marine and Environmental Technology, Baltimore, MD, USA
c
School of Biological Sciences, University of Aberdeen, Aberdeen, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Cartilaginous fishes, comprising the chimeras, sharks, skates, and rays, split from the common ancestor with
Cartilaginous fish other jawed vertebrates approx. 450 million years ago. Being the oldest extant taxonomic group to possess an
Shark immunoglobulin (Ig)-based adaptive immune system, examination of this group has taught us much about the
Antibody
evolution of adaptive immunity, as well as the conserved and taxon-specific characteristics of Igs. Significant
IgM
progress has been made analyzing sequences from numerous genomic and transcriptomic data sets. These
IgNAR
Memory findings have been supported by additional functional studies characterizing the Igs and humoral response of
B cells sharks and their relatives. This review will summarize what we have learned about the genomic organization,
protein structure, and in vivo function of these Ig isotypes in cartilaginous fishes and highlight the areas where
our knowledge is still lacking.

1. Introduction
Since adaptive immunity arose in the common ancestor of all
vertebrate lineages, it has diversified into a complex arsenal of cellular
and molecular defenses that each phylogenetic branch of both the ec­
totherms (cold-blooded organisms) and endotherms (warm-blooded
organisms) has preserved or altered to varying degrees. However, the
fundamental molecular armament of humoral immunity in all gna­
thostomes (jawed vertebrates) are immunoglobulins (Igs) or antibodies
(Abs). It is these molecules, expressed as heterodimers of heavy (H) and
light (L) chains on the surface of B cells and secreted by plasma cells, that
provide the host adaptive immune system with exquisite specificity to
protect against pathogens via a myriad of functional roles.
Cartilaginous fish (Chondrichthyes) split from the common ancestor
with other jawed vertebrates approx. 450 million years ago (Inoue et al.,
2010) and have two extant subclasses, the holocephalans (chimeras,
such as elephant sharks and rat fishes) and the better known elasmo­
branchs (sharks, rays, and skates). Being the oldest extant taxonomic
group to possess an Ig-based adaptive immune system (Flajnik and
Rumfelt, 2000) examination of this group has taught us much about the
evolution of adaptive immunity, as well as the conserved and
taxon-specific characteristics of Igs. Sharks and their relatives have three
IgH chain isotypes: the first two, IgM (μ) (Clem et al., 1967; Marchalonis
and Edelman, 1966) and IgW (ω) (Anderson et al., 1999; Berstein et al.,
1996; Greenberg et al., 1996; Kunihiko and Susumu, 1988), are
orthologous to IgM and IgD (respectively) in other vertebrate groups
(Ohta and Flajnik, 2006). In addition to these ‘conventional’ IgH-IgL
isotypes elasmobranchs possess a third isotype IgNAR, an IgH chain
homodimer that does not associate with L chain (Greenberg et al., 1995;
Roux et al., 1998). Further, cartilaginous fishes have four IgL chain
isotypes: kappa (κ), lambda (λ), sigma (σ), and sigma-2 (σ-2; also called
σ-cart prior to its discovery in lobe-finned fishes) (Criscitiello and Flaj­
nik, 2007; Saha et al., 2014). This review will summarize what we have
learned about the genomic organization, protein structure, and in vivo
function of these Ig isotypes in cartilaginous fishes in the context of
immune system evolution.
2. Cartilaginous fish immune organs
Like mammals, T cell maturation occurs in the cartilaginous fish
thymus, which is paired and located dorsomedial to the gills (Fig. 1)
(Luer et al., 1995; Miracle et al., 2001). Lacking bone marrow, carti­
laginous fish B cell lymphopoiesis primarily occurs in special sites
known as the epigonal organ, associated with the gonads, and the Leydig
organ, embedded within the wall of the esophagus (Fig. 1). Not all
elasmobranch species have a Leydig organ, for example, while found in
little skate (Leucoraja erinacea) it is not present in nurse sharks (Gin­
glymostoma cirratum). Histologically both organs are similar in structure,

* Corresponding author. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.
E-mail address: hdooley@som.umaryland.edu (H. Dooley).

https://doi.org/10.1016/j.dci.2020.103873
Received 2 July 2020; Received in revised form 12 September 2020; Accepted 16 September 2020
Available online 23 September 2020
0145-305X/© 2020 Elsevier Ltd. All rights reserved.
H. Matz et al.
with the spongy appearance of mammalian bone marrow (Luer et al.,
2004). Cartilaginous fishes, like other ectothermic vertebrates, lack
lymph nodes (Zapata and Amemiya, 2000). Consequently, adaptive
immune responses occur primarily in the spleen, although there is some
evidence of lymphocyte activity in the gills, gut, and several other tis­
sues (Hart et al., 1986; Rumfelt et al., 2002; Tomonaga et al., 1986). The
shark spleen is highly vascularized and contains both red pulp and
defined white pulp regions. Organized zones of B cells are evident and
may facilitate clonal selection and somatic hypermutation (SHM) (Cas­
tro et al., 2013; Rumfelt et al., 2002), although true germinal centers
(GCs) have not been identified in any ectotherms to date (Zapata and
Amemiya, 2000; Zapata et al., 1996). As the spleen houses both naïve B
cells and differentiated plasma cells, it is the major site of Ig expression
and secretion (Castro et al., 2013; Rumfelt et al., 2002). While the epi­
gonal organ is hypothesized to be the home of long-lived plasma cells
(Castro et al., 2013) secretory Ig transcript has also been found in the
pancreas, gill, liver, kidney, and olfactory organ of nurse sharks (Rum­
felt et al., 2004a). Thus, as in mammals, Igs seemingly play a defensive
role throughout all cartilaginous fish tissues.
3. Genomic organization of cartilaginous fish Igs and repertoire
generation
Most vertebrate Ig genes are organized in a translocon configuration
(Fig. 2), in which multiple variable (V), diversity (D; only present in IgH
chains), and joining (J) segments are located upstream of constant (C)
domains for all Ig isotypes. In contrast, cartilaginous fish IgH and IgL
chain genes are arranged in a cluster configuration (Fig. 2), in which a
single V segment, one or more D segments (again, IgH chains only), and
a single J segment lie upstream of C region exons of a single isotype
(Hinds and Litman, 1986). The number of clusters varies between iso­
types and between cartilaginous fish species: for example, nurse sharks
have ~15 IgM clusters (each having V, D, J segments and a set of Cμ
domains) and 3 IgNAR clusters (V, multiple Ds, J, and CNAR domains)
while the spiny dogfish (Squalus acanthias) has >100 IgM and 10–20
IgNAR clusters (Malecek et al., 2005; Smith et al., 2012). While most Ig
segments are unjoined in the cartilaginous fish germline genome, a small
portion are found to be partially (VD-J) or fully (VDJ or VJ)
pre-rearranged or “germline-joined”; e.g. nurse sharks have both
germline-joined IgM and IgNAR clusters (Diaz et al., 2002; Kokubu et al.,
1988; Rumfelt et al., 2001). These germline-joined nurse shark IgH
genes are preferentially expressed early in ontogeny and are hypothe­
sized to play unique protective and/or homeostatic roles in neonates
(Diaz et al., 2002; Rumfelt et al., 2001).
Cartilaginous fishes use the same recombination mechanisms as
mammals to generate a primary Ig repertoire, with recombination-
activation gene (RAG1 and RAG2) enzymes active in the
Fig. 1. The major immunological organs of cartilaginous fishes and their relative positions.
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Developmental and Comparative Immunology 115 (2021) 103873
lymphopoietic organs (epigonal and Leydig organ) along with terminal
deoxynucleotidyl transferase (TdT), which inserts N-nucleotides at the
rearrangement junctions (Rumfelt et al., 2002). Rearrangement is pri­
marily restricted within a cluster, and rarely occurs between different
clusters (Malecek et al., 2008). Unlike mice and humans, where there is
a strict order of segmental rearrangement (always D to J first, then V to
DJ for Ig heavy chains) the V, D, and J segments of shark Ig clusters can
recombine in any order (e.g. V to D1, D1 to D2, and D3 to J first round
rearrangements have all been observed; H. Dooley, unpublished data),
and multiple Ig genes are accessible for recombination at one time
(Malecek et al., 2008). Despite this, single cell PCRs amplified only one
IgH V region transcript per B cell in the clearnose skate (Raja eglanteria)
(Eason and Litman, 2002) and no overlap is observed between IgM- and
IgNAR-secreting cells in nurse sharks (Rumfelt et al., 2002), indicating
both isotypic and allelic exclusion occur. More recent studies have
shown that shark IgM clusters operate autonomously during VDJ
recombination. This stochastic activation of IgH genes for rearrange­
ment initiation, combined with a restricted time window of accessibility,
likely help maintain IgH chain exclusion (Zhu et al., 2011). It is hy­
pothesized that once a productive V region is produced from any of the
accessible clusters it prevents rearrangement and expression of the
remaining Ig clusters, thereby maintaining B cell clonality (Zhu et al.,
2011).
Based upon the limited number of V, (D) and J segments within a
single cartilaginous fish cluster it might be concluded that the shark
primary immune repertoire is restricted in diversity. However, several
mechanisms compensate for this. First, as noted above, every Ig isotype
has multiple clusters with extensive germline-encoded heterogeneity
across complementarity determining regions (CDR)1 and 2 (Rast et al.,
1998; Rumfelt et al., 2004b). Second, each heavy chain cluster contains
multiple D segments and thus requires multiple rearrangement events
(e.g. 4 for IgNAR), with associated trimming and N/P-addition, to
generate a functional V region (Malecek et al., 2008). Finally, cartilag­
inous fish have four IgL chain isotypes that are among the most heter­
ogenous of any vertebrate studied to date (Criscitiello and Flajnik, 2007;
Fleurant et al., 2004). Thus, the weight of evidence suggests the primary
repertoire of cartilaginous fishes is just as diverse (or possibly even more
diverse) than that of mammals. Study of random IgNAR cDNAs obtained
from peripheral blood lymphocyte samples showed a much lower mu­
tation rate in transmembrane transcripts compared to secretory tran­
scripts, and only mutations in the latter were biased towards
replacement mutations in the CDRs (Diaz et al., 1998). This indicates
that shark Igs undergo activation induced cytidine deaminase (AID)-­
mediated somatic diversification following antigen stimulation akin to
mammals, rather than using mutation to generate the primary repertoire
like birds and rabbits. Interestingly, the mutation frequency of IgNAR is
exceptionally high, surpassing even the upper limits reported for
H. Matz et al.
Fig. 2. Schematic showing the different Ig gene arrangements in mammals and sharks. Mammalian Igs are found in a translocon arrangement, with multiple V
segments, D segments, and J segments arranged upstream of the C regions for all the different Ig isotypes. In contrast, cartilaginous fish Igs are found in a cluster
configuration, where a single V segment, two or more D segments, a J segment, and the C regions for a single isotype are present in each cluster, and multiple clusters
are present for each isotype. C regions are shown in grey, the segments that make up the variable region are colored by isotype.
mammalian V regions (Diaz et al., 1999). Study of random nurse shark
IgNAR and IgL transcripts showed mutations accumulate in a pattern
similar to mammalian IgG, targeting AGC/T hotspots, but with a high
proportion of tandem mutations rather than singlets (Diaz et al., 1999;
Greenberg et al., 1995; Lee et al., 2002). The fact that high replacement
to synonymous (R/S) ratios were observed in the CDRs of some clones
suggests positive selection can occur despite the apparent absence of
GCs (Diaz et al., 2002). Thus, the ability of cartilaginous fishes to
generate and diversify a primary Ig repertoire is by no means
sub-optimal. Indeed, by all measures, sharks are as capable as mammals
of combating the potential universe of pathogens they may encounter.
The cluster organization of cartilaginous fish Ig genes and absence of
recognizable classical (i.e. mammalian-like) switch (S) regions (Zhu
et al., 2012) suggests this group lacks tetrapod-like class switch
recombination, which so far has only been identified as far back as
amphibians (Muβmann et al., 1997). However, recent studies of nurse
shark Ig transcripts have shown that the V regions from one cluster may
be expressed fused to the C domains of another, despite being separated
by large (>120 Kb) distances (Zhang et al., 2013; Zhu et al., 2012).
Unlike mammals this form of ‘switching’ is adirectional, i.e. IgW V re­
gions can be expressed with IgM C regions and vice versa (Zhu et al.,
2012). As the number of switched transcripts is higher in immunized
animals it is hypothesized to occur via AID-initiated DNA lesions that
prompt recombination between the clusters (Zhu et al., 2012). We do
not yet know how this process is directed or its biological consequence;
however, it is possible this is the predecessor of conventional CSR as
found in other vertebrates. Thus, most of the fundamental components
of Ig repertoire diversification, including V(D)J recombination, somatic
hypermutation (SHM), and a potential precursor of CSR were all present
in the common ancestor of jawed vertebrates.
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Developmental and Comparative Immunology 115 (2021) 103873
4. The Ig isotypes of cartilaginous fishes
4.1. IgM
A protein with characteristics similar to IgM was first discovered in
the plasma of cartilaginous fishes in the mid-1960s (Clem and Small Jr,
1967; Clem et al., 1967; Marchalonis and Edelman, 1966) and was
originally thought to be the only isotype present. Indeed, in nurse shark
IgM makes up roughly half of the total serum protein and is present in
two forms, a monomeric form (mIgM or 7S) and a pentameric (pIgM or
19S) form (Fig. 3) in approximately equal amounts (Clem et al., 1967;
Small et al., 1970). The two forms do not interconvert (Small et al.,
1970) and are hypothesized to be produced by different B cell lineages
(Dooley and Flajnik, 2005).
Secretion of pIgM is coordinated with J chain expression in adult
nurse sharks, and it has also been found that pIgM/J chain + nurse shark
splenic B cells do not express the transcription factor Blimp-1, which
controls plasma cell differentiation, and express lower levels of IgM
transcript than mIgM B cells (Castro et al., 2013). In contrast, mIgM is
expressed in Blimp-1+/J chain- B cells (Castro et al., 2013) and is likely
produced in a T cell-dependent manner (Clem et al., 1967; Dooley and
Flajnik, 2005; Marchalonis and Edelman, 1966), with antigen specific
titers of mIgM rising over the course of an immune response. Coupled
with its non-specific nature, pIgM is thought to act as an innate-like,
‘first line of defense’ against invading pathogens until a strong
antigen-specific response can be developed. A lack of serum albumin in
sharks suggests pIgM may also act as a carrier molecule or in osmo­
regulation, in addition to acting as an innate defender (Dooley and
Flajnik, 2006; Fellows and Hird, 1981; Fellows et al., 1980). Like IgM in
other vertebrates, the large size of pIgM restricts it to the vasculature,
while mIgM is capable of penetrating tissues similar to mammalian IgG
(Small et al., 1970).
Both the transmembrane and secreted forms of IgM contain 4 C do­
mains (Rumfelt et al., 2004a) but, interestingly, a third form with only 3
C domains (expressed from a separate cluster where Cμ2 is deleted,
H. Matz et al.
Fig. 3. Illustration of the transmembrane and secretory immunoglobulin isoforms found in cartilaginous fishes to date. Constant domains are shown in grey while the
variable domains of each are colored by isotype. Secreted IgM pentamers are stabilized by joining (J) chain.
making it structurally convergent with mammalian IgG) predominates
in the plasma of neonatal animals and pups (Rumfelt et al., 2001). This
IgM form, IgM1gj, forms both monomers and dimers, has an entirely
germline-joined (VDJ) V domain (Rumfelt et al., 2001) and preferen­
tially associates with a germline-joined L chain ((Lee et al., 2000) Flajnik
& Hsu, unpublished data), giving this Ig a completely predetermined
binding site. As pups age the levels of pIgM and mIgM gradually increase
to become the dominant Ig forms. Like pIgM secreting cells, IgM1gj B
cells are J chain+/Blimp-1-, and while they can be found in the spleen of
nurse shark pups, they are only found in the epigonal organ of adult
animals (Castro et al., 2013).
There are two proposed models for how the pIgM and mIgM B cells
arise in the shark: model 1 suggests that these cells types are determined
at the stem cell stage, and that pIgM and mIgM-secreting cells are
derived from distinct lineages akin to B1 and B2 cell populations in
mammals. Model 2 suggests that pIgM cells are a distinct developmental
stage preceding mIgM cells, akin to plasmablasts (Castro et al., 2013;
Dooley and Flajnik, 2005). The early appearance of pIgM B cells in
ontogeny coupled with their lack of Blimp-1 expression, suggesting a
potential for self-renewal, is similar to the phenotype of mammalian B1
cells which do not require Blimp-1 upregulation for antibody secretion
(Tumang et al., 2005), supporting model 1. However, mouse
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Developmental and Comparative Immunology 115 (2021) 103873
preplasmablasts also lack Blimp-1 expression (Kallies et al., 2007), and
thus the absence of Blimp-1 in pIgM B cells would support model 2.
However, if pIgM are truly a transitional stage preceding mIgM cells
their high proportion and presence in the shark spleen regardless of
immune state would suggest that this stage is more long-lived than seen
in mammals (Castro et al., 2013). More work is required to prove which
of these two models, or a combination of both, explains the role of pIgM
and mIgM shark B cell lineages.
4.2. IgNAR
First reported in 1995, IgNAR is a novel Ig isotype that has so far only
been found in elasmobranchs. Like the novel heavy chain antibodies
found in Camelidae (camels, llamas, and their relatives), IgNAR is also a
homodimer of IgH chains that does not associate with IgL chains
(Greenberg et al., 1995; Roux et al., 1998), although the two isotypes
evolved convergently (Nguyen et al., 1998, 1999). In nurse shark there is
a single secretory form of IgNAR where an N-terminal V domain (VNAR)
is joined to five C (C1–C5) domains (Fig. 3) (Greenberg et al., 1996;
Rumfelt et al., 2004a). A second form of secretory IgNAR, where C1 is
spliced directly to C4 (i.e. ΔC2-C3), has recently been found in the
whitespotted bamboo shark (Chiloscyllium plagiosum) (Zhang et al.,
H. Matz et al. Developmental and Comparative Immunology 115 (2021) 103873

2020). Membrane bound IgNAR is also expressed as long (five C do­
mains) and short forms (three C domains), however here the short form
is missing the C4 and C5 domains (Rumfelt et al., 2004a). Phylogenetic
and structural analyses revealed that VNARs share greater structural
homology with T cell receptor (TCR) and IgL V domains than conven­
tional IgH V regions (Greenberg et al., 1995; Richards and Nelson, 2000;
Stanfield et al., 2004). VNAR-like domains are also found in NAR-TCR, a
doubly rearranging T cell receptor unique to cartilaginous fishes, sug­
gesting a common ancestral origin for these domains (Criscitiello et al.,
2006). As the constant domains (C2–C5) of IgNAR are homologous to
those of IgW, it is hypothesized that IgNAR originated through the in­
vasion of an IgW cluster by the V region of NAR-TCR (Criscitiello et al.,
2006; Greenberg et al., 1996). Lacking light chains IgNAR does not have
the combinatorial diversity generated through VH-VL pairing in con­
ventional antibodies. However, as mentioned above, this deficiency is
more than compensated by the multiple rearrangement events required
to assemble their three D regions (Fig. 2). This generates CDR3s that are
highly diverse in both length (7–34 aa) and sequence composition,
bringing considerable heterogeneity to the primary repertoire (Diaz
et al., 1998; Flajnik and Dooley, 2009; Greenberg et al., 1995). Upon
exposure to antigen VNARs are further diversified by extremely high
levels of SHM (Diaz et al., 1999).
IgNAR levels in nurse shark serum are approximately 10x lower than
those observed for IgM, likely due to the presence of fewer IgNAR
clusters and the apparent susceptibility of IgNAR proteins to proteolysis
(Dooley and Flajnik, 2005; Roux et al., 1998). In nurse shark, IgNAR is
found only as a monomer, but in spiny dogfish and small spotted cat­
shark (Scyliorhinus canicula) it has been found as both a monomer and a
multimer (Crouch et al., 2013; Smith et al., 2012).
The role of IgNAR in the adaptive response is undoubtedly enhanced
by the diversity of VNAR structures which enable this isotype to bind a
wide array of epitopes. VNAR domains act as independent soluble units,
that are attached to the C1 domain by an extended flexible tether rather
than a conventional Ig hinge region (Roux et al., 1998). While VNARs
adopt the typical conformation of Ig superfamily domains, a large
portion of framework region 2 (FR2) and CDR2 is deleted (Greenberg
et al., 1995) giving this domain its characteristically small size (approx.
12 kDa). The loss of CDR2 is compensated by two loops of high amino
acid diversity, hypervariable region 2 (HV2) that forms a belt-like
structure around the VNAR domain and hypervariable region 4 (HV4)
that lies at the top of the domain next to CDR1 (Stanfield et al., 2004). In
addition to the cysteines that typify Ig domains, VNARs also contain
non-canonical cysteines that can form additional disulphide bonds and
dramatically alter the structural topology of the variable loops. Based
upon the number and position of non-canonical cysteine residues,
VNARs are categorized into several ‘types’ (Fig. 4). Type I VNARs, so far
identified only in nurse sharks (Greenberg et al., 1995; Matz and Dooley,
2019), carry an even number of cysteines (2, 4, or 6) in CDR3 and one
each in FR2 and FR4. Disulphide bond formation between these residues
causes the CDR3 loop to be pinned tightly against the side of the
molecule (Stanfield et al., 2004). Type II VNAR domains, identified in
several shark species (Liu et al., 2007; Nuttall et al., 2001; Zielonka
et al., 2014), carry additional cysteines in CDR1 and CDR3 which results
in an intra-molecular disulphide bond that brings the loops closer
together, forming a protrusive ‘finger-like’ structure at the top of the
domain (Streltsov et al., 2005). Type III VNARs, so far found only in
nurse sharks, also carry extra non-canonical cysteines in CDR1 and
CDR3. However, because two of the three diversity regions of type III
VNARs are germline joined, they show less diversity in CDR3 compared
to type I or II VNARs (Diaz et al., 2002). Like IgM1gj, type III VNARs are
mainly expressed during neonatal development, before maturation of
the antigen-driven response; however, some expression is maintained in
the epigonal organ into adulthood (Diaz et al., 2002). Type IV domains
(alternately called type IIb) only have the canonical cysteines possessed
by all Ig domains, which may make their paratope topology more flex­
ible (Kovalenko et al., 2013). The VNAR-antigen complexes crystalized
thus far indicate that VNARs have a propensity to target clefts and re­
cesses on their target antigens. Indeed, VNARs appear to exploit this
binding strategy to increase their surface area of interaction with a
target, burying as much surface area in the interaction as a conventional
H-L chain antibody, and facilitating high affinity interactions (Fig. 4).
Finally, VNARs exist that do not conform to any of the classical types
described above; for example, in a recent analysis of 1.2 million VNAR
transcripts isolated from naïve nurse sharks, a small percentage (~5%)
could not be classified into any of the traditional types based upon
cysteine number or location (Feng et al., 2019). Further structural and
functional characterization is needed to understand the biophysical
properties of these previously undescribed VNAR types.
The inherent attributes of VNARs, i.e., their small size, unique
structural topologies, propensity to bind clefts and pockets on target
antigens, and their high thermostability, make VNARs interesting
prospects as future diagnostic and therapeutic agents (reviewed in (Matz
and Dooley, 2019).

Fig. 4. Structural analyses show that VNARs (blue) can complex their target antigens (gold) using a range of different binding modalities. The VNAR CDR1 is shown
in yellow, CDR3 in red, HV2 in green, and HV4 in cyan in all panels. As can be seen in (a) the type I VNAR 5A7 contacts HEL via CDR1 and a folded CDR3 that is
constrained by disulfide bonding to the FR2 and FR4 (PDB 1T6V) while (b) the type II VNAR PBLA8 contacts hen egg lysozyme (HEL) via CDR1 and an extended
CDR3 loop projecting deep into HELs enzymatic cleft (PDB 2I25). In contrast, (c) the type IV/IIb VNAR E06 binds its target human serum albumin (HSA) through
CDR3 and HV2 (PDB 4HGK), and (d) the synthetic VNAR D01 contacts Aurora-A kinase via CDR1, CDR3, and HV2 (PDB 5L8L). From Matz and Dooley (2019). (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

5
H. Matz et al.
4.3. IgW
IgW was first identified in ocellate spot skate (Raja kenojei) and little
skate (Raja erinacea) (Harding et al., 1990; Kunihiko et al., 1984) where
it was named IgR and IgX, and later in nurse shark as IgNARC (Green­
berg et al., 1996) then sandbar shark (Carcharhinus plumbeus) as IgW
(Berstein et al., 1996). With the discovery of an additional ortholog in
lungfish (Ota et al., 2003) the cartilaginous fish Igs were reconciled into
one isotype, IgW, and recognized as orthologs of tetrapod IgD (Ohta and
Flajnik, 2006). Thus, both IgM-like and IgW/D-like molecules must have
been present in the ancestor of all jawed vertebrates (Ohta and Flajnik,
2006). Curiously, IgW appears to be lost from the elephant shark (Cal­
lorhinchus milii) genome, a holocephalan and ancient chondrichthyan
(Venkatesh et al., 2014), but has been found in every elasmobranch
species yet examined.
Several forms of IgW have been discovered. Based on transcript and
protein S35 labeling, there exists both long (6 and 8Cω) and short (2 and
4Cω) secreted forms of IgW (Fig. 3) (Greenberg et al., 1996; Rumfelt
et al., 2004a; Zhang et al., 2013). Transcript data has also confirmed the
existence of two transmembrane forms of IgW (2 and 4Cω) (Rumfelt
et al., 2004a; Zhang et al., 2013). The short secretory form (2Cω) has a
long, cysteine-rich tail, unlike that of any other Ig, suggesting its effector
function is different from other isoforms (Rumfelt et al., 2004a). It is
likely that the different forms are generated through alternative splicing
(Zhang et al., 2013). Additionally, transcript for a 6Cω form in which the
leader is spliced directly to the C1, resulting in a molecule lacking the V
domain (IgWΔV), was discovered in spiny dogfish and nurse shark
(Smith et al., 2012). The existence of IgWΔV in two species of shark
separated by ~200 million years of evolution, at least at the transcript
level, suggests this form fulfils a unique functional role. It has been
speculated that the C domains of IgW/D can act as pattern recognition
molecules (Edholm et al., 2010; Seifert et al., 2009), binding directly to
pathogens then triggering antimicrobial and immunostimulatory re­
sponses (Chen et al., 2009). The discovery of IgWΔV in sharks can help
us put this hypothesis to the test.
A lack of monoclonal Abs (mAbs) for IgW has made it difficult to
study its function at the protein level, but the plethora of transcript
forms suggests IgW (along with IgNAR) has several diverse functions.
Serum protein levels of IgW are low in adult nurse shark, however
transcript levels are high in spleen, epigonal, pancreas, thymus, and gill,
suggesting IgW plays a role in mucosal protection (Rumfelt et al., 2004a;
Smith et al., 2012; Zhang et al., 2013). The short secreted form of IgW
varies greatly in expression levels, both between juvenile and adult
nurse sharks, and between individuals in the same cohort (Rumfelt et al.,
2004a). In situ hybridization shows transcription of the IgW long
secretory form overlaps with that of J chain in the spleen and epigonal
organ of nurse sharks, suggesting it may form multimers (Castro et al.,
2013).
We hypothesize that the diverse forms of secretory IgW generated
through splicing provide a means to control the effector functions trig­
gered (as has been observed for the full-length and short (ΔFc) forms of
mallard duck IgY (Humphrey et al., 2004)), however this requires
formal testing. The presence of two IgW Tm forms also remains a mys­
tery, although the Tm forms have fewer flexible hinge regions, possibly
helping reduce their proteolytic shedding from the B cell surface (Smith
et al., 2012; Zhang et al., 2013). Thus, it is possible cells expressing
different versions of IgW-Tm have different activation requirements
(Zhang et al., 2013). So far, no germline-joined versions of IgW have
been discovered, suggesting there are not innate roles early in ontogeny
for this isotype (Rumfelt et al., 2004a). Unlike IgM and IgD in mammals,
IgM and IgW do not appear to be expressed simultaneously on the sur­
face of cartilaginous fish B cells (Eason et al., 2004). While the role of
IgD is not fully understood even in mammals, comparative studies have
already increased our knowledge of this enigmatic isotype (Chen et al.,
2009; Edholm et al., 2010). We believe that further investigation of
shark IgW, which would be greatly facilitated by the development of
6
Developmental and Comparative Immunology 115 (2021) 103873
antibodies for this isotype, will yield further insights regarding the
evolution of this isotype and its contribution to humoral immunity.
4.4. IgL chains
Early immunization studies recognized that shark Igs contained L
chains akin to canonical mammalian Igs (Clem et al., 1967). It is now
known that cartilaginous fishes have four L chain isotypes. The
nomenclature of Ig L chains in chondrichthyans was confusing for many
years, as it was difficult to determine the classification of L chains across
vertebrate taxa due to large phylogenetic distances (Dooley and Flajnik,
2006; Smith et al., 2014). It is now established that sharks possess λ
(formerly Type II/NS3), κ (formerly Type III/NS4), σ, and σ-cart/σ-2
(formerly Type I/NS5) L chains (Criscitiello and Flajnik, 2007). Like the
H chains, shark Ig L chain genes are organized in a cluster configuration
(Greenberg et al., 1993; Shamblott and Litman, 1989). Of these loci in
nurse shark, κ and σ-cart contain both germline-joined and unjoined or
split clusters, σ contains no germline-joined clusters, and all λ clusters
are germline-joined (Fig. 5) (Criscitiello and Flajnik, 2007; Fleurant
et al., 2004; Lee et al., 2000; Rast et al., 1994). The number of clusters of
each isotype, like Ig H chains, varies between shark species, as does the
proportion of split versus germline-joined clusters (Fleurant et al., 2004;
Hohman et al., 1992; Rast et al., 1994).
It has been proposed that germline-joined Igs arose as a consequence
of RAG activity in the germline (Lee et al., 2000), however, it should be
noted that other scenarios have not been entirely ruled out; for example,
retrotransposition of a rearranged VDJ transcript back into the germline
is a possibility, but the presence of a split leader (Anderson et al., 1995)
makes this explanation far less likely. While it might be thought that
germline-joined L chains would restrict the antibody repertoire, λ L
chain loci hypermutate at high rates in an antigen-driven fashion that
results in tandem mutations, leading to diverse sequences despite a lack
of junctional diversity (Lee et al., 2002). For isotypes with split clusters
that undergo V-J rearrangement (e.g. κ and σ-cart) the majority of clones
examined carry N nucleotide additions, vastly increasing light chain
CDR3 repertoire diversity (Fleurant et al., 2004).
The major outstanding question from L chain research in shark is, if
these four clades are present in the oldest phylogenetic group with Ig-
based immunity, what was the model for their evolution and the bio­
logical significance of each isotype? As might be predicted, neither of
these questions has been simple to answer. Several models are proposed
for the origins of these L chain isotypes and how Ig antigen receptors
evolved (Criscitiello and Flajnik, 2007), but what is apparent is that
σ-cart is a “dead-end” isotype found only in cartilaginous fishes, σ is
maintained only in ectothermic vertebrate lineages (Criscitiello and
Flajnik, 2007; Schwager et al., 1991), and only κ and λ are maintained in
the endothermic vertebrate lineages. The functional difference between
κ and λ is not even clear in mammals (Hershberg and Shlomchik, 2006;
Sverremark et al., 2000; Townsend et al., 2016), so the benefit of four
different L chain isotypes in cartilaginous fish is equally unclear.
5. Ig responses in sharks
Immunization studies performed in the mid-1960s suggested that the
antibody responses of cartilaginous fishes differed substantially from
those of mammals; following a long (2–3 month) lag period serum IgM
levels climbed slowly. While the proportion of mIgM to pIgM changed in
some studies, binding affinities did not appear to increase appreciably
over the course of most responses (Clem and Small Jr, 1967; Clem et al.,
1967; Marchalonis and Edelman, 1966). Thus, for a long time, it was
thought that the antibody response in cartilaginous fishes was somewhat
‘lacking’ when compared to mammals. However, pioneering work in
nurse sharks determined how to generate true antigen-specific humoral
responses and revealed that sharks are capable of affinity maturing their
Ig repertoires (Dooley and Flajnik, 2005). While the affinity maturation
observed was 10 fold rather than the ~100 fold change seen in mice
H. Matz et al.
Fig. 5. Like IgH chains, cartilaginous fish IgL chains can be found in both split and germline-joined configurations. Notably, the λ chains of all species examined to
date are all germline-joined and σ are split, while the other isotypes are present in varying ratios of split and joined in different species.
(Dooley et al., 2006), it is presumed to be no less effective at protecting
the host against pathogens.
Functional studies indicate distinct functional roles for pIgM and
mIgM in the shark humoral response. Low affinity, high avidity pIgM
can be found prior to injection of antigen and affinities of antigen-
specific pIgM do not increase during an antigen-driven immune
response, suggesting that affinity maturation does not occur in this Ig
form (Dooley and Flajnik, 2005; Leslie and Clem, 1970; Voss and Sigel,
1972). It has been shown that pIgM serum levels can increase exclu­
sively in response to bacterial carbohydrate, suggesting a T cell inde­
pendent response (Clem and Leslie, 1971; Shankey and Clem, 1980a, b).
In contrast, mIgM affinity matures in an antigen-driven manner during
the humoral response. This response includes a delay in mIgM produc­
tion following antigen exposure, an increase in antibody affinity over
the duration of the response, and maintenance of long term,
antigen-specific mIgM memory (Dooley and Flajnik, 2005; Eve et al.,
2020; Rumfelt et al., 2002). That mIgM undergoes affinity maturation in
an antigen-driven manner suggests there is a mechanism in sharks for B
cell selection reminiscent of mammalian GCs; however, as noted above,
true GCs that would mediate this process have not yet been observed in
cartilaginous fishes (Zapata and Amemiya, 2000).
Akin to mIgM, antigen specific IgNAR titers also rise slowly after
immunization and exhibit affinity maturation over the course of a
response. The lag-period necessary to generate an antigen-driven IgNAR
response, coupled with a lack of IgNAR in neonatal serum, strongly
suggests that IgNAR responses require T cell help (Dooley and Flajnik,
2005; Rumfelt et al., 2002).
The Ig titers achieved through immunization in nurse sharks persist
up to 1–3 years before returning to initial pre-immunization levels,
suggesting long-term circulating protection against antigens. Further,
antigen-specific Ig expression can be re-stimulated long (>8 years) after
primary antigen exposure, clearly demonstrating B cell memory (Dooley
and Flajnik, 2005; Eve et al., 2020). Collectively, this data demonstrates
that sharks are capable of Ig responses of the same caliber as endo­
therms, albeit on longer timescales.
6. Perspective and future directions
From what has been learned about Igs in sharks and their relatives,
what conclusions can we draw about the evolution of adaptive
7
Developmental and Comparative Immunology 115 (2021) 103873
immunity? The discovery that cartilaginous fishes employ V(D)J
recombination and somatic hypermutation to generate a highly diverse
repertoire of binding specificities, can affinity mature their Ig pool, and
maintain antigen-specific clones to provide long-term immunological
memory (as detailed earlier) indicates these traits - long-associated with
Ig-mediated protection in mammals - were almost certainly present in
the common ancestor of all jawed vertebrates. The apparent division of
function between cartilaginous fish innate-like Igs (pIgM and IgM1gj)
and adaptive Igs (mIgM and IgNAR) suggests B1-like and B2-like cell
lineages arose early in vertebrate evolution. Segregation of these cells
followed by sequencing of their V region repertoires would allow us to
test this hypothesis, not a simple task when species-specific monoclonal
antibodies are lacking.
Further, the differential use of multiple Ig isotypes, combined with
the presence of multiple forms of some isotypes (Dooley and Flajnik,
2005; Rumfelt et al., 2004a), indicates that cartilaginous fishes can
tailor their response to achieve different immunological outcomes like
other vertebrates. However, significant work is required to understand
the effector functions mediated by the different shark Ig isotypes. The
development of additional isotype-specific mAbs would greatly advance
these studies; currently, the research tools available only allow for
limited study of IgM and IgNAR. Once the in vivo roles of each isotype
have been established it should also become easier to understand the
functional consequences of the atypical form of CSR found in sharks.
Such mAbs would also facilitate flow cytometry experiments, permitting
the isolation of isotype-specific B cells from their native sites for further
characterization.
The ability of cartilaginous fishes to affinity mature their Ig reper­
toire (Dooley and Flajnik, 2005; Dooley et al., 2006) in the apparent
absence of GCs is also a point that requires further exploration. We
hypothesize the presence of alternative selective environments (perhaps
evolutionary precursors to mammalian-like GCs?) that facilitate the
selection and expansion of antigen-specific B cell clones. The cloning of
cartilaginous fish AID (Conticello et al., 2005; Quinlan et al., 2017) and
orthologs of other molecules key to the germinal center reaction in
mammals, will allow this hypothesis to be put to the test.
Now that long term memory has unequivocally been proven in
cartilaginous fishes (Eve et al., 2020), we next need to confirm where
long-lived memory B cells reside while awaiting recall and the signaling
molecules required to recruit and sustain them. It is hypothesized that
H. Matz et al.
long-lived memory B cells reside in the epigonal organ of nurse shark
(Castro et al., 2013) however the work performed so far has been unable
to prove this. It may be that the frequency and diversity of the
antigen-specific B cells retained for memory are few in number, as has
previously been observed in studies of mammalian immunological
memory (Mesin et al., 2020) making these cells difficult to find. It would
also be interesting to establish if there is a division in B cell fates in
sharks, for example, between long-lived plasma cells and memory B cells
as observed in mammals.
Moving forward we anticipate the growing number of sequence
datasets for cartilaginous fishes and application of new technologies,
such as single cell sequencing and high-throughput proteomics, will
greatly facilitate our work. Such approaches should help overcome the
paucity of molecular tools available for cartilaginous fishes and better
our understanding of adaptive immunity in this key lineage. As our
knowledge increases, it will become easier to distinguish lineage-
specific traits from those shared across jawed vertebrates, and un­
doubtedly result in a better understanding of Ig-mediated adaptive im­
munity in both human and non-human animals.
Acknowledgements
HM is supported by NIH-NIAID predoctoral fellowship
F31AI147532. DM was supported by an Elphinstone PhD Scholarship
awarded by the University of Aberdeen.
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