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*For correspondence: [email protected] Competing interests: The authors declare that no competing interests exist. Funding: See page 25 Received: 09 May 2017 Accepted: 16 April 2018 Published: 17 April 2018 Reviewing editor: David G Schatz, Yale University School of Medicine, United States Copyright Ott et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Somatic hypermutation of T cell receptor a chain contributes to selection in nurse shark thymus Jeannine A Ott 1 , Caitlin D Castro 2 , Thaddeus C Deiss 1 , Yuko Ohta 2 , Martin F Flajnik 2 , Michael F Criscitiello 1,3 * 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 Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, United States; 3 Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, Texas A&M University, Texas, United States Abstract 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 a 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 TcRa 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 TcRa utilizes SHM to broaden diversification of the primary ab T cell repertoire in sharks, the first reported use in vertebrates. DOI: https://doi.org/10.7554/eLife.28477.001 Introduction All jawed vertebrates share fundamental components of the adaptive immune system. The cartilagi- nous 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 iso- types 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 recep- tors 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 recep- tor 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 pro- cesses 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). Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 1 of 31 RESEARCH ARTICLE

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Page 1: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

*For correspondence:

[email protected]

Competing interests: The

authors declare that no

competing interests exist.

Funding: See page 25

Received: 09 May 2017

Accepted: 16 April 2018

Published: 17 April 2018

Reviewing editor: David G

Schatz, Yale University School of

Medicine, United States

Copyright Ott et al. This article

is distributed under the terms of

the Creative Commons

Attribution License, which

permits unrestricted use and

redistribution provided that the

original author and source are

credited.

Somatic hypermutation of T cell receptora chain contributes to selection in nurseshark thymusJeannine A Ott1, Caitlin D Castro2, Thaddeus C Deiss1, Yuko Ohta2,Martin F Flajnik2, Michael F Criscitiello1,3*

1Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology,College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,Texas, United States; 2Department of Microbiology and Immunology, University ofMaryland at Baltimore, Baltimore, United States; 3Department of MicrobialPathogenesis and Immunology, College of Medicine, Texas A&M Health ScienceCenter, Texas A&M University, Texas, United States

Abstract 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 a 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 TcRa 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 TcRa utilizes SHM to broaden diversification of the primary ab T cell

repertoire in sharks, the first reported use in vertebrates.

DOI: https://doi.org/10.7554/eLife.28477.001

IntroductionAll jawed vertebrates share fundamental components of the adaptive immune system. The cartilagi-

nous 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 iso-

types 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 recep-

tors 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 recep-

tor 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 pro-

cesses 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).

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 1 of 31

RESEARCH ARTICLE

http://creativecommons.org/licenses/by/4.0/
http://creativecommons.org/licenses/by/4.0/
https://doi.org/10.7554/eLife.28477.001
https://doi.org/10.7554/eLife.28477
https://creativecommons.org/
https://creativecommons.org/
http://elifesciences.org/
http://elifesciences.org/
http://en.wikipedia.org/wiki/Open_access
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Page 2: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

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 TcRa rearrangements in

hybridomas (Marshall et al., 1999), TcRb sequences from HIV-positive individuals (Cheynier et al.,

1998), and reports of SHM in TcRa 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 d and g in the dromedary camel and TcR g in the sandbar shark somatically hypermutate

(see below) (Antonacci et al., 2011; Chen et al., 2012). Despite these findings, the general consen-

sus 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 TcRg translocon in C. plumbeus showed definitively that SHM is occur-

ring at that locus (Chen et al., 2009). Shark TcRg SHM occurs in two distinct patterns: point muta-

tions 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 TcRg locus. Chen et al. (2012) examined ratios of replace-

ment (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

TcRg uses SHM to generate a more diverse repertoire rather than for affinity maturation. SHM-

induced changes to TcRd in camels showed similar results.

Early work in our lab also suggested that SHM occurs in the less restricted gd T cells in nurse shark

(Ginglymostoma cirratum) and perhaps in the alpha chain of MHC-restricted ab 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 thymo-

cyte clones containing the same unique third complementarity-determining region (CDR3). Our data

suggest that SHM of TcRa is involved in primary T cell repertoire diversification and the enhance-

ment 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).

Results

Somatic hypermutation in TcR gV and TcR dVWe assessed the presence of possible SHM within TcR V segments from g , d, and b chains. Using

neighbor-joining consensus trees, we grouped sequences from each chain into V families based on

85% nucleotide identity and into V subfamilies based on 90% nucleotide identity. We then examined

each V subfamily for the possibility of SHM. However, since none of the sequences from these chains

contained CDR3 regions, we did not rigorously analyze mutations within these chains.

We first corroborated the original finding of SHM in TcRg variable regions (V) in sandbar shark

spleen (Chen et al., 2012; Chen et al., 2009) using peripheral lymphoid tissue from the spiral valve

(intestine) of nurse shark. We also examined mutation in clones from the thymus. We conservatively

assigned 69 sequences to 9 V genes from three TcRg families (Figure 1). Even with a conservative

assignment of clones to predicted germline V sequences, we found nearly twice as many V genes as

in the sandbar shark locus (which only contains five Vs). Only TcR g4 did not display mutations in the

nurse shark, but since we found only two g4 sequences, it is possible that it occurs but our sample

was too small to observe it.

We then investigated the possibility of mutation occurring at the TcRd locus. We analyzed muta-

tion in clones from nurse shark thymus, peripheral blood leukocytes, and spiral valve, conservatively

grouping 111 clones into 12 V genes from 7 TcRd families (Figure 2). Only one of these families (TcR

d10) lacked mutation. We found that in five of the seven dV families, the same V-d segments used to

generate d cDNA sequences also generated a cDNA sequences. The sequence diversity at TcRg and

TcRd was in contrast to TcRb, where we found no such evidence for mutation in 56 sequences repre-

senting six V segments from six different V families (Figure 3). Limited existing data also do not

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 2 of 31

Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477
Page 3: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

support mutation at NAR-TcR, a distinct TcR containing a NAR V domain supported by a more

canonical V d domain, each resulting from independent V(D)J rearrangements (Criscitiello et al.,

2006) (data not shown).

Identification of TcR Va genes in the nurse shark genomeWe identified 17 germline a/d V gene sequences corresponding to 12 unique V segments. Unfortu-

nately, these segments matched only two groups of sequences in our TcRa dataset (TcRa/d V4 and

V9), likely due to inter-individual polymorphisms. In the absence of a complete germline sequence

for this locus, we limited our database for TcRa to thymocyte clones with the same unique CDR3 sig-

nature. The CDR3 region results from the somatic recombination and assembly of variable (V) and

joining (J) gene segments during lymphocyte development in the thymus (Kuklina, 2006;

Lantelme et al., 2008). The recombination process cleaves DNA and initiates repair mechanisms

that result in the random insertion of non-template (N) nucleotides within the join, forming a unique

binding sequence that contributes to the diversity and specificity of a TcR (Gellert, 2002;

[1.1] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|gV1.1 Consensus— - CAGATTGTCCCATCCATTACCAAAAATGAGGGTAAAACCGCTCGATTCAAATGCGAAGTCGAAGGTGCAACTATTACCGCCACTGATCGGCTTCACTGGTACCAGCAAAAGCCCGGCCAGGCACCCACCCGAATCCTCTATTATGGAGCAGGCACATCGCAGGATCCGGGATTTGGTGAACGGTTCAAAGCTGGGAAATCTAAAGACAAAGAATTCTACCTCACAATCGACAAAATAAAGACGGAGGACAConsensus gV1.1— -..Q..I..V..P..S..I..T..K..N..E..G..K..T..A..R..F..K..C..E..V..E..G..A..T..I..T..A..T..D..P..L..H..W..Y..Q..Q..K..P..G..Q..A..P..T..R..I..L..Y..Y..G..A..G..T..S..Q..D..P..G..F..G..E..R..F..K..A..G..K..S..K..D..K..E..F..Y..L..T..I..D..K..I..K..T..E..D..gV1.1J_PBL14_150831 ..........................................................................................................................................................................................................................................................gV1.1J_THY01_150408 .........................................................................................C................................................................................................................................................................

[1.3] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|gV1.3-Consensus - CAGATTGTCCCATCCATTACCAAAAATGAGGGTAAAACCGCTCGATTCAAATGCGAAGTCGAAGGTGTAACTGTCAGCAACGATGATCCCCTTCACTGGTACCAGCAAAAGCCCGGCCAGGCACCCACCCGAATCCTCTATTATGGAGCAAGCACATCGCGGGATCCGGGATTTGGTGAACGGTTCAAAGCTAGGAAATTTAAAGACAAAGATTTCTACCTCACAATCGACAAAATAAAGACAGAGGACAConsensus gV1.2- - .Q..I..V..P..S..I..T..K..N..E..G..K..T..A..R..F..K..C..E..V..E..G..V..T..V..S..N..D..D..P..L..H..W..Y..Q..Q..K..P..G..Q..A..P..T..R..I..L..Y..Y..G..A..S..T..S..R..D..P..G..F..G..E..R..F..K..A..R..K..F..K..D..K..D..F..Y..L..T..I..D..K..I..K..T..E..D..gV1.3J_PBL05_150824 ..........................................................................................................................................................................................................................................................gV1.3J_THY12_150415 ..........................................................................................................................................................................................................................................................

[1.4] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|gV1.4—Consensus CAGATTGTCCCATCCATTACCAAAAATGAGGGTAAAACCGCTCGATTCAAATGCGAAGTCGAAGGTGTAACTATCAGCAGCAATAACCCCCTTCACTGGTACCAGCAAAAGCCCGGCCAGGCACCCACCCGAATCCTCTATTATGGAGCAGGCACATCGCGGGATCCGGGATTTGGTGAACGGTTCAAAACTGGGAAATCTAAAGACAAAGAATTCTACCTCGCAATCGACAAACTAAAGACAGAGGACAConsensus gV1.3—- .Q..I..V..P..S..I..T..K..N..E..G..K..T..A..R..F..K..C..E..V..E..G..V..T..I..S..S..N..N..P..L..H..W..Y..Q..Q..K..P..G..Q..A..P..T..R..I..L..Y..Y..G..A..G..T..S..R..D..P..G..F..G..E..R..F..K..T..G..K..S..K..D..K..E..F..Y..L..A..I..D..K..L..K..T..E..D..gV1.4J_PBL*02 seqs* .........................................................................................................................................................................................................................................................gV1.4J_THY*02 seqs* ..........................................................................................................................................................................................................................................................gV1.4J_PBL10_150824 .....................................................................................................................................................................................................................................................A....gV1.4J_THY09_150415 ---------------------................................................................................................................................................................................................................................A....

gV1.3. .

[1.5] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|gV1.5—Consensus . CAGATTATCCCATCCATTACCAAAGACGTGAGCAAAATCGGTATATTCAAATGCGAAGTCGATGGTGTAAAGATCAGGAATAGTTATCCCCTTCACTGGTACCAGCACAAGCCTGGCCAGGCACCCACCCGAATCCTCTATTATGGATCAGGCACATCGCGAGATCCGGGATTTGGTGAACGGTTCAAAGCTGGGAAATCTAAAGGCAACGAATTCTACCTAACAATCGACAAAGTAAAGACGGAGGACAConsensus gV1.4—. .Q..I..I..P..S..I..T..K..D..V..S..K..I..G..I..F..K..C..E..V..D..G..V..K..I..R..N..S..Y..P..L..H..W..Y..Q..H..K..P..G..Q..A..P..T..R..I..L..Y..Y..G..S..G..T..S..R..D..P..G..F..G..E..R..F..K..A..G..K..S..K..G..N..E..F..Y..L..T..I..D..K..V..K..T..E..D..gV1.5J_PBL*07 seqs* ..........................................................................................................................................................................................................................................................

[3.1] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...gV3.1 Consensus CAGTCTCCAATATCAATAACCAGAGCACCGGGGAAGACTGCGCGGTTTGAATGTAAGTATGAAGGGAGCGATTTCGACAATACAGTTATCCACTGGTACCGGCACATCCCGGGTCAACGGATACAGCGCATTTTATATTTCAAGAGTCCGTCTGAAACTGAGCAAGATATCACCGACAAAATACTATTTGCTGACAAAAATTCTGCGACCAAAACGTGCAGCTTGGCTATGAAGAAAATCAAAConsensus gV2.1-- ..Q..S..P..I..S..I..T..R..A..P..G..K..T..A..R..F..E..C..K..Y..E..G..S..D..F..D..N..T..V..I..H..W..Y..R..H..I..P..G..Q..R..I..Q..R..I..L..Y..F..K..S..P..S..E..T..E..Q..D..I..T..D..K..I..L..F..A..D..K..N..S..A..T..K..T..C..S..L..A..M..K..K..I..K.gV3.1J_SPV*02 seqs* ...................................................................................................................................................................................................................................................gV3.1J_THY*04 seqs* ...................................................................................................................................................................................................................................................gV3.1J_PBL*03 seqs* --------------------------------...................................................................................................................................................................................................................gV3.1J_THY*04 seqs* --------------------------------...................................................................................................................................................................................................................

[3.2] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...gV3.2-Consensus - CAGTCTCCAATATCAATAACCAGAGCACCGGGGAAGACTGCGCGGTTTGAATGTAAGTATGAAGGGAGCGATTTCGACAATACGGTTATCCACTGGTACCGGCACATCCCGGGTCAACGGATACAGCGCATTTTATATTTCAAGAATCCGTCTGAAACTCGGCAAGATATCGCCGACAAAATACTATTGGCTGATAAAAATTCTGCGACCAAAACGTGCAGCTTGGCTATGAAGAAAATCAAAConsensus gV2.2-- ..Q..S..P..I..S..I..T..R..A..P..G..K..T..A..R..F..E..C..K..Y..E..G..S..D..F..D..N..T..V..I..H..W..Y..R..H..I..P..G..Q..R..I..Q..R..I..L..Y..F..K..N..P..S..E..T..R..Q..D..I..A..D..K..I..L..L..A..D..K..N..S..A..T..K..T..C..S..L..A..M..K..K..I..KgV3.2J_THY08_150408 ...................................................................................................................................................................................................................................................gV3.2J_SPV04_151014 ...................................................................................................................................................................................................................................................gV3.2J_PBL*08 seqs* --------------------------------...................................................................................................................................................................................................................gV3.2J_THY04_150514 --------------------------------...................................................................................................................................................................................................................

[3.3] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...gV3.3 Consensus - CAGTCTCCAATATCAACAACCAGAGCTCCGGGGAAGACTGCGCGGTTTGAATGTAAGTTTGAAGGGAGCAATTTCGACAATACGGTTATCCACTGGTACCGGCACATCCCGGGTCAACGGATACAGCGCATTTTATATTTCAAGAGTCAGTTTGAAACTCAGCAAGATATCACCGACAAAGTACTAGTTGCTGAAAAAAATTCTGAGTCGAAAACCTGCAGCTTGCTCATGAGGAATATCAAGConsensus gV2.3-- .Q..S..P..I..S..T..T..R..A..P..G..K..T..A..R..F..E..C..K..F..E..G..S..N..F..D..N..T..V..I..H..W..Y..R..H..I..P..G..Q..R..I..Q..R..I..L..Y..F..K..S..Q..F..E..T..Q..Q..D..I..T..D..K..V..L..V..A..E..K..N..S..E..S..K..T..C..S..L..L..M..R..N..I..K.gV3.3J_SPV03_151014 ...................................................................................................................................................................................................................................................gV3.3J_THY*02 seqs* ...................................................................................................................................................................................................................................................gV3.3J_PBL*02 seqs* --------------------------------...................................................................................................................................................................................................................gV3.3J_SPV01_151014 .....................................T.............................................................................................................................................................................................................gV3.3J_THY10_150420 ............................T......................................................................................................................................................................................................................

[3.4] ....|....|....|....|....|... CDR1 ....|....|....-------------- CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...Consensus gV3.4-- CAGTCTCCAATGTCAATGATCAGAGCTCCGGGGAACACAATTTGGTTGGACTGTAAGTATGAAGGGAGAGACTTCGACAATACGGTTATCCACTGGTACCGGCACATCCCGGGTCAACGGATACAGCGCATTTTATATTTCAAGAATCCGTCTGAAACTCAGTCCGATATCGCCGACATAACAGTACTTGCTGAAAAAAATTCTGAGACCAAAACATGCAGCTTGCTCATGAGGAATATCAAGConsensus gV3.4-- .Q..S..P..M..S..M..I..R..A..P..G..N..T..I..W..L..D..C..K..Y..E..G..R..D..F..D..N..T..V..I..H..W..Y..R..H..I..P..G..Q..R..I..Q..R..I..L..Y..F..K..N..P..S..E..T..Q..S..D..I..A..D..I..T..V..L..A..E..K..N..S..E..T..K..T..C..S..L..L..M..R..N..I..K.gV3.4J_SPV*03 seqs* ...................................................................................................................................................................................................................................................gV3.4J_THY*02 seqs* ...................................................................................................................................................................................................................................................gV3.4J_THY01_150520 --------------------------------...................................................................................................................................................................................................................gV3.4J_THY06_150408 ................................................................................................................................................................................................................................A..................

[4] ....|....|....|....|....| CDR1 ....|....|....---- - - CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|gV4-Consensus --- CTATCGGTCAGCAGGGCTCGGACAAAAATGGCCCGTTTTCGGTGCATCTTGGAGGATGTGGATTTCGCGAGTGTGATCGTGCACTGGTACCGGCACACTCCGAGCCAGGGTTTCCGCCGCATTCTCTACGTGACTGGAGCCCGACCAGTTTACGACGACGATCTCGCCGACAGATTCACATCCGAAAACGTGGAGAAAGAGAAAAGCAGTCTGCTCGACATAAGAAACATConsensus gV4----. .L..S..V..S..R..A..R..T..K..M..A..R..F..R..C..I..L..E..D..V..D..F..A..S..V..I..V..H..W..Y..R..H..T..P..S..Q..G..F..R..R..I..L..Y..V..T..G..A..R..P..V..Y..D..D..D..L..A..D..R..F..T..S..E..N..V..E..K..E..K..S..S..L..L..D..I..R..N...gV4J_PBL*08 seqs* ......................................................................................................................................................................................................................................gV4J_THY*06 seqs* ......................................................................................................................................................................................................................................

Figure 1. Alignment of Gamma V clones suggests minimal somatic hypermutation. Thymocyte clones for nine gV groups from three different predicted

V genes. CDR regions are marked above the scale for each gV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots

represent identity to this sequence. We highlighted nonsynonymous changes in black; synonymous changes are underlined. Gaps are used for

alignment purposes and indicate a shortened sequence (at the beginning or end). Sequences are identified by a single clone number or a group of

identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘THY’ are from thymus, ‘PBL’ are from

peripheral blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We deposited all 69 sequences into GenBank under accession numbers

KY351639 – KY351707.

DOI: https://doi.org/10.7554/eLife.28477.002

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 3 of 31

Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.002
https://doi.org/10.7554/eLife.28477
Page 4: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

Kuklina, 2006). Once recombination ceases and the thymocyte proliferates, this distinctive CDR3

sequence is perpetuated in all daughter cells (Murphy and Weaver, 2017). Since it is unlikely that

two thymocytes would generate identical CDR3 sequences during VJ recombination, we predict that

amplicons containing identical CDR3 sequences derived from the same progenitor and thus must

contain the same germline V and J segments. Alpha CDR3s exhibited substantial variation within our

shark sequences, despite the absence of diversity (D) segments. For example, TcRa V1 sequences

using the same V and J segments had CDR3 lengths that differed by as many as six amino acids (18

nucleotides), and few CDR3s shared more than one amino acid within this V-J join (Figure 4). Fur-

ther, the majority of sequences in our overall dataset do contain N and palindromic (P) nucleotides

within this join. Using clones containing the same V segment from different sharks, we determined

the putative end of each V segment. We first aligned sequences containing the same V segment.

Then, assuming that any nucleotide present in the same position within more than one shark must

be germline, we determined which nucleotides within a join belong to the V segment. We then

[3.2] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...dV3.2 Consensus CCGCAGTCCAGCTGTACAAACCTCTGTTACCGGTGAGACTGTTACTTTAAGCTGTGAATATTCAGGATTCTGCCAGTACACGATTTACTGGTACCGTCAGTCCCCAGGAGAGGGTCTGAAATACCTGCTTCAGCGTTACACGTCAGGAGAGCAGAATAAAAAAAATGCAACTGGAGGCAGAATTTCTGCTGCTATTGACTCCTCAGCAAAAATCAGTCGATTAAAGATCTCCACAATACAACTGAGCGACTCT

dV03 Consensus .. R..S..P..A..V..Q..T..S..V..T..G..E..T..V..T..L..S..C..E..Y..S..G..F..C..Q..Y..T..I..Y..W..Y..R..Q..S..P..G..E..G..L..K..Y..L..L..Q..R..Y..T..S..G..E..Q..N..K..K..N..A..T..G..G..R..I..S..A..A..I..D..S..S..A..K..I..S..R..L..K..I..S..T..I..Q..L..S..D..S.

dV3.2_SPV*04 seqs* .............................................................................................................................................................................................................................................................

dV3.2_THY19_041510 .............................................................................................................................................................................................................................................................dV3.2_THY14_041510 .................................................................................A................................................................................C.........................................G.........................................T......

[5.1] CDR1 CDR2

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..dV5.1 Consensus ATTGTGTGGGCAGCAATTCTAACAGACTTGTGTCATGGAGATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGGACGGAAGGAGAGACGATGATGCTAAGCTGTACCTATGATACCACATCAACTTCCTATACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATATACTGTTGAGAAGTACCACTGGCTATGAGGATAAAGCAGATTTTGCCAGAAATCGCTTCTTTGCGGAACTGCAGACATCGAATAAACTCACCAGTTTGACTGTCACTGGGTTACAACTGACCGACGCTGdV05 Consensus .I..V..W..A..A..I..L..T..D..L..C..H..G..D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..T..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..T..T..G..Y..E..D..K..A..D..F..A..R..N..R..F..F..A..E..L..Q..T..S..N..K..L..T..S..L..T..V..T..G..L..Q..L..T..D..A...dV05_THY*02 seqs* ----------------------------------------..............................................................................................................................................................................................................................................................T............dV05_THY*02 seqs* ----------------------------------------....................................T......................................................................................................................................................................................................................................dV05_SPV09_101409 ....................................................................................................................T............G.G...............................................................................................................................................................................dV05_SPV11_101409 ..............C..............................................................A......................................T....T...........G..........................................................G...A..............................................................................................................dV05_SPV12_101409 ..............C..............................................................A.............C........................T....T...........G..........................................................G...A..............................................................................................................dV05_THY*04 seqs* ----------------------------------------.............................................................................................................................................................................................................T.............................................................dV05_SPV*02 seqs* .....................................................................................................................................................................................................................................................T.............................................................

[7.2] CDR1 CDR2

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.dV7.2 Consensus AGAAGTTCGGACAGAAGGAGACGTGGTTACTCTAAGTTGTACCTATGAAGCTACAGTGAGCTACTATACACTCTTCTGGTACCGACAGCAACCAGACACACGTCCTGACTTTATTATGCTCAAAGATACTGGTGGTTCTAATGATAAAGCAAATTTTGCACAAGATCGTTTCTCTATGGAACTTCAGACCTCGAAGAAATTCACAAdV07 Consensus ..E..V..R..T..E..G..D..V..V..T..L..S..C..T..Y..E..A..T..V..S..Y..Y..T..L..F..W..Y..R..Q..Q..P..D..T..R..P..D..F..I..M..L..K..D..T..G..G..S..N..D..K..A..N..F..A..Q..D..R..F..S..M..E..L..Q..T..S..K..K..F..T..dV07_PBL*03 seqs* .............................................................................................................................................................................................................. dV07_THY*04 seqs* ..............................................................................................................................................................................................................dV07_PBL20_072310 ------------------------------------------------------------------.........T..................................................................................................................................dV07_THY06_051410 .................................................A............................................................................................................................................................dV07_PBL*02 seqs* ...................................................................................................................................T..........................................................................dV07_THY05_052010 ...................................................................................................................................T..........................................................................dV07_THY26_062110 ..............................................................................................................C....................T..........................................................................

[10.1] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|dV10.1 Consensus CCAGAGTCCACCTTTACAGACCTCGGCTGTTGGCGACACTGTTGCTTTAAGCTGTCAATATTCAGGCTTCTGTCAGTATGCAGTTTACTGGTACAGTCAGTCCACAACACATGCGCCAAAATACCTACTTCAGGGACATACTTCAGGAGAAGGGAAGAAAGAAAATGCTG dV10 Consensus . .Q..S..P..P..L..Q..T..S..A..V..G..D..T..V..A..L..S..C..Q..Y..S..G..F..C..Q..Y..A..V..Y..W..Y..S..Q..S..T..T..H..A..P..K..Y..L..L..Q..G..H..T..S..G..E..G..K..K..E..N..A.. dV10_SPV*04 seqs* ..........................................................................................................................................................................

dV10_THY19_040810 . ..........................................................................................................................................................................

[12.1] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...dV12.1_Consensus CGGTTGAGGAAGGACAAACGGTAATGTTGAACTGTAGTTACAAATCGTTGGTTGCTACTGCTCTCTTCTGGTACGTCCAACATCCTGGGGAAGCTCTGAGATATTTACTGAGGGCGTACAAGGATGAAGAGTGGGAGAGCTCACCAGCTTTCCGTGACCGGTTTTCTGCCAATTTGGACAAAGTCAAAAAAGTGGTCCCTTTGAGAATTAATGAAACCCGTCTGTCTGACTCAdV12.1_Consensus ...V..E..E..G..Q..T..V..M..L..N..C..S..Y..K..S..L..V..A..T..A..L..F..W..Y..V..Q..H..P..G..E..A..L..R..Y..L..L..R..A..Y..K..D..E..E..W..E..S..S..P..A..F..R..D..R..F..S..A..N..L..D..K..V..K..K..V..V..P..L..R..I..N..E..T..R..L..S..D..S.dV12.1_THY*14 seqs* .........................................................................................................................................................................................................................................

dV12.1_PBL*02 seqs* .........................................................................................................................................................................................................................................

[12.2] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...dV12.2_Consensus CGGTTGAGGAAGGACAAACTGTAATGTTGAACTGTAGTTACAAAACGTCGGGTGCTGCTACTCTCTTCTGGTACGTCCAATATCCTGGGGAAGCTCCGAGATATTTACTGAGGGCGTACAAGGATGAAGAGAGGAAGAGCTCACCAGATTTCCGTGACCGGTTTTCTGCCAATTTGGACAAAGTCAAAAAAGTGGTCCCTTTGAGAATTAATGAAACCCGTCTGTCTGACTCAdV12.2_Consensus ...V..E..E..G..Q..T..V..M..L..N..C..S..Y..K..T..S..G..A..A..T..L..F..W..Y..V..Q..Y..P..G..E..A..P..R..Y..L..L..R..A..Y..K..D..E..E..R..K..S..S..P..D..F..R..D..R..F..S..A..N..L..D..K..V..K..K..V..V..P..L..R..I..N..E..T..R..L..S..D..S..dV12.2_THY*04 seqs* .........................................................................................................................................................................................................................................

dV12.2_PBL28_072310 .........................................................................................................................................................................................................................................

[12.3] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|dV12.3_Consensus CGGTTGAGGAAGGACAAACTGTAATGTTGAACTGTAGTTACAAAACGTCGGGTGCTGCTACTCTCTTCTGGTACGTCCAATATCCTGGGGAAGCTCCGAGATATTTATTGAGGGCGTACAAGGATGAAGAGTGGGAGAGCTCACCAGATTTCCGTGACCGGTTTTCTGCCAATTTGGACAAAGTCAAAAACGTGGTCCCTTTGAGAATTAATGAAACCCGTCTGTCTGACTCAdV12.3_Consensus ...V..E..E..G..Q..T..V..M..L..N..C..S..Y..K..T..S..G..A..A..T..L..F..W..Y..V..Q..Y..P..G..E..A..P..R..Y..L..L..R..A..Y..K..D..E..E..W..E..S..S..P..D..F..R..D..R..F..S..A..N..L..D..K..V..K..N..V..V..P..L..R..I..N..E..T..R..L..S..D..S.dV12.3_THY09_052010 .........................................................................................................................................................................................................................................

dV12.3_PBL*02 seqs* .........................................................................................................................................................................................................................................dV12.3_PBL30_072310 ...........................................................T...............................................C............................................................................................................................. dV12.3_THY12_052010 ........................................................A..G....................C...............T..........C.......................................C..........................................A..........................................

[16.1] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..dV16.1 Consensus AGAGAGGGAAAAGGTCACTTTAACCTGCACGTACACCGATGATGTTGATTACTTGTTCTGGTACCGCCAGCACCCTGGCAGAAAGCCCGATTTCGCAGTCCGTAGGCACAAGACCGGCAGGGCTGAATCCAAGGCAGATTTTGCTCAGAAGCGGTTTTCTGACACAGTCCAGCAGTCAGACAAGTCATACCGdV16.1 Consensus ..E..R..E..K..V..T..L..T..C..T..Y..T..D..D..V..D..Y..L..F..W..Y..R..Q..H..P..G..R..K..P..D..F..A..V..R..R..H..K..T..G..R..A..E..S..K..A..D..F..A..Q..K..R..F..S..D..T..V..Q..Q..S..D..K..S..Y..dV16.1_PBL*03 seqs* ................................................................................................................................................................................................

dV16.1_THY13_052010 ................................................................................................................................................................................................

[16.2] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..dV16.2_Consensus AGAGAAGGAAAATGTCACTTTAACCTGCACATACACCGATGATGTTGGTTACTTGTTCTGGTACCGCCAGCACCCTGGCAGAAAGCCCGAGTTCGCAGTCCGTACGTCCAAGAGCAGCAGTGCTGAATCCAAGGCAGATTTTGCTCAGAAGCGGTTTTCTAGCACAGTCCAGCAGTCAGACAAGTCATATCGdV16.2_Consensus ..E..K..E..N..V..T..L..T..C..T..Y..T..D..D..V..G..Y..L..F..W..Y..R..Q..H..P..G..R..K..P..E..F..A..V..R..T..S..K..S..S..S..A..E..S..K..A..D..F..A..Q..K..R..F..S..S..T..V..Q..Q..S..D..K..S..Y...dV16.2_PBL*02 seqs* ................................................................................................................................................................................................

dV16.2_THY42_062110 ................................................................................................................................................................................................

[16.3] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|dV16.3_Consensus AGAGAAGGAAAATGTCACTTTAACCTGCACATACACCGATGATGTTGATTACTTGTTCTGGTACCGCCAGCACCCTGGCAGAAAGCCCGAGTTCGCAGTCCTTACGTACAAGAGCAGCAGTGATGAATACAAGGCAGATTTTGCTAAGAAGCGGTTTTCTAGCACAGTCCAGCAGTCAGACAAGTCATATCGdV16.3_Consensus ..E..K..E..N..V..T..L..T..C..T..Y..T..D..D..V..D..Y..L..F..W..Y..R..Q..H..P..G..R..K..P..E..F..A..V..L..T..Y..K..S..S..S..D..E..Y..K..A..D..F..A..K..K..R..F..S..S..T..V..Q..Q..S..D..K..S..Y..dV16.3_THY*06 seqs* ................................................................................................................................................................................................ dV16.3_THY17_062110 .......................................................................T..................................................C.....................................................................dV16.3_THY37_062110 ..............................................A...............................................A.G.........................C.....C................C..............................................dV16.3_THY*03 seqs* ..............................................A......................................................G...........C........C.....................................................................

[16.4] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|dV16.4_Consensus AGAGAGGGAAAATATCACTTTAACCTGTACGTACACCGATGATGTTGATTACTTGTTCTGGTACCGCCAGCACGCAGGCAGAAAACCCGATTTCGCAGTCCGTAGGCGGAAGACCGGCGGTGCTGTATTCAAGGCAGATTTTGCTCAGAAGCGGTTTTCTGACACAGTCCAGCAGTCAGACAAATCATACCGdV16.4_Consensus ..E..R..E..N..I..T..L..T..C..T..Y..T..D..D..V..D..Y..L..F..W..Y..R..Q..H..A..G..R..K..P..D..F..A..V..R..R..R..K..T..G..G..A..V..F..K..A..D..F..A..Q..K..R..F..S..D..T..V..Q..Q..S..D..K..S..Y..dV16.4_PBL33_072310 ................................................................................................................................................................................................

dV16.4_THY*05 seqs* ................................................................................................................................................................................................

[17.1] CDR1 CDR2 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...dV17.1 Consensus TTGAAAGCAGGTCAAACTTTCACAACGCAATGCAGCTATTCAACAACAGTAAGATCGTATGATCTGTACTGGTACCGAAAGTCTCCTGATGCTCCCCTGGAGATGATAGTGTGGAGTAGTGCGAGTGGCAGTCGGGGAAAAGCGAAAAATATTGGAACTCGCTTCTCTTCTGAGATAAACACAACAACAAAAACATTTGTTTTAAdV17 Consensus .L..K..A..G..Q..T..F..T..T..Q..C..S..Y..S..T..T..V..R..S..Y..D..L..Y..W..Y..R..K..S..P..D..A..P..L..E..M..I..V..W..S..S..A..S..G..S..R..G..K..A..K..N..I..G..T..R..F..S..S..E..I..N..T..T..T..K..T..F..V..L..dV17_PBL*06 seqs* .............................................................................................................................................................................................................

dV17_THY*17 seqs* .............................................................................................................................................................................................................dV17_THY19_052010 ..........................................................C..................................................................................................................................................

Figure 2. Alignment of Delta V clones suggests somatic hypermutation. Thymocyte clones for 12 dV groups from seven different predicted V genes.

CDR regions are marked above the scale for each dV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots represent

identity to this sequence. We highlighted nonsynonymous changes in black; synonymous changes are underlined. Gaps are used for alignment

purposes and indicate a shortened sequence (at the beginning or end). Sequences are identified by a single clone number or a group of identical

clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘THY’ are from thymus, ‘PBL’ are from peripheral

blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We deposited all 112 sequences into GenBank under accession numbers KY346705 –

KY346816.

DOI: https://doi.org/10.7554/eLife.28477.003

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 4 of 31

Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.003
https://doi.org/10.7554/eLife.28477
Page 5: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

repeated this process for each J segment. Of 290 clones, we found 197 (68%) unique sequences

within this join (0–34 nucleotides in length), suggesting that most sequences contain N/P nucleotides

[1] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.bV1 Consensus---- GAAGGAGAGTCGAAGACACTCTGGTGCAGCCTGAAGGATACGAGCTATCTCCAAATGTACTGGTACAGACAGCAACCAAACCGGACAATAGAAGCTTTGTTTTACTCTGCTGCAACGGGAGCTAGTACAAATTACACATCTGGGTACTATCAGGCCAAACGACCCAGCCAAACACTATTTTCTTTGGATCTGAGGAGCGCCACTTCGTCTCACACGConsensus bV1----.. E..G..E..S..K..T..L..W..C..S..L..K..D..T..S..Y..L..Q..M..Y..W..Y..R..Q..Q..P..N..R..T..I..E..A..L..F..Y..S..A..A..T..G..A..S..T..N..Y..T..S..G..Y..Y..Q..A..K..R..P..S..Q..T..L..F..S..L..D..L..R..S..A..T..S..S..H..T..bV1J_PBL*05 seqs* ........................................................................................................................................................................................................................bV1J_THY*09 seqs* ........................................................................................................................................................................................................................ bV1J_THY01_030210 .........................................................................................................................................................A.............................AA...............................

[2] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...bV2-Consensus --- ATTGTGTCGATTTCAGAAGGACATTCGTCTACACTCAGATGCACGCTGAAGGATACAGCTTACAGATACATGTCCTGGTACAGACAGAAACCGGACCAGGCAATAGAAGCTTTATTCTACTCTTCTGGAAAAGATTTCGTTACAAATTACACATCCGAGCCTTACCAGGCTAAAAGAGAAGATGAAACGCTGTTTAGTTTGGAACTGAGAGAGGCCGCTCCATCTCACConsensus bV2----. .I..V..S..I..S..E..G..H..S..S..T..L..R..C..T..L..K..D..T..A..Y..R..Y..M..S..W..Y..R..Q..K..P..D..Q..A..I..E..A..L..F..Y..S..S..G..K..D..F..V..T..N..Y..T..S..E..P..Y..Q..A..K..R..E..D..E..T..L..F..S..L..E..L..R..E..A..A..P..S..H.bV2J_PBL*06 seqs* ....................................................................................................................................................................................................................................bV2J_THY*13 seqs* ....................................................................................................................................................................................................................................

[3] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..bV3 Consensus ATTGTTTCGATTACTAGAGGGAACTCAGATACACTTAAATGCATGCTGAAGAATACAGTTTACCGCTCTATGTACTGGTACAGACAGCAACACAGCCGGGCAATAGAAGCTTTATACTACTCTGCTGGTGAAGGTTTAATTACAAATTACACATCCGAATCTTATCAGGCTAAAAGACCCAGTGAAACGACGTTTACTTTAGAACTGAAAGGTGCCTCTCCATCTCACACGGConsensus bV3 .. I..V..S..I..T..R..G..N..S..D..T..L..K..C..M..L..K..N..T..V..Y..R..S..M..Y..W..Y..R..Q..Q..H..S..R..A..I..E..A..L..Y..Y..S..A..G..E..G..L..I..T..N..Y..T..S..E..S..Y..Q..A..K..R..P..S..E..T..T..F..T..L..E..L..K..G..A..S..P..S..H..T..bV3J_PBL*05 seqs* ........................................................................................................................................................................................................................................bV3J_THY*07 seqs* ........................................................................................................................................................................................................................................

[3] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.bV4-Consensus --- ATCGTGTCGATTTCAGGAGGACACTCAGCTACACTCAAGTGCACGCTGAAAGATACTGGCTATCTCCGAATGTACTGGTACAGACAGCAAACAGGCCGGGCAGTAGAAGCTTTATTCTTTTCAGTTGCAGCAGGGGTTGTTACAAATTACACATTTGAATCTCATCAGGCCACAAGACCCACTGATACTCTGTTCATTCTGGAACTGAGAACTGCCTTACCGTCTCACACGConsensus bV4----- I..V..S..I..S..G..G..H..S..A..T..L..K..C..T..L..K..D..T..G..Y..L..R..M..Y..W..Y..R..Q..Q..T..G..R..A..V..E..A..L..F..F..S..V..A..A..G..V..V..T..N..Y..T..F..E..S..H..Q..A..T..R..P..T..D..T..L..F..I..L..E..L..R..T..A..L..P..S..H..T.bV4J_PBL14_072310 .......................................................................................................................................................................................................................................

bV4J_THY05_030210 ....................................................................................................................................................................................................................................

[5] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...bV5-Consensus -- GCGCTCGTTTCCCAATGGGAGAACCGCTTGTCTGTTGGTGAAGGGAAGATGGCAGAGATGCAATGTTACCAAAACGACACTAGTGAAAACTGGATGTACTGGTACAGCCAGAGCAGAGGAGCAGGGCTGCAGCTCATAGCAACCTCCATGTTCGGATCTGAACCCACGCTTGAAGAAGGTTTCAAGGGGAGGTTTGAAGTCACTCGATCCAGTCAGAAGTCGTGCAGCCTGAAAATACAGAGCConsensus bV5--- . .A..L..V..S..Q..W..E..N..R..L..S..V..G..E..G..K..M..A..E..M..Q..C..Y..Q..N..D..T..S..E..N..W..M..Y..W..Y..S..Q..S..R..G..A..G..L..Q..L..I..A..T..S..M..F..G..S..E..P..T..L..E..E..G..F..K..G..R..F..E..V..T..R..S..S..Q..K..S..C..S..L..K..I..Q..SbV5J_THY15_030210 ...................................................................................................................................................................................................................................................bV5J_THY23_031210 ...................................................................................................................................................................................................................................................

[9] ....|....|....|....|. CDR1 ....|....|....|----------------. . CDR210 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...bV9-Consensus -- ACTCTCGTCTCCCAGTGGGACAACCACTTGTCTGTTGGAGAAGGGAAGACGGCGGAGGTGCACTGTTACCAAAACGACACTAGCAATAGCTACATGTATTGGTACCGCCAGGTTGGAGGAGCAGGTCTTCAGCTCCTTACCACCTCCATTTTCGGCTCTGGTAGCACGCCAGAAGACAGTTTCAAGGAGCGGACTGAAGTCATTCGACGCAGTCAAAAGTTGTGCAGCCTAAAAATACTGAACConsensus bV9--- .T..L..V..S..Q..W..D..N..H..L..S..V..G..E..G..K..T..A..E..V..H..C..Y..Q..N..D..T..S..N..S..Y..M..Y..W..Y..R..Q..V..G..G..A..G..L..Q..L..L..T..T..S..I..F..G..S..G..S..T..P..E..D..S..F..K..E..R..T..E..V..I..R..R..S..Q..K..L..C..S..L..K..I..L..N.bV9J_PBL10_082410 ...................................................................................................................................................................................................................................................bV9J_THY*05 seqs* ...................................................................................................................................................................................................................................................

Figure 3. Alignment of Beta V clones illustrates a lack of somatic hypermutation. Thymocyte clones for bV groups from six different predicted V genes.

CDR regions are marked above the scale for each bV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots represent

identity to this sequence. We observed three nonsynonymous changes within a single sequence (highlighted in black). Sequences are identified by a

single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘PBL’

are from peripheral blood leukocytes and ‘THY’ are from thymus. We deposited all 57 sequences into GenBank under accession numbers KY351708 –

KY351764.

DOI: https://doi.org/10.7554/eLife.28477.004

< alpha V1 region >< CDR3 >< J Region > 10 20 30 40 50 60 70 80

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....αV1 Consensus-- LFWYRQKTSGSVEYLVQRSKYIRNQERFPGDRLSSDFDHVNHNIQLKVLKTELTDSAVYYCALALW---NDGTYKLIFGGGTRLTVEPKαV1J_V08_102810 ...............................................................ALW---NDGTYKLIFGGGTRLTVEPKαV1J_T17_100710 ...............................................................CR----....................αV1J_V09_110210 ...............................................................RSEYDG----................

αV1J_V07_091410 ...............................................................SP------DNNRLIFGGGTRLTVEPKαV1m_T13_060512 ...............................................................QKN-----..................

αV1m_T17_071212 ...............................................................SR----TGVADQLIFGEGTKLTVEPKαV1J_V07_090110 ...............................................................RPG---...................S

αV1J_V13_101310 ...............................................................RAGL-----ASKLIFGEGTKLTVEPKαV1J_V05_091010 ...............................................................RPVSES---.................

αV1J_V18_090110 ...............................................................RPGA---GGNDKLIFGSGTKVTVEPKαV1m_T14_060512 ..............................................................VN.T----...................

Figure 4. CDR3s of TcR Alpha chain are diverse. Amino acid (aa) alignment of TcR aV1 thymocyte clones illustrating diversity of the third

complementarity-determining region (CDR3). All clones contain identical variable (V) region sequence (aa 1–61). We grouped clones by shared,

identical joining (J) regions (purple boxes) and highlight the differences in the V-J join (CDR3 region) in red boxes.

DOI: https://doi.org/10.7554/eLife.28477.005

The following source data is available for figure 4:

Source data 1. CDR3 regions diversified by exonuclease activity and addition of N and P nucleotides.

DOI: https://doi.org/10.7554/eLife.28477.006

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Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.004
https://doi.org/10.7554/eLife.28477.005
https://doi.org/10.7554/eLife.28477.006
https://doi.org/10.7554/eLife.28477
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(see Figure 4—source data 1). Finally, we never observed the same CDR3 sequence in more than

one shark, suggesting both exonuclease activity and addition of N and P nucleotides help diversify

alpha CDR3s in nurse shark. Therefore, even in the absence of an assembled locus, we were able to

evaluate mutation to germline aV segments by considering changes within only those thymocyte

clones containing identical CDR3s. Our extremely conservative approach of using only clones con-

taining the same CDR3 encoding rearrangement and N and P nucleotide sequences provided us the

assurance that we had distinct aVs descendant from clonal T cells, since it would be extremely

unlikely that two T cells created receptors that contained the exact same nucleotide sequence by

chance.

Somatic hypermutation in nurse shark TcR aVWith SHM confirmed in g and d TcR chains but apparently not the TcR beta chain of nurse shark, we

checked for mutation of the TcRa locus. One might expect mutation in gd T cells since antigen bind-

ing more closely mirrors that of B cells. However, mutations to receptors of MHC-restricted ab T

cells would be surprising given that even minor modifications to these receptors could risk incompat-

ibility with MHC.

Our preliminary V a dataset contained 539 TcRa clones (encoding 286 unique amino acid sequen-

ces representing nine V a families) from three tissues (PBL, spleen, thymus) of two sharks (Joanie,

Mary Junior). Of this total, only 447 sequences contained complete CDR3-J junctions (including all

bases after the last predicted nucleotide of the V segment to the last predicted nucleotide of the J

segment; see Table 1). We observed 239 (53.5%) sequences with unique V regions (from the first

predicted nucleotide of the V segment to the last predicted nucleotide of the J segment) and 179

Table 1. Summary of sequence data used in this paper.

Putative subfamilies within each TcR alpha V family share at least 85% nucleotide identity using nearest-neighbor consensus trees of V

segments. Number of TcR alpha nucleotide (NUC), amino acid (AA) sequences or sequence groups within each category. Highlighted

columns specifically refer to data used in this study. (See results for detailed descriptions of sequences included within each column.)

Alpha Vsegment

Putative #Sub-families

All clonedsequences

CompleteCDR3-Jjunction*

Unique VRegion †

Unique Vsegment‡

UniqueCDR3-J§

Groups withidentical CDR3-J#

CDR3-J groupsin Study**

Sequences ineach dataset††NUC AA NUC AA NUC AA

TRA V1 1 40 40 35 34 24 16 34 32 7 4 2, 3, 3, 5

TRA V2 3 18 18 13 13 13 10 12 12 3 1 5

TRA V3 3 217 194 55 52 35 34 51 50 5 1 4

TRA V4 3 60 28 22 22 21 21 21 21 6 2 2, 2

TRA V5 4 35 34 15 13 9 8 13 13 3 1 2

TRA V6 2 9 9 7 7 5 6 7 7 1 0 0

TRA V7 5 96 60 49 48 39 38 48 48 9 2 2, 2

TRA V9 3 19 19 14 14 12 11 13 13 4 0 0

TRA V10 2 45 45 29 26 21 19 27 26 10 2 4, 9

539 447 239 229 179 163 226 224 48 13 45

*A full list of these sequences can be found in Table 1—source data 1.†V Region includes all bases between the 1 st predicted nucleotide of the V segment to the last predicted nucleotide of the J segment (V and J).

‡V Segment includes all bases between the 1 st predicted nucleotide of the V segment to the last predicted nucleotide of the V segment (V only).§CDR3-J includes all bases after the last predicted nucleotide of the V segment to the last predicted nucleotide of the J segment.

#Number of groups with identical CDR3-J sequences, which we used to determine sequence relatedness (see text for details).

**Number of groups with identical CDR3-J sequences used in this study. (Those not used contained no mutation with V segments.)

††Total number of sequences for each alpha V used to assess somatic hypermutation within this study (e.g., for aV1, 4 different clonal groups contained 2,

3, 3, and 5 identical CDR3-J regions, respectively).

DOI: https://doi.org/10.7554/eLife.28477.007

The following source data available for Table 1:

Source data 1. Tab-delimited text file containing sequences used in this paper for analysis of somatic hypermutation of TcR alpha chain.

DOI: https://doi.org/10.7554/eLife.28477.008

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Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.007
https://doi.org/10.7554/eLife.28477.008
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[1.1] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV1 G1 Consensus GATTCTGTATCACAAACACCGCGTGGAATCATAGTGACTGAGGGAGATGATGTCCAGTTGTTTTGTAATTACACTGCTGCAGCAAGCAACGATCCCAACCTGTTCTGGTACCGTCAGAAAACCAGCGGATCCGTGGAGTATCTAGTACAGAGAAGCAAATATATCAGGGACCAAGAAAGAaV1 G1 Consensus .D..S..V..S..Q..T..P..R..G..I..I..V..T..E..G..D..D..V..Q..L..F..C..N..Y..T..A..A..A..S..N..D..P..N..L..F..W..Y..R..Q..K..T..S..G..S..V..E..Y..L..V..Q..R..S..K..Y..I..R..D..Q..E..R.aV1.1J_SPV18_101310 ................G.....T..T..GT..............................C...............................C..........................................................................A............aV1.1J_SPV08_110210 .....................G...................................A..................T....................................................A..............A.......................A...........aV1.1J_SPV01_101310 ......................CACA.......AC............................................G..................T........................................................T............C...........

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV1 G1 Consensus TTTCCAGGAGATCGGCTTTCCTCGGATTTTGACCATGTGAACCACAATATCCAGCTGAAAGTGCTGAAAACAGAACTGACAGACTCCGCAGTGTATTACTGCGCTCTGAGACGGGGCCACTACGGAACTGGTACTAAGTTTATCTTTGGAGAGGGGACAAAGTTAACTGTGGAGCCAAAAaV1 G1 Consensus. .F..P..G..D..R..L..S..S..D..F..D..H..V..N..H..N..I..Q..L..K..V..L..K..T..E..L..T..D..S..A..V..Y..Y..C..A..L..R..R..G..H..Y..G..T..G..T..K..F..I..F..G..E..G..T..K..L..T..V..E..P..K.aV1.1J_SPV18_101310.....................................................T...T........GC......................................................................GG......................GG...G.G.........GG..aV1.1J_SPV08_110210.............................................................................................................................T......................................AC.................aV1.1J_SPV01_101310.....................................................A...........................................................................GA..AG...................................C............

[1.2] < FR1 > CDR1 < FR2 > CDR2 < 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV1 G2 Consensus GATTCTGTATCACAAACACCACTTGTAATCATAGTGACTGAGGGAGATGATGTCCAGTTGTTTTGTAATTACACTGCTGCAGTAAGCAACGATCCCAACCTGTTCTGGTACCGTCAGAAAACCAGCGGATCCGTGGATTATCTAGTACAGAGAACCAAATATATCAGGAACCAGGAAAGAaV1 G2 Consensus .D..S..V..S..Q..T..P..L..V..I..I..V..T..E..G..D..D..V..Q..L..F..C..N..Y..T..A..A..V..S..N..D..P..N..L..F..W..Y..R..Q..K..T..S..G..S..V..D..Y..L..V..Q..R..T..K..Y..I..R..N..Q..E..R.aV1.1J_SPV *2 seqs* ...C.............GG...................G......................................T........A..........TG.......................................A......C..........T..........T............aV1.1J_SPV05_102810 ........T......................GT......................................T..CA..........................................C..................G.................G........................aV1.1J_SPV23_090110 ....................G.............................................................CG...........G..........................................................G...C.......A.............aV1.1J_SPV18_110210 .C.............GT...G.............................................................CG..T........G......................CGT.................................G.CCC............A........

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..aV1 G2 Consensus TTTCCAGGAGATCGGCTTTCCTCGGATTTTGACCATGTGAACCACAATATCCAGCTGAAAGTGCTGAAAACAGAACTGACAGACTCCGCAGTGTATTACTGCGCTCTGAGACCACGGGCTGAATCAGCTTCTAAGCTTATCTTTGGAGAGGGGACAAAGTTAACCGTGGAGCCAAAAaV1 G2 Consensus .F..P..G..D..R..L..S..S..D..F..D..H..V..N..H..N..I..Q..L..K..V..L..K..T..E..L..T..D..S..A..V..Y..Y..C..A..L..R..P..R..A..E..S..A..S..K..L..I..F..G..E..G. T..K..L..T..V..E..P..K.aV1.1J_SPV *2 seqs* ......................................................T..........................................................................................................GT.....A........aV1.1J_SPV05_102810 ...................................................G...........................................................G.................................................................aV1.1J_SPV23_090110 ..............................................................C.............................C........................T..........C..................................T.............aV1.1J_SPV18_110210 ..................................................T............................................................G.....T............................T................T.............

[1.4] < FR1 > CDR1 < FR2 > CDR2 < 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV1 G4 Consensus GATTCTGTATCACAAACACCGCTTGTAATCATAGTGACTGAGGGAGATGATGTCCAGTTGTTTTGTAATTACACTGCTGCAGCAAGCAACGATCCCAACCTGTTCTGGTACCGTCAGAAAACCAGCGGATCCGTGGAGTATCTAGTAGAGAGAAGCAAATATATCAGGAACCAGGAAAGAaV1 G4 Consensus ..D..S..V..S..Q..T..P..L..V..I..I..V..T..E..G..D..D..V..Q..L..F..C..N..Y..T..A..A..A..S..N..D..P..N..L..F..W..Y..R..Q..K..T..S..G..S..V..E..Y..L..V..Q..R..S..K..Y..I..R..N..Q..E..R.aV1.1J_SPV08_101310 ........................................................................................C..............................................C.TC........C................................aV1.1J_SPV17_090110 ....................................................................................................................................................................................aV1.1J_SPV07_090110 ............................................................C.......................................................................................................................

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..aV1 G4 Consensus TTTCCAGGAGATCGGCTTTCCTCGGATTTAGACCATGTGAACCACAATATCCAACTGAAAGTGCTGAAAACAGAACTGACAGACTCCGCAGTGTATTACTGCGCTCTGAGGCCGGGTACTGGAATAGCTGATCAGCTTATCTTTGGAGAGGGGACAAGTATGACCGTGGAGCCAAAAaV1 G4 Consensus. .F..P..G..D..R..L..S..S..D..L..D..H..V..N..H..N..I..Q..L..K..V..L..K..T..E..L..T..D..S..A..V..Y..Y..C..A..L..R..P..G..T..G..I..A..D..Q..L..I..F..G..E..G..T..K..L..T..V..E..P..K.aV1.1J_SPV08_101310 A..AT..CC...............................................A......................................................................................C...........CG....................aV1.1J_SPV17_090110 .....................................................G........................................................................A..................................................aV1.1J_SPV07_090110 .............................T....................T........................................................................G...................................................GT

[2] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV2 G1 Consensus GATTCTGTCTCACAAACAGCGGCTGCAATTGCAGTGAATGAGGGTGATGATGTCCTGTTGTCCTGTAATTACTCTACAACAAGTAGCAACCCTTACCTGTTCTGGTACCGCCAACATTCCAGGGGGTCCATGCATTTTCTACTCCAGAAAAACCGATATAGTGAGAAACCGGCGACTTTTaV2 G1 Consensus. .D..S..V..S..Q..T..A..A..A..I..A..V..N..E..G..D..D..V..L..L..S..C..N..Y..S..T..T..S..S..N..P..Y..L..F..W..Y..R..Q..H..S..R..G..S..M..H..F..L..L..Q..K..N..R..Y..S..E..K..P..A..T..F.aV2.1m_THY *4 seqs* ....................................................................................................................................................................................aV2.1m_THY05_072012 .........................................................................................G...................................................G......................................

FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.aV2 G1 Consensus ATCAAGGACAGGCTTTCCTTGGATTTTGATCATGTGAACCATACTATCCAGATGGGATTGTTGAAAACAGAACTATCAGACTCCGCAGTGTATTTCTGCGCACTGATTCGCCGGTATGATAACACCAAGCTTATCTTTGGAGAAGGGACCAGACTAACAGTGGAGCCAAAAaV2 G1 Consensus- .I..K..D..R..L..S..L..D..F..D..H..V..N..H..T..I..Q..M..G..L..L..K..T..E..L..S..D..S..A..V..Y..F..C..A..L..I..R..R..Y..D..N..T..K..L..I..F..G..E..G..T..R..L..T..V..E..P..K.aV2.1m_THY *4 seqs* ...........................................................................................................................................................................aV2.1m_THY05_072012 ...........................................................................................................................................................................

[3] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV3_Consensus GATCCAGTTTCCCAGAGCCCACCTCAGTTACCGGTTGAGGAAGGACAAACTGTAAGTTTGAACTGTAGTTACAAAACGTCGGGTGCTGCTACTCTCTTCTGGTACGTCCAATATCCTGGGGAAGCTCCGAGATATTTACTGAGGGCGTACAAGGATGAAGAGAGGAAGAGCTCACCAGATaV3.1_Consensus .D..P..V..S..Q..S..P..P..Q..L..P..V..E..E..G..Q..T..V..S..L..N..C..S..Y..K..T..S..G..A..A..T..L..F..W..Y..V..Q..Y..P..G..E..A..P..R..Y..L..L..R..A..Y..K..D..E..E..R..K..S..S..P..D.aV3J_SPV18_091010 ....................................................................................................................................................................................aV3J_SPV19_101310 ....................................................................................................................................................................................aV3J_SPV19_091010 .......................................................TG.....................................................................................................................G.....aV3J_SPV22_102810 .......................................................TG..................G..GG....................................................................................................

-- FR3 > < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..aV3_Consensus TTCCGTGACCGGTTTTCTGCCAATTTGGACAAAGTCAAAAAAGTGGTCCCTTTGAGAATTAATGAAACCCGTCTGTCTGACTCAGCCATCTATTACTGCGCAGCGGATGGCACCTACAAGCTTATCTTTGGAGGTGGGACCAGACTGACAGTGGAGCCAAAAaV3.1_Consensus .F..R..D..R..F..S..A..N..L..D..K..V..K..K..V..V..P..L..R..I..N..E..T..R..L..S..D..S..A..I..Y..Y..C..A..A..D..G..T..Y..K..L..I..F..G..G..G..T..R..L..T..V..E..P..K.aV3J_SPV18_091010 ..................................................................................................................................................................aV3J_SPV19_101310 ...........................................................................................................................................................A..C...aV3J_SPV19_091010 ..................................................................................................................................................................aV3J_SPV22_102810 ............................................................................................................CA....................................................

[4.1] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV4 G1 Consensus -----GATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGGACGGAAGGAGAGACGATGATGCTAAACTGTACCTATGATACCACATCAAGGTCCTCTACATTATACTGGTATCGGCTGCACCCAGGCACACAGCCCGAGTATATACTGTACAGAAGTACCCCTGGCTATGAGGATACCGCAGATaV4_consensus -----.D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..N..C..T..Y..D..T..T..S..R..S..S..T..L..Y..W..Y..R..L..H..P..G..T..Q..P..E..Y..I..L..Y..R..S..T..P..G..Y..E..D..T..A..D.aV4J_SPV01_102810 ........................................................A..........................C..A....G..................................AG....................................................aV4J_SPV23_110210 ....................................................................T...A......................C............................................................G........A.A............

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.aV4 G1 Consensus -----TTTGCCAGAAATCGCTTCTTTGCGGAACTGGAGACATCGAATAAACTCACCAGTTTGACTGTCACTGGGTTACAATTGACCGACACTGCTCTGTATTACTGTGCTTTCTGGAAACCTAGTGGTTACAGTCTTATCTTTGGAGGTGGGACCAGACTGACAGTGGAGCCCCAAaV4 GI Consensus -----.F..A..R..N..R..F..F..A..E..L..E..T..S..N..K..L..T..S..L..T..V..T..G..L..Q..L..T..D..T..A..L..Y..Y..C..A..F..W..K..P..S..G..Y..S..L..I..F..G..G..G..T..R..L..T..V..E..P..Q.aV4J_SPV01_102810 .........CCA.....T..............T...................................AC.....................................T...........C..C..................................GC............aV4J_SPV23_110210 -..............A....................T............................GC...........C.......................C..GA.......................................................TC........

[4.2] < FR1 > CDR1 < FR2 > CDR2 < 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV4 G2_consensus -- GATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGGACGGAAGGAGAGACGATGATGCTAAGATGTACCTATGATACCACATCAACGTCCTATACATTATACTGGTACCGGCAGCACCCAGGGACACAGCCCGAGTATATACTGTTGAGAAGTAGCCCTGGCTATGAGGATATCGCAGATaV4.1_consensus --- .D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..R..C..T..Y..D..T..T..S..T..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..S..P..G..Y..E..D..I..A..D.aV4J_SPV10_110210 .......................................................................................A.......................................................A............................AA......aV4J_SPV14_090110 ......................................................................................T...............................................C.............................................

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.aV4 G2_consensus --- TTTGCCAGAAATCGCTTCTTTGCGGAACTGCAGACATCGAATAAACTCACCAGTTTGACTGTCACTGGGTTACAATTGACCGACGCTGCTCTGTATTATTGTGCTTTCTGGAAACCTAGTGGTTACAACCTTATCTTTGGAGGTGGGACCAGACTGACAGTGGAGCCAAAAaV4.1_consensus ---.F..A..R..N..R..F..F..A..E..L..Q..T..S..N..K..L..T..S..L..T..V..T..G..L..Q..L..T..D..A..A..L..Y..Y..C..A..F..W..K..P..S..G..Y..N..L..I..F..G..G..G..T..R..L..T..V..E..P..K.aV4J_SPV10_110210 .......................................................................................................................................................................A...aV4J_SPV14_090110 .......A......A..............................GAT....A......................A...........................................................................................A...

[5] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV5 G1 Consensus- GATTCAATTTCACAACACCCTGGCAATTTGTGGATTCGTGACGGAGAGACGGCAATGTTGAACTGCAGTTATACAGCAGAACAC---ACTGGAGAAACTCTCTTCTGGTATATCCAGCATCCTGGCAAATCTCCAAAATATATACTGTCGAGAGATGGCAAGGCGCAAGAGGAGACATCTaV5 G1 Consensus- .D..S..I..S..Q..H..P..G..N..L..W..I..R..D..G..E..T..A..M..L..N..C..S..Y..T..A..E..H.---.T..G..E..T..L..F..W..Y..I..Q..H..P..G..K..S..P..K..Y..I..L..S..R..D..G..K..A..Q..E..E..T..S.aV5J_SPV20_091010 ......................C..................G..............A.............T..........T..---.............................A..............................AA........C....AT................aV5J_SPV17_102810 .....................A....A...................................................C.....TAT..........G...................T............................A......T..........................

-- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..aV5 G1 Consensus TCAGAGTTTTCCAACCGCTTTAGTGCAAACTTGGACACAGACAAGAAAACAATTCCACTATATATTGTAAGGGCTCGTTTCTCTGACTCTGCCATCTATCTCTGCGCGCTGAGACGTGATGAAGCTGGTTACAAGCTAATCTTTGGAGAGGGGACAAGGTTAACCGTGAGCCCAAAAaV5 G1 Consensus- .S..E..F..S..N..R..F..S..A..N..L..D..T..D..K..K..T..I..P..L..Y..I..V..R..A..R..F..S..D..S..A..I..Y..L..C..A..L..R..R..D..E..A..G..Y..K..L..I..F..G..E..G..T..R..L..T..V..S..P..K.aV5J_SPV20_091010 .................................................................................................................................................................................aV5J_SPV17_102810 ...........................................................................................G...T...T......................................G..............................TG......

[7.2] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV7 G2 Consensus GTTTCTGTGATTCAGGGGGAGACCTCGCTCACGCAACGAGAGAAGGAAAATGTCACTTTAACCTGCACATACACCGATGATGTTGATTACTTGTTCTGGTACCGCCAGCACCCTGGCAGAAAGACCGAGTTCGCGGTCCTTACGTACAAGAGCAACAGTGCTGAATCCAAGGCAGATTTTaV7 G8 Consensus .V..S..V..I..Q..G..E..T..S..L..T..Q..R..E..K..E..N..V..T..L..T..C..T..Y..T..D..D..V..D..Y..L..F..W..Y..R..Q..H..P..G..R..K..T..E..F..A..V..L..T..Y..K..S..N..S..A..E..S..K..A..D..F.aV7J_SPV04_102810 ..................................GG.....A..........................................................................................................................................aV7J_SPV14_102810 ..........C.............C....G.AC...TT.............................A........................................T........TTC..................................TA..............T.........

FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...aV7 G2 Consensus GCTCAGAAGCGGTTTTCTAGCACAGTCCAGCAGTCAGACAAGTCATATCGGTTGACCATAACGGTGCTGCAACTGTCTGACACCGCTGTCTATTACTGTGCAGTGAGAGACAGCGGAGATAGAAGGTTGATCTTTGGCCGAGGGACCCAGTTAACGGTGGAACCAAAAaV7 G8 Consensus .A..Q..K..R..F..S..S..T..V..Q..Q..S..D..K..S..Y..R..L..T..I..T..V..L..Q..L..S..D..T..A..V..Y..Y..C..A..V..R..D..S..G..D..R..R..L..I..F..G..R..G..T..Q..L..T..V..E..P..K.aV7J_SPV04_102810 ....................................C...................................G...................................................T..........................................aV7J_SPV14_102810 .............................TG.......................................G............G.................................A.................................................

[10.1] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV10 G1 Consensus GACTCGATCTCCCAGGAGCCGTTTTCAGCGATGAAATTTGAAGATGAATTGGTGACCATTAGTTACAATTATTCGACCACTGCTAGTACATATTCTTTGCAACTATATCGTCAGGATCACGACAAAACCCTAACATTCTTGATCTACATCCCTAACTATGGCGATGCTATCAGAGCTAAGaV10 G2 Consensus .D..S..I..S..Q..E..P..F..S..A..M..K..F..E..D..E..L..V..T..I..S..Y..N..Y..S..T..T..A..S..T..Y..S..L..Q..L..Y..R..Q..D..H..D..K..T..L..T..F..L..I..Y..I..P..N..Y..G..D..A..I..R..A..K.aV10J_SPV *3 seqs* ....................................................................................................................................................................................aV10J_SPV08_090110 ....................................................................................................................................................................................aV10J_SPV23_101210 .................................................................................................................A..................................................................aV10J_SPV *3 seqs* ...................................................................................A................................................................................................aV10J_SPV21_091410 ................................................................................................................G...................................................................

FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....aV10 G1 Consensus GGTGTGGGGCCTCGATTTTCTGCTAATTTCGACGATGTGAAAAGTGAAGGGAATTTCACCATCCGTGATCTGCGACTGTCTGACAATGCCGTGTATTACTGCGGAGTGAAATCTGGAGCAGCTGGTTTTAAGCTGATGTTTGGAGAGGGTACAAAGTTAACTGTGGAGCCAAAAaV102 Consensus .G..V..G..P..R..F..S..A..N..F..D..D..V..K..S..E..G..N..F..T..I..R..D..L..R..L..S..D..N..A..V..Y..Y..C..G..V..K..S..G..A..A..G..F..K..L..M..F..G..E..G..T..K..L..T..V..E..P..K.aV10J_SPV *3 seqs* ..............................................................................................................................................................................aV10J_SPV08_090110 ............................................................................................C.................................................................................aV10J_SPV23_101210 ..............................................................................................................................................................................aV10J_SPV *3 seqs* ...........................................................................................................TC.................................................................aV10J_SPV21_091410 ..........................................................................................................................................................................CC..

[10.2] < FR1 > CDR1 < FR2 > CDR2 <10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|aV10 G2 Consensus GACTCGATCTCCCAGGAGCCGTTTTCAGCGCTGAAATTTGAAGAAGAATCGGTGACCATTAGTTACAATTATTCGACCACTGCTAGGATATATTCTTTGCAACTATATCGTCAGGATCACGACAAAACCCTAACATTCTTGATCTACATCCCTAACTATGGCGATGCTATCAGAGCTAAGGGaV10G1 Consensus - . .D..S..I..S..Q..E..P..F..S..A..L..K..F..E..E..E..S..V..T..I..S..Y..N..Y..S..T..T..A..R..I..Y..S..L..Q..L..Y..R..Q..D..H..D..K..T..L..T..F..L..I..Y..I..P..N..Y..G..D..A..I..R..A..K..GaV10J_SPV13_090110 -..............................A..........G..T....T.............................AA..G....C........................A...................................................C................aV10J_SPV13_091010 -..............................A.............T....T..................A................T..GC.T.................................GT.......C..........................GAG..T...............aV10J_SPV16_110210 -...............................A................A................................................................A............CT.A...................................A................aV10J_SPV22_091410 -.......................C.......A................A....................................T..............................................................................C.....G...........

- FR3 > CDR3 < J >190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..aV10 G2 Consensus TGTGGGGCCTCGATTTTCTGCTAATTTCGACGATGTGAAAAGTGAAGGGAATTTCACCATCCGTGATCTGCGACTGTCTGACAATGCCGTGTATTACTGCGGACTGCTCGAGTATGATGGCTTCAAGCTTATCTTTGGAGGTGGGACCAGACTGACAGTGGAGCCAAAAaV10 G1 Consensus ..V..G..P..R..F..S..A..N..F..D..D..V..K..S..E..G..N..F..T..I..R..D..L..R..L..S..D..N..A..V..Y..Y..C..G..L..L..E..Y..D..G..F..K..L..I..F..G..G..G..T..R..L..T..V..E..P..K.aV10J_SPV13_090110 .............G..............................................T....C...................................C...................................................................aV10J_SPV13_091010 ......................................................................................G................T...........................G.....................................aV10J_SPV16_110210 .........................A........................................................GCC..................T.......................G...........TC............T...........G...aV10J_SPV22_091410 .............................................G........................................................C........................G..................................C......

CDR3

Figure 5. Alignment of Alpha V cDNA clones suggest somatic hypermutation at shark TCRa. Thymocyte clones for all 11 aV groups with the same

CDR3 from six different predicted V genes. Locations of framework regions (FR), complementarity determining regions (CDR), joining regions (J), and

constant (C) regions are marked above the scale for each aV. In absence of germline sequence information, we used a Geneious-derived nucleotide

consensus sequence for analysis of nucleotide changes in thymocyte clones. Amino acids are shown under the consensus sequence, and dots represent

Figure 5 continued on next page

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(40%) with unique V segments (from the first to the last predicted nucleotide of the V segment only).

We found 226 sequences (50%) containing unique CDR3-J regions (all bases after the last predicted

nucleotide of the V to the last predicted nucleotide of the J). However, we found 48 groups contain-

ing identical CDR3-J sequences across all nine V a families (suggesting they bear the V-J rearrange-

ment from a single founder thymocyte), each V a family containing anywhere from one to ten clonal

groups (Table 1). The majority of these groups contained no mutation within V, J or C regions. For

example, one a V3 sequence group occurred 131 times, the most numerous sequence in the data-

set, yet contained no mutation in any sequence. We did observe mutation in 12 of these 48 groups

belonging to seven different V a families. Each family contained between one and four clonal

groups, and each group contained two to nine sequences with identical CDR3-J regions (45 total

sequences; see Table 1). We include these 45 sequences in our TcR V a dataset (see Table 1 –

source data for sequence data).

Using these 45 sequences from both thymus and peripheral immune tissues, we found evidence

for SHM acting on the TcRa genes. We divided our 45 clones into 13 CDR3-sharing groups from

seven different a V families and then analyzed sequences within groups for potential mutation (Fig-

ure 5). We excluded two groups (four sequences) from analyses (aV1.3 and aV7.1) that contained

no mutations within FR or CDR regions (leaving 41 clones for analysis). Two sequences (aV7.4m_-

THY09_051410 and aV5J_SPV17_102810) contained one 3-base insertion and one sequence

(aV1.4J_SPV07_090110) contained an 18-base insertion; although SHM can result in insertions and

deletions (Diaz et al., 2002), we did not include insertion nucleotides in mutation counts. All sequen-

ces were in-frame and contained no internal stop codons, suggesting functionality of cells. Average

lengths of CDRs were as follows: CDR1: 7.0 amino acids (range: 5–8); CDR2: 5.7 amino acids (range:

5–7); and CDR3: 6.0 amino acids (range: 1–10). Naming of families and subfamilies followed

Criscitiello et al. (2010). However, with the accumulation of sequence data over our previous analy-

sis of nurse shark TcRa (Criscitiello et al., 2010), we expanded our nomenclature considerably.

Additional annotation followed the IMGT guidelines for TcRs (Lefranc et al., 2003).

Figure 5 shows all 12 aV CDR3 groups exhibiting mutation. The overall TcRa mutation frequency

was 0.0226 substitutions per nucleotide (S/N), with 66% of all substitutions (187 of 283) resulting in

amino acid replacements. The CDRs accumulated significantly more mutations than FRs (CDR:

0.0352 S/N; FR: 0.0188 S/N; p=0.0373), and substitutions in CDRs were twice as likely to be nonsy-

nonymous changes (NSYN) than those in FRs (CDR: 0.0235 S/N; FR: 0.0122 S/N; p=0.0312; Table 2).

There was no difference in frequency of synonymous (SYN) mutations between regions (CDR: 0.0117

S/N, FR: 0.0066 S/N; p=0.0705). Finally, although we found more tandemly mutated bases in CDRs

(41 of 81, or 50.6% of all CDR mutations) than in FRs (73 of 192, or 38.0% of all FR mutations), this

difference was not significant; p=0.721; Table 3). Tandem mutations ranged from two to four bases

in length (mean = 2.78 bases). That this feature of SHM, specific of cartilaginous fish Ig

(Rumfelt et al., 2001; Lee et al., 2002; Diaz et al., 1999), also occurs in the TcR strongly supports

the validity of our analyses.

Mutation frequency also varied by region (CDR or FR). The highest mutation frequency occurred

in CDR1 and accumulated significantly more mutations overall than other regions (�X=4.48%; see Fig-

ures 6 and 7a, Table 2). CDR3 displayed an unusually low mean mutation frequency (2.97%). How-

ever, these frequencies may be artificially low since our groups were based only on clones

containing the same CDR3 sequence, and clones whose CDR3s deviated markedly from the consen-

sus would have been excluded by our conservative grouping approach. We observed the lowest

mean mutation frequency in FR3 (1.64%). These results are consistent with what is known about

human TcR binding to MHC:Ag structures. CDR1 and CDR3 make more contacts with Ag while

Figure 5 continued

identity to this sequence. Nonsynonymous changes are highlighted in black; synonymous changes are underlined. Gaps are used for alignment

purposes and indicate either a shortened sequence (at the beginning or end) or insertions or deletions within the sequence. Sequences are identified

by a single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain

‘THY’ are from thymus, ‘PBL’ are from peripheral blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We did not use clones from aV1.3 and

aV7.1 because they did not contain mutations in FR or CDR regions. We deposited all 42 sequences into GenBank under accession numbers KY189332

– KY189354 or KY366469 – KY366487.

DOI: https://doi.org/10.7554/eLife.28477.009

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Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.009
https://doi.org/10.7554/eLife.28477
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Table

2.Frequenciesofso

matichyp

erm

utationin

nurseshark

alphaVgroups(aVG)co

ntainingthesameCDR3.

Mutationfrequency

isthetotalnumberofnucleotidech

angesto

aGeneious-derive

dco

nsensussequence

dividedbythetotalnumberofnucleotides.

Weco

unted

synonym

ous(S)and

nonsynym

ous(N

)mutationsseparately

foreach

FR

and

CDR

for11differentCDR3groupsin

seve

npredicted

alphaV

genes.

[FR:framework

region;CDR:co

mplementarity-determ

iningregion;Seqs:sequence

s;Nuc:

nucleotides;Freq:frequency]

FR1

FR2

FR3

FRMeans

aV

Gro

up

#

Seqs

#

Codons

Total

Mutations

Total

Mutation

#

Codons

Total

Mutations

Total

Mutation

#

Codons

Total

Mutations

Total

Mutation

Nuc

SN

Mutation

Freq(%

)Nuc

SN

Mutation

Freq(%

)Nuc

SN

Mutation

Freq(%

)S

NALL

aV1.1

325

225

212

14

6.222

17

153

02

21.307

41

369

44

82.168

618

24

aV1.2

525

375

10

818

4.800

17

255

18

93.529

41

615

55

10

1.626

16

21

37

aV1.4

325

225

10

10.444

17

153

13

42.614

41

369

45

92.439

68

14

aV2.1

525

375

00

00.000

17

255

10

10.392

42

630

00

00.000

10

1

aV3.1

425

300

04

41.333

17

204

00

00.000

40

480

01

10.208

05

5

aV4.1

225

150

12

32.000

17

102

02

21.961

43

258

11

718

6.977

12

11

23

aV4.2

225

150

00

00.000

17

102

11

21.961

43

258

27

93.488

38

11

aV5.1

225

150

06

64.000

17

102

23

54.902

42

252

12

31.190

311

14

aV7.2

225

150

47

11

7.333

17

102

13

43.922

41

246

16

72.846

616

22

aV10.1

425

300

00

00.000

17

204

11

20.980

41

492

40

40.813

51

6

aV10.2

925

675

211

13

1.926

17

459

44

81.743

40

1080

311

14

1.296

926

35

Sum

41

275

3075

20

50

70

187

2091

12

27

39

455

5049

35

48

83

67

125

192

Mean

MutationFreq(%

)0.65

1.63

2.28

0.57

1.29

1.87

0.69

0.95

1.64

0.66

1.22

1.88

StandardDeviation

2.994

4.547

6.531

1.136

2.252

2.841

3.125

3.414

5.429

4.742

8.201

11.861

CDR1

CDR2

CDR3

CDRMeans

AlphaV

Gro

up

#

Seqs

#

Codons

Total

Mutations

Total

Mutation

#

Codons

Total

Mutations

Total

Mutation

#

Codons

Total

Mutations

Total

Mutation

Nuc

SN

Mutation

Freq(%

)Nuc

SN

Mutation

Freq(%

)Nuc

SN

Mutation

Freq(%

)S

NALL

aV1.1

38

72

22

45.556

545

10

12.222

10

90

16

77.778

48

12

aV1.2

58

120

54

97.500

575

12

34.000

8120

14

54.167

710

17

aV1.4

38

72

01

11.389

545

00

00.000

545

02

24.444

03

3

aV2.1

57

105

01

10.952

575

00

00.000

345

00

00.000

01

1

aV3.1

46

72

03

34.167

672

00

00.000

112

00

00.000

03

3

aV4.1

27

42

31

49.524

636

01

12.778

636

20

25.556

52

7

aV4.2

27

42

11

24.762

636

00

00.000

636

00

00.000

11

2

aV5.1

27

42

03

37.143

636

07

719.444

742

00

00.000

010

10

aV7.2

25

30

00

00.000

742

11

24.762

636

02

25.556

13

4

aV10.1

47

84

30

33.571

672

00

00.000

784

03

33.571

33

6

aV10.2

97

189

18

94.762

6162

22

42.469

7189

30

31.587

610

16

Sum

41

77

870

15

24

39

63

696

513

18

66

735

717

24

27

54

81

Mean

MutationFreq(%

)1.72

2.76

4.48

0.72

1.87

2.59

0.95

2.31

3.27

1.17

2.35

3.52

StandardDeviation

1.690

2.316

2.979

0.688

2.089

2.248

1.027

2.067

2.272

2.659

3.754

5.626

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 9 of 31

Research article Immunology and Inflammation

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CDR2 interacts primarily with non-polymorphic regions of MHC (Buslepp et al., 2003; Garcia and

Adams, 2005), making mutation in CDR2 less favorable; further, FR regions are important for the

structural stability of the domain and mutations to these regions may affect the ability of the Ig

superfamily domain to fold properly (Mantovani et al., 2002; Reinherz et al., 1999).

HotspotsWe found 1327 G/C base pairs within DGYW/WRCH hotspot motifs (4,402 G/C base pairs occurred

outside these motifs) and 2931 A/T base pairs within WA/WT hotspot motifs (3690 A/T base pairs

occurred outside these motifs; see Table 4). Mutations of G:C nucleotides were strongly associated

with DGYW/WRCH hotspots (Figure 7b,c). Overall, G:C mutations occurred 4.3x as often within hot-

spots as those outside hotspots (p=0.0267). However, G:C mutations were 1.4x more likely within

CDRs than within FRs (p<0.0001). A:T nucleotides did not appear to prefer WA/TW hotspots

(p=0.2248), although they were 2.3x as likely to mutate within hotspots than outside hotspots. Fur-

ther, while A:T mutations occurred more often than expected in both FRs and CDRs (p=0.0015), the

frequency of A:T mutations were 1.9x more likely in CDRs than FRs. Chen et al. (2012) observed

similar results in TcR gV sequences of sandbar shark. The authors suggested that A:T mutations still

are likely the result of DNA polymerase h use during mismatch repair mechanisms due to the lack of

A:T point mutations in that study (the majority occurring in tandem with other mutations)

(Rogozin et al., 2001; Chen et al., 2012; Wei et al., 2015). In nurse shark Ig light chain genes, the

majority of mutation to A:T nucleotides (55%) also occurred in tandem (Alder et al., 2005). How-

ever, 63% (76 of 121) of A:T mutations occurred as point mutations in our study, which is inconsis-

tent with DNA polymerase h use during mismatch repair (Rogozin et al., 2001; Chen et al., 2012;

Wei et al., 2015). This result suggests that an alternate A:T motif is targeted or that shark TcRs

employ a different mechanism to alter A:T nucleotides.

Base substitution indicesWe identified 283 mutations within the eleven CDR3 groups. There was a bias toward mutations of

G and C nucleotides (p=0.0022) and G:C changes comprised 57% of all mutations (Table 5a). More

mutations to both G and C nucleotides occurred in FRs (p=0.0037) while only C nucleotides showed

greater mutation than expected in CDRs (p=0.1136). There were fewer mutations of A and T

Table 3. Number and frequency of DNA mutations that occur in tandem within framework regions (FR) and complementarity

determining regions (CDR) in nurse shark alpha V (aV) groups.

All mutations include both tandem and point mutations within a region. [Seqs: sequences]

FR CDR

# NucleotidesTandemlyMutated

All mutation Frequency of tandem mutation

# NucleotidesTandemlyMutated

All mutation Frequency of tandem mutationaV Group # Seqs 2 3 4 Sum 2 3 4 Sum

aV1.1 3 1 0 1 6 24 25.0 3 0 0 6 12 50.0

aV1.2 5 4 1 0 11 37 29.7 5 1 0 13 17 76.5

aV1.4 3 3 0 0 6 14 42.9 0 0 0 0 3 0.0

aV2 5 0 0 0 0 1 0.0 0 0 0 0 1 0.0

aV3 4 2 0 0 4 5 80.0 1 0 0 2 3 66.7

aV4.1 2 4 1 0 11 23 47.8 0 1 0 3 7 42.9

aV4.2 2 1 1 0 5 11 45.5 0 1 0 3 2 150.0

aV5 2 1 0 0 2 14 14.3 1 1 0 5 10 50.0

aV7.2 2 4 1 0 11 22 50.0 1 0 0 2 4 50.0

aV10.1 4 3 0 0 6 6 100.0 0 0 0 0 6 0.0

aV10.2 9 4 1 0 11 35 31.4 2 1 0 7 16 43.8

Total 41 27 5 1 73 192 38.0 13 5 0 41 81 50.6

DOI: https://doi.org/10.7554/eLife.28477.011

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Research article Immunology and Inflammation

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nucleotides in FRs (p=0.0082; Table 5b), but mutations of A and T nucleotides in CDRs did not differ

from random (p=0.3620; Table 5c). The frequency of mutated nucleotides also varied by region: In

FR1, more A and T nucleotides mutated while fewer C nucleotides mutated. Mutations of G and C

nucleotides were lower in FR2 and FR3, respectively (Table 5b,c). In both CDR1 and CDR2, there

were more A mutations and fewer G mutations than expected. However, more G nucleotides

mutated in CDR3. We saw a bias toward G:A and C:T transitions (42.8%) among the TcR a muta-

tions. Transition mutations appeared only slightly more often in CDRs (45.1%) than in FRs (41.7%).

Transversions of C:A or G:T mutations occurred only 25.1% of the time (Table 5).

Mutations, in situ hybridization, and AID expression in thymusSince we cloned TcR aV sequences in both central (thymus) and peripheral (blood, spiral valve) lym-

phoid tissues, we analyzed mutation frequencies by tissue type. Although data were limited to only

six sequences in two CDR3 groups (aV2, aV7.2), we found more mutations to FRs of peripheral

sequences, though this was not significant (p=0.0541; Table 6). We found no differences between

tissues within CDRs (p=0.2) from this limited data set. It is possible that positive and negative selec-

tion pressures remove more clonal sequences within the thymus, but with so few sequences to com-

pare it is difficult to determine.

The mutated sequences in the primary T lymphoid tissue suggests the activity of activation-

induced cytidine deaminase (AID) in the thymus. We confirmed the expression of AID in the thymus

through real-time RT-qPCR, where thymus tissue expressed AID at more than half (0.7x) the levels

found in spleen (positive control, where B cell SHM is known to occur) and nearly 6x the levels

0

1

2

3

4

5

6

FR1 CDR1 FR2 CDR2 FR3 CDR3

Mu

ta�

on

Fre

qu

en

cy (

%)

alpha V region

ALL

NSYN

SYN

Figure 6. Mutation frequencies differed within TcR V regions. Mutability of complementarity determining regions

(CDR), especially CDR1, exceeded that of framework regions (FR) for all mutations together (black bars) and for

nonsynonymous mutations alone (NSYN, hatched bars). We found no statistical difference in synonymous

mutations (SYN, white bars) between CDRs and FRs.

DOI: https://doi.org/10.7554/eLife.28477.012

The following source data is available for figure 6:

Source data 1. Frequency of mutation in TcR alpha V framework and complementarity determining regions for all

mutation, nonsynonymous (NSYN) mutation only, or synonymous (SYN) mutation only.

DOI: https://doi.org/10.7554/eLife.28477.013

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Research article Immunology and Inflammation

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https://doi.org/10.7554/eLife.28477.013
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observed in forebrain (negative control; Figure 8a). Further, colorimetric in situ hybridization (CISH)

of nurse shark thymus revealed a diffuse signal of TcR b (Figure 8b, panels 1,2) and AID (Figure 8b,

panels 3,4) mRNA expression throughout the thymic cortex. However, AID expression was greatest

in the central cortex and cortico-medullary junction (CMJ), while TcR b expression was highest in the

outer cortex. The thymic sequence data combined with the tissue localization of AID message

strongly suggest that TcRa loci undergo AID-dependent SHM in the shark thymus.

We refined our CISH results with RNA fluorescence in situ hybridization (FISH) using probes

against the TcR alpha constant region (aC) and exons 2 and 3 of AID. Since Stellaris RNA FISH uses

a mixture of shorter probes (~20 bp in length) that hybridize along the length of the target RNA, the

resulting signal is detectable only when tens of probes hybridize to the target. This makes the tech-

nology very specific – only those transcripts bound to numerous probes are visible – and limits the

potential for false-positive or false-negative results (Orjalo et al., 2011; Raj and Tyagi, 2010). Our

FISH results indicated a more specific TcR aC signal within the inner cortex and medulla adjacent to

the CMJ (Figure 9; Figure 9—figure supplement 1), regions where, in mammals, developing cells

actively rearrange their alpha chain genes and where mature ab T cells are found. AID expression

0

1

2

3

41 6

11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

10

1

10

6

11

1

11

6

12

1

12

6

13

1

13

6

14

1

14

6

15

1

15

6

16

1

16

6

17

1

17

6

18

1

18

6

19

1

19

6

20

1

20

6

21

1

21

6

22

1

22

6

23

1

23

6

24

1

24

6

25

1

25

6

26

1

26

6

27

1

27

6

28

1

28

6

29

1

29

6

30

1

30

6

31

1

31

6

32

1

32

6

# M

uta

!o

ns

G:C A:T

0

1

2

3

4

5

6

7

8

9

10

11

12

1 6

11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

10

1

10

6

11

1

11

6

12

1

12

6

13

1

13

6

14

1

14

6

15

1

15

6

16

1

16

6

17

1

17

6

18

1

18

6

19

1

19

6

20

1

20

6

21

1

21

6

22

1

22

6

23

1

23

6

24

1

24

6

25

1

25

6

26

1

26

6

27

1

27

6

28

1

28

6

29

1

29

6

30

1

30

6

31

1

31

6

32

1

32

6

# M

o!

fs

Sequence Posi!on

A

B

Figure 7. Mutation and motif locations within individual domains of TcR aV sequences. (A) Number of mutations (both single and tandem, 283 total) to

A:T nucleotides (black bars, 122 mutations) or G:C nucleotides (blue bars, 161 mutations) observed at each position along the sequence length of TcR

aV sequences. We counted mutations to Geneious-derived consensus sequences within framework and complementarity-determining region (CDR)

domains (41 sequences within 11 aV groups). (B) Number of WA/TW motifs (black bars, indicating possible polymerase h action) or DGYW/WRCH

motifs (blue bars, indicating hotspots for AID activity, and thus possible mutation) at each position along the sequence length. Position indicates the

forward (3’ to 5’) location of the mutable base of each motif within a Geneious-derived consensus sequence for each TcR aV group (eleven groups).

Red boxes indicate the location of CDR1 (positions 76–99), CDR2 (positions 151–171), and CDR3 (positions 300–328) within each panel. [WA/TW: A:T is

the mutable position; DGYW/WRCH: G:C is the mutable position; W = A/T, D = A/G/T, Y = C/T, R = A/G, and H = T/C/A].

DOI: https://doi.org/10.7554/eLife.28477.014

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occurred in ‘rings’ around areas of expressed TcR aC messages within the inner cortex and CMJ

(where positive selection occurs in mammals) and the medulla (where negative selection occurs in

mammals). Further, AID always co-localized with TcR aC (Figure 9). Thus, we observed a consistent

pattern of expression where a ‘ring’ of cells expressing both TcR aC and AID surround a central cell

expressing only TcR aC. The more specific signal generated by FISH may suggest that, once a T cell

completes RAG-mediated somatic rearrangement of its alpha chain locus, it clonally expands to

form a ring of daughter cells around it. These daughter T cells then express AID (and TcR aC), pro-

moting somatic hypermutation within their TcR alpha sequences during times when cells also

undergo positive and negative selection.

DiscussionThe 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 (Alvarez-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 pre-

dict 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 ab 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

Table 4. Target nucleotide mutation frequency in DGYW/WRCH or WA/TW mutation hotspots within framework regions (FR) and

complementarity determining regions (CDR).

[DGYW/WRCH (G:C is the mutable position; D = A/G/T, Y = C/T, W = A/T, R = A/G, and H = T/C/A); WA/TW (A:T is the mutable posi-

tion; W = A/T); ‘ALL’ refers to nucleotides within a hotspot motif; ‘All other’ refers to nucleotides outside a hotspot motif]

Mutated base Hotspot motif Region Total nucleotides Observed # Mutations Expected # Mutations Mutation freq (%) c2 p T-test P

G/C DGYW/WRCH FR 927 56 18.16 6.04 0.0000 0.0267

CDR 400 35 26.22 8.75

ALL 1327 91 37.52 6.86 0.0000

Outside Motif 4402 71 124.48 1.61

A/T WA/TW FR 2531 54 36.07 2.13 0.0015 0.2248

CDR 400 26 21.12 4.03

ALL 2931 80 55.97 2.52 0.0000

Outside Motif 3690 41 65.03 1.11

*T-test analysis was used to compare mutations within hotspot motifs to those outside hotspot motifs. Mutations to G and C nucleotides occurred signifi-

cantly more often within DGYW/WRCH motifs than outside these motifs, while mutations to A and T nucleotides showed no preference for WA/TW

motifs.

†c2 analysis was used to compare observed and expected numbers of mutations between FR and CDR regions and between mutations inside and outside

hotspot motifs. More mutations to all nucleotides occurred within hotspots than outside hotspots, and significantly more mutations occurred to nucleoti-

des within CDRs than FRs.

DOI: https://doi.org/10.7554/eLife.28477.015

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Table 5. Bias in base substitution during somatic hypermutation of TcR alpha V genes within all sequence regions (ALL), framework

regions (FR), or complementarity determining regions (CDR).

Probability of occurrence is the proportion of that base out of the total nucleotides. [Nuc: nucleotides; OBS: Observed; EXP: expected;

MI: mutability index; ChiSq: Chi squared]

a ALL Base Occurrence Probability of occurrence OBS EXP MI* ChiSq

G 2895 0.230 77 65.05 1.18 0.0022

C 2834 0.225 85 63.68 1.33

A 3498 0.278 64 78.60 0.81 0.0068

T 3368 0.267 57 75.68 0.75

Total 12595 1.00 283 283

GC Mutation: 57.0%; Transitions: 42.8%; Transversions: 25.1%

b FR Base Occurrence Probability of Occurrence OBS EXP MI* ChiSq

G 2378 0.23 58 44.50 1.30 0.0037

C 2268 0.22 56 42.44 1.32

A 2779 0.27 38 52.00 0.73 0.0082

T 2835 0.28 40 53.05 0.75

Total 10260 1.00 192 192

GC Mutation: 59.0%; Transitions: 41.7%; Transversions: 24.0%

c CDR Base Occurrence Probability of Occurrence OBS EXP MI ChiSq

G 517 0.22 19 20.15 0.94 0.1336

C 566 0.24 29 22.06 1.31

A 719 0.31 26 28.02 0.93 0.3620

T 533 0.23 17 20.77 0.82

Total 2335 1.00 91 91

GC Mutation: 52.9%; Transitions: 45.1%; Transversions: 27.5%

*Mutability Index, as first defined in Chen et al., 2012. c2 analysis was used to compare observed and expected numbers of mutations. G and C mutated

significantly more often than expected, while A and T mutated significantly less often than expected. Base composition: 23.0% G, 22.5% C 27.8% A, 26.7%

T.

DOI: https://doi.org/10.7554/eLife.28477.016

Table 6. Frequencies of somatic hypermutation in nurse shark thymus and peripheral lymphoid tissue (blood and spiral valve).

Mutations were analyzed only in alpha V groups containing the same third complementarity-determining region (CDR). Mutation fre-

quency was measured as the total number of nucleotide changes to a Geneious-derived consensus sequence divided by the total

number of nucleotides in all sequences. Nonsynymous (N) and synonymous (S) mutations (mut) were counted separately for each

framework (FR) and CDR for two predicted alpha V genes. [FR1, FR2, FR3, CDR1, CDR2, and CDR3 refer to the first, second, or third

FR or CDR region, respectively.]

Tissue type Mut type

FR Mutations (#) CDR Mutations (#)FR mutationfrequency

CDR mutationfrequency

FR1 FR2 FR3 All FR CDR1 CDR2 CDR3 All CDR FR1 FR2 FR3 CDR1 CDR2 CDR3

Thymus(6 sequences)

N 8 8 7 23 1 3 3 7 0.570 0.871 0.317 0.071 0.327 0.136

S 8 2 6 16 1 0 0 1 0.570 0.218 0.272 0.071 0.000 0.000

ALL 16 10 13 39 2 3 3 8 1.140 1.089 0.590 0.529 1.075 1.235

Total Nucleotides 1404 918 2205 4527 378 279 243 900

Periphery(2 sequences)

N 7 4 6 17 0 2 2 4 1.496 1.307 0.833 0.000 1.852 1.587

S 4 0 1 5 0 0 0 0 0.855 0.000 0.139 0.000 0.000 0.000

ALL 11 4 7 22 0 2 2 4 2.350 1.307 0.972 0.000 1.852 1.587

Total Nucleotides 468 306 720 1494 126 108 126 360

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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 g chain of gd T cells of sandbar shark

(Chen et al., 2012; Chen et al., 2009) and in both g and d chains of dromedary camel

(Antonacci et al., 2011; Ciccarese et al., 2014; Vaccarelli et al., 2012). In each study, SHM mir-

rored the mutational patterns observed in B cells during affinity maturation. However, in both sand-

bar shark g and dromedary camel g and d 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 ab T

cells, gd 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 gd T cells typically bind Ag in a manner more similar to that of Ig than to ab 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 gd T cells earlier in an immune response, releasing pro-

inflammatory cytokines and killing infected macrophages. Thus, gd T cells combine an innate-like

Figure 8. Shark thymus expresses AID. (A) Expression of AID in shark spleen (black bar), thymus (hashed bar), and forebrain (gray bar) using real-time

quantitative PCR. Expression was measured using the delta delta Cq method and are normalized against shark muscle tissue using b2M as a reference

gene. Data represent expression fold-change differences at four cDNA concentrations. (B) In situ hybridization of mRNA using ribo-probes on adult

shark thymus sections. 1,2: Two different fields probing for TcRb antisense. 3,4: AID antisense. 5,6: TcRb sense. 7,8: AID sense. All micrographs at 10X

magnification; black scale bar in lower right of each panel is 100 uM. Anatomical structures are designated on the top panels [c: cortex; m: medulla; sc:

subcapsular region; cmj: corticomedullary junction].

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Figure 9. AID expression localized to inner cortex and cortico-medullary junction. (a) H and E staining of fixed shark thymus tissue illustrating thymic

architecture (10x). The densely packed cells at the margins of the image comprise the cortex (cor), while the less densely packed cells in the center

constitute the medulla (med). The region at the junction between cortex and medulla incorporates the corticomedullary junction (CMJ), delineated

generally by a hashed white circle. [b-z] Single molecule RNA fluorescence in situ hybridization (FISH) probing fixed thymus sections simultaneously for

Figure 9 continued on next page

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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 Gar-

boczi, 2002; Allison et al., 2001). It is evident that how SHM presents a useful solution for accom-

plishing these tasks by creating a more diverse repertoire of these antibody-like gd 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 g and d chains of gd T cells in both thy-

mic and peripheral immune tissue of nurse shark. Remarkably, we also detected SHM occurring in

the a chain of ab T cells. We observed mutational characteristics within a chain of nurse sharks simi-

lar 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 hot-

spots. Out of curiosity, we compared counts of AID hotspot motifs within CDR and FR regions

between our 11 nurse shark TcR aV consensus sequences and 6 human TcR aV 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 TcRa

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 tis-

sues (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 TcRa takes place in mammals. Positive selection on self-MHC/self pep-

tide 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

TcRa locus, with up to 100 J segments depending on species, permits multiple successive rearrange-

ments 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;

Figure 9 continued

AID (probes labeled with Quasar 670; pseudo colored red) and TcRa (probes labeled with CalFluor Red 610; pseudo colored green) and counterstained

with DAPI (blue). (b) Composite of seven Z-stacked images (10x) depicting overall thymic architecture and the localization of AID expression to the inner

cortex and cortico-medullary junction regions of shark thymus. We superimposed (and minimally adjusted) the outlined CMJ boundaries from (a) onto

(b) to elucidate the junction between cortex and medulla. [c-z] We obtained images of each fluorophore using 10x, 20x, and 63x magnification and

merged Z-stacked images together. Individual (10x) fluorophore images of DAPI [c-f], TcRa [g-j], and AID [k-n] and Z-stacked merged images [o-r]

illustrate AID and TcRa expression in four locations of shark thymus. White boxes indicate the magnified regions of the 10x and 20x images shown in

the 20x [s-v] and 63x [w-z] images, respectively. Scale bars [a,b,c,g,k,o] 150 mm, [s] 75 mm, and [w] 30 mm. [cor: cortex; med: medulla; CMJ:

corticomedullary junction].

DOI: https://doi.org/10.7554/eLife.28477.019

The following figure supplements are available for figure 9:

Figure supplement 1. Localization of AID and TcRa probes is independent.

DOI: https://doi.org/10.7554/eLife.28477.020

Figure supplement 2. Lack of AID and TcRa probe hybridization in shark brain.

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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 TcRa loci in conjunction with receptor editing (note that

sharks, like all other gnathostomes, have a large number of TcRa 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 upre-

gulated in cells undergoing RAG-mediated alpha rearrangement (and thus in cells also expressing

RAG). However, if SHM occurs after rearrangement of TcR a and thus used for rescuing cells during

negative selection, the same T cell would not express both AID and RAG. While we cannot deter-

mine conclusively without RAG expression data, the patterns of AID and TcRa 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 ‘profli-

gate 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 eluci-

date the timing of AID-catalyzed SHM in relation to T cell development.

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

Figure 10. Model predicting how AID acts on T cells in the thymus. CD4/CD8 double negative (DN) thymocytes in the subcapsular region (SC) and

cortex rearrange the b chain, using a surrogate pTa receptor to test for expression signaling. Cells with productive b arrangements then proliferate and

express both CD4 and CD8, becoming double positive thymocytes (DPs). As DPs move toward the inner cortex and cortico-medullary junction (CMJ)

where a chain rearranges, cells may begin to express AID. Non-productive rearrangements can be rescued from apoptosis by receptor editing or by

receptor salvaging, in which AID catalyzes SHM to produce cells with improved affinity to MHC:Ag complexes (to pass positive selection). Salvaged

thymocytes then proliferate and express either CD4 or CD8 on their surface as single-positive (SP) cells. AID-mediated receptor salvaging may also

reduce recognition of self-peptide, rescuing self-reactive thymocytes from apoptosis (to pass negative selection). [sc: subcapsular region; cmj:

corticomedullary junction; green shading indicates region of AID expression].

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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 diversifica-

tion and honing, perhaps mechanisms convergent with the thymic process we describe here. How-

ever, 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 thy-

mus did not co-localize with AID expression.

Shark TcRs are capable of a wide variety of diversification mechanisms. In addition to RAG-medi-

ated combinatorial and junctional diversity from complex loci, TcRd (and possibly a) rearrange Ig-

TcR chimera using IgM and IgW V exons with TcR D-J-C (Criscitiello et al., 2010). The TcR d locus

also encodes the doubly rearranging NAR-TcR, a d 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 dV of the NAR-TcR dV 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 g , d, and a 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 muta-

tion (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 immunoglobu-

lin gene conversion in some birds and mammals (Rogozin et al., 2007; Zheng et al., 1994). One

study suggested that VLRA (the analog to ab TcR in jawed vertebrates) also might use CDA to affin-

ity 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 lym-

phocyte receptor development in a thymus-like organ. The expression of AID in the thymus of primi-

tive 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

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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 verte-

brate 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.

Materials and methods

Study animalsTCR sequence data used in this study came from two adult female nurse sharks (‘Joanie’ and ‘Mary

Junior’; Ginglymostoma cirratum) delivered by caesarian section off the Florida Keys and matured in

the aquatic vivarium of the University of Maryland’s Center of Marine Biotechnology. We used pub-

lished T cell aV sequences (Criscitiello et al., 2010) as a baseline for aV locus numbering (sharks Yel-

low and 1299), though we did not analyze any of these sequences for mutation.

Total RNA isolation and cDNA synthesisWe harvested tissues from animals after MS-222 (Argent, Redmond, WA) overdose, and immediately

purified RNA with TRIzol reagent (Life Technologies, Carlsbad, CA). Nurse shark thymi are located

dorsomedial to the gills (Luer et al., 1995) in the crevasse between the epaxial and brachial constric-

tor muscle groups. We used 5 ug total RNA from spiral valve, spleen, thymus, and peripheral blood

leukocytes (PBL) for oligo-dT primed cDNA generation with Superscript III First Strand Synthesis Sys-

tem (Thermo Fisher Scientific, Inc., Waltham, MA). (Criscitiello et al., 2010) We estimated cDNA

concentration using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc.).

RACE PCR, Cloning, and Sanger SequencingWe generated a 5’ RACE (Rapid Amplification of cDNA Ends) library using the GeneRacer Kit (Life

Technologies) and reverse primers designed to the end of the shark TcRb, TcRg, or TcRd variable (V)

region or to the middle of the shark TcRa constant (C) region. We amplified RACE products using

Phusion High-Fidelity DNA polymerase (New England Bio Labs, Inc., Ipswich, MA) to minimize PCR

errors under these specific PCR conditions: primary denaturing at 94˚C for 2 min; 30 cycles at 94˚Cfor 30 s and 78˚C for 1 min; and a final extension at 72˚C for 10 min. Using this RACE library, we

then amplified a specific a V region using a gene-specific primer to its leader region and the follow-

ing PCR conditions: 98˚C for 1 min; 25 cycles of 98˚C for 5 s, 49–60˚C for 30 s, 72˚C for 150 s; 72˚Cfor 10 min. Annealing temperatures varied for each amplified aV (see Table 7). We visualized PCR

products with agarose gel (8%) electrophoresis and then excised bands of correct size. We then iso-

lated amplified bands from agarose gels using the PureLink Quick Gel Extraction Kit (Life Technolo-

gies) or RICO chips (TaKaRa Bio USA, Mountain View, CA).

We transformed PCR amplicons into One-Shot Top10-competent cells (Thermo Fisher) using a

pCR4-TOPO TA blunt end vector and cloning kit (Thermo Fisher) followed by a Zyppy plasmid mini-

prep kit (Zymo Research, Irvine, CA) for plasmid purification of individual clones (Criscitiello et al.,

2012). We checked insert size using an Eco RI restriction enzyme (Promega Corp, Madison, WI),

then amplified and purified the sequencing reaction using BigDye xTerminator Sequencing and Puri-

fication Kit (Thermo Fisher Scientific, Inc.). We submitted samples for sequencing to the DNA Tech-

nologies Core Lab on the Texas A and M University campus (College Station, TX). We deposited

sequences in GenBank with the following accession numbers: Alpha KY189332-KY189354 and

KY366469-KY355487; Beta KY351708-KY366487; Gamma KY351639-KY351707; Delta KY346705-

KY346816.

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Table 7. List of forward (F) and reverse (R) primers used to generate T cell receptor (TcR) sequences and expression data.

[AID: Activation induced cytidine deaminase; B2M: beta-2 microglobulin; a: alpha; b: beta; g : gamma; d: delta; V: variable region; C:

constant region].

Primer F/R ID Location Nucleotide sequence (5’ to 3’) Amino acid Tm

TcR aV1 F MFC370 leader region of aV1 ATG TTG CCT GAA GCT C MLPEA 55

R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR aV4 F MFC122 beginning of aV4 GTC TCC TCA GTT GTT CGT AC VSSVVR 58

R MFC123 end of aV4 CAG TAA TAC ACA GCA GCG TC DAAVYY 58

F MFC374 leader region of aV4 TGG ATT GTG TGG GCA GTA WIVWAV 54

R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR aV5 F MFC124 beginning of aV5 CTC AGG AAG GAG AGA TTA TCA C QEGEII 60

R MFC125 end of aV5 CAA TGA TAC ACG GCG GAG TC DSAVYH 60

F MFC124 beginning of aV5 CTC AGG AAG GAG AGA TTA TCA C QEGEII 60

R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR aV7 F MFC376 end of leader aV7 AGC GAT GGA GTT TCT GTG ATT SDGVSVI 58

R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR aV10 F MFC378 leader region of aV10 CTA TTT CTT CAC TAC CGC AG YFFTTA 56

R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR a 5’ F GeneRacer 5’ Nested homologous to RNA oligo GGA CAC TGA CAT GGA CTG AAG GAG TA – 78

TcR a 3’ R MFC191 alpha C region CAT TGG TGG ATA GCA AGC CCT TCG AT SKGLLSTN 76

TcR bV1 F MFC126 beginning of bV1 CTC CGT ACA TCG TCT CTA TTG PYIVSI 60

R MFC127 end of bV1 CAC GCA CAG AAA TAG ACA GC AVYFCA 58

TcR bV2 F MFC128 beginning of bV2 CTA CGT GGA GCA GTC TCC ATC YVEQSP 63

R MFC129 end of bV2 GCA CGC ACA ATA ATA GAC AGC C AVYYCAC 62

TcR bV3 F MFC130 beginning of bV3 CTA CGT GGA ACA GTC TCC TTC YVEQSP 61

R MFC131 end of bV3 CAC GCG CAG AAA TAG ACA G VYFCA 57

TcR bV5 F MFCb50 beginning of bV5 GTT CGG TGC TCT TTC TCT GC MFGALSLH 60

R MFCb54 end of bV5 GAC TGC AGT ATC AGT CGG CAC C LVPTDTAV 66

TcR gV1 F MFCg56 beginning of gV1 GTC GCT GTA TTA CTG GCT CAT TG MSLYYWL 63

R MFCg59 end of gV1 GAG CGC ACA GTA ATA GGT GGC AG TATYYCAL 67

TcR gV3 F MFCg58 beginning of gV3 GAA GGG TCA CGT CCT TGC G MKGHVLA 62

R MFCg61 end of gV3 GAT CCC AGA GTC ATC CTC EDDSGI 56

F MFC170 beginning of gV3 CAA TAA CCA GAG CAC CGG G ITRAP 56

R MFC171 end of gV3 AGA TCC CAG AGT CGT CCT C EDDSGI 56

TcR dV3 F MFCd62 beginning of dV3 GAT TCC CCG TCC CTG GTG TC DSPSLVS 65

R MFCd66 end of dV3 CAG TGC ACA GTG ATA CAC AGC AVYHCAL 61

TcR dV5 F MFCd63 beginning of dV5 GCA GCT ACT CAG TAT CTG G MQLLSIW 57

R MFCd67 end of dV5 GAA AGC ACA GTA ATA CAG AG ALYYCAF 54

TcR dV7 F MFC172 beginning of dV7 CTG TCA CTC AGT TAT TCT CCT C VTQLFS 60

R MFC173 end of dV7 GCA GCC CAG TTA TAG TCA AAC LTITGL 60

TcR dV12 F MFC174 beginning of dV12 CAG AGC CCA CCT CAG TTA C QSPPQ; 60

R MFC175 end of dV12 GAG CGC AGT AAT AGA TGG C AIYYCA 57

TcR dV16 R MFC176 end of dV16 GCA GCT CCG AGA TAG ACA AC LSISEL 60

F MFC177 beginning of dV16 GAG TCC TGG CTC ACG CAA TC ESWLTQ 63

TcR dV17 F MFC178 beginning of dV17 CAG TCT TGG TCA GAA ATA ACC QSWSEIT 57

R MFC179 end of dV17 CAA CTG AAG ATA AGT GAT CG ITYLQL 54

Table 7 continued on next page

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Sequence alignment and tree buildingWe used Geneious and BioEdit (v7.2.5, Ibis BioSciences, Carlsbad, CA) software to manage DNA

sequence data. We aligned nucleotide and amino acid sequences using the ClustalW Multiple Align-

ment tool in Geneious with a gap penalty of 15, a gap extension penalty of 6.66, and free end gaps.

We manually adjusted the alignments as necessary. We determined sequence relationships phyloge-

netically using the Geneious tree builder with default settings. We grouped sequences into unique V

families based on 70% nucleotide sequence identity and 75% amino acid sequence identity

(Brodeur and Riblet, 1984; Rumfelt et al., 2004) using the same a V numbering scheme as in

Criscitiello et al. (2010). We created graphical alignments in BioEdit and imported these files into

Microsoft Word to generate figures. Our preliminary dataset contained 564 TcRa clones (encoding

286 unique amino acid sequences representing nine Va families) from three tissues (PBL, spleen, thy-

mus) of two sharks (Joanie, Mary Junior). Using this dataset, we separated sequences containing

identical CDR3 rearrangements and counted mutations within each TcR ‘clone family’ bearing the

V-J rearrangement from single founder thymocytes.

Identification of TcR Va genes in the nurse shark genomeWe probed the filter sets for the G. cirratum BAC library (Arizona Genomics Institute, Tucson, AZ) of

shark ‘Yellow’ and screened with variable segment and constant region probes for TcRa and TcRd.

We cultured several positive clones and isolated BAC DNA according to manufacturer’s protocol

with the Qiagen Large Construct Kit (Qiagen, Valencia, CA). We sent purified BAC DNA to the Duke

University Center for Genomic and Computational Biology (Durham, NC) for PacBio SMRT (Menlo

Park, CA) large insert (15–20 kb) library preparation, sequenced on the PacBio RSII platform with P6-

C4 chemistry. Read correction and contig assembly were performed with the PBcR software

(Koren et al., 2012), using the BLASR error correction method and the Celera Assembler 8.2. We

annotated the resulting sequencing within the Geneious software suite (v9.1.5, Biomatters Ltd.,

Auckland, NZ) using a custom BLAST database of all TcR and IgH sequences for G. cirratum in the

IMGT database (Montpellier, France).

Our search yielded 17 a/d V germline segments, significantly fewer than expected based on TcR

aV segment numbers in other species (Murphy and Weaver, 2017). Of these 17 segments, only 13

contained unique nucleotide sequences, and all V segments were highly similar to each other (69–

100% nucleotide and 52–100% amino acid identity). Twelve germline V segments shared >93%

nucleotide identity (>85% amino acid identity), with three segments differing by only a single nucleo-

tide (Figure 11). Based on the variability we observed in our sequence data, these 17 germline a/d

V segments must represent only a small portion of the available Vs in the nurse shark genome.

We compared these 17 germline a/d V segments to our TcR aV database containing all nine

potential V families from two different sharks. All 17 germline a/d V segments aligned to our aV4

data with >75% nucleotide identity, while 15 segments shared >93% nucleotide identity to at least

one sequence in our aV4 dataset. Of the 60 sequences in our aV4 dataset, 37 sequences aligned

specifically to eight germline a/d V segments, with alignments containing one to 17 aV4 sequences

per germline segment (nucleotide alignments shared >97% identity; Figure 12.). While we did

observe nucleotide differences within alignments, most differed by fewer than four nucleotides from

the germline a/d V segments. Because several germline segments differed by only a single nucleo-

tide and we are certain that we have not found all a/d V segments in the genome, these differences

could represent variation in alleles or individuals rather than mutation. Thus, we chose not to rely on

these data for mutational analysis.

Table 7 continued

Primer F/R ID Location Nucleotide sequence (5’ to 3’) Amino acid Tm

AID F MFC342 beginning of AID exon 1 AGG CAC GAG ACC TAC ATG TTG RHETYML 61

R MFC347 end of AID exon 2 TGA ACC AGG TGA GGC GGT A YRLTWF 60

B2M F MFC211 first cysteine AAC GTG TTG CTC TGT CAT GC NVLLCHA 58

R MFC212 before second cysteine GGG GTG AAC TCC ACA TAA CG RYVEFTP 60

DOI: https://doi.org/10.7554/eLife.28477.023

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Mutation frequencyWe defined mutation frequency as the number of nucleotide changes divided by the total number

of nucleotides within a particular region (e.g. FR, CDR, J, C) based on differences to a consensus

sequence. We classified all nucleotide changes as either synonymous (SYN) or non-synonymous

(NSYN) mutations based on whether or not the codon was unaltered or altered, respectively. For

tandem base changes, we assessed the effect of each nucleotide change independent of its neigh-

boring mutation(s). We then compared mutation frequencies between CDR and FR regions for all

clone families that contained mutations using a Student’s 1-tailed t-test unless otherwise noted.

Determination of hotspotsWe searched for the ProSite motifs DGYW/WRCH (G:C mutable target) and WA/TW (A:T mutable

target) using the motif search function in Geneious. These motifs serve as common ‘hotspots’ for

SHM within Ig variable regions, where AID favors the G/C bases within DGYW/WRCH motifs during

the first phase of SHM while low-fidelity polymerases (i.e., polymerase h) preferentially target A/T

bases within WA/TW motifs during the second phase of SHM (Wei et al., 2015; Rogozin and Diaz,

[A]

.. 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TCR α/δV5 ..D..L..C..H..G..D..S..V..T..Q..T..V..S..S..V..V..R..T..G..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..N..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..T..L..L..R..S..T..T..G.TCR α/δV5 --AGACTTGTGTCATGGAGATTCTGTTACTCAGACAGTCTCCTCAGTTGTTCGGACAGGAGGAGAGACGATGATGCTAAGCTGTACCTATGATACTACATCAAATTCCTATACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATACACTGTTGAGAAGTACCACTGGCTCR α/δV7.1 --...........................................................................................................................................................T......................TCR α/δV7.2 --...........................................................................................................................................................T......................TCR α/δV7.3 --...........................................................................................................................................................T......................TCR α/δV10 --...........................................................................................................................................................T......................TCR α/δV8 --...............................T......................G.A............................................C......................G..............................T......................TCR α/δV6.1 --...............................T......................G.A....................................C.......C.....................................................T......................TCR α/δV6.2 --...............................T......................G.A....................................C.......C.....................................................T......................TCR α/δV6.3 --...............................T......................G.A....................................C.......C.....................................................T......................TCR α/δV9 --...............................T......................G.A............................................C....G.G..............................................T......................TCR α/δV11 --...............................T...................T..G.A..................C.........................C...TG...G............................................T......................TCR α/δV4 --...............................T........................A.........................................T..C........G............................................T.............G...A....TCR α/δV2 --....C......G............C......T..C.........ACA..G.C....A......AG.AG.T.CT.........................TG.GCAA.....T...............A..........A..AG...TT........T.....G..A......TG.....TCR α/δV13 TC....C..A.............A..C.....AT..C.T.......A.....A....CAG....C.G.AGCA.CT.....T....A....A.GG.....CAC..C..T...TGGA....T............T.........TG........T....T....AG..A......GGGA.A.TCR α/δV3 ------C..A.C.....CA.......C......T.GC.G.......A...........A......CGT.G.A.CTA................G.......GG..C.A.C..GT.................G....T...A.................T.....G..A..A..G..A..C.TCR α/δV12 --...GC..A.............C..C......T..CAG.......AC.....A....A........A.G.A.CCA....T.....T.....C......CAG.GCAG.....G......T........A.....T......T.T.CA.T........T.....C..A...C.TG.A...TTCR α/δV1 --....A..A................C......T..CCT......TA...C.A..A..A....A.AG.TG..GCT....A...C..............G..G.GCGAT..C.................T.......A..A.G...G........T..TT....G.CA.GA...TGG...T

190 200 210 220 230 240 250 260 270 280 290 300 310....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....

TCR α/δV5 .Y..E..D..E..A..D..F..A..R..S..R..F..S..A..E..L..Q..T..S..N..K..F..T..N..L..T..V..T..G..L..Q..L..T..D..S..A..V..Y..Y..C..A..F..R..N...TCR α/δV5 TATGAGGATGAAGCAGATTTTGCCAGAAGTCGCTTCTCTGCAGAACTGCAGACCTCGAATAAATTCACCAATTTGACTGTCACTGGGTTACAGCTGACCGACTCTGCTGTCTATTACTGTGCTTTCAGGAACCTTCR α/δV7.1 ...................................................................................................................................... 99.2%TCR α/δV7.2 ...................................................................................................................................... 99.2%TCR α/δV7.3 ...................................................................................................................................... 99.2%TCR α/δV10 ..........................................................................T........................................................... 98.9%TCR α/δV8 .........A............................................................G............................................................... 97.0%TCR α/δV6.1 .........A..................A........T...G...........A.........C......G.....................A.........G.....C.G...............T......A 93.5%TCR α/δV6.2 .........A..................A........T...G...........A.........C......G.....................A.........G.....C.G...............T......A 93.5%TCR α/δV6.3 .........A..................A........T...G...........A.........C......G.....................A.........G.....C.G...............T......A 93.5%TCR α/δV9 .........A..................A........T...G...........A.........C......G.....................A.........G.....C.G......................A 93.5%TCR α/δV11 .........................A................A..........A.........C............T.........................G.......G...........A..........A 93.5%TCR α/δV4 .........A..................A........T...G...........A.........C......G.....................A.........G.....C.G......................A 93.2%TCR α/δV2 ....GT...A.....A......TT.A..A...A.......T......T.G..AG.G...G..........G......GA..T.AT..C..........T...A....CA.G...............CA...... 78.5%TCR α/δV13 A........A...............A.GA...T.......T......T.T.........G...C......G...A...A.......A...G...........G.A.....G..................G.--- 77.3%TCR α/δV3 A........A..........C..T.A......T..............T.....T.G...G.....T....G.......A.....C..C.GG.AT....T...A.......G........C...C...CTGG--- 77.3%TCR α/δV12 A........A.............T.A.GA..A............C..T...G..................G.......A...A....C..G.A..C..T...A.......G........C...A..T..G...G 76.9%TCR α/δV1 AG...T.G.A.....AG.....TGCA.GA...T......ATGC....TG..G.......A....CG..G.G.......A.......AC..........T...A....C..G........C.G...TG.TGG.TG 70.9%

[B]

10 20 30 40 50 60 70 80 90 100

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR adV5 -DLCHGDSVTQTVSSVVRTGGETMMLSCTYDTTSNSYTLYWYRQHPGTQPEYTLLRSTTGYEDEADFARSRFSAELQTSNKFTNLTVTGLQLTDSAVYYCAFRNTCR adV7.1 -...................................................I................................................... 99.2%TCR adV7.2 -...................................................I................................................... 99.2%TCR adV7.3 -...................................................I................................................... 99.2%TCR adV10 -...................................................I...............................F................... 98.9%TCR adV8 -..........S.......E..............T.......G.........I..........K...................S.................... 97.0%TCR adV6.1 -..........S.......E..............T.................I..........K.....N..F........L.S..........A.L.....W. 93.5%TCR adV6.2 -..........S.......E..............T.................I..........K.....N..F........L.S..........A.L.....W. 93.5%TCR adV6.3 -..........S.......E..............T.................I..........K.....N..F........L.S..........A.L.....W. 93.5%TCR adV9 -..........S.......E..............T.E...............I..........K.....N..F........L.S..........A.L....... 93.5%TCR adV11 -..........S.......E..............T.DR..............I...............K.....K......L...I........A......... 93.5%TCR adV4 -..........S.......E.............LT..R..............I....AN....K.....N..F........L.S..........A.L....... 93.2%TCR adV2 -...R......SL..DIG.E..AVT........LSN.I........DK.F..I.WK..A..G.K.N.VKN...V..RKWK...S..ISW.....T.M.....Q. 78.5%TCR adV13 S..S.......SL..D.Q.Q.AAAT...N.KA.H...WI.....Y..M..D.I.RK..GDN..K....KD...V..L..K.L.S..I...E...A........ 77.3%TCR adV3 --.S..N....SL..D...E.DVVTI....E..W.YHV.....R.SD.....I.WKNSNAN..K....K.........WK...S..I.R.E...T......LT 77.3%TCR adV12 -E.S.......SQ..D...E..KVTI.......QS..R.........SPL..I.SK.MN.N..K....KDH...D..A.....S..IN..E...T......IWD 76.9%TCR vdV1 -.MS.......SP..Y.QKE.KAVA.N.......SD.........QDAR..FI.WQD.G.SDGK.S.VQD...MQ.EA.K.S.S..I.......T.....G.GG 70.9%

Figure 11. Observed TcR Alpha/Delta germline Vs exhibit high sequence identity. Nucleotide (A) and amino acid (B) alignments of 17 germline variable

(V) region gene segments. Two V groups contained three identical germline gene segments each (highlighted in gray), leaving only 13 unique V gene

segments. Boxes surround conserved amino acids. Numbers at the ends of sequences indicate percent identity to the first germline sequence (a/d V5)

within the alignment.

DOI: https://doi.org/10.7554/eLife.28477.024

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2004; Chen et al., 2012). We counted only motifs present in the consensus sequence (rather than

those created by the mutation) as hotspots. For each domain, we first counted the number of target

nucleotides within DGYW/WRCH or WA/TW hotspots. We then examined each mutation to deter-

mine if it occurred inside or outside a hotspot. We counted changes from the consensus sequence

to a target nucleotide within its respective motif as a hotspot mutation. We defined the frequency of

hotspot mutation as the number of mutations (of target nucleotides) occurring within hotspots

divided by the total number of mutations for each region. From these data, we compared the muta-

bility of bases between FR and CDR regions using c2 Analysis.

Base substitution indicesWe calculated a mutability index for each nucleotide using methods similar to Chen et al. (2012). In

our case, we derived the expected number of mutations by multiplying the frequency of a particular

nucleotide within a family of sequences (e.g. a V2) by the total number of observed mutations within

that family. We then defined the mutability index as in Chen et al. (2012) [the observed number of

mutations of a specific nucleotide divided by the expected number of mutations of that nucleotide,

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|TCR α/δV5 germ --AGACTTGTGTCATGGAGATTCTGTTACTCAGACAGTCTCCTCAGTTGTTCGGACAGGAGGAGAGACGATGATGCTAAGCTGTACCTATGATACTACATCAAATTCCTATACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATACACTGTTGAGAAGTACCACTGGCTCR α/δV5 germ ....D..L..C..H..G..D..S..V..T..Q..T..V..S..S..V..V..R..T..G..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..N..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..T..L..L..R..S..T..T..G.aV4J_V11_101310 --..............................................................................T............................................................................................A......aV4m_T11_060612 --.................................................................................................A.........................................................................A......

190 200 210 220 230 240 250 260 270 280 290 300 310

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR α/δV5 germ TATGAGGATGAAGCAGATTTTGCCAGAAGTCGCTTCTCTGCAGAACTGCAGACCTCGAATAAATTCACCAATTTGACTGTCACTGGGTTACAGCTGACCGACTCTGCTGTCTATTACTGTGCTTTCAGGAACCTTCR α/δV5 germ .Y..E..D..E..A..D..F..A..R..S..R..F..S..A..E..L..Q..T..S..N..K..F..T..N..L..T..V..T..G..L..Q..L..T..D..S..A..V..Y..Y..C..A..F..R..N...aV4J_V11_101310 ....................................................A........................................A........................................--------aV4m_T11_060612 .............................A...................................................................................................CCG.C-------

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|TCR α/δV6 germ --AGACTTGTGTCATGGAGATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGGACGGAAGGAGAGACGATGATGCTAAGCTGTACCTATGATACCACATCAACTTCCTATACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATATACTGTTGAGAAGTACCACTGGCTCR α/δV6 germ ....D..L..C..H..G..D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..T..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..T..T..G.aV4m_T04_061812 --.T................................................................................................................................................................................aV4m_T09_061812 --.............................................................................................T....................................................................................aV4m_T07_061812 - -.............................................................................................T....................................................................................aV4m_T03_061812 --CAGA.........................................................................................T....................................................................................

190 200 210 220 230 240 250 260 270 280 290 300 310

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR α/δV6 germ TATGAGGATAAAGCAGATTTTGCCAGAAATCGCTTCTTTGCGGAACTGCAGACATCGAATAAACTCACCAGTTTGACTGTCACTGGGTTACAACTGACCGACGCTGCTCTGTATTACTGTGCTTTCTGGAACCATCR α/δV6 germ .Y..E..D..K..A..D..F..A..R..N..R..F..F..A..E..L..Q..T..S..N..K..L..T..S..L..T..V..T..G..L..Q..L..T..D..A..A..L..Y..Y..C..A..F..W..N...aV4m_T04_061812 ................................................................................T....................................................G --------aV4m_T09_061812 ..............................................................................................................................A.....AC --------aV4m_T07_061812 ............................................................................................................................CTGC..GAACaV4m_T03_061812 ............................................................................................................................CTGC..GAAC ----------

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|TCR α/δV7 germ --AGACTTGTGTCATGGAGATTCTGTTACTCAGACAGTCTCCTCAGTTGTTCGGACAGGAGGAGAGACGATGATGCTAAGCTGTACCTATGATACTACATCAAATTCCTATACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATATACTGTTGAGAAGTACCACTGGCTCR α/δV7 germ......D..L..C..H..G..D..S..V..T..Q..T..V..S..S..V..V..R..T..G..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..N..S..Y..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..T..T..G.TCR α/δV10 germ --..................................................................................................................................................................................TCR α/δV10 germ.-.. .............................................................................................................................................. ..................................aV4m_T12_060612 --CAGA.........................................................................................................................................................A....................

190 200 210 220 230 240 250 260 270 280 290 300 310

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR α/δV7 germ TATGAGGATGAAGCAGATTTTGCCAGAAGTCGCTTCTCTGCAGAACTGCAGACCTCGAATAAATTCACCAATTTGACTGTCACTGGGTTACAGCTGACCGACTCTGCTGTCTATTACTGTGCTTTCAGGAACCTTCR α/δV7 germ .Y..E..D..E..A..D..F..A..R..S..R..F..S..A..E..L..Q..T..S..N..K..F..T..N..L..T..V..T..G..L..Q..L..T..D..S..A..V..Y..Y..C..A..F..R..N...TCR α/δV10 germ ..........................................................................T...........................................................TCR α/δV7 germ .........................................................................F............................................................aV4m_T12_060612 .....................................................................................................................................G--------

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|TCR α/δV11 germ --AGACTTGTGTCATGGAGATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGTACGGAAGGAGAGACGATGATGCTCAGCTGTACCTATGATACTACATCAACTTCTGATAGATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATATACTGTTGAGAAGTACCACTGGCTCR α/δV11 germ ....D..L..C..H..G..D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..T..S..D..R..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..T..T..G.aV4B_A_419.33 --CAGA..............................................................................................................................................................................aV4J_V03_110210 --..................................................................................................................................................................................aV4m_T02_070612 --..................................................................................................................................................................................aV4m_T09_080212 --..................................................................................................................................................................................aV4m_T11_071612 --..................................................................................................................................................................................

190 200 210 220 230 240 250 260 270 280 290 300 310

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR α/δV11 germ TATGAGGATGAAGCAGATTTTGCCAAAAGTCGCTTCTCTGCAAAACTGCAGACATCGAATAAACTCACCAATTTGATTGTCACTGGGTTACAGCTGACCGACGCTGCTGTGTATTACTGTGCATTCAGGAACCATCR α/δV11 germ .Y..E..D..E..A..D..F..A..K..S..R..F..S..A..K..L..Q..T..S..N..K..L..T..N..L..I..V..T..G..L..Q..L..T..D..A..A..V..Y..Y..C..A..F..R..N...aV4B_A_419.33 ......................................................................................................................................--------aV4J_V03_110210 ...........G..........................................................................................................................--------aV4m_T02_070612 ...........................................................................................................................AAAGCCGGG.. ------------aV4m_T09_080212 ...........................................................................................................................AAAGCCGGG..------------aV4m_T11_071612 ...........................................................................................................................AAAGCCGGG..-----------

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|TCR α/δV9 germ --AGACTTGTGTCATGGAGATTCTGTTACTCAGTCAGTCTCCTCAGTTGTTCGGACGGAAGGAGAGACGATGATGCTAAGCTGTACCTATGATACTACATCAACTTCCGAGACATTATACTGGTACCGGCAGCACCCAGGCACACAGCCCGAGTATATACTGTTGAGAAGTACCACTGGCTCR α/δV9 germ ....D..L..C..H..G..D..S..V..T..Q..S..V..S..S..V..V..R..T..E..G..E..T..M..M..L..S..C..T..Y..D..T..T..S..T..S..E..T..L..Y..W..Y..R..Q..H..P..G..T..Q..P..E..Y..I..L..L..R..S..T..T..G.aV4m_T04_062512 --..................................................................................................................................................................................aV4m_T09_062512 --..................................................................................................................................................................................aV4m_T13_070612 ------------------------------------------------------------------------------------------------..TGA.GGAGTA..A.....................................................................

190 200 210 220 230 240 250 260 270 280 290 300 310

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....TCR α/δV9 germ TATGAGGATAAAGCAGATTTTGCCAGAAATCGCTTCTTTGCGGAACTGCAGACATCGAATAAACTCACCAGTTTGACTGTCACTGGGTTACAACTGACCGACGCTGCTCTGTATTACTGTGCTTTCAGGAACCATCR α/δV9 germ .Y..E..D..K..A..D..F..A..R..N..R..F..F..A..E..L..Q..T..S..N..K..L..T..S..L..T..V..T..G..L..Q..L..T..D..A..A..L..Y..Y..C..A..F..R..N...aV4m_T04_062512 ......................................................................................................................................--------aV4m_T09_062512 ......................................................................................................................................--------aV4m_T13_070612 ..............................................................................................................................T.......-------

Figure 12. Observed germline sequences align only to TcR aV4 clones. Nucleotide alignments of TcR aV4 thymocyte clones to known germline V

segments. Highlighted and underlined bases denote nonsynonymous and synonymous differences (respectively) to the germline V segment. Boxed

regions represent nucleotides of the third complementarity-determining region (CDR3) according to IMGT guidelines, accounting for differences

between clone and germline sequences. We highlight the single nucleotide/amino acid change between a/d V7 and a/d V10 germline segments in red.

DOI: https://doi.org/10.7554/eLife.28477.025

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 24 of 31

Research article Immunology and Inflammation

https://doi.org/10.7554/eLife.28477.025
https://doi.org/10.7554/eLife.28477
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with a value of 1.00 indicating random mutation]. We used c2 analysis to compare mutability indices

between FR and CDR regions.

In situ hybridizationWe used thymus tissue from an adult nurse shark for in situ hybridization as previously described

(Criscitiello et al., 2010). We generated a probe for G. cirratum AID mRNA with primers NSAIDEH2

and NSAIDEH1 (Table 7) designed to amplify 211 base pairs of cDNA sequence of the first two AID

exons (sequence and primers kindly shared by Ellen Hsu). We acquired images on an Axioscop2

microscope with AxioCam MRc5 (Zeiss, Thornwood CT) using Zeiss Axio Vision software.

Additionally, we performed fluorescence in situ hybridization (FISH) on adult nurse shark (‘Black’)

thymus tissue. Slides contained two 8 mm thick sections of flash frozen thymus tissue preserved in

OCT. We designed custom Stellaris FISH Probes against the TcR alpha constant region (aC) for T

cell identification and exons 1 and 2 of AID by utilizing the Stellaris RNA FISH Probe Designer (Bio-

search Technologies Inc., Petaluma, CA) available online at www.biosearchtech.com/stellarisdesigner

(Version 2). We hybridized TcR aC with the CalFluor Red 610 fluorophore and the AID sequence

with the Quasar 670 fluorophore for the Stellaris RNA FISH Probe set (Biosearch Technologies, Inc.).

We followed all manufacturer’s instructions for frozen tissue (available online at www.biosearchtech.

com/stellarisprotocols), allowing hybridization probes to incubate for 16 hr. We counterstained slides

with wash buffer containing 5 ng/mL of DAPI (Sigma-Aldrich, St. Louis, MO). We obtained 10x, 20x,

and 63x images using a Zeiss Stallion Digital Imaging Workstation including a 2x CoolSnap HQ Cam-

era and Zeiss Stallion software. We merged Z-stacked images of each fluorophore together and

edited and processed images using ImageJ software, version 1.47 (Schneider et al., 2012).

Real-time qPCR for AID expressionWe synthesized cDNA from nurse shark spleen, thymus, muscle, and forebrain RNA (see RNA purifi-

cation and extraction methods above) using SuperScript III First-strand Synthesis System

(Thermo Fisher Scientific, Inc.) and a 1:1 mixture of oligo-dT and random hexamer primers. We then

amplified cDNA using touchdown PCR on an MJ mini thermal cycler (Bio-Rad, Hercules, CA) and

GoTaq colorless DNA polymerase (Promega Corp) using the following conditions: primary denatur-

ing at 94˚C for 2 min, five cycles at 94˚C for 30 s and 56˚C for 4 min; five cycles at 94˚C for 30 s and

54˚C for 4 min; 20 cycles at 94˚C for 30 s, and 52˚C for 30 s, and 72˚C for 4 min; with a final exten-

sion at 72˚C for 10 min. We visualized PCR products using agarose gel electrophoresis (as described

above) to verify presence of AID in each tissue. We then cloned and sequenced the resulting PCR

products to confirm the sequence was AID.

We looked for relative AID expression in shark spleen (positive control, where B cell AID-medi-

ated SHM is known to occur), thymus, and forebrain (negative control) at four tissue concentrations

(50 ng, 25 ng, 12.5 ng, and 6.25 ng) using the SYBR-green RT-PCR reagents kit (Thermo Fisher Sci-

entific, Inc.) on a LightCycler 480 System (Roche Diagnostics Corp, Indianapolis, IN). We analyzed

relative quantification using the LightCycler480 software and quantified relative AID expression

using the DDCq method. We normalized results against shark muscle tissue using beta2-microglobu-

lin (b2M) as a reference gene. We present data as expression-fold changes of AID to b2M.

AcknowledgementsWe acknowledge expert technical assistance from Pat Chen and Ferenc Livak for helpful advice and

comments on the manuscript. Ellen Hsu kindly shared a partial nurse shark AID sequence.

Additional information

Funding

Funder Grant reference number Author

National Science Foundation IOS 1257829 Michael F Criscitiello

National Institute of Allergyand Infectious Diseases

R01OD0549 Martin F Flajnik

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 25 of 31

Research article Immunology and Inflammation

http://www.biosearchtech.com/stellarisdesigner
http://www.biosearchtech.com/stellarisprotocols
http://www.biosearchtech.com/stellarisprotocols
https://doi.org/10.7554/eLife.28477
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The funders had no role in study design, data collection and interpretation, or the

decision to submit the work for publication.

Author contributions

Jeannine A Ott, Conceptualization, Data curation, Software, Formal analysis, Validation, Investiga-

tion, Visualization, Methodology, Writing—original draft; Caitlin D Castro, Yuko Ohta, Formal analy-

sis, Investigation, Methodology; Thaddeus C Deiss, Software, Formal analysis, Investigation,

Methodology, Writing—review and editing; Martin F Flajnik, Formal analysis, Funding acquisition,

Investigation, Methodology, Project administration, Writing—review and editing; Michael F Crisci-

tiello, Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Project

administration, Writing—review and editing

Author ORCIDs

Jeannine A Ott http://orcid.org/0000-0002-3537-8631

Michael F Criscitiello http://orcid.org/0000-0003-4262-7832

Ethics

Animal experimentation: These studies were carried out in strict accordance with the recommenda-

tions in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

The protocol was approved by the Animal Care and Use Committees at Texas A&M University and

University of Maryland School of Medicine. Experiments in the Criscitiello lab were performed under

Texas A&M University Institutional Biosafety Committee permit IBC 2014-293 and Animal Use Proto-

col 2015-0374.

Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.28477.032

Author response https://doi.org/10.7554/eLife.28477.033

Additional files

Supplementary files. Transparent reporting form

DOI: https://doi.org/10.7554/eLife.28477.026

Data availability

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.

ReferencesAdams EJ, Chien YH, Garcia KC. 2005. Structure of a gammadelta T cell receptor in complex with thenonclassical MHC T22. Science 308:227–231. DOI: https://doi.org/10.1126/science.1106885, PMID: 15821084

Alder MN, Rogozin IB, Iyer LM, Glazko GV, Cooper MD, Pancer Z. 2005. Diversity and function of adaptiveimmune receptors in a jawless vertebrate. Science 310:1970–1973. DOI: https://doi.org/10.1126/science.1119420, PMID: 16373579

Alitheen NB, McClure S, McCullagh P. 2010. B-cell development: one problem, multiple solutions. Immunologyand Cell Biology 88:445–450. DOI: https://doi.org/10.1038/icb.2009.119, PMID: 20084079

Allison TJ, Garboczi DN. 2002. Structure of gammadelta T cell receptors and their recognition of non-peptideantigens. Molecular Immunology 38:1051–1061. DOI: https://doi.org/10.1016/S0161-5890(02)00034-2,PMID: 11955597

Allison TJ, Winter CC, Fournie JJ, Bonneville M, Garboczi DN. 2001. Structure of a human gammadelta T-cellantigen receptor. Nature 411:820–824. DOI: https://doi.org/10.1038/35081115, PMID: 11459064

Anderson MK, Shamblott MJ, Litman RT, Litman GW. 1995. Generation of immunoglobulin light chain genediversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 26 of 31

Research article Immunology and Inflammation

http://orcid.org/0000-0002-3537-8631
http://orcid.org/0000-0003-4262-7832
https://doi.org/10.7554/eLife.28477.032
https://doi.org/10.7554/eLife.28477.033
https://doi.org/10.7554/eLife.28477.026
https://doi.org/10.1126/science.1106885
http://www.ncbi.nlm.nih.gov/pubmed/15821084
https://doi.org/10.1126/science.1119420
https://doi.org/10.1126/science.1119420
http://www.ncbi.nlm.nih.gov/pubmed/16373579
https://doi.org/10.1038/icb.2009.119
http://www.ncbi.nlm.nih.gov/pubmed/20084079
https://doi.org/10.1016/S0161-5890(02)00034-2
http://www.ncbi.nlm.nih.gov/pubmed/11955597
https://doi.org/10.1038/35081115
http://www.ncbi.nlm.nih.gov/pubmed/11459064
https://doi.org/10.7554/eLife.28477
Page 27: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

cell immunity. Journal of Experimental Medicine 182:109–119. DOI: https://doi.org/10.1084/jem.182.1.109,PMID: 7790811

Antonacci R, Mineccia M, Lefranc MP, Ashmaoui HM, Lanave C, Piccinni B, Pesole G, Hassanane MS, Massari S,Ciccarese S. 2011. Expression and genomic analyses of Camelus dromedarius T cell receptor delta (TRD) genesreveal a variable domain repertoire enlargement due to CDR3 diversification and somatic mutation. MolecularImmunology 48:1384–1396. DOI: https://doi.org/10.1016/j.molimm.2011.03.011, PMID: 21511341

Archer OK, Sutherland DER, Good RA. 1963. Appendix of the RABBIT: A homologue of the bursa in thechicken? Nature 200:337–339. DOI: https://doi.org/10.1038/200337a0, PMID: 14087882

Alvarez-Prado AF, Perez-Duran P, Perez-Garcıa A, Benguria A, Torroja C, de Yebenes VG, Ramiro AR. 2018. Abroad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets. The Journal ofExperimental Medicine 215:761–771. DOI: https://doi.org/10.1084/jem.20171738, PMID: 29374026

Bachl J, Wabl M. 1995. Hypermutation in T cells questioned. Nature 375:285–286. DOI: https://doi.org/10.1038/375285c0, PMID: 7753192

Barreto VM, Magor BG. 2011. Activation-induced cytidine deaminase structure and functions: a speciescomparative view. Developmental & Comparative Immunology 35:991–1007. DOI: https://doi.org/10.1016/j.dci.2011.02.005, PMID: 21349283

Becker RS, Knight KL. 1990. Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence forsomatic gene conversion in rabbits. Cell 63:987–997. DOI: https://doi.org/10.1016/0092-8674(90)90502-6,PMID: 2124176

Bedel R, Matsuda JL, Brigl M, White J, Kappler J, Marrack P, Gapin L. 2012. Lower TCR repertoire diversity inTraj18-deficient mice. Nature Immunology 13:705–706. DOI: https://doi.org/10.1038/ni.2347, PMID: 22814339

Bernstein RM, Schluter SF, Lake DF, Marchalonis JJ. 1994. Evolutionary conservation and molecular cloning ofthe recombinase activating gene 1. Biochemical and Biophysical Research Communications 205:687–692.DOI: https://doi.org/10.1006/bbrc.1994.2720, PMID: 7999099

Borgulya P, Kishi H, Uematsu Y, von Boehmer H. 1992. Exclusion and inclusion of alpha and beta T cell receptoralleles. Cell 69:529–537. DOI: https://doi.org/10.1016/0092-8674(92)90453-J, PMID: 1316241

Brodeur PH, Riblet R. 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. Onehundred Igh-V genes comprise seven families of homologous genes. European Journal of Immunology 14:922–930. DOI: https://doi.org/10.1002/eji.1830141012

Buslepp J, Wang H, Biddison WE, Appella E, Collins EJ. 2003. A correlation between TCR Valpha docking onMHC and CD8 dependence: implications for T cell selection. Immunity 19:595–606. DOI: https://doi.org/10.1016/S1074-7613(03)00269-3, PMID: 14563323

Butler JE, Santiago-Mateo K, Sun XZ, Wertz N, Sinkora M, Francis DH. 2011. Antibody repertoire developmentin fetal and neonatal piglets. XX. B cell lymphogenesis is absent in the ileal Peyer’s patches, their repertoiredevelopment is antigen dependent, and they are not required for B cell maintenance. The Journal ofImmunology 187:5141–5149. DOI: https://doi.org/10.4049/jimmunol.1101871, PMID: 22013126

Cantaert T, Schickel JN, Bannock JM, Ng YS, Massad C, Oe T, Wu R, Lavoie A, Walter JE, Notarangelo LD, Al-Herz W, Kilic SS, Ochs HD, Nonoyama S, Durandy A, Meffre E. 2015. Activation-Induced cytidine deaminaseexpression in human b cell precursors is essential for central B cell tolerance. Immunity 43:884–895.DOI: https://doi.org/10.1016/j.immuni.2015.10.002, PMID: 26546282

Chen H, Bernstein H, Ranganathan P, Schluter SF. 2012. Somatic hypermutation of TCR g V genes in the sandbarshark. Developmental & Comparative Immunology 37:176–183. DOI: https://doi.org/10.1016/j.dci.2011.08.018,PMID: 21925537

Chen H, Kshirsagar S, Jensen I, Lau K, Covarrubias R, Schluter SF, Marchalonis JJ. 2009. Characterization ofarrangement and expression of the T cell receptor gamma locus in the sandbar shark. PNAS 106:8591–8596.DOI: https://doi.org/10.1073/pnas.0811283106, PMID: 19439654

Cheynier R, Henrichwark S, Wain-Hobson S. 1998. Somatic hypermutation of the T cell receptor V beta gene inmicrodissected splenic white pulps from HIV-1-positive patients. European Journal of Immunology 28:1604–1610. DOI: https://doi.org/10.1002/(SICI)1521-4141(199805)28:05<1604::AID-IMMU1604>3.0.CO;2-R, PMID:9603466

Choudhary M, Tamrakar A, Singh AK, Jain M, Jaiswal A, Kodgire P. 2018. AID Biology: A pathological andclinical perspective. International Reviews of Immunology 37:37–56. DOI: https://doi.org/10.1080/08830185.2017.1369980, PMID: 28933967

Ciccarese S, Vaccarelli G, Lefranc MP, Tasco G, Consiglio A, Casadio R, Linguiti G, Antonacci R. 2014.Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) anddelta (TRD) variable domains. Developmental & Comparative Immunology 46:300–313. DOI: https://doi.org/10.1016/j.dci.2014.05.001, PMID: 24836674

Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS. 2005. Evolution of the AID/APOBEC family ofpolynucleotide (deoxy)cytidine deaminases. Molecular Biology and Evolution 22:367–377. DOI: https://doi.org/10.1093/molbev/msi026, PMID: 15496550

Criscitiello MF, Flajnik MF. 2007. Four primordial immunoglobulin light chain isotypes, including lambda andkappa, identified in the most primitive living jawed vertebrates. European Journal of Immunology 37:2683–2694. DOI: https://doi.org/10.1002/eji.200737263, PMID: 17899545

Criscitiello MF, Kamper SM, McKinney EC. 2004a. Allelic polymorphism of TCRalpha chain constant domaingenes in the bicolor damselfish. Developmental & Comparative Immunology 28:781–792. DOI: https://doi.org/10.1016/j.dci.2003.12.004, PMID: 15043946

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 27 of 31

Research article Immunology and Inflammation

https://doi.org/10.1084/jem.182.1.109
http://www.ncbi.nlm.nih.gov/pubmed/7790811
https://doi.org/10.1016/j.molimm.2011.03.011
http://www.ncbi.nlm.nih.gov/pubmed/21511341
https://doi.org/10.1038/200337a0
http://www.ncbi.nlm.nih.gov/pubmed/14087882
https://doi.org/10.1084/jem.20171738
http://www.ncbi.nlm.nih.gov/pubmed/29374026
https://doi.org/10.1038/375285c0
https://doi.org/10.1038/375285c0
http://www.ncbi.nlm.nih.gov/pubmed/7753192
https://doi.org/10.1016/j.dci.2011.02.005
https://doi.org/10.1016/j.dci.2011.02.005
http://www.ncbi.nlm.nih.gov/pubmed/21349283
https://doi.org/10.1016/0092-8674(90)90502-6
http://www.ncbi.nlm.nih.gov/pubmed/2124176
https://doi.org/10.1038/ni.2347
http://www.ncbi.nlm.nih.gov/pubmed/22814339
https://doi.org/10.1006/bbrc.1994.2720
http://www.ncbi.nlm.nih.gov/pubmed/7999099
https://doi.org/10.1016/0092-8674(92)90453-J
http://www.ncbi.nlm.nih.gov/pubmed/1316241
https://doi.org/10.1002/eji.1830141012
https://doi.org/10.1016/S1074-7613(03)00269-3
https://doi.org/10.1016/S1074-7613(03)00269-3
http://www.ncbi.nlm.nih.gov/pubmed/14563323
https://doi.org/10.4049/jimmunol.1101871
http://www.ncbi.nlm.nih.gov/pubmed/22013126
https://doi.org/10.1016/j.immuni.2015.10.002
http://www.ncbi.nlm.nih.gov/pubmed/26546282
https://doi.org/10.1016/j.dci.2011.08.018
http://www.ncbi.nlm.nih.gov/pubmed/21925537
https://doi.org/10.1073/pnas.0811283106
http://www.ncbi.nlm.nih.gov/pubmed/19439654
https://doi.org/10.1002/(SICI)1521-4141(199805)28:05%3C1604::AID-IMMU1604%3E3.0.CO;2-R
http://www.ncbi.nlm.nih.gov/pubmed/9603466
https://doi.org/10.1080/08830185.2017.1369980
https://doi.org/10.1080/08830185.2017.1369980
http://www.ncbi.nlm.nih.gov/pubmed/28933967
https://doi.org/10.1016/j.dci.2014.05.001
https://doi.org/10.1016/j.dci.2014.05.001
http://www.ncbi.nlm.nih.gov/pubmed/24836674
https://doi.org/10.1093/molbev/msi026
https://doi.org/10.1093/molbev/msi026
http://www.ncbi.nlm.nih.gov/pubmed/15496550
https://doi.org/10.1002/eji.200737263
http://www.ncbi.nlm.nih.gov/pubmed/17899545
https://doi.org/10.1016/j.dci.2003.12.004
https://doi.org/10.1016/j.dci.2003.12.004
http://www.ncbi.nlm.nih.gov/pubmed/15043946
https://doi.org/10.7554/eLife.28477
Page 28: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

Criscitiello MF, Ohta Y, Graham MD, Eubanks JO, Chen PL, Flajnik MF. 2012. Shark class II invariant chainreveals ancient conserved relationships with cathepsins and MHC class II. Developmental & ComparativeImmunology 36:521–533. DOI: https://doi.org/10.1016/j.dci.2011.09.008, PMID: 21996610

Criscitiello MF, Ohta Y, Saltis M, McKinney EC, Flajnik MF. 2010. Evolutionarily conserved TCR binding sites,identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark. TheJournal of Immunology 184:6950–6960. DOI: https://doi.org/10.4049/jimmunol.0902774, PMID: 20488795

Criscitiello MF, Saltis M, Flajnik MF. 2006. An evolutionarily mobile antigen receptor variable region gene:doubly rearranging NAR-TcR genes in sharks. Proceedings of the National Academy of Sciences 103:5036–5041. DOI: https://doi.org/10.1073/pnas.0507074103, PMID: 16549799

Criscitiello MF, Wermenstam NE, Pilstrom L, McKinney EC. 2004b. Allelic polymorphism of T-cell receptorconstant domains is widespread in fishes. Immunogenetics 55:818–824. DOI: https://doi.org/10.1007/s00251-004-0652-7, PMID: 14985877

Crouch EE, Li Z, Takizawa M, Fichtner-Feigl S, Gourzi P, Montano C, Feigenbaum L, Wilson P, Janz S,Papavasiliou FN, Casellas R. 2007. Regulation of AID expression in the immune response. The Journal ofExperimental Medicine 204:1145–1156. DOI: https://doi.org/10.1084/jem.20061952, PMID: 17452520

Deng L, Velikovsky CA, Xu G, Iyer LM, Tasumi S, Kerzic MC, Flajnik MF, Aravind L, Pancer Z, Mariuzza RA. 2010.A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey. PNAS 107:13408–13413. DOI: https://doi.org/10.1073/pnas.1005475107, PMID: 20616002

Diaz M, Stanfield RL, Greenberg AS, Flajnik MF. 2002. Structural analysis, selection, and ontogeny of the sharknew antigen receptor (IgNAR): identification of a new locus preferentially expressed in early development.Immunogenetics 54:501–512. DOI: https://doi.org/10.1007/s00251-002-0479-z, PMID: 12389098

Diaz M, Velez J, Singh M, Cerny J, Flajnik MF. 1999. Mutational pattern of the nurse shark antigen receptor gene(NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesionsynthesis model of somatic hypermutation. International Immunology 11:825–833. DOI: https://doi.org/10.1093/intimm/11.5.825, PMID: 10330287

Flajnik MF. 2002. Comparative analyses of immunoglobulin genes: surprises and portents. Nature ReviewsImmunology 2:688–698. DOI: https://doi.org/10.1038/nri889, PMID: 12209137

Flajnik MF. 2014. Re-evaluation of the immunological Big Bang. Current Biology 24:R1060–R1065. DOI: https://doi.org/10.1016/j.cub.2014.09.070, PMID: 25517375

Garcia KC, Adams EJ. 2005. How the T cell receptor sees antigen–a structural view. Cell 122:333–336.DOI: https://doi.org/10.1016/j.cell.2005.07.015, PMID: 16096054

Gellert M. 2002. V(D)J recombination: RAG proteins, repair factors, and regulation. Annual Review ofBiochemistry 71:101–132. DOI: https://doi.org/10.1146/annurev.biochem.71.090501.150203, PMID: 12045092

Guo J, Hawwari A, Li H, Sun Z, Mahanta SK, Littman DR, Krangel MS, He YW. 2002. Regulation of the TCRalpharepertoire by the survival window of CD4(+)CD8(+) thymocytes. Nature Immunology 3:469–476. DOI: https://doi.org/10.1038/ni791, PMID: 11967541

Guo P, Hirano M, Herrin BR, Li J, Yu C, Sadlonova A, Cooper MD. 2009. Dual nature of the adaptive immunesystem in lampreys. Nature 459:796–801. DOI: https://doi.org/10.1038/nature08068, PMID: 19474790

Hansen VL, Miller RD. 2015. The Evolution and Structure of Atypical T Cell Receptors. In: Pathogen-HostInteractions: Antigenic Variation v. Somatic Adaptations. Results and Problems in Cell Differentiation. Cham:Springer. p. 265–278. DOI: https://doi.org/10.1007/978-3-319-20819-0_11

Hirano M. 2015. Evolution of vertebrate adaptive immunity: immune cells and tissues, and AID/APOBEC cytidinedeaminases. BioEssays 37:877–887. DOI: https://doi.org/10.1002/bies.201400178, PMID: 26212221

Huesmann M, Scott B, Kisielow P, von Boehmer H. 1991. Kinetics and efficacy of positive selection in the thymusof normal and T cell receptor transgenic mice. Cell 66:533–540. DOI: https://doi.org/10.1016/0092-8674(81)90016-7, PMID: 1868548

Jerne NK. 1971. The somatic generation of immune recognition. European Journal of Immunology 1:1–9.DOI: https://doi.org/10.1002/eji.1830010102, PMID: 14978855

Kasahara M, Vazquez M, Sato K, McKinney EC, Flajnik MF. 1992. Evolution of the major histocompatibilitycomplex: isolation of class II A cDNA clones from the cartilaginous fish. PNAS 89:6688–6692. DOI: https://doi.org/10.1073/pnas.89.15.6688, PMID: 1495958

Kazen AR, Adams EJ. 2011. Evolution of the V, D, and J gene segments used in the primate gammadelta T-cellreceptor reveals a dichotomy of conservation and diversity. PNAS 108:E332–E340. DOI: https://doi.org/10.1073/pnas.1105105108, PMID: 21730193

Koren S, Schatz MC, Walenz BP, Martin J, Howard JT, Ganapathy G, Wang Z, Rasko DA, McCombie WR, JarvisED. 2012. Hybrid error correction and de novo assembly of single-molecule sequencing reads. NatureBiotechnology 30:693–700. DOI: https://doi.org/10.1038/nbt.2280, PMID: 22750884

Kreslavsky T, Kim HJ, Koralov SB, Ghitza D, Buch T, Cantor H, Rajewsky K, von Boehmer H. 2013. Negativeselection, not receptor editing, is a physiological response of autoreactive thymocytes. The Journal ofExperimental Medicine 210:1911–1918. DOI: https://doi.org/10.1084/jem.20130876, PMID: 23980099

Kuklina EM. 2006. Revision of the antigen receptor of T-lymphocytes. Biochemistry 71:827–837. DOI: https://doi.org/10.1134/S0006297906080025, PMID: 16978144

Kuraoka M, Holl TM, Liao D, Womble M, Cain DW, Reynolds AE, Kelsoe G. 2011. Activation-induced cytidinedeaminase mediates central tolerance in B cells. PNAS 108:11560–11565. DOI: https://doi.org/10.1073/pnas.1102571108, PMID: 21700885

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 28 of 31

Research article Immunology and Inflammation

https://doi.org/10.1016/j.dci.2011.09.008
http://www.ncbi.nlm.nih.gov/pubmed/21996610
https://doi.org/10.4049/jimmunol.0902774
http://www.ncbi.nlm.nih.gov/pubmed/20488795
https://doi.org/10.1073/pnas.0507074103
http://www.ncbi.nlm.nih.gov/pubmed/16549799
https://doi.org/10.1007/s00251-004-0652-7
https://doi.org/10.1007/s00251-004-0652-7
http://www.ncbi.nlm.nih.gov/pubmed/14985877
https://doi.org/10.1084/jem.20061952
http://www.ncbi.nlm.nih.gov/pubmed/17452520
https://doi.org/10.1073/pnas.1005475107
http://www.ncbi.nlm.nih.gov/pubmed/20616002
https://doi.org/10.1007/s00251-002-0479-z
http://www.ncbi.nlm.nih.gov/pubmed/12389098
https://doi.org/10.1093/intimm/11.5.825
https://doi.org/10.1093/intimm/11.5.825
http://www.ncbi.nlm.nih.gov/pubmed/10330287
https://doi.org/10.1038/nri889
http://www.ncbi.nlm.nih.gov/pubmed/12209137
https://doi.org/10.1016/j.cub.2014.09.070
https://doi.org/10.1016/j.cub.2014.09.070
http://www.ncbi.nlm.nih.gov/pubmed/25517375
https://doi.org/10.1016/j.cell.2005.07.015
http://www.ncbi.nlm.nih.gov/pubmed/16096054
https://doi.org/10.1146/annurev.biochem.71.090501.150203
http://www.ncbi.nlm.nih.gov/pubmed/12045092
https://doi.org/10.1038/ni791
https://doi.org/10.1038/ni791
http://www.ncbi.nlm.nih.gov/pubmed/11967541
https://doi.org/10.1038/nature08068
http://www.ncbi.nlm.nih.gov/pubmed/19474790
https://doi.org/10.1007/978-3-319-20819-0_11
https://doi.org/10.1002/bies.201400178
http://www.ncbi.nlm.nih.gov/pubmed/26212221
https://doi.org/10.1016/0092-8674(81)90016-7
https://doi.org/10.1016/0092-8674(81)90016-7
http://www.ncbi.nlm.nih.gov/pubmed/1868548
https://doi.org/10.1002/eji.1830010102
http://www.ncbi.nlm.nih.gov/pubmed/14978855
https://doi.org/10.1073/pnas.89.15.6688
https://doi.org/10.1073/pnas.89.15.6688
http://www.ncbi.nlm.nih.gov/pubmed/1495958
https://doi.org/10.1073/pnas.1105105108
https://doi.org/10.1073/pnas.1105105108
http://www.ncbi.nlm.nih.gov/pubmed/21730193
https://doi.org/10.1038/nbt.2280
http://www.ncbi.nlm.nih.gov/pubmed/22750884
https://doi.org/10.1084/jem.20130876
http://www.ncbi.nlm.nih.gov/pubmed/23980099
https://doi.org/10.1134/S0006297906080025
https://doi.org/10.1134/S0006297906080025
http://www.ncbi.nlm.nih.gov/pubmed/16978144
https://doi.org/10.1073/pnas.1102571108
https://doi.org/10.1073/pnas.1102571108
http://www.ncbi.nlm.nih.gov/pubmed/21700885
https://doi.org/10.7554/eLife.28477
Page 29: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

Lanning D, Sethupathi P, Rhee KJ, Zhai SK, Knight KL. 2000. Intestinal microflora and diversification of the rabbitantibody repertoire. The Journal of Immunology 165:2012–2019. DOI: https://doi.org/10.4049/jimmunol.165.4.2012, PMID: 10925284

Lantelme E, Orlando L, Porcedda P, Turinetto V, De Marchi M, Amoroso A, Mantovani S, Giachino C. 2008. Anin vitro model of T cell receptor revision in mature human CD8+ T cells. Molecular Immunology 45:328–337.DOI: https://doi.org/10.1016/j.molimm.2007.06.153, PMID: 17659780

Lee SS, Tranchina D, Ohta Y, Flajnik MF, Hsu E. 2002. Hypermutation in shark immunoglobulin light chain genesresults in contiguous substitutions. Immunity 16:571–582. DOI: https://doi.org/10.1016/S1074-7613(02)00300-X, PMID: 11970880

Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. 2003. IMGTunique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Developmental & Comparative Immunology 27:55–77. DOI: https://doi.org/10.1016/S0145-305X(02)00039-3,PMID: 12477501

Li Z, Woo CJ, Iglesias-Ussel MD, Ronai D, Scharff MD. 2004. The generation of antibody diversity throughsomatic hypermutation and class switch recombination. Genes & Development 18:1–11. DOI: https://doi.org/10.1101/gad.1161904, PMID: 14724175

Luer CA, Walsh CJ, Bodine AB, Wyffels JT, Scott TR. 1995. The elasmobranch thymus: Anatomical, histological,and preliminary functional characterization. Journal of Experimental Zoology 273:342–354. DOI: https://doi.org/10.1002/jez.1402730408

Malecek K, Brandman J, Brodsky JE, Ohta Y, Flajnik MF, Hsu E. 2005. Somatic hypermutation and junctionaldiversification at Ig heavy chain loci in the nurse shark. The Journal of Immunology 175:8105–8115.DOI: https://doi.org/10.4049/jimmunol.175.12.8105, PMID: 16339548

Mantovani S, Palermo B, Garbelli S, Campanelli R, Robustelli Della Cuna G, Gennari R, Benvenuto F, Lantelme E,Giachino C. 2002. Dominant TCR-alpha requirements for a self antigen recognition in humans. The Journal ofImmunology 169:6253–6260. DOI: https://doi.org/10.4049/jimmunol.169.11.6253, PMID: 12444131

Marshall B, Schulz R, Zhou M, Mellor A. 1999. Alternative splicing and hypermutation of a nonproductivelyrearranged TCR alpha-chain in a T cell hybridoma. Journal of Immunology 162:871–877.

Martin AP. 1999. Substitution rates of organelle and nuclear genes in sharks: implicating metabolic rate (again).Molecular Biology and Evolution 16:996–1002. DOI: https://doi.org/10.1093/oxfordjournals.molbev.a026189,PMID: 10406116

Miller RD. 2010. Those other mammals: the immunoglobulins and T cell receptors of marsupials andmonotremes. Seminars in Immunology 22:3–9. DOI: https://doi.org/10.1016/j.smim.2009.11.005,PMID: 20004116

Miracle AL, Anderson MK, Litman RT, Walsh CJ, Luer CA, Rothenberg EV, Litman GW. 2001. Complexexpression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate uniquelymphoid tissues in generating an immune repertoire. International Immunology 13:567–580. DOI: https://doi.org/10.1093/intimm/13.4.567, PMID: 11282996

Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. 2000. Class switch recombination andhypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553–563. DOI: https://doi.org/10.1016/S0092-8674(00)00078-7, PMID: 11007474

Murphy K, Weaver C. 2017. Janeway’s Immunology. 9th ed. New York: Garland Science. ISBN: 9780815345053Nakagawa Y, Ohigashi I, Nitta T, Sakata M, Tanaka K, Murata S, Kanagawa O, Takahama Y. 2012. Thymic nursecells provide microenvironment for secondary T cell receptor a rearrangement in cortical thymocytes. PNAS109:20572–20577. DOI: https://doi.org/10.1073/pnas.1213069109, PMID: 23188800

Nielsen J, Hedeholm RB, Heinemeier J, Bushnell PG, Christiansen JS, Olsen J, Ramsey CB, Brill RW, Simon M,Steffensen KF, Steffensen JF. 2016. Eye lens radiocarbon reveals centuries of longevity in the Greenland shark(Somniosus microcephalus). Science 353:702–704. DOI: https://doi.org/10.1126/science.aaf1703,PMID: 27516602

Odegard VH, Schatz DG. 2006. Targeting of somatic hypermutation. Nature Reviews Immunology 6:573–583.DOI: https://doi.org/10.1038/nri1896, PMID: 16868548

Orjalo A, Johansson HE, Ruth JL. 2011. StellarisÔ fluorescence in situ hybridization (FISH) probes: a powerfultool for mRNA detection. Nature Methods 8:i–0. DOI: https://doi.org/10.1038/nmeth.f.349

Parra ZE, Mitchell K, Dalloul RA, Miller RD. 2012. A second TCRd locus in Galliformes uses antibody-like Vdomains: insight into the evolution of TCRd and TCRm genes in tetrapods. The Journal of Immunology 188:3912–3919. DOI: https://doi.org/10.4049/jimmunol.1103521, PMID: 22407916

Parra ZE, Ohta Y, Criscitiello MF, Flajnik MF, Miller RD. 2010. The dynamic TCRd: TCRd chains in the amphibianXenopus tropicalis utilize antibody-like V genes. European Journal of Immunology 40:2319–2329. DOI: https://doi.org/10.1002/eji.201040515, PMID: 20486124

Pavri R, Nussenzweig MC. 2011. AID targeting in antibody diversity. Advances in Immunology 110:1–26.DOI: https://doi.org/10.1016/B978-0-12-387663-8.00005-3, PMID: 21762814

Perera J, Huang H. 2015. The development and function of thymic B cells. Cellular and Molecular Life Sciences72:2657–2663. DOI: https://doi.org/10.1007/s00018-015-1895-1, PMID: 25837998

Perera J, Meng L, Meng F, Huang H. 2013. Autoreactive thymic B cells are efficient antigen-presenting cells ofcognate self-antigens for T cell negative selection. PNAS 110:17011–17016. DOI: https://doi.org/10.1073/pnas.1313001110, PMID: 24082098

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 29 of 31

Research article Immunology and Inflammation

https://doi.org/10.4049/jimmunol.165.4.2012
https://doi.org/10.4049/jimmunol.165.4.2012
http://www.ncbi.nlm.nih.gov/pubmed/10925284
https://doi.org/10.1016/j.molimm.2007.06.153
http://www.ncbi.nlm.nih.gov/pubmed/17659780
https://doi.org/10.1016/S1074-7613(02)00300-X
https://doi.org/10.1016/S1074-7613(02)00300-X
http://www.ncbi.nlm.nih.gov/pubmed/11970880
https://doi.org/10.1016/S0145-305X(02)00039-3
http://www.ncbi.nlm.nih.gov/pubmed/12477501
https://doi.org/10.1101/gad.1161904
https://doi.org/10.1101/gad.1161904
http://www.ncbi.nlm.nih.gov/pubmed/14724175
https://doi.org/10.1002/jez.1402730408
https://doi.org/10.1002/jez.1402730408
https://doi.org/10.4049/jimmunol.175.12.8105
http://www.ncbi.nlm.nih.gov/pubmed/16339548
https://doi.org/10.4049/jimmunol.169.11.6253
http://www.ncbi.nlm.nih.gov/pubmed/12444131
https://doi.org/10.1093/oxfordjournals.molbev.a026189
http://www.ncbi.nlm.nih.gov/pubmed/10406116
https://doi.org/10.1016/j.smim.2009.11.005
http://www.ncbi.nlm.nih.gov/pubmed/20004116
https://doi.org/10.1093/intimm/13.4.567
https://doi.org/10.1093/intimm/13.4.567
http://www.ncbi.nlm.nih.gov/pubmed/11282996
https://doi.org/10.1016/S0092-8674(00)00078-7
http://www.ncbi.nlm.nih.gov/pubmed/11007474
https://doi.org/10.1073/pnas.1213069109
http://www.ncbi.nlm.nih.gov/pubmed/23188800
https://doi.org/10.1126/science.aaf1703
http://www.ncbi.nlm.nih.gov/pubmed/27516602
https://doi.org/10.1038/nri1896
http://www.ncbi.nlm.nih.gov/pubmed/16868548
https://doi.org/10.1038/nmeth.f.349
https://doi.org/10.4049/jimmunol.1103521
http://www.ncbi.nlm.nih.gov/pubmed/22407916
https://doi.org/10.1002/eji.201040515
https://doi.org/10.1002/eji.201040515
http://www.ncbi.nlm.nih.gov/pubmed/20486124
https://doi.org/10.1016/B978-0-12-387663-8.00005-3
http://www.ncbi.nlm.nih.gov/pubmed/21762814
https://doi.org/10.1007/s00018-015-1895-1
http://www.ncbi.nlm.nih.gov/pubmed/25837998
https://doi.org/10.1073/pnas.1313001110
https://doi.org/10.1073/pnas.1313001110
http://www.ncbi.nlm.nih.gov/pubmed/24082098
https://doi.org/10.7554/eLife.28477
Page 30: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

Petrie HT, Livak F, Schatz DG, Strasser A, Crispe IN, Shortman K. 1993. Multiple rearrangements in T cellreceptor alpha chain genes maximize the production of useful thymocytes. Journal of Experimental Medicine178:615–622. DOI: https://doi.org/10.1084/jem.178.2.615, PMID: 8393478

Qin H, Suzuki K, Nakata M, Chikuma S, Izumi N, Huong leT, Maruya M, Fagarasan S, Busslinger M, Honjo T,Nagaoka H. 2011. Activation-induced cytidine deaminase expression in CD4+ T cells is associated with aunique IL-10-producing subset that increases with age. PLoS ONE 6:e29141. DOI: https://doi.org/10.1371/journal.pone.0029141, PMID: 22216188

Raj A, Tyagi S. 2010. Chapter 17 - Detection of Individual Endogenous RNA Transcripts In Situ Using MultipleSingly Labeled Probes. In: WALTER N. G (Ed). Methods in Enzymology. Academic Press. p. 365–386.DOI: https://doi.org/10.1016/S0076-6879(10)72004-8

Rast JP, Anderson MK, Strong SJ, Luer C, Litman RT, Litman GW. 1997. a, b, g , and d T Cell Antigen ReceptorGenes Arose Early in Vertebrate Phylogeny. Immunity 6:1–11. DOI: https://doi.org/10.1016/S1074-7613(00)80237-X

Reinherz EL, Tan K, Tang L, Kern P, Liu J, Xiong Y, Hussey RE, Smolyar A, Hare B, Zhang R, Joachimiak A, ChangHC, Wagner G, Wang J. 1999. The crystal structure of a T cell receptor in complex with peptide and MHC classII. Science 286:1913–1921. DOI: https://doi.org/10.1126/science.286.5446.1913, PMID: 10583947

Reynaud CA, Anquez V, Grimal H, Weill JC. 1987. A hyperconversion mechanism generates the chicken lightchain preimmune repertoire. Cell 48:379–388. DOI: https://doi.org/10.1016/0092-8674(87)90189-9,PMID: 3100050

Reynaud CA, Garcia C, Hein WR, Weill JC. 1995. Hypermutation generating the sheep immunoglobulinrepertoire is an antigen-independent process. Cell 80:115–125. DOI: https://doi.org/10.1016/0092-8674(95)90456-5, PMID: 7813007

Reynaud CA, Mackay CR, Muller RG, Weill JC. 1991. Somatic generation of diversity in a mammalian primarylymphoid organ: the sheep ileal Peyer’s patches. Cell 64:995–1005. DOI: https://doi.org/10.1016/0092-8674(91)90323-Q, PMID: 1900459

Reynolds JD, Morris B. 1983. The evolution and involution of Peyer’s patches in fetal and postnatal sheep.European Journal of Immunology 13:627–635. DOI: https://doi.org/10.1002/eji.1830130805, PMID: 6884422

Rogozin IB, Diaz M. 2004. Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ighypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. The Journal of Immunology 172:3382–3384. DOI: https://doi.org/10.4049/jimmunol.172.6.3382, PMID: 15004135

Rogozin IB, Iyer LM, Liang L, Glazko GV, Liston VG, Pavlov YI, Aravind L, Pancer Z. 2007. Evolution anddiversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosinedeaminase. Nature Immunology 8:647–656. DOI: https://doi.org/10.1038/ni1463, PMID: 17468760

Rogozin IB, Pavlov YI, Bebenek K, Matsuda T, Kunkel TA. 2001. Somatic mutation hotspots correlate with DNApolymerase eta error spectrum. Nature Immunology 2:530–536. DOI: https://doi.org/10.1038/88732,PMID: 11376340

Rumfelt LL, Avila D, Diaz M, Bartl S, McKinney EC, Flajnik MF. 2001. A shark antibody heavy chain encoded by anonsomatically rearranged VDJ is preferentially expressed in early development and is convergent withmammalian IgG. PNAS 98:1775–1780. DOI: https://doi.org/10.1073/pnas.98.4.1775, PMID: 11172027

Rumfelt LL, Lohr RL, Dooley H, Flajnik MF. 2004. Diversity and repertoire of IgW and IgM VH families in thenewborn nurse shark. BMC Immunology 5:8–15. DOI: https://doi.org/10.1186/1471-2172-5-8, PMID: 15132758

Rumfelt LL, McKinney EC, Taylor E, Flajnik MF. 2002. The development of primary and secondary lymphoidtissues in the nurse shark Ginglymostoma cirratum: B-cell zones precede dendritic cell immigration and T-cellzone formation during ontogeny of the spleen. Scandinavian Journal of Immunology 56:130–148. DOI: https://doi.org/10.1046/j.1365-3083.2002.01116.x, PMID: 12121433

Saini J, Hershberg U. 2015. B cell variable genes have evolved their codon usage to focus the targeted patternsof somatic mutation on the complementarity determining regions. Molecular Immunology 65:157–167.DOI: https://doi.org/10.1016/j.molimm.2015.01.001, PMID: 25660968

Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. NatureMethods 9:671–675. DOI: https://doi.org/10.1038/nmeth.2089, PMID: 22930834

Thompson CB, Neiman PE. 1987. Somatic diversification of the chicken immunoglobulin light chain gene islimited to the rearranged variable gene segment. Cell 48:369–378. DOI: https://doi.org/10.1016/0092-8674(87)90188-7, PMID: 3100049

Vaccarelli G, Antonacci R, Tasco G, Yang F, Giordano L, El Ashmaoui HM, Hassanane MS, Massari S, Casadio R,Ciccarese S. 2012. Generation of diversity by somatic mutation in the Camelus dromedarius T-cell receptorgamma variable domains. European Journal of Immunology 42:3416–3428. DOI: https://doi.org/10.1002/eji.201142176, PMID: 22961631

Wagner SD, Milstein C, Neuberger MS. 1995. Codon bias targets mutation. Nature 376:732. DOI: https://doi.org/10.1038/376732a0, PMID: 7651532

Wang X, Miller RD. 2012. Recombination, transcription, and diversity of a partially germline-joined VH in amammal. Immunogenetics 64:713–717. DOI: https://doi.org/10.1007/s00251-012-0627-z, PMID: 22710822

Wang X, Parra ZE, Miller RD. 2011. Platypus TCRm provides insight into the origins and evolution of a uniquelymammalian TCR locus. The Journal of Immunology 187:5246–5254. DOI: https://doi.org/10.4049/jimmunol.1101113, PMID: 21976776

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 30 of 31

Research article Immunology and Inflammation

https://doi.org/10.1084/jem.178.2.615
http://www.ncbi.nlm.nih.gov/pubmed/8393478
https://doi.org/10.1371/journal.pone.0029141
https://doi.org/10.1371/journal.pone.0029141
http://www.ncbi.nlm.nih.gov/pubmed/22216188
https://doi.org/10.1016/S0076-6879(10)72004-8
https://doi.org/10.1016/S1074-7613(00)80237-X
https://doi.org/10.1016/S1074-7613(00)80237-X
https://doi.org/10.1126/science.286.5446.1913
http://www.ncbi.nlm.nih.gov/pubmed/10583947
https://doi.org/10.1016/0092-8674(87)90189-9
http://www.ncbi.nlm.nih.gov/pubmed/3100050
https://doi.org/10.1016/0092-8674(95)90456-5
https://doi.org/10.1016/0092-8674(95)90456-5
http://www.ncbi.nlm.nih.gov/pubmed/7813007
https://doi.org/10.1016/0092-8674(91)90323-Q
https://doi.org/10.1016/0092-8674(91)90323-Q
http://www.ncbi.nlm.nih.gov/pubmed/1900459
https://doi.org/10.1002/eji.1830130805
http://www.ncbi.nlm.nih.gov/pubmed/6884422
https://doi.org/10.4049/jimmunol.172.6.3382
https://doi.org/10.4049/jimmunol.172.6.3382
http://www.ncbi.nlm.nih.gov/pubmed/15004135
https://doi.org/10.1038/ni1463
http://www.ncbi.nlm.nih.gov/pubmed/17468760
https://doi.org/10.1038/88732
http://www.ncbi.nlm.nih.gov/pubmed/11376340
https://doi.org/10.1073/pnas.98.4.1775
http://www.ncbi.nlm.nih.gov/pubmed/11172027
https://doi.org/10.1186/1471-2172-5-8
http://www.ncbi.nlm.nih.gov/pubmed/15132758
https://doi.org/10.1046/j.1365-3083.2002.01116.x
https://doi.org/10.1046/j.1365-3083.2002.01116.x
http://www.ncbi.nlm.nih.gov/pubmed/12121433
https://doi.org/10.1016/j.molimm.2015.01.001
http://www.ncbi.nlm.nih.gov/pubmed/25660968
https://doi.org/10.1038/nmeth.2089
http://www.ncbi.nlm.nih.gov/pubmed/22930834
https://doi.org/10.1016/0092-8674(87)90188-7
https://doi.org/10.1016/0092-8674(87)90188-7
http://www.ncbi.nlm.nih.gov/pubmed/3100049
https://doi.org/10.1002/eji.201142176
https://doi.org/10.1002/eji.201142176
http://www.ncbi.nlm.nih.gov/pubmed/22961631
https://doi.org/10.1038/376732a0
https://doi.org/10.1038/376732a0
http://www.ncbi.nlm.nih.gov/pubmed/7651532
https://doi.org/10.1007/s00251-012-0627-z
http://www.ncbi.nlm.nih.gov/pubmed/22710822
https://doi.org/10.4049/jimmunol.1101113
https://doi.org/10.4049/jimmunol.1101113
http://www.ncbi.nlm.nih.gov/pubmed/21976776
https://doi.org/10.7554/eLife.28477
Page 31: Somatic hypermutation of T cell receptor a chain ...vetmed.tamu.edu/media/2081983/ott et al 2018 shm t cells.pdf · as high as that seen at B cell receptor loci (BcR) in sharks and

Wei L, Chahwan R, Wang S, Wang X, Pham PT, Goodman MF, Bergman A, Scharff MD, MacCarthy T. 2015.Overlapping hotspots in CDRs are critical sites for V region diversification. PNAS 112:E728–E737. DOI: https://doi.org/10.1073/pnas.1500788112, PMID: 25646473

Zheng B, Xue W, Kelsoe G. 1994. Locus-specific somatic hypermutation in germinal centre T cells. Nature 372:556–559. DOI: https://doi.org/10.1038/372556a0, PMID: 7990929

Zhu C, Lee V, Finn A, Senger K, Zarrin AA, Du Pasquier L, Hsu E. 2012. Origin of Immunoglobulin IsotypeSwitching. Current Biology 22:872–880. DOI: https://doi.org/10.1016/j.cub.2012.03.060

Ott et al. eLife 2018;7:e28477. DOI: https://doi.org/10.7554/eLife.28477 31 of 31

Research article Immunology and Inflammation

https://doi.org/10.1073/pnas.1500788112
https://doi.org/10.1073/pnas.1500788112
http://www.ncbi.nlm.nih.gov/pubmed/25646473
https://doi.org/10.1038/372556a0
http://www.ncbi.nlm.nih.gov/pubmed/7990929
https://doi.org/10.1016/j.cub.2012.03.060
https://doi.org/10.7554/eLife.28477

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