Patterns of Receptor Revision in the Immunoglobulin Heavy Chains of a Teleost Fish

This information is current as Miles D. Lange, Geoffrey C. Waldbieser and Craig J. Lobb of September 26, 2021. J Immunol 2009; 182:5605-5622; ; doi: 10.4049/jimmunol.0801013 http://www.jimmunol.org/content/182/9/5605 Downloaded from

References This article cites 77 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/182/9/5605.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 26, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Patterns of Receptor Revision in the Immunoglobulin Heavy Chains of a Teleost Fish1,2

Miles D. Lange,* Geoffrey C. Waldbieser,† and Craig J. Lobb3*

H chain cDNA libraries were constructed from the RNA derived from seven different organs and tissues from the same individual catfish. Sequence analysis of >300 randomly selected clones identified clonal set members within the same or different tissues, and some of these represented mosaic or hybrid sequences. These hybrids expressed VH members of the same or different VH families within different regions of the same clone. Within some clonal sets multiple hybrids were identified, and some of these represented the products of sequential VH replacement events. Different experimental methods confirmed that hybrid clones identified in the cDNA library from one tissue could be reisolated in the cDNA pool or from the total RNA derived from the same or a different tissue, indicating that these hybrids likely represented the products of in vivo receptor revision events. Murine statistical recom-

bination models were used to evaluate cryptic recombination signal sequences (cRSS), and significant cRSS pairs in the predicted Downloaded from

VH donor and recipient were identified. These models supported the hypothesis that seamless revisions may have occurred via hybrid joint formation. The heptamers of the cRSS pairs were located at different locations within the coding region, and different events resulted in the replacement of one or both CDR as well as events that replaced the upstream untranslated region and the leader region. These studies provide phylogenetic evidence that receptor revision may occur in clonally expanded B cell lineages, which supports the hypothesis that additional levels of somatic H chain diversification may exist. The Journal of Immunology,

2009, 182: 5605–5622. http://www.jimmunol.org/

he structural diversity of the VH,DH, and JH gene seg- deaminase (AID)-dependent processes of somatic mutation, class ments that undergo somatic recombination to form the switch recombination, and gene conversion (5–11). T rearranged VDJ provides the inherent structural diversity There is an additional level of postrearrangement Ig diversifi- of Ig H chains (1). Recombination requires the presence of the cation that has been termed receptor replacement (12–16). Recep- recombination signal sequence (RSS)4 located at the ends of these tor replacement is divided into two major forms. The first is termed segments. Each RSS is composed of a heptamer (e.g., CA receptor editing. These events appear to principally occur in naive CAGTG), a 12- or 23-bp spacer (denoted 12-RSS or 23-RSS, re- B cells that present with an Ig receptor that reacts with self-Ags. spectively), and an A-rich or T-rich nonamer (e.g., ACAAAAACA Studies have shown that the RSS at the usual 3Ј-end of an up- by guest on September 26, 2021 or TGTTTTTTG). Recombination is mediated by RAG1 and stream VH segment is partnered in a RAG-dependent manner with RAG2 and occurs more efficiently when one segment has a 12- a cryptic RSS (cRSS) located near the end of the framework region RSS and the other has a 23-RSS (the 12/23 rule). The combina- (FR) 3-encoded region of the VDJ rearrangement. This secondary torial diversity provided by the recombination of these different rearrangement replaces most of the original VH segment, usually segments is extended by the processes of junctional diversity and resulting in a lengthened CDR3 and altered receptor reactivity N-region nucleotide additions at the coding region junctions (2–4). (15–20). The potential to further increase the diversity of H chains post- The second type of receptor replacement is termed receptor re- VDJ rearrangement may occur by the activation-induced cytidine vision (21–29). In contrast to receptor editing, receptor revision appears to occur in B cells that may have undergone clonal ex- pansion as the result of Ag stimulation (26–29). In addition, the B *Department of Microbiology, University of Mississippi Medical Center, Jackson, cells in these populations may have been the targets of somatic MS 39216; and †U.S. Department of Agriculture, Catfish Genetics Research Unit, mutation. Revision events also differ from those known in receptor Thad Cochran National Warmwater Aquaculture Center, Stoneville, MS 38776 editing because the cRSS within both the VH donor and the tar- Received for publication March 28, 2008. Accepted for publication February geted VDJ recipient appear to be used. In these cases an unusual 24, 2009. situation is presented. These alternative cRSS must be located in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance nearly identical positions in homologous regions within both the with 18 U.S.C. Section 1734 solely to indicate this fact. donor and recipient coding regions if Ig function is to be preserved. 1 This work was supported in part by National Institutes of Health Grant AI23052. If not, nucleotides essential to coding region structure and function 2 The sequence(s) presented in this article has been submitted to GenBank under may be deleted during the replacement rearrangement. These re- accession number(s) EU492547-EU492867. quirements have led to the hypothesis that if receptor revision is 3 Address correspondence and reprint requests to Dr. Craig J. Lobb, University of RAG dependent, then the resulting replacement is dependent upon Mississippi Medical Center, Department of Microbiology, 2500 North State Street, the formation of a hybrid joint (26–28). As opposed to the standard Jackson, MS 39216-4505 USA. E-mail address: [email protected] recombination event that joins the coding ends from two rearrang- 4 Abbreviations used in this paper: RSS, recombination signal sequence; AID, acti- vation-induced cytidine deaminase; AK, anterior kidney; BLAST, basic local align- ing gene segments, a hybrid joint recombines the signal end con- ment search tool; cRSS, cryptic recombination signal sequence; CS, clonal set; FR, taining the RSS from one segment with the coding end from the framework region; GL, gill lamellae; I, intestine; LDR, leader region; RIC, recom- other segment (30). As suggested by earlier studies, if the heptam- bination signal information content; SK, skin; SP, spleen; UT, untranslated region. ers in both the donor cRSS and the recipient cRSS are in the same Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 orientation and the same relative coding region locations, then a www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801013 5606 RECEPTOR REVISION IN TELEOST H CHAINS

RAG-mediated recombination event that results in a hybrid joint cDNA libraries were also hybridized with ␥-32P-end-labeled oligonucleo- could yield a functional and apparently seamless revision product tides designed to be specific for the CDR3-encoded region in various hy- (26–28). Alternatively, it has been postulated that receptor revi- brid sequences defined in this study. Filters were prehybridized for at least 2 h at 65°C in 3 ϫ SSC, 0.1% SDS, 5ϫ Denhardt’s solution, and 0.1% sion might result from an AID-dependent, gene conversion-type sodium pyrophosphate. Hybridizations were conducted overnight at a tem- ϫ event (12, 27, 28, 31). The mechanisms, the frequency, and the perature dependent upon the Tm of the oligonucleotide in 3 SSC, 0.2% location where the revisions may occur present major challenges in SDS, 1ϫ Denhardt’s solution, and 0.1% sodium pyrophosphate. The mem- understanding the potential biological significance of receptor re- branes were washed once in 3 ϫ SSC for 5 min at the same wash tem- perature as that used for hybridization, with the last two 5-min washes in vision in B cell populations. 0.5 ϫ SSC at a probe-specific temperature. The respective hybridization In early phylogeny at the level of the bony fish there was an and final wash temperatures used were 50°C and 54°C for the explosion of structural diversity that occurred in the VH,DH, and VH2CS1CDR3 primer, 39°C and 42°C for the VH7CS1DHJHRev primer, 50°C and 54°C for the V 1CS2V D Rev primer, and 47°C and 50°C for JH gene segments that undergo rearrangement to encode the Ig H H H H the VH2CS2DJRev primers. The sequences of these primers are shown in chain V region. In our studies with the channel catfish, different VH ϳ Figs. 2B,3C,4C, and 6A, respectively. Plasmids were sequenced using families representing 200 VH segments have been defined, and ABI PRISM BigDye Terminators chemistry on an ABI 3100 DNA ana- each of these 13 VH families have been shown to be expressed in lyzer (Applied Biosystems) at the U.S. Department of Agriculture, Agri- the splenic repertoire of an individual animal. The genomic se- cultural Research Services, Mid-South Area Genomic Laboratory in quences of representative members from these families have been Stoneville, MS. H chain FR and CDR were assigned using the nomencla- ture of IMGT, the international ImMunoGeneTics information system (40). characterized, and the sequences of at least five functional DH and The sequences of 308 clones from these cDNA libraries and 13 confirma- ␮ nine functional JH segments located upstream from C have been tory clones directly derived from the cDNA pools (see below) have been determined (32–38). We have recently focused our studies upon submitted to GenBank under the accession numbers EU492547-EU492867 Downloaded from the utilization patterns of the H chain gene segments in the adult (www.ncbi.nlm.nih.gov/GenBank/). Nucleotide substitutions are desig- 3 repertoire as we seek to better understand the relationships be- nated using IMGT nomenclature (ImMunoGeneTics), e.g., A322 G de- notes that the A at position 322 is substituted by a G. The nomenclature for tween the systemic and mucosal immune systems. Toward this catfish germline DH and JH segments is from earlier studies (36–37). Da- goal, separate Ig H chain cDNA libraries were constructed from tabase searches were conducted using the basic local alignment search tool different organs and tissues derived from an individual adult chan- (BLAST; Ref. 41). The accession numbers for additional sequences in this article are NG22 (M58670), 3D09AV 2 (DQ230574), 1A11AV 5 nel catfish. The analyses on these cDNA libraries have led to this H H http://www.jimmunol.org/ (DQ230591), 3B12AV 5 (DQ230597), and the germline segments V 2-6, report in which we observe that some members of clonal sets rep- H H VH3-47, VH7-12, and VH11-2 (DQ400445; Ref 38). resented hybrids that expressed VH members of the same or a different family within different regions of the same clone. The Direct amplification of hybrid sequences from H chain cDNA experimental design of these studies allowed the results to be con- pools derived from different tissues firmed in cDNA libraries, cDNA pools, or total RNA derived from Experiments were done to confirm the presence of hybrid clones in the H the same or different tissues. These results, coupled with structural chain cDNA pools from the different tissues. Forward primers were de-

analyses of the donor and recipient partners, provide new insight signed to be restricted to the VH member expressed in the hybrid, whereas the reverse primers corresponded to nucleotides in the CDR3-encoded re- into VH replacement events.

gion. PCR amplifications were done in a volume of 50 ␮l containing 1 ␮l by guest on September 26, 2021 of the H chain cDNA pool diluted 1/25 (the same cDNA pools as used to

Materials and Methods construct the cDNA libraries), 20 mM Tris-HCl (pH 8.4), 1 mM MgCl2,10 Construction of H chain cDNA libraries, hybridization, and pmol of each primer, and1UofTaq polymerase (Invitrogen). The am- sequence analysis plification parameters used an initial denaturation of 5 min at 94°C fol- lowed by cycles of 30 s at 94°C, 30 s at an annealing temperature depen-

An individual adult outbred channel catfish (Ictalurus punctatus) was ex- dent on the Tm of the primer pair, 30 s at 72°C, and a final extension of 7 sanguinated and the following tissues were excised: anterior kidney (AK); min at 72°C. The primer pairs (primer sequences are shown in the figures), gill lamellae (GL); spleen (SP); skin (SK; ϳ2cm2 from the dorsolateral annealing temperature, number of cycles, and tissue source of the H chain surface); and intestine (ϳ30 cm). The mesenteries and blood vessels were cDNA were as follows: VH7FFor and VH7CS1DHJHRev, 58°C for 32 cy- ␮ removed from the intestine, and the organ was thoroughly washed in 90% cles using I2 and I3 cDNA; VH1AFor and C 1Rev, 58°C for 30 cycles RPMI 1640 (diluted with sterile water). The intestine was cut into three using I3 cDNA; VH1EFor and VH1CS2VHDHRev, 60°C for 35 cycles us- equal sections (each was ϳ10 cm), and 2-cm samples were taken from the ing I3 cDNA; VH6BFor and VH2CS2DJRev, 63°C for 30 cycles using SP middle of each section. These sections were designated as intestine (I) 1 and AK cDNA; VH2HFR1For and DH2/JH1Rev, 65°C for 30 cycles using (I1; from the proximal third), I2 (from the middle third), and I3 (from the SP and AK cDNA; and VH6MFor and VH2CS2DJRev, 65°C for 30 cycles distal third). The PBL were isolated as described (39). These experiments using AK cDNA. After 30 cycles, faint bands of the correct size were were conducted under an approved animal protocol from the Institutional present for the VH6B and VH6M products. These gel-excised products Animal Care and Use Committee of the University of Mississippi Medical were reamplified for an additional 30 cycles using the same primers before Center (Jackson, MS). cloning. Our earlier analyses had determined that under similar amplifica- Ϫ Total RNA was extracted from each tissue using TRIzol (Invitrogen), tion conditions the resultant Taq polymerase error rate was 0.30 ϫ 10 4 and first-strand cDNA synthesis was initiated with a C␮2 domain primer as mutations per base pair per cycle. Therefore, Taq misincorporation errors previously described (35). The products were tailed with poly(C) at the within the VH-encoded region of the clones from the cDNA libraries in this 5Ј-end. Following RNase digestion and spin column purification, the prod- report would be expected to be no more than 0.32–0.42 mutations per ucts were amplified using a C␮1 domain primer and the abridged adapter sequence (42). The confirmatory PCR clones from 30 or 60 rounds of direct primer that is supplied with the 5Ј-RACE kit (Invitrogen). PCR amplifi- amplification from the cDNA pools would be expected to be no more than cation was conducted with Taq polymerase (Invitrogen) using the follow- 0.26–0.34 or 0.44–0.67 errors per sequence, respectively. Therefore, it is ing parameters: 1 min at 94°C, 45 s at 52°C, and 45 s at 72°C for 30 cycles unlikely that any one sequence has a mutation resulting from amplification with the terminal cycle given a 7-min final extension time. PCR products error. between 450 and 550 bp in length were gel excised (subsequently termed the cDNA pool), aliquots were ligated into cloning vector pCR2.1, and Additional experiments to confirm hybrid products TOP10FЈ competent cells were transformed (Invitrogen). Five hundred can be amplified from RNA pools derived from fourteen to 534 H chain cDNA colonies derived from each of these tissues different tissues except for I1 were arrayed onto Luria-Bertani agar plates. Replicate lifts of the colonies were made using Nytran membranes (Schleicher & Schu¨ll) Additional experiments were conducted to determine whether the hybrid that were separately hybridized with radiolabeled probes specific for the products identified in the cDNA libraries and cDNA pools could also be

VH1, VH2, VH6, or VH7 families under high stringency conditions that did directly amplified from the total RNA derived from these tissues. The not permit these probes to cross-hybridize (35), a conclusion also con- experiments focused upon four hybrids: 18E02SK (Fig. 1B); 19E02I2 (Fig. firmed in this study by sequencing of hybridization positive clones. The 4C); 21F01AK (Fig. 6B); and 19E10SP (Fig. 6C). For these experiments, The Journal of Immunology 5607

the various total RNA pools derived from the tissues of the animal were the hybrid but express the full-length sequence of the VH mem- used, and first-strand cDNA was synthesized using a primer designed to be ber used in the initial rearrangement. specific for the CDR3-encoded region. PCR then used sense and nested The I2 hybrid 21D02I2 expressed members of the V 3 and antisense primers designed to specifically amplify the hybrid sequence H identified in the cDNA libraries. The sequences of these primers are shown VH1 families with the junctional boundary located in the CDR1/ in the figures unless otherwise listed. To confirm 18E02SK, VH2CS3Rev3 FR2-encoded region (Fig. 1A). Another CS member, clone (5Ј-GTAGTCAAAAGCATTATAAC-3Ј) was used for cDNA synthesis 15A10V 1GL from the GL library, was not a hybrid because it ␮ H with 5 g of GL or SK RNA, and the VH6For and VH2CS3Rev primer pair expressed the full-length V 1 segment. This sequence from FR2 was used for amplification (60°C for 30 cycles with one-tenth of the re- H action product reamplified for an additional 30 cycles). To confirm through CDR3/FR4 was identical, except at one position, to that in Ј the hybrid. BLAST analyses (41) indicated that the V 3 donor was 19E02I2, VH1CS2DHJHRev (5 -TCCCCAGTAGTCGAAGTAGCTGA- H Ј ␮ 3 ) was used for cDNA synthesis using 5 g of I1 or I3 RNA, and the similar to germline segment VH3-47, which differed from the hy- VH1EFor and VH1CS2VHDHRev primer pair was used for amplification brid by 2 nt in the region extending from the 5Ј-untranslated region (60°C for 30 cycles with one-tenth of the reaction product reamplified for Ј (UT) downstream through CDR1. an additional 30 cycles). To confirm 21F01AK, VH2CS2DHRev3 (5 - AAAGCCCGCTAGATCC-3Ј) was used for cDNA synthesis using 5 ␮gof Hybrid SK clone 18E02SK expressed members of the VH6 and

AK RNA, and the VH2HFR1For and VH2CS2DJRev primer pair was used VH2 families with the junctional boundary in FR2 (Fig. 1B). Clone for amplification (65°C for 30 cycles with one-fifth of the reaction product 18E03VH2SK, also from the SK library, expressed the full-length reamplified for an additional 30 cycles). To confirm 19E10SP, V 2 but was a member of the same CS, as its sequence from near V 2CS2D Rev3 was also used for cDNA synthesis using 5 ␮gofAKor H H H the beginning of FR2 through C␮ was almost identical to that of SP RNA, and the VH6MFor and the VH2CS2VHDHRev primer pair was used for amplification (67°C for 30 cycles with one-tenth of the reaction the hybrid (2-nt differences). The predicted donor was a VH6 mem- Downloaded from product reamplified for an additional 30 cycles). Products were gel excised, ber similar to that expressed in 18H12VH6I3 (Fig. 1B). This VH6 cloned, and sequenced as described above. sequence, extending from the 5Ј-UT through FR2, differed by 2 nt from that in the hybrid. cRSS analysis in recipient and donor partners This hybrid (18E02SK) was also of interest because VH2 and VH6 represented two of the four families chosen for library hy- To determine the presence of cRSS in likely donor and recipient VH sequences, the statistical models for defining murine RSS developed by bridization and sequencing. If this hybrid resulted from PCR error

Cowell et al. were used (43). These models, which have been made due to template switching or bridging during cDNA library con- http://www.jimmunol.org/ available online at www.dulci.org/ric, provide scores for the recombi- struction (44–47), then the probability of its detection would be nation information content (RIC) of potential cRSS at 28- or 39-bp expected to be related to the proportion of template partners avail- nucleotide intervals (i.e., 12-bp RSS and 23-bp RSS, respectively). In these models, the CA dinucleotide at positions 1 and 2 of the heptamer able for amplification. Hybridization with a VH6 family-specific is required for scoring by RIC analysis. The applicability of these mod- probe showed that only 16 of the 514 clones in the SK library were els was evaluated using the functional catfish germline VH,DH, and JH VH6 positive (3.1%), and none of the nine clones that were se- sequences (see Results). quenced used the VH6 member expressed in the hybrid. The clonally related nonhybrid clone (18E03VH2SK) was one of only

three VH2-positive rearrangements in this library (0.2% of the total

Results by guest on September 26, 2021 library). VH primers were not used during cDNA construction, Hybrid H chain cDNA clones express members of different VH which also made it less likely that these specific VH6 and VH2 families cDNA templates underwent PCR bridging. RNA was derived from eight different tissues from the same Hybrid clones 16A08GL and 20F03I2 were derived from two individual adult channel catfish, and seven of these were used to different libraries (GL and I2, respectively), and both are members construct different H chain cDNA libraries. These seven tissues of the same CS because they shared the identical CDR3/FR4. The were anterior kidney (a major hematopoietic organ in bony fish; upstream and downstream regions are encoded by segments from denoted AK throughout), PBL, spleen (denoted SP throughout), the VH2 and VH5 families, with the junctional boundary located in gill lamellae (denoted GL throughout), skin (denoted SK the CDR1/FR2 region (Fig. 1C). These hybrids differ by 9 nt, throughout), and two nonadjacent regions of the intestine des- indicating that somatic mutation had occurred. BLAST results in- ignated I2 and I3. Probes specific for the catfish VH families dicated that the VH5 recipient was similar to that expressed in VH1, VH2, VH6, and VH7 were separately hybridized with each 3B12AVH5, whereas the VH2 donor was similar to that expressed library, representative positive clones were sequenced, and se- in 16E05VH2SP. The probability that both hybrids resulted from quence alignments were constructed. From these analyses PCR errors that occurred in virtually the same positions of the clones were identified and assigned to clonal sets (CS). These same partner templates when the RNA was derived from different related sequences, some of which were found in different cDNA tissues and the cDNA libraries were constructed in independent libraries, expressed the same VDJ and a characteristic CDR3 reactions seemed unlikely. likely specific to that rearrangement. Within a CS, the pattern of The sequences of two additional hybrids are shown (Fig. 1, D the somatic mutations identified in the VH-, DH-, and/or JH- and E). Hybrid clone 21H06I3 represented an event in which a encoded regions allowed likely genealogies of clonal lineages VH2 member replaced the upstream region of the VH5 member to be determined. used in the initial rearrangement. Hybrid clone 16D11AK repre-

During these analyses, hybrid clones were identified that ex- sents an event where a VH2 donor replaced the upstream region of pressed members of different VH families in different regions of the VH11 member used in the original rearrangement. In these the same sequence. The sequences of six hybrid clones are hybrids the junctional boundary occurred near or within the FR2 shown in Fig. 1. These clones were isolated from five different region. cDNA libraries, and each encoded an open reading frame. This Hybrid cDNA clones express different members of the same VH suggested that a donor segment from a different VH family had replaced the upstream coding region in a recipient rearrange- family ment. Hence, we sought to determine whether likely recipients The above results suggested that hybrid formation may result from were also present in the H chain libraries. These recipients VH replacement events involving donors from VH families differ- would be expected to share the same CDR3/FR4 sequence as ent from those expressed in the original VDJ rearrangement. These 5608 RECEPTOR REVISION IN TELEOST H CHAINS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Hybrid Ig H chain sequences express members of different VH families in different regions of the same clone. Ig H chain cDNA libraries were separately constructed from seven different tissues derived from the same individual adult catfish. The hybrids shown in each panel (designated as revisions) were identified by random sequencing following library hybridization with probes specific for the VH1, VH2, VH6, or VH7 family. Tissue origin is designated by the last two letters of the clone name: AK, GL, I2 (from the middle third of the intestine), I3 (from the distal third of the intestine), PBL,

SK, and SP. The arrows demarcate regions within the hybrid that were encoded by members of different VH families, with the upstream and downstream regions assigned to sequences most similar to the potential donor and recipient respectively. The sequence shared in common by the hybrid, recipient, and donor lies between the arrows. The sequences are demarcated into FR and CDR regions. In A and B, the hybrid and recipient were clonally related because each encoded the identical CDR3/FR4 regions. In B the clone 25A09GL (designated as ConfrmRNA) was derived by PCR from the GL total RNA pool using experimental methods and primer pairs different from those used in H chain cDNA construction (see Materials and Methods). Other PCR clones derived from GL or SK total RNA pools were either identical to 25A09GL or differed by one or two nucleotides (positions of difference are shown by an see Results). The underlined recipients in C–E were not clonally related to the hybrid but were identified in BLAST searches as sequences with ;ء) asterisk the highest similarity to the downstream VH-encoded region in the hybrid. Dots indicate sequence identity and dashes indicate gaps introduced to maximize nucleotide similarity. The heptamer (7-mer) and nonamer (9-mer) of the cRSS predicted to be recombinogenic when evaluated using the statistical models of Cowell et al. (43) are boxed in A and B (see also Table III).

observations were pursued to determine whether VH members of were usually located at different sites within the encoded region. the same family might also serve as sequence donors. Sequence These results allowed VH consensus sequences to be constructed alignments indicated that the VH-encoded regions could be as- that were generally derived from three or more independent rear- signed to different groups. Each group shared between 95 and rangements. The consensus sequence shared ϳ98% or greater nu-

100% nucleotide identity and exhibited coding region similarities cleotide identity to the VH-encoded region of the clones assigned likely restricted to a specific germline gene. Nucleotide differences to that group. A summary of the consensus sequences pertinent to were principally single positions, and these infrequent differences this study is shown in Table I. The Journal of Immunology 5609

Table I. Summary of catfish VH consensus sequences expressed in H chain cDNA libraries

Total Number of Nucleotide Total Number Differences Number of of cDNA Mean Ϯ SD, and Range Between Independent VDJ Number of cDNA Clones of the Percentage of Representative Rearrangements Clones in Clonal Expressing Nucleotide Identities in cDNA Clone VH Consensus Expressing the Sets Expressing the the VH cDNA Clones when Representative cDNA Clone and the VH Sequence VH Consensus Consensus VH Consensus Aligned with the VH Expressing the VH Consensus Consensus (length) Sequence Sequence Sequence Consensus Sequence Sequence (accession no.) Sequence

a Ϯ VH1A (400 nt) 7 31 (CS1) 2 (CS6) 38 98.9 0.4 (97.8–99.7) 20A06VH1SK_CS1 (EU492754) 1 Ϯ b VH1B (398 nt) 10 10 (CS2) 19 99.6 0.5 (97.7–100) 15C07VH1PBL (EU492557) 0 Ϯ VH1E (400 nt) 4 0 4 99.2 0.7 (98.5–100) 15E06VH1SP (EU492641) 0 Ϯ VH2B (369 nt) 4 0 4 98.8 0.2 (98.6–98.9) 16C02VH2PBL (EU492565) 4 Ϯ VH2D (369 nt) 4 4 (CS1) 7 99.7 0.2 (99.4–100) 16H03VH2I3_CS1 (EU492821) 0 c Ϯ VH2H (350 nt) 3 3 (CS7) 5 98.8 0.4 (98.2–99.1) 16E08VH2SP (EU492651) 3 Ϯ VH2N (369 nt) 3 0 3 98.7 1.0 (97.5–99.5) 21H05VH2I3 (EU492846) 2 d Ϯ VH2O (369 nt) 20299.5 0.4 (99.2–99.7) 16C03VH2PBL (EU492566) 1 Ϯ e VH6B (366 nt) 11 2 (CS5) 12 99.3 0.5 (98.6–100) 17B09VH6GL (EU492717) 0 Ϯ VH6M (366 nt) 4 0 4 99.3 0.7 (98.4–100) 17C11VH6PBL (EU492572) 0 V 7F (399 nt) 4 2 (CS2) 5 99.5 Ϯ 0.4 (99.0–99.7) 18A02V 7GL_CS2 (EU492727) 1

H H Downloaded from a Clonal sets expressed the identical VDJ rearrangements. The number of clones analyzed in these sets precedes the CS designation. b The VH-encoded region in clones 15E09VH1SP (EU492643), 19C10VH1I3_CS2 (EU492834), and 21D06VH1I3 (EU492842) was also identical to the VH1B consensus sequence. c The VH2H consensus sequence was derived by including one clone from a previous study (M58670; Ref. 33). d The VH2O consensus sequence was derived by including one clone from a previous study (DQ230574; Ref. 42). e The VH-encoded region in clones 18B05VH6SP (EU492664) and 18F08VH6SP_CS5 (EU492675) was also identical to the VH6B consensus sequence. http://www.jimmunol.org/

When known germline and consensus sequences were aligned had the same T2493 C and A3223 G mutations that were with the library sequences, some CS members appeared to repre- present in the I3 hybrid (16H07I3), which supports a common sent hybrids. The hybrids shared the same CDR3/FR4 and se- ancestry (VH2-CS1H1 in Fig. 2A). quence identity with other CS members continued upstream to- A different CS (VH7-CS1) was initially identified in I2, and the ward the leader region (LDR) until an unexpected change in cDNA libraries were hybridized with a CDR3 probe to determine sequence occurred. At that point the sequence matched the se- the locations of other CS members. These results showed that 40 quence of a different V member. These observations suggested by guest on September 26, 2021 H of the 42 VH7-positive I2 clones as well as one of two VH7 PBL three different possibilities: 1) these hybrids were PCR artifacts; 2) clones were members of VH7-CS1. A PBL clone and 34 of the I2 the nucleotide differences were due to multiple somatic mutations clones were sequenced and multiple alignments were constructed. resulting in substitutions identical to the nucleotides in potential VH7-CS1 likely resulted from the recombination of germline seg- donors; or 3) the hybrids represented bona fide in vivo VH replace- ments VH7-12, DH1, and JH6 (Fig. 3). Each clonal member carried ment events. These hypotheses were pursued to determine the most 3 3 two VH mutations, G25 A and G85 C (differences that might likely explanation. also represent polymorphisms). VH7-CS1 had undergone expan- Clonal lineage VH2-CS1 likely resulted from rearrangement of sion and somatic mutation because clonal members differed at one V 2 member V 2D with germline segments D 2 and J 2 (Fig. H H H H or two additional positions (Fig. 3A). I2 clonal member 20C07I2, 2). An A to G mutation (A3223 G) in the D 2-encoded region H however, represented a hybrid in which a V 7 member similar to was present in each descendant, and this was the only example H V 7F had replaced the 5Ј-UT downstream through the end of FR2 of this mutation in the Ͼ20 independent rearrangements iden- H (Fig. 3B). Each of the 24 nt differences in this region were identical tified in this fish that used D 2. The I3 clone 16H07I3 had the H to nucleotides in V 7F. This event maintained the open reading identical CDR3/FR4 as well as the A3223 G mutation; how- H frame but replaced the upstream point mutations that distinguished ever, this clone represented a hybrid. The sequence extending various clonal members (e.g., C-373T, T-273C, G253A, etc.). from the 5Ј-UT downstream through CDR2 differed by 14 nt from clonal precursor 16H03I3, but nine of these 14 nt were Thus, it could not be determined which sublineage was the likely recipient. Because it was unlikely this hybrid would be represented identical to those in the VH2 member VH2O. Four of the re- maining 5 nt differences occurred in the GCT trinucleotide, a in other library clones, PCR approaches were used to obtain con- known catfish somatic mutation hotspot (see Ref. 42 and Fig. firmatory evidence. 2B). The arrayed cDNA libraries were hybridized with a CDR3 The I2 and I3 H chain cDNA pools were separately amplified with a VH7F-restricted FR1 forward primer and the VH7-CS1 probe designed to be specific for VH2-CS1 to determine whether there were other hybrids in this CS. Five additional CDR3 reverse primer. Products of the expected size were cloned, clones were identified: four from I3 and one from SP (Fig. 2B). and three clones from each product were sequenced. Although Of the I3 clones, three were identical to the clonal precursor none of the six matched the 20C07I2, a different hybrid was iden- 16H03I3, and 20E03I3 differed by 1 nt (Fig. 2A). The SP clone tified. A VH member similar to VH7F appeared to have replaced Ј (20E01SP) differed at five positions in the 5Ј-UT from the VH7-12 from the 5 -UT downstream through the beginning of the clonal precursor; however, each of these positions was identical FR3 region. The hybrid sequence was identical in clones 22A02I2 to nucleotides in VH2 member VH2N (Fig. 2C). This regional from I2 and 22A04I3 from I3 (Fig. 3C). Sequences of additional difference suggested that 20E01SP might represent another hy- clones indicated that somatic mutation had occurred in this hybrid Ј brid where the 5 -UT was replaced by VH2N. This SP hybrid lineage (Fig. 3A). 5610 RECEPTOR REVISION IN TELEOST H CHAINS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Genealogical relationships between clonally related cDNA sequences in clonal set VH2-CS1. A, The dashed circle indicates the common progenitor VH2D/DJ-CS1, which likely resulted from the recombination of VH2 member VH2D with the germline segments DH2 and JH2. The dotted circle represents the hypothetical intermediate VH2-CS1H1, and the solid circles represent various cDNA library clones designated by their names and tissue source (see legend to Fig. 1). Mutations that occurred during clonal expansion are designated using standard IMGT nomenclature (e.g., T1983 C means that the nucleotide “T” at position 198 of the V region has been mutated to “C”). Thus, for example, clone 21H07I3 had accumulated two additional mutations from 16H03I3 that were different from those found in each of the other clones (A3223 G was present in all clonal descendants). B and C, Sequence alignments of hybrid clones (REV) with sequences most similar to the likely donor (DNR; Ј the VH2O and VH2N consensus sequences are shown in B and C, respectively) and recipient (REC). The sequences are demarcated into the 5 -UT Ј ␮ (5 -UT), LDR, FR, CDR, and partial C 1 region. Arrows indicate the regions within the hybrid that were assigned to different VH2 members, with the upstream region assigned to the donor and the downstream region assigned to the recipient. The utilized regions of the germline DH2 and JH2 segments are shown. Nucleotides designated as V/N could have been derived from either the VH2D member or from the N region additions. Dots indicate nucleotide identity to the hybrid sequence. The black triangle designates mutation T2493 C, which was present in both 16H07I3 and

20E01SP and thus likely arose in a common hypothetical intermediate. The sequence of the VH2CS1 CDR3 probe used to hybridize the cDNA libraries is underlined. The designated heptamer (7-mer) and nonamer (9-mer) of cRSS predicted to be recombinogenic (43) are boxed, underlined, or overlined (see also Table III).

Hybrids may result from sequential VH donor replacements CS-specific probe, and multiple I3 clones, an I2 clone, and a These results indicated that different hybrids may be present in the PBL clone were sequenced. The I3 and PBL clones differed by same CS and that a hybrid identified in the H chain cDNA popu- 1–4 mutations (Fig. 4A). However, clone 19E02I2 from the I2 lation from one tissue may be present in a different tissue. Addi- library represented a hybrid with 23-nucleotide differences in Ј tional studies to support these results were pursued during the anal- the 5 -UT through FR2 regions when compared with VH1B ysis of VH1-CS2 (initially identified in I3). VH1-CS2 resulted (Fig. 4C). When aligned with other VH1 members, these dif- from the recombination of member VH1B with germline seg- ferences grouped into two subregions, with each assigned to a ments DH1 and JH2. The libraries were hybridized with a CDR3 different VH1 member. Eleven nucleotide differences (located The Journal of Immunology 5611 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Genealogical relationships between clonally related cDNA sequences in clonal set VH7-CS1. A, The dashed circle indicates the common progenitor VH7-12/DJ-CS1, which likely resulted from the recombination of germline VH7 member VH7-12 with the germline segments DH1 and JH6. The solid circles represent various cDNA library clones designated by their names and tissue source (see legend to Fig. 1). The dotted circle represents the hypothetical clonal intermediate (VH7F/V7-12 H1), and the double circles represent PCR clones amplified from the H chain cDNA pool derived from the indicated tissues using the primers underlined in C. The mutations that occurred during clonal expansion are shown using standard IMGT nomenclature (see Fig. 2 legend). B and C, Multiple sequence alignments of the hybrid (REV), the likely donor (DNR; the consensus sequence for VH7 member 7F is shown), and the recipient (REC). Arrows demarcate the regions within the hybrids that were assigned to different VH7 members, with the upstream and downstream regions assigned to the donor and recipient respectively. The utilized regions of the germline DH1 and JH6 segments are shown. N refers to N region additions and dots indicate nucleotide identity. The heptamer (7-mer) and nonamer (9-mer) of cRSS predicted to be recombinogenic (43) are boxed (see also Table III). between the 5Ј-UT and the end of the LDR) were identical to 4B), was used in conjunction with a C␮1 reverse primer. A those in member VH1E (Fig. 4C). Nine other differences product of the expected size was amplified and two clones were (between the end of CDR1 and the end of FR2) were each sequenced. These results showed that both clones (21E01I3 and identical to nucleotides in member VH1A. The remaining three 21E07I3) had the same CDR3/FR4 as the other clonal members, nucleotide differences were located near the end of FR1, and and the sequences matched that of the predicted hybrid inter- two of these were present in both VH1A and VH1E (positions 60 mediate (Fig. 4B). Two nucleotide differences between these and 78; G683 T was a likely mutation; Fig. 4C). clones indicated that this intermediate lineage had undergone These results suggested that 19E02I2 might be the product of somatic mutation. sequential replacements wherein the upstream region was ini- Another primer pair was used to determine whether the se- tially replaced by a segment similar to VH1A, and this inter- quential hybrid could be identified in the I3 H chain cDNA mediate hybrid was secondarily replaced with a member similar pool. A primer designed to amplify VH1E, but not VH1A or to VH1E. We reasoned that if this hypothesis was to receive VH1B (VH1Efor; Fig. 4C), was used with the CDR3 primer, and further consideration, then the hypothetical intermediate as well a product of the expected size was cloned and sequenced. This as the secondary hybrid would have to be identified in the I3 product, 22A07I3, was identical except for two nucleotide cDNA population from a different tissue. The I3 cDNA pool mutations to the hybrid clone 19E02I2 from I2. Thus, these was chosen for these experiments and different primer pairs results confirmed the presence of this hybrid in a different tissue were used for amplification. A CDR1 primer designed to am- and supported the hypothesis that sequential VH replacements plify member VH1A, but not VH1B or VH1E (VH1Afor; Fig. may occur. 5612 RECEPTOR REVISION IN TELEOST H CHAINS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Genealogical relationships between clonally related cDNA sequences in clonal set VH1-CS2. A, The dashed circle indicates the common progenitor VH1B/DJ, which likely resulted from the recombination of VH1 member VH1B with the germline segments DH1 and JH2. The sequences of the predicted common progenitor and the I3 library clone 19C10I3 were identical. The solid circles represent cDNA library clones

designated by their names and tissue source (see legend to Fig. 1). The dotted circle represents the hypothetical clonal intermediate VH1A/B which was the initial hybrid product (subsequently retargeted with donor member VH1E). The double circles represent PCR clones amplified from I3 H chain cDNA pool using the underlined primers shown in B and C. The triple circles represent PCR clones derived from the total RNA pool derived from either I1 or I3 using different experimental methods and primers than those used for H chain cDNA construction (see Materials and Methods). The mutations that occurred during clonal expansion are shown beside the arrows (see Fig. 2 legend). B and C, Multiple sequence alignments of hybrid clones (REV; the clones were from cDNA libraries, cDNA H chain pools, or total RNA pools) with the likely donor (DNR; the consensus Ј sequence for members VH1A and VH1E, respectively, are shown) and the recipient (REC). The sequences are demarcated into the 5 -UT, LDR, FR, ␮ CDR, and partial C 1 region. In C the consensus sequences for member VH1A and VH1B are shown to provide clarity. Arrows demarcate the Œ upstream and downstream regions encoded by different VH1 members. The filled triangle ( ) designates T267 in VH1B or A267 in VH1A and serves to demarcate the boundary between VH1B- and VH1A-encoded regions. The utilized regions of the germline DH1 and JH2 segments are shown. N refers to N region additions and dots indicate nucleotide identity. The heptamer (7-mer) and nonamer (9-mer) of cRSS predicted to be recombi- nogenic (43) are boxed, underlined, or overlined (see also Table III). The Journal of Immunology 5613 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Genealogical relationships between clonally related cDNA sequences in clonal set VH2-CS2. A, The dashed circle indicates the common progenitor VH2/DJ-CS2, which resulted from recombination of a VH2 member with germline DH2 and JH2 gene segments. The solid circles represent H chain cDNA library clones designated by their names and tissue source (see legend to Fig. 1), and the dotted circles represent hypothetical clonal intermediates. Clonal intermediate VH2H/VH2 represents a hybrid that was sequentially targeted for VH replacement by donor member VH6M of the VH6 family. The double circles represent PCR clones amplified from the H chain cDNA pool of the indicated tissues (see the legend to Fig. 1) using the underlined primers shown in Fig. 6. The triple circles represent PCR clones derived from the total RNA pool of the indicated tissues using experimental methods and primer pairs different from those used during H chain cDNA construction (see Materials and Methods). The mutations that occurred during clonal expansion are shown beside the arrows (see Fig. 2 legend). B, The predicted amino acid sequences of different hybrids within VH2-CS2 aligned with the clonal progenitor 16C12AK. The coding region is demarcated into the LDR, FR, CDR and partial C␮1 region. Dots indicate sequence identity and dashes indicate gaps that were introduced to maximize sequence similarity.

Different hybrids in the same CS may result from independent ample of this mutation in the Ͼ20 independent rearrangements and sequential events and may be isolated from different tissues from this fish that expressed DH2. Hybridization of the libraries

with a CS-specific CDR3 probe (VH2CS2DJRev; Fig. 6A) iden- The last CS examined was VH2-CS2, which arose by the re- tified seven other clones; two AK clones differed from the combination of a member of the VH2 family with the germline clonal precursor by only 1 or 2 nt. However, the other five segments DH2 and JH1 (Fig. 5). The clonal precursor was des- ignated as clone 16C12AK, with identical clones identified in clones (two from SP and one each from the AK, I2, and PBL the PBL and SP libraries. Each clone had the A3283 C somatic libraries) represented different hybrids, two of which expressed mutation in the DH2-encoded region, and this was the only ex- members of different VH families (Figs. 5 and 6). 5614 RECEPTOR REVISION IN TELEOST H CHAINS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Sequence alignments of the hybrid clones in clonal set VH2-CS2 (as depicted in Fig. 5). In all five panels (A–E), the sequences of hybrid clones (REV) with arrows demarcating regions within the hybrids that were assigned to different donor (DNR) and recipient (REC) sequences are shown.

The consensus sequence for VH members VH6B, VH2H, VH6M, VH2B, or germline VH2-6 are respectively shown in A–E as the donor (see also Table I). Ј VH6B and VH6M are members of the VH6 family, whereas the others are members of VH2. The hybrids are demarcated into the 5 -UT, LDR, FR, CDR, ␮ Ј Ј Ј and partial C 1 regions. The 5 - and 3 -ends of some library clones as well as the 5 -ends of some VH consensus sequences are not shown because their full-length sequence is not necessary to indicate the derived donor or recipient regions. The utilized regions of the germline DH2 and JH1 segments are shown in A. Nucleotides designated as V/N were either derived from the germline VH2 segment or from N region additions. Dots indicate sequence identity and dashes indicate gaps introduced to maximize nucleotide similarity. The heptamer (7-mer) and nonamer (9-mer) of cRSS predicted to be recombinogenic (43) are boxed (see also Table III). The sequences of the primers used to derive the PCR clones from the H chain cDNA pools or the total RNA pools (shown as double or triple circles, respectively, in Fig. 5) are underlined (see also Materials and Methods). The Journal of Immunology 5615

Table II. Estimated percentage of potential recipients and donors in cDNA libraries where hybrid clones were identifieda

Estimated Percentage of Estimated Percentage of Potential Recipients in Potential Donors in Clonal Set Name Figure Shown Hybrid cDNA Clone cDNA Library (%) cDNA Library (%)

VH2-CS3 1B 18E02SK 0.2 1.4 VH2-CS1 2B 16H07I3 1.7 0.7 b VH2-CS1 2C 20E01SP 0.2 13.5 VH7-CS1 3B 20C07I2 7.3 0.4 c VH1-CS2 4B 19E02I2 (primary) 0.2 10.5 4C 19E02I2 (secondary)c 0.2b 4.5

VH2-CS2 5, 6A 19E06AK 1.7 17.6 c VH2-CS2 5, 6B 19E10SP (primary) 0.4 13.5 5, 6C 19E10SP (secondary)c 0.2 24.2 b VH2-CS2 5, 6D 20C02PBL 0.2 7.6 VH2-CS2 5, 6D 16G06I2 0.2 3.6 VH2-CS2 5, 6E 20C04SP 0.4 13.7 a The number of potential recipients in the H chain cDNA library where the hybrid was identified (designated by the last two letters of the clone name) was determined by hybridization with CS-specific CDR3 probes. The estimated percentage of recipients was calculated by dividing the number of potential recipients by the total number of clones in the cDNA library (all cDNA libraries were composed of 534 clones except SK, which was composed of 514 clones). Similarly, the number of clones in the VH family of the donor member (as shown in the designated figures) was determined by library Downloaded from hybridization with VH family-specific probes. Various clones were sequenced and the number of clones that potentially expressed the donor VH member was determined. Clones that expressed the VH family but were not sequenced were assumed to be potential donors (an improbable assumption). The estimated percentage of potential donors was determined by dividing the number of potential donors by the total number of H chain clones in the respective cDNA library. The number of potential recipients shown for hybrid clone 18E02SK was determined by directly sequencing the VH2 positive SK clones. b A potential recipient was not identified. The estimated frequency was calculated as described above by assuming that there was one additional clone in the cDNA library that represented this hypothetical recipient. c Clones 19E02I2 and 19E10SP represented hybrids that appeared to be the products of sequential VH replacement events. These events are designated as primary and secondary (see Results). http://www.jimmunol.org/

AVH6 member similar to VH6B was the likely replacement ferences were identical to nucleotides in member VH2B (Fig. 6D), donor expressed in hybrid clone 19E06AK. The 5Ј-UT down- which suggested that this regional difference was the result of an stream through the beginning of FR2 differed extensively from CS additional replacement event resulting in a sublineage that had precursor 16C12AK, yet differed by only 1 nt when aligned with undergone somatic mutation.

VH6B. Downstream from the FR2 boundary the sequence was Lastly, the fifth hybrid in VH2-CS2 was represented by SP li- identical to 16C12AK. To determine whether this hybrid was brary clone 20C04SP (Fig. 5). This clone differed from clonal pre- present in other libraries, the AK and SP H chain cDNA pools cursor 16C12AK at nine positions in the region extending from the were used in separate PCR with a FR2/CDR1 primer designed to 5Ј-UT downstream through the beginning of FR1. Three of these by guest on September 26, 2021 Ј be restricted to VH6B and the CDR3 primer (Fig. 6A). Products of nine nucleotides represented a deletion within the 5 -UT (Fig. 6E). the expected size were obtained from both reactions, and the prod- Yet, when aligned with other VH2 members, eight of these nine ucts were cloned and sequenced. The sequences of two AK clones positions were identical to those in germline VH2 member VH2-6. and the sequence of a SP clone were either identical or differed at Thus, 20C04SP likely represented the product of another event.

1 position to this region in hybrid 19E06AK (Fig. 5A). Thus, these combined results indicated that VH2-CS2 was the The second hybrid, 19E10SP, appeared to be the product of likely target of multiple VH replacements, each resulting in an sequential replacement events (similar to results observed in the open reading frame that preserved the CDR3 coding region of the previous Results section). The initial or primary donor was sim- original rearrangement and served to modestly or extensively di- ilar to VH2 member VH2H (Figs. 5A and 6B), and the secondary versify the CDR1 and/or CDR2 combining sites (Fig. 5A). donor was a VH6 member similar to VH6M (Figs. 5A and 6C). Experiments were conducted to confirm the existence of the primary intermediate by separately amplifying AK and SP Frequency of potential recipients and donors in libraries where hybrid clones were identified cDNA with a VH2H member-restricted LDR/FR1 primer and a CDR3 primer designed to not encompass the hallmark DH2 As noted earlier, if the hybrids identified in this study were the A3283 C mutation (Fig. 6B). A product of the expected size result of PCR error due to template switching or bridging during was amplified from both tissues. The SP and AK sequences cDNA construction, the probability of hybrid formation would be (21F01AK, and 21E08SP) matched that predicted for the inter- expected to be related to the proportion of template partners avail- mediate hybrid, and A3283 C was present (Figs. 5A and 6B). able for amplification. This probability was evaluated by estimat- Studies were then done to determine whether the secondary ing the percentage of donors and recipients that could serve as hybrid product could be amplified from a different tissue. The potential PCR hybridization partners in the cDNA pools where the

AK cDNA pool was amplified using the VH6M LDR/FR1 hybrids were identified. These results are summarized in Table II primer and the CDR3 primer. The sequence of this product for the 10 hybrid cDNA library clones where the assigned recipient

(22C03AK) was identical to the hybrid SP library clone and donor were members of one of the four VH families charac- 19E10SP (Fig. 6C). Thus, these experiments also supported the terized in this study. These analyses indicated that the potential hypothesis of sequential donor replacements. recipients were minor populations in all but one of the cDNA

The fourth hybrid in VH2-CS2 was represented by clones from libraries where the hybrids were found. The percentage of potential the PBL and I2 cDNA libraries, 20C02PBL and 16G06I2. These recipients generally represented Ͻ0.5% of the total cDNA popu- two clones shared the hallmark CDR3 features of the CS, yet the lation. The highest percentage of potential recipients for these hy- Ј 5 -UT downstream through CDR1 had 6 or 10 nt differences, re- brids was represented by members of VH7-CS1 in the I2 library spectively, from the clonal precursor. Five or seven of these dif- (7.3%). The range in the percentage of potential donors was higher 5616 RECEPTOR REVISION IN TELEOST H CHAINS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 The Journal of Immunology 5617 than the number of potential recipients, as these values were cal- to the 19E10SP sequential product were also sequenced. All culated by assuming that any nonsequenced clone expressed the four of the AK clones and one of the SP clones were identical same VH member as that assigned to the replacement donor (an in sequence to 19E10SP (representative clone 25C01AK is improbable assumption). The donor values ranged from 0.4 to shown in Fig. 6C). The other two SP clones had two (A173 G 24.2% in the various libraries. These percentages of potential re- and A2403 G) or one (A1523 G) nucleotide difference. Thus, cipients and donors within the libraries indicate that it was unlikely the hybrids studied in these experiments could be independently that these specific templates underwent PCR bridging to result in amplified from the total RNA derived from one or more differ- hybrid formation. ent tissues. Hybrids identified in H chain cDNA libraries can be amplified Presence of cryptic recombination signals within junctional from total RNA derived from these different tissues boundaries

An additional series of experiments were done to determine As noted in the Introduction, mammalian VH revisions occur in a whether the hybrids from the H chain cDNA libraries were the region where the recipient and the donor share a common se- result of in vivo VH replacement events. For these experiments an quence. The occurrence of cRSS within this common region has aliquot of the original total RNA pool derived from these various suggested that receptor revision may be RAG mediated and occurs tissues was used. Rather than initiating cDNA synthesis with a C␮ via hybrid joint formation (26–28). The hybrid clones identified in primer (as was done in the previous phase of this study), first- this study were examined to evaluate this possibility. strand cDNA synthesis was initiated with a CDR3/FR4 primer Cowell and colleagues (43) developed statistical models to com- designed to be specific to the rearrangement expressed in the hy- pute RIC values that estimated the recombinogenic potential of Downloaded from brid. A sense primer corresponding to the VH member used in the murine RSS. These models were also used to define potential cRSS replacement was then paired with an internally nested CDR3/FR4 within their genomic contexts (cRSS are also composed of a hep- primer to determine whether the hybrid sequence could be ampli- tamer, a 12- or 23-bp spacer, and a nonamer). To evaluate whether fied (see Materials and Methods). these models could identify catfish cRSS, RIC values for the 23- These experiments focused upon four hybrids: 18E02SK (Fig. RSS located at the conventional positions of 34 previously char-

1B), 19E02I2 (Fig. 4C), 21F01AK (Fig. 6B), and 19E10SP (Fig. acterized functional germline catfish VH and JH segments were http://www.jimmunol.org/ 6C). Hybrid clone 18E02SK from the SK H chain cDNA library determined (34–36, 38). These RIC scores ranged from Ϫ34.3 to Ϫ Ϫ expressed members of the VH6 and VH2 families. The PCR ex- 64.5, and each was below the 69.69 value defined for func- periments amplified a band of the expected size from both GL and tional murine 23-RSS (43). Similarly, RIC scores for the conven-

SK total RNA pools. The excised products were cloned, and four tional 12-RSS that flank the functional catfish DH segments (37– clones from GL and three from SK were sequenced. Two of the 38) ranged from Ϫ16.0 to Ϫ31.0, and each was below the Ϫ48.16 GL clones and two of the SK clones were identical to each other RIC value defined for functional murine 12-RSS (43). These re- as well as identical to 18E02SK (representative clone 25A09GL is sults on the conventional catfish RSS indicated that RIC murine shown in Fig. 1B). When compared with 18E02SK, the other two models should provide a reasonable approach to evaluate whether GL clones had either one (C1463T) or two (T1473G and potentially functional cRSS may be present. by guest on September 26, 2021 A3163G) nucleotide differences. The other SK clone differed by The formation of a functional revision via hybrid joint formation one nucleotide (G1883A). requires the heptamers of the cRSS in the recipient and the cRSS Hybrid clone 19E02I2 from the I2 H chain library appeared to in the donor to exist in the same orientation and coding region result from sequential replacements (initially by member VH1A location in order for a seamless revision to occur and thus preserve and secondarily by member VH1E). Following first-strand syn- H chain functionality (26–28). Revisions can theoretically occur thesis, a VH1E member-restricted primer was used in PCR and with recipient/donor cRSS pairs in any of four possible spacer bands of the expected size were amplified from the total RNA region combinations, i.e., 12-RSS in both, 23-RSS in both, 12- pools derived from the other two intestinal regions, I1 and I3. RSS/23-RSS in recipient and donor, or 23-RSS/12-RSS in recip- The products were gel excised, and four I1 clones and four I3 ient and donor. Thus, eight possible cRSS pair combinations might clones were sequenced. Three of the I1 clones and three of the lead to receptor revision: four combinations of spacers in one of I3 clones were identical in sequence to 19E02I2 (representative two orientations (the “positive” (ϩ) orientation where the 9-mer is sequence 25A03I1 is shown; Fig. 4C). The other I1 and I3 downstream of the 7-mer and the “negative” (Ϫ) orientation where clones had one (A153 C) or two (C593 T and T1163 C) nu- the 9-mer is upstream of the 7-mer). There were 13 hybrids in this cleotide differences, respectively. study in which both recipient and donor partners could be as- The last two experiments focused on hybrid clone 19E10SP, signed. RIC analyses predicted that the total number of spatially which also appeared to be the result of sequential events in which correct cRSS pairs that had recombinogenic potential in the VH- VH2H had initially replaced the VH2 member expressed in the encoded region of these partners ranged from a low of nine to a original rearrangement and, in turn, the upstream region of VH2H high of 37 (Table III). Potential cRSS pairs were also identified had been replaced by a member of a different VH family, VH6M. where the recipient and donor were members of different families. For these experiments, first strand cDNA was synthesized with a The number of possible cRSS pairs located only in the sequence

CDR3/FR4 primer and PCR subsequently used either a VH2H or a region shared in common by the predicted recipient and donor VH6M member-restricted primer with an internally nested CDR3 used in hybrid formation were then determined. In 12 of the 13 primer. In both experiments, products of the expected size were hybrids, potential recipient and donor cRSS pairs were identified. recovered from AK RNA and the VH6M product was also recov- The number of potential cRSS recombinogenic pairs generally in- ered from SP RNA. Three AK clones corresponding to the inter- creased as the length of the common sequence region increased mediate VH2H product were sequenced, and two were identical to (Table III). The 7-mer and 9-mer sequences that reflect those cRSS the intermediate hybrid amplified earlier from the H chain cDNA pairs with the highest RIC scores in Table III are boxed in Figs. pool (representative clone 25B07AK is shown aligned with 1–4, and Fig. 6. Thus, cRSS pairs that may be functional in re- 21F01AK in Fig. 6B). The other AK clone had one mismatch combination events appear to be present in both the recipient and (T2043 C). Four AK clones and three SP clones corresponding the donor in the required orientations and locations. 5618 RECEPTOR REVISION IN TELEOST H CHAINS

Presence of mutational targets within junctional boundaries these alternative sources. If such a hybrid existed and had the same Earlier studies determined the mutability indexes of mononucle- donor/recipient junctional boundaries as the original, then the otides, dinucleotides, and trinucleotides and defined AGCT and probability that this second hybrid resulted from an identical PCR AGCA as the significant RGYW tetranucleotide targets for so- amplification error would seem so unlikely that the best conclusion matic mutation in catfish H chains (42). The high concentration of would be that the hybrid likely resulted from in vivo receptor somatic mutations in these few RGYW motifs indicated that the revision. ϳ motifs likely targeted by AID are restricted in the catfish compared In this study 15 clones from the cDNA libraries, 4% of the with those known in mammals. These results allowed us to pursue clones that were randomly sequenced, represented hybrids, and at the alternative hypothesis that hybrid formation was mediated by least one hybrid was identified from each of the seven different H AID (12, 27–28, 31). If revision occurred via an initiating AID- chain cDNA libraries (five from I2, two from I3, three from SP, induced DNA lesion, the targeted site would be expected to be two from AK, and one each from the SK, GL, and PBL libraries). within the junctional boundary where the recipient and donor These hybrids involved different VH members, with seven of these shared the same sequence. The frequency of these targets in the 15 hybrids expressing VH donors from VH families that were dif- recipient was determined and compared with the frequency of ferent from those expressed in the original rearrangement. Overall, nine of the 140 sequenced clones from the six different clonal sets these targets within the complete VH coding region. These results showed that these mutational targets were not within the junctional hybridized with CS-specific CDR3 probes represented hybrids. boundary of five of the 13 recipients (Table III). In the eight re- The percentage of hybrids varied within a CS, and not all clonal cipients that contained these targets within the junctional bound- sets contained hybrids. The largest clonal set sequenced in this Downloaded from ary, the frequency of these targets was generally higher than that study (VH1-CS1, not shown) was represented by 60 clonal mem- bers, and none represented a hybrid. In contrast, five of the 10 within the total VH coding region. Thus, these results do not uni- formly support the hypothesis that receptor revision is mediated by different VH2-CS2 clonal members (Figs. 5 and 6) represented four AID (see Discussion). different hybrids, and these were identified in the H chain cDNA libraries derived from four different tissues. The structural features of the H chain sequences that resulted from sequential events were

Discussion also distinct from those that resulted from a single event. In a http://www.jimmunol.org/ These studies have sought to provide insight into the patterns and primary replacement event the 5Ј-end of the recipient was replaced potential mechanisms of VH replacements that may serve to di- with the sequence from the incoming donor resulting in two dis- versify Ig H chains at the phylogenetic level of the bony fish. In the tinct regions: the 1) upstream region extending into the 5Ј-UT/ experimental design of this study, RNA was isolated from eight LDR encoded by the donor; and 2) the downstream region, includ- different organs and tissues from the same individual adult catfish ing the CDR3/FR4 region encoded by the recipient. In the and seven of these were used to construct C␮ H chain cDNA sequential replacements there were three regions, with each region libraries. These libraries were screened with probes specific for encoded by a different VH member. The upstream region (includ- Ͼ Ј four VH families and 300 clones were randomly sequenced. ing the 5 UT/LDR region) was encoded by the donor member used by guest on September 26, 2021 These analyses identified clonally related sequences that expressed in the secondary event, a middle region was encoded by the VH the same VDJ and encoded a characteristic CDR3, including de- member used in the primary event, and the downstream region was letions and N regions likely specific to that clonal rearrangement. encoded by the VH member used in the original rearrangement. Different clonal members were present in the same as well as in The supporting series of experiments to confirm the presence of different tissues, and differences between some members appeared the hybrids in the H chain cDNA libraries focused upon four li- to extend beyond somatic mutation to include the formation of brary clones, two of which appeared to represent sequential VH hybrid sequences. If hybrid sequences had been rare, an occasional replacements. The approach taken was 2-fold. First, the same H clone might have been discarded with its origin attributed to PCR chain cDNA pools used for library construction were directly an- amplification error (e.g., PCR splicing by overlapping extension or alyzed by PCR with primers designed to specifically amplify the bridging; Refs. 44–47). However, the observation that multiple hybrids identified in the libraries. Secondly, the original total RNA hybrids were present in these cDNA libraries, which were con- pools used to construct H chain cDNA were directly analyzed by structed without the use of VH family-specific primers, had to be RT-PCR, with first-strand cDNA synthesized using primers de- pursued. This question became central when cDNA clones ex- signed to be CS specific. PCR then used sense and nested antisense pressed members of different VH families within different regions primers designed to specifically amplify the hybrids identified in of the same clone. Because PCR hybridization error is dependent the libraries. The RNA or H chain cDNA pools that were used in upon the relative proportion of available templates and requires the these experiments were derived from either the same or a different shortened end from a damaged template to anneal to a comple- tissue than that from which the library hybrid was initially mentary sequence within a secondary template, sequence disparity identified. between members of different families would be expected to min- The results showed that hybrid library clone 18E02SK (Fig. 1B) imize the formation of stable hybrids. Although it is not possible was confirmed to be present in the total RNA pools from both GL to ensure that any of the hybrids identified in this study did not and SK. Hybrid 20C07I2 (Fig. 3B) was not confirmed, but another result from PCR amplification error, the relative proportions of hybrid within this same CS that used the same VH7F donor mem- potential H chain cDNA donor and recipient hybridization partners ber was confirmed in the H chain cDNA pools from both I2 and I3 did not generally support the conclusion that hybrids resulted by (Fig. 3C). Separate experiments were done to confirm the inter- experimental chance (summarized in Table II). Yet, as shown in mediate and sequential VH events that likely resulted in hybrid Fig. 1 and Fig. 6, A, and C, hybrids were identified that expressed library clone 19E02I2 from the I2 library (Fig. 4). The predicted different VH families in different regions of the same clone. The intermediate hybrid (VH1A/VH1B) was confirmed to be present in experimental design of these studies allowed different cDNA li- the cDNA H chain pool derived from I3, and the sequential hybrid braries, cDNA H chain pools, and total RNA derived from these corresponding to 19E02I2 was identified in the I3 H chain cDNA various tissues to be analyzed to determine whether a hybrid iden- pool as well as in both the I1 and the I3 total RNA pool. It is noted tified in one library could be confirmed to exist in one or more of that this sequential hybrid was thus identified in each of the three The Journal of Immunology 5619 nonadjacent sections of the intestine that were analyzed in these signal, and it is estimated to be responsible for as many as 25% of experiments (see Materials and Methods). Library hybrid the VDJ rearrangements in mammalian B cells (56). 19E06AK (Figs. 5A and 6A) was confirmed to be present in the H The seamless replacement products observed by Koralov et al. chain cDNA pool from both SP and AK. Lastly, separate experi- (52) were also observed in the revisions identified in earlier studies ments were done to confirm the intermediate and sequential events (27–29). These events likely occurred in mature B cells, which that led to the library hybrid 19E10SP (Figs. 5A and 6, B and C). suggested that RAG reactivation led to these seamless revision The predicted intermediate hybrid product was identified in both products via hybrid joint formation. A hybrid joint is a composite the AK and the SP H chain cDNA pool as well as the AK total of signal and coding joints, such that the signal end from one RNA pool. The sequential hybrid product resulting from replace- recombinant is located adjacent to the coding end of the other. ment events involving members of different VH families was con- Hybrid joint formation requires that a pair of RSS participate in firmed in the AK H chain cDNA pool and in both the AK and the the recombination event (30, 57) and that certain criteria are met. SP total RNA pool. Thus, these combined experiments support the The pair of RSS (one RSS from the donor and the other from the hypothesis that in vivo VH receptor revision occurred. recipient) can be in one of two relative orientations. If the RSS of

The donor sequences in the hybrids were assigned to 13 VH the pair are in opposite orientations (relative to each other), dele- members representing five different VH families, whereas the re- tion of the intervening DNA is the usual outcome. This is observed cipients were assigned to eight VH members from five different in receptor editing where the FR3 heptamer of the recipient un- families. Although the size and number of VH genes in the catfish dergoes recombination (see above). In contrast, hybrid joint for- ϳ IgH locus have been estimated, only 25% of the germline VH mation requires that the RSS in the donor and the recipient reside genes have been sequenced (34–35, 38, 49). Thus, the location of in the same orientation. Thus, the RSS heptamers in the recipient Downloaded from ϩ donor segments within the locus relative to the VH recipients used and the donor must both be either upstream (the positive ( ) ori- in the primary rearrangement cannot be presently addressed. It is entation) or downstream (the negative (Ϫ) orientation) of their noted, however, that hybrid germline VH genes similar to those respective nonamers (30). known in pigs (50) have not been identified in any of the germline The analyses of the catfish hybrid sequences allowed us to de- analyses conducted in catfish or, to our knowledge, in the VH fine boundaries that delineated the regions assigned to the donor genes identified in any other bony fish. But it is known that the and the recipient. The common sequence region (located in be- http://www.jimmunol.org/ germline members of the different catfish VH families are exten- tween recipient- and donor-assigned regions) was then evaluated sively interspersed and closely linked (34–35, 49). Thus, hybrids to determine whether a heptamer might be present. It had been that result using donors from families different from that of the shown that the first three nucleotides of the heptamer (CAC) are recipient would not be inconsistent with the organization of the required for efficient recombination (58–60). Inspection showed IgH locus. It was also generally evident that nonhybrid clonal that CA (GT) motifs were present, but our initial efforts to define members remained present within the B cell population isolated criteria necessary to identify a cRSS were problematic. This same from these tissues. This is one of the potential features that serves difficulty led Cowell and colleagues (43, 61) to conclude that con-

to distinguish receptor revision from receptor editing, as the latter sensus RSS sequences may not provide sufficient insight into re- by guest on September 26, 2021 mechanism would be expected to replace the original and poten- combination efficiencies (62). This led to their development of the tially autoreactive rearrangement before clonal expansion (14–20). RIC algorithm which scans for 28- or 39-bp sequences within their It is noted, however, that editing has been found to occur in the genomic context (12-RSS or 23-RSS, respectively). The recombi- absence of self-reactivity (51–53). nogenic potential of these motifs is then correlated to known func- Different hybrids resulting from different replacement events tional murine RSS and values are assigned (43). This theoretical were also identified in the same CS, suggesting that replacement model, when used to evaluate catfish RSS in their conventional events may be a targeted occurrence. Multiple VH replacement locations within functional VH,DH, and JH segments, defined RIC events within the same clonal set had also been identified in the values within the threshold limits defined for functional RSS in analysis of revised clonal H chains derived from human B cells murine systems. It is noted that RIC scoring of human VH seg- (27–29). The consecutive progression of replacement events tar- ments was lower than that demonstrated for mouse segments (48, geted toward a specific and preceding VDJ rearrangement had also 53, 55, 61); thus, the predictive value of RIC scores when applied been observed during receptor editing, where it was referred to as to a new and untested system such as catfish VH genes will need sequential (54) or serial replacement (18). In the primary event, the to be confirmed experimentally. embedded cryptic FR3 heptamer of the recipient (which partially These results, however, laid the foundation for determining encodes Cys104) was recombined with the RSS located at the con- whether potentially functional cRSS may be present within the Ј ventional 3 -end of the donor VH segment. This resulted in re- sequence region shared in common by the recipient, the donor, and placement of the entire upstream V region, leaving only a few of the hybrid. The analyses on the 13 hybrid clones where both donor the original V/N nucleotides upstream of those assigned to DJ. and recipient partners could be defined are summarized in Table When the primary event was sequentially targeted, the above steps III. Although many of these cRSS pairs would likely have low again occurred, leaving V/N nucleotides from the primary revision recombinogenic potential if tested experimentally, these results in- adjacent to those few nucleotides from the original V/N junction. dicated that these cRSS pairs have the potential to undergo recom- CDR3 lengthening occurred, and a characteristic footprint was de- bination via hybrid joint formation and form seamless revision fined (18). However, as shown in the studies of Koralov et al. (52), products. These cRSS pairs were distributed over multiple coding nucleotide deletion and N region insertion at the site of replace- region locations and ranged in number from a low of nine to a high ment events are not necessarily observed, indicating to these au- of 37 (Table III). In 12 of the 13 hybrids a cRSS pair was identified thors that there is strong selection for replacement joints without N within the region shared in common by the recipient and the donor. region additions. They further postulate that this may be due to RIC significant cRSS pairs were in both orientations in five of the sequence homology in the involved spacer sequences, a conclusion hybrids. The positive orientation was the only orientation identi- further supported by functional analyses (55). Receptor editing is fied in two of the hybrids, and the negative orientation was the only known to be RAG mediated and dependent upon a recombination orientation found in five of the hybrids. 5620 RECEPTOR REVISION IN TELEOST H CHAINS

Davila et al. (53) evaluated high RIC scoring cRSS in the coding or greater than that found in the entire VH region. Thus, the regions of murine VH germline segments. Their analyses of 12- hypothesis that receptor revision is due to AID-dependent, gene cRSS in the negative orientation identified conserved sites desig- conversion-like mechanisms becomes less likely (although it nated as site I through site V. Sites I and II essentially flank the cannot be discarded because less favored nucleotide motifs are upstream and downstream borders of CDR1, whereas sites III and also the targets of somatic mutations; Ref. 42). However, these IV flank and extend through CDR2. Site V encompasses the FR3 results do not preclude the possibility that an AID-induced, per- Cys104 cryptic heptamer that is usually associated with RAG-de- haps crippling somatic mutation occurred during clonal expan- pendent receptor editing in B cell precursors as discussed above sion that led to RAG reactivation (see below). (12–14, 18). The authors subsequently evaluated four of these pre- Receptor editing in immature B cells is dependent upon RAG dicted cRSS sites and showed that each was active in recombina- (18, 67), and studies indicate that RAG may also be reactivated tion assays, leading the authors to postulate that cRSS sites adja- in mature B cells. RT-PCR approaches showed that RAG tran- cent to CDR1 and CDR2 might be phylogenetically conserved to scripts were induced in splenic B cells from immunized mice provide somatic CDR reassortment. The present study supports and that RAGϩ lymphocytes were present in germinal centers this general hypothesis. Of the hybrids in this study where a high from spleen as well as regional lymph nodes (21–22). In addi- RIC scoring cRSS heptamer was identified within the region tion, RAG-dependent, double-stranded RSS signal breaks have shared in common by the recipient and donor (Table III), four had been identified in peritoneal B cells and splenic B cells from undergone replacement of both CDR1 and CDR2, five had under- immunized animals. They have also been shown to be present in gone replacement of CDR1 only, and three resulted in the replace- splenic, PBL, and tonsillar B cells following appropriate in ment of the 5Ј-UT/LDR. The other four (those where a high scor- vitro stimulation (23–24, 68–70). GFP marker studies with the Downloaded from ing RIC cRSS could not be assigned because the recipient or RAG enzymes, however, have found little evidence to support donors partners were either unassigned (Fig. 1, C–E) or the nearest RAG reactivation in mature B cells (26, 63, 71, 72) suggesting RIC-significant cRSS pair was outside the common boundary (Fig. that immature B cells that expressed RAG may have migrated 3B)) each resulted in the replacement of CDR1. Thus, the hypoth- to germinal centers or that RAG reactivation occurred below the esis that revision provides for somatic CDR reassortment is sup- level of detection used in these assays. Recently, however,

ported. However, as discussed below, some of the hybrid lineages Wang and Diamond, studying a murine lupus-like disease, http://www.jimmunol.org/ also appear to have undergone somatic mutation. Thus, receptor found that RAG was reinduced during an autoimmune response revision may function beyond repertoire diversification to include in Ag-activated, early memory, or preplasma B cells. They also the rescue of B cells from lethal mutations or their rescue from showed that L chain receptor revision had occurred (73). replacement mutations that promote autoreactivity. It may even Our studies would not have identified the number of hybrids provide a CDR-independent mechanism that replaces the 5Ј-UT, defined if it were not for the fact that multiple tissues were thus altering VH promoter-regulated Ig expression (53, 63–65). examined. This suggests possibilities that extend beyond rep- The cumulative results of this study indicate that hybrid for- ertoire diversification. B cells, upon migration to a different mation is occurring within expanded, presumably Ag-stimu- tissue, might be subjected to adverse conditions that promote lated, clonal B cells. It was also observed that in CS where these RAG reactivation. Migrating B cells might encounter “new” by guest on September 26, 2021 hybrids were identified, unrevised clonal members had under- autoantigens that were previously either not expressed or ex- gone somatic mutation (Figs. 2A,3A,4A, and 5A). It cannot be pressed below activation thresholds. RAG reactivation and re- concluded from sequence information alone whether somatic ceptor revision may occur in these B cells to escape clonal mutation preceded hybrid formation, because mutations within deletion, similar to that known in B cells that undergo receptor the distal 5Ј-end of a targeted recipient would have been re- editing to achieve peripheral tolerance (14, 74, 75). Alterna- placed by the donor. However, there is evidence to indicate that tively, the Ag or the required cellular interactions that led to hybrid lineages occurred after a clonal precursor had undergone clonal expansion in the primary tissue may be absent from the somatic mutation. Examples of this are the CDR3 A3223 G secondary tissue. If mature B cells require tonic signals to main- mutation found in all clonal members of VH2-CS1 (Fig. 2) or tain homeostasis (76, 77), the lack of or diminished signaling the CDR3 A3283 C mutation within all clonal members of (perhaps due to somatic mutation) in these new environments

VH2-CS2 (Figs. 5 and 6), where mutations occurred within the may result in RAG reactivation, leading to altered specificities germline encoded region of DH2 (see Results). There was ad- and potential autoreactivity. Experimental analyses are now re- ditional evidence that somatic mutation had occurred after re- quired to determine the developmental B cell stage and ana- vision, as many of the confirmatory clones derived from either tomical locations where H chain receptor revision may occur. H chain cDNA pools or the RNA from these different tissues had different mutational patterns. Somatic mutation had also Acknowledgments been observed to occur both before and after receptor editing We extend appreciation to Mary Duke for high throughput cDNA sequenc- (66). This interwoven relationship between somatic mutation ing support and Dr. Feixue Yang for assistance and helpful discussions. and replacement events had suggested that receptor revision might be AID mediated (13, 27–28, 31, 63). Earlier studies had shown that AGCT and AGCA were the Disclosures principal RGYW targets for somatic mutation in catfish (42). If The authors have no financial conflict of interest. AID was involved in hybrid formation, these targets would be expected to be present within the region shared in common by References the donor and the recipient. The presence of these targets within 1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302: 575–581. this region was determined and compared with their frequency 2. Gellert, M. 2002. V(D)J recombination: RAG proteins, repair factors, and regu- within the entire VH coding region. These results showed that lation. Annu. Rev. Biochem. 71: 101–132. these motifs were not present in almost 40% of the hybrids (five 3. Schatz, D. G., and E. Spanopoulou. 2005. Biochemistry of VDJ recombination. Curr. Top. Microbiol. Immunol. 290: 49–85. of 13; Table III). In the other eight hybrids the frequency of 4. Jung, D., and F. W. Alt. 2004. Unraveling V(D)J recombination; insights into these targets within the region shared in common was equal to gene regulation. Cell 116: 299–311. The Journal of Immunology 5621

5. Muramatsu, M., H. Nagaoka, R. Shinkura, N. A. Begum, and T. Honjo. 2007. 36. Hayman, J. R., Ghaffari, S. H., and C. J. Lobb. 1993. Heavy chain joining region Discovery of activation-induced cytidine deaminase, the engraver of antibody segments of the channel catfish. J. Immunol. 151: 3587–3596. memory. Adv. Immunol. 94: 1–36. 37. Hayman, J. R., and C. J. Lobb. 2000. Heavy chain diversity region segments of 6. Chaudhuri, J., U. Basu, A. Zarrin, C. Yan, S. Franco, T. Perlot, B. Vuong, the channel catfish: structure, organization, expression and phylogenetic impli- J. Wang, R. T. Phan, A. Datta, et al. 2007. Evolution of the immunoglobulin cations. J. Immunol. 164: 1916–1924. heavy chain class switch recombination mechanism. Adv. Immunol. 94: 157–214. 38. Bengten, E., S. Quiniou, J. Hikima, G. Waldbieser, G. W. Warr, N. W. Miller, 7. McCormack, W. T., and C. B. Thompson. 1990. Chicken IgL variable region and M. Wilson. 2006. Structure of the catfish IGH locus: analysis of the region gene conversions display pseudogene donor preference and 5Ј to 3Ј polarity. including the single functional IGHM gene. Immunogen 58: 831–844. Genes Dev. 4: 548–558. 39. van Ginkel, F. W., N. W. Miller, C. J. Lobb, and L. W. Clem. 1992. Character- 8. Weill, J. C., and C. A. Reynaud. 1996. Rearrangement/hypermutation/gene con- ization of anti-hapten antibodies generated in vitro by channel catfish peripheral version: when, where and why? Immunol. Today 17: 92–97. blood lymphocytes. Dev. Comp. Immunol. 16: 139–151. 9. Winstead, C. R., S. K. Zhai, P. Sethupathi, and K. L. Knight. 1999. Antigen- 40. Lefranc, M. P., V. Giudicelli, C. Ginestoux, J. Bodmer, W. Muller, R. Bontrop, induced somatic diversification of rabbit IgH genes: gene conversion and point M. Lemaitre, A. Malik, V. Barbie´, and D. Chaume. 1999. IMGT, the international mutation. J. Immunol. 162: 6602–6612. ImMunoGeneTics database. Nucleic Acids Res. 27: 209–212. 10. Arakawa, H., and J. M. Buerstedde. 2004. Immunoglobulin gene conversion: 41. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic insights from bursal B cells and the DT40 cell line. Dev. Dyn. 229: 458–464. local alignment search tool. J. Mol. Biol. 215: 403–410. 11. Tang, E. S., and A. Martin. 2007. Immunoglobulin gene conversion: synthesizing 42. Yang, F., G. C. Waldbieser, and C. J. Lobb. 2006. The nucleotide targets of antibody diversification and DNA repair. DNA Repair 6: 1557–1571. somatic mutation and the role of selection in immunoglobulin heavy chains of a 12. Kleinfeld, R., R. R. Hardy, D. Tarlinton, J. Dangl, L. A. Herzenberg, and teleost fish. J. Immunol. 176: 1655–1667. M. Weigert. 1986. Recombination between an expressed immunoglobulin heavy- ϩ 43. Cowell, L. G., M. Davila, T. B. Kepler, and G. Kelsoe. 2002. Identification and chain gene and a germline variable gene segment in a Ly 1 B-cell lymphoma. utilization of arbitrary correlations in models of recombination signal sequences. Nature 322: 843–846. Genome Biol. 3: research 0072.1–0072.20. 13. Reth, M., P. Gehrmann, E. Petrac, and P. Wiese. 1986. A novel VH to VHDJH 44. Horton, R. M., H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease. 1989. joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy- Engineering hybrid genes without the use of restriction enzymes: gene splicing by

chain production. Nature 322: 840–842. overlap extension. Gene 77: 61–68. Downloaded from 14. Chen, C., Z. Nagy, E. L. Prak, and M. Weigert. 1995. Immunoglobulin heavy 45. Paabo, S., D. M. Irwin, and A. C. Wilson. 1990. DNA damage promotes jumping chain gene replacement: a mechanism of receptor editing. Immunity 3: 747–755. between templates during enzymatic amplification. J. Biol. Chem. 265: 15. Gay, D., T. Saunders, S. Camper, and M. Weigert. 1993. Receptor editing: an 4718–4721. approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177: 46. Liu, S., D. S. Thaler, and A. Libchaber. 2002. Signal and noise in bridging PCR. 999–1008. BMC Biotechnol. 2: 13. 16. Tiegs, S. L., D. M. Russell, and D. Nemazee. 1993. Receptor editing in self- 47. Darlow, J. M., and D. I. Stott. 2005. VH replacement in rearranged immunoglob- reactive bone marrow B cells. J. Exp. Med. 177: 1009–1020. ulin genes. Immunology 114: 155–165. 17. Seagal, J., and D. Melamed. 2002. Role of receptor revision in forming a B cell 48. Murray, J. M., J. P. O’Neill, T. Messier, J. Rivers, V. E. Walker, B. McGonagle, http://www.jimmunol.org/ Clin. Immunol. repertoire. 105: 1–8. L. Trombley, L. G. Cowell, G. Kelsoe, F. McBlane, and B. A. Finette. 2006. 18. Zhang, Z., M. Zemlin, Y. Wang, D. Munfus, L. E. Huye, H. W. Findley, V(D)J recombinase-mediated processing of coding junctions at cryptic recombi- S. L. Bridges, D. B. Roth, P. D. Burrows, and M. D. Cooper. 2003. Contribution nation signal sequences in peripheral T cells during human development. J. Im- of VH gene replacement to the primary B cell repertoire. Immunity 19: 21–31. munol. 177: 5393–5404. 19. Nemazee, D. 2006. Receptor editing in lymphocyte development and central 49. Ventura-Holman, T., and C. J. Lobb. 2002. Structural organization of the immu- tolerance. Nat. Rev. Immunol. 6: 728–740. noglobulin heavy chain locus in the channel catfish: the IgH locus represents a 20. Pelanda, R., and R. M. Torres. 2006. Receptor editing for better or for worse. composite of two gene clusters. Mol. Immunol. 38: 557–564. Curr. Opin. Immunol. 18: 184–190. 50. Butler, J. E., P. Weber, and N. Wertz. 2006. Antibody repertoire development in 21. Hikida, M., M. Mori, T. Takai, K. Tomochika, K. Hamatani, and H. Ohmori. fetal and neonatal piglets. XIII. Hybrid VH genes and the preimmune repertoire 1996. Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B revisited. J. Immunol. 177: 5459–5470. cells. Science 274: 2092–2094. 51. Koralov, S. B., T. I. Novobrantseva, K. Hochedlinger, R. Jaenisch, and 22. Han, S., B. Zheng, D. G. Schatz, E. Spanopoulou, and G. Kelsoe. 1996. Neoteny K. Rajewsky. 2005. Direct in vivo V to J rearrangement violating the 12/23 by guest on September 26, 2021 Science H H in lymphocytes: Rag 1 and Rag 2 expression in germinal center B cells. rule. J. Exp. Med. 201: 341–348. 274: 2094–2097. 52. Koralov, S. B., T. I. Novobrantseva, J. Ko¨nigsmann, A. Ehlich, and K. Rajewsky. 23. Papavasiliou, P., R. Casellas, H. Suh, X. F. Qin, E. Besmer, R. Pelanda, 2006. Antibody repertoires generated by V replacement and direct V to J D. Nemazee, K. Rajewsky, and M. C. Nussenzweig. 1997. V(D)J recombination H H H joining. Immunity 25: 45–53. in mature B cells: a mechanism for altering antibody responses. Science 278: 53. Davila, M., F. Liu, L. G. Cowell, A. E. Lieberman, E. Heikamp, A. Patel, and 298–301. G. Kelsoe. 2007. Multiple conserved cryptic recombination signals in V gene 24. Han, S., S. R. Dillon, B. Zheng, M. Shimoda, M. S. Schlissel, and G. Kelsoe. H segments: detection of cleavage products only in pro-B cells. J. Exp. Med. 204: 1997. V(D)J recombinase activity in a subset of germinal center B lymphocytes. 3195–3208. Science 278: 301–305. 54. Kleinfield, R. W., and M. G. Weigert. 1989. Analysis of V gene replacement 25. Meffre, E., F. Papavasiliou, P. Cohen, O. de Bouteiller, D. Bell, H. Karasuyama, H events in a B cell lymphoma. J. Immunol. 142: 4475–4482. C. Schiff, J. Banchereau, Y. J. Liu, and M. C. Nussenzweig. 1998. Antigen re- ceptor engagement turns off the V(D)J recombination machinery in human tonsil 55. Lee, A. I., S. D. Fugmann, L. G. Cowell, L. M. Ptaszek, G. Kelsoe, and B cells. J. Exp. Med. 188: 765–772. D. G. Schatz. 2003. A functional analysis of the spacer of V(D)J recombination 26. Nemazee, D., and M. Weigert. 2000. Revising B cell receptors. J. Exp. Med. 191: signal sequences. PLoS Biol. 1: 56–69. 1813–1817. 56. Casellas, R., T. A. Shih, M. Kleinewietfeld, J. Rakonjac, D. Nemazee, 27. Wilson, P. C., K. Wilson, Y. J. Liu, J. Banchereau, V. Pascual, and J. D. Capra. K. Rajewsky, and M. C. Nussenzweig. 2001. Contribution of receptor editing to 2000. Receptor revision of immunoglobulin heavy chain variable region genes in the antibody repertoire. Science 291: 1541–1544. normal human B lymphocytes. J. Exp. Med. 191: 1881–1894. 57. Melek, M., M. Gellert, and D. C. van Gent. 1998. Rejoining of DNA by the 28. Itoh, K., E. Meffre, E. Albesiano, A. Farber, D. Dines, P. Stein, S. E. Asnis, RAG1 and RAG2 proteins. Science 280: 301–303. R. A. Furie, R. I. Jain, and N. Chiorazzi. 2000. Immunoglobulin heavy chain 58. Hesse, J. E., M. R. Lieber, K. Mizuuchi, and M. Gellert. 1989. V(D)J recombi- variable region gene replacement as a mechanism for receptor revision in rheu- nation: a functional definition of the joining signals. Genes Dev. 3: 1053–1061. matoid arthritis synovial tissue B lymphocytes. J. Exp. Med. 192: 1151–1164. 59. Ramsden, D. A., K. Baetz, and G. E. Wu. 1994. Conservation of sequence in 29. Lenze, D., A. Greiner, C. Kno¨rr, I. Anagnostopoulos, H. Stein, and M. Hummel. recombination signal sequence spacers. Nucleic Acids Res. 22: 1785–1796. 2003. Receptor revision of immunoglobulin heavy chain genes in human MALT 60. Lewis, S. M., E. Agard, and L. Czyzyk. 1997. Cryptic signals and the fidelity of lymphomas. Mol. Pathol. 56: 249–255. V(D)J joining. Mol. Cell. Biol. 17: 3125–3136. 30. Lewis, S. M., J. E. Hesse, K. Mizuuchi, and M. Gellert. 1988. Novel strand 61. Cowell, L. G., M. Davila, K. Yang, T. B. Kepler, and G. Kelsoe. 2003. Prospec- exchanges in V(D)J recombination. Cell 55: 1099–1107. tive estimation of recombination signal efficiency and identification of functional 31. Darlow, J. M., and D. I. Stott. 2006. Gene conversion in human rearranged im- cryptic signals in the genome by statistical modeling. J. Exp. Med. 197: 207–220. munoglobulin genes. Immunogen 58: 511–522. 62. Marculescu, R., T. Le, P. Simon, U. Jaeger, and B. Nadel. 2002. V(D)J-mediated 32. Ghaffari, S. H., and C. J. Lobb. 1989. Nucleotide sequence of channel catfish translocations in lymphoid neoplasms: a functional assessment of genomic in- heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of stability by cryptic sites. J. Exp. Med. 195: 85–98. Ig heavy chains. J. Immunol. 143: 2730–2739. 63. Monroe, R. J., K. J. Seidl, F. Gaertner, S. Han, F. Chen, J. Sekiguchi, J. Wang, 33. Ghaffari, S. H., and C. J. Lobb. 1991. Heavy chain variable region gene families R. Ferrini, L. Davidson, G. Kelsoe, and F. W. Alt. 1999. RAG2:GFP knockin evolved early in phylogeny. Ig complexity in fish. J. Immunol. 146: 1037–1046. mice reveal novel aspects of RAG2 expression in primary and peripheral lym- 34. Ventura-Holman, T., J. C. Jones, S. H. Ghaffari, and C. J. Lobb. 1994. Structure phoid tissues. Immunity 11: 201–212. and genomic organization of VH gene segments in the channel catfish: members 64. Brard, F., M. Shannon, E. L. Prak, S. Litwin, and M. Weigert. 1999. Somatic of different VH gene families are interspersed and closely linked. Mol. Immunol. mutation and light chain rearrangement generate autoimmunity in anti-single- 31: 823–832. stranded DNA transgenic MRL/lpr mice. J. Exp. Med. 190: 691–704. 35. Yang, F., T. Ventura-Holman, G. C. Waldbieser, and C. J. Lobb. 2003. Structure, 65. Sekiguchi, D. R., L. Yunk, D. Gary, D. Charan, B. Srivastava, D. Allman, genomic organization, and phylogenetic implications of six new VH families in M. G. Weigert, and E. T. Prak. 2006. Development and selection of edited B cells the channel catfish. Mol. Immunol. 40: 247–260. in B6.56R mice. J. Immunol. 176: 6879–6887. 5622 RECEPTOR REVISION IN TELEOST H CHAINS

66. de Wildt, R. M., R. M. Hoet, W. J. van Venrooij, I. M. Tomlinson, and G. Winter. 72. Kuwata, N., H. Igarashi, T. Ohmura, S. Aizawa, and N. Sakaguchi. 1999. 1999. Analysis of heavy and light chain pairings indicates that receptor editing Absence of expression of RAG1 in peritoneal B-1 cells detected by knocking shapes the human antibody repertoire. J. Mol. Biol. 285: 895–901. into RAG1 locus with green fluorescent protein gene. J. Immunol. 163: 67. Lutz, J., W. Mu¨ller, and H. M. Ja¨ck. 2006. VH replacement rescues progenitor B 6355–6359. cells with two nonproductive VDJ alleles. J. Immunol. 177: 7007–7014. 73. Wang, Y.-H., and B. Diamond. 2008. B cell receptor revision diminishes the 68. Qin, X. F., S. Schwers, W. Yu, F. Papavasiliou, H. Suh, A. Nussenzweig, autoreactive B cell response after antigen activation in mice. J. Clin. Invest. 118: K. Rajewsky, and M. C. Nussenzweig. 1999. Secondary V(D)J recombination in 2896–2907. B-1 cells. Nature 397: 355–359. 74. Rice, J. S., J. Newman, C. Wang, D. J. Michael, and B. Diamond. 2005. Receptor 69. Hillion, S., A. Saraux, P. Youinou, and C. Jamin. 2005. Expression of RAGs in editing in peripheral B cell tolerance. Proc. Natl. Acad. Sci. USA 102: peripheral B cells outside germinal centers is associated with the expression of 1608–1613. CD5. J. Immunol. 174: 5553–5561. 75. Zhang, Z. 2007. VH replacement in mouse and humans. Trends Immunol. 28: 70. Hillion, S., M. Dueymes, P. Youinou, and C. Jamin. 2007. IL-6 contributes to the 132–137. expression of RAGs in human mature B cells. J. Immunol. 179: 6790–6798. 76. Kraus, M., M. B. Alimzhanov, N. Rajewsky, and K. Rajewsky. 2004. Survival of 71. Yu, W., H. Nagaoka, M. Jankovic, Z. Misulovin, H. Suh, A. Rolink, F. Melchers, resting mature B lymphocytes depends on BCR signaling via the Ig␣/␤ het- E. Meffre, and M. C. Nussenzweig. 1999. Continued RAG expression in late erodimer. Cell 117: 787–800. stages of B cell development and no apparent re-induction after immunization. 77. Monroe, J. G. 2006. ITAM-mediated tonic signalling through pre-BCR and BCR Nature 400: 682–687. complexes. Nat. Rev. Immunol. 4: 283–294. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021