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Antibody Repertoire Development in Fetal
and Neonatal Piglets. XI. The Relationship of
Variable Heavy Chain Gene Usage and the
Genomic Organization of the Variable Heavy
Chain Locus
Tomoko Eguchi-Ogawa, Nancy Wertz, Xiu-Zhu Sun,
Francois Puimi, Hirohide Uenishi, Kevin Wells, Patrick
Chardon, Gregory J. Tobin and John E. Butler
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http://www.jimmunol.org/content/suppl/2010/03/05/jimmunol.090361
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The Journal of Immunology is published twice each month by
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1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:3734-3742; Prepublished online 5
March 2010;
doi: 10.4049/jimmunol.0903616
http://www.jimmunol.org/content/184/7/3734
The Journal of Immunology
Antibody Repertoire Development in Fetal and Neonatal
Piglets. XI. The Relationship of Variable Heavy Chain Gene
Usage and the Genomic Organization of the Variable Heavy
Chain Locus
Tomoko Eguchi-Ogawa,* Nancy Wertz,† Xiu-Zhu Sun,† Francois Puimi,‡
Hirohide Uenishi,* Kevin Wells,x Patrick Chardon,‡ Gregory J. Tobin,{ and
John E. Butler†
T
he H chain Ig variable locus is organized similarly in all
mammals that have been studied. As few as 12 to .1000 H
chain V region (VH) genes are thought to be arrayed 59 of
multiple H chain diversity (DH) segments that in turn are found 59 of
up to nine H chain joining (JH) segments (1, 2). VH genes characterized to date include many pseudogenes (3), such as those with
nonconsensus signal sequences, stop codons, frameshift mutations,
or the absence of functional leader sequences. In humans, for example, only 38–46 of the .100 VH genes (haplotype dependent) are
*National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan; †Department
of Microbiology, University of Iowa, Iowa City, IA 52242; ‡National Institute for Agronomic Research, Institute of Molecular Biology, Jouy-en-Josas, France; xDivision of
Animal Sciences, University of Missouri, Columbia, MO 65211; and {Biological Mimetics, Frederick, MD 21702
Received for publication November 11, 2009. Accepted for publication February 1,
2010.
This work was supported by National Science Foundation-Integrative Organismal
Systems Grant 15203800 and Grant 58-1265-5-053 from the National Pork Board
and subaward C080090-UI from Biological Mimetics.
The sequences presented in this article have been submitted to GenBank under
accession numbers AB513624 and AB513625.
Address correspondence and reprint requests to Dr. John E. Butler, Interdisciplinary
Immunology Program, Department of Microbiology, University of Iowa, 51 Newton
Road, Iowa City, IA 52242. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: BAC, bacterial artificial chromosome; CH, H chain
constant; DG, day of gestation; DH, H chain diversity; FR, framework region; IMGT,
ImMunoGeneTics; IPP, ileal Peyer’s patches; JH, H chain joining; LINE, long interspersed nucleotide element; RSS, recombination signal sequence; VH, H chain V
region.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903616
functional, whereas 27 DH segments can be expressed, but only six of
the nine JH segments are functional (2, 4). The VH genes of vertebrates belong to different families that have been grouped into three
clans based on sequence homologies (5). There are large differences
in the number of VH genes and VH gene families among species. In
swine, ,30 genes have been described, all belonging to the ancestral
VH3 family (6, 7) and have identical or nearly identical framework
regions but differ in their CDR1 and CDR2 regions (8, 9). However,
CDR regions are often shared among different porcine VH genes,
suggesting they could be a consequence of gene duplication combined with a type of genomic gene conversion (10, 11).
The distribution of VH gene segment usage in fetal and newborn
mice, humans, rabbits, and swine is nonrandom, and usage patterns
vary during ontogeny. In the fetal liver of mice, members of the VH5
and VH2 families (especially 7183 and Q52, respectively) are
preferentially used (12, 13), but after birth, splenic B cells use the
VH1 family (J558) (14). The VH2 and VH5 family genes are the most
39 in the locus, whereas the J558 family is midway (more 59) in the
locus, suggesting that VH usage in the preimmune repertoire favors
39 VH genes but that usage shifts upstream, presumably due to exposure to environmental Ag because this shift does not occur in
germ-free mice (14, 15). A preferential usage of the most JH proximal VH3 family genes has also been reported in humans (16, 17).
Early VH usage in rabbits favors the most 39 VH gene (VH1) in 90%
of VDJ rearrangements, but this preferential usage declines during
development and with exposure to environmental Ag (18). However, developmental repertoire changes in rabbits are compounded
by somatic gene conversion, a phenomenon rarely seen in mice (19).
The pattern of preferential usage of 39 VH genes is not supported in
all studies. In humans, V3-23 and V3-30 are preferentially used in the
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
In this study, we have mapped the 39 H chain V region (VH) genes and those in the H chain diversity, H chain joining, and 59 portion of
the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain
joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal
the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region
containing Enh, Cm, and Cd while revealing that the ancestral IgG3 is the most 59 Cg gene. We also observed a recent duplication
(∼1.6 million years ago) in the VH locus that contains six of the seven VH genes that comprise 75% of the preimmune repertoire.
Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal VH usage must
be intrinsically regulated. Therefore, we quantified VH usage in fetal piglets and demonstrated that usage is independent of the
position of VH genes in the genome; the most 39 functional VH gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14
and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three VH genes account for
40% of the repertoire and six for ∼70%. This limited combinatorial diversity of the porcine VH repertoire further emphasizes the
dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species. The Journal of Immunology, 2010, 184:
3734–3742.
The Journal of Immunology
3735
region of the H chain locus that includes 15 DH proximal VH genes
and the 59 portion of the H chain constant (CH) genome. Our map
shows that swine possess only one functional JH gene segment, apparently two functional DH segments, and that preferential VH usage
in primary B cell tissues of fetal animals cannot be explained as
a stochastic process dependent on the location of VH genes in the
locus. In contrast, the dominant early expression of porcine IgG3 in
late-term fetuses in an organ believed to develop an Ag-independent
repertoire appears correlated with its position in the H chain C region
of the locus.
Materials and Methods
Nomenclature
The porcine VH genes were originally named according to their order of
discovery and given vernacular names like VHA and VHB (Table I) (9).
The intention was that once their location in the genome was known, the
nomenclature would be changed to the familiar VH1, VH2, etc., system or
the ImMunoGeneTics (IMGT) system (IGHV1, IGHV2). In this report, we
have maintained the vernacular nomenclature in certain cases and used it in
parenthesis in the text. Both the vernacular and IMGT system are used in
Table II. Because the IMGT system contains an unfortunate redundancy
(IGHD is used for both IgD and DH diversity segments), we have used the
conventional Cm, Cd, and Cg for CH regions.
Screening of bacterial artificial chromosome clones and
construction of bacterial artificial chromosome contigs
All bacterial artificial chromosome (BAC) clones were isolated from
a genomic library constructed by using a Large White boar resulting from
intrafamilial matings at Institute Nationale Recherche Agronomique (Jouy
en Josas, France) (42). BAC clones containing the porcine IGHV, IGHD,
and IGHJ regions and 59 IGHC genes (Cm, Cd, Cg3, and Cg5) were
identified by using a PCR-based screening system. Selected BAC clones
were cultured overnight in Luria-Bertani broth containing 12.5 mM
chloramphenicol. BAC clones 850A09 and 747A01 that hybridized with
polynucleotide probes specific for VH genes, JH, and Cm (Table I) were
selected for further study. DNA from relevant clones was extracted with
a Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany).
Sequencing of BAC clones
BAC DNAs were extracted by using a Qiagen Large Construction Kit
(Qiagen). The DNAwere cleaved into 2- to 4-kb fragments by sonication and
then subcloned into the plasmid vector pUC118 (Takara, Otsu, Japan) at the
HincII site to construct a shotgun library. The inserts obtained were sequenced by an ABI 3730xl sequencer with a Big Dye Terminator version 3
cycle sequencing kit (Applied Biosystems, Foster City, CA) using universal
primers annealing to vector sequences. Sequence data were processed with
Phred base caller and assembled with Phrap (CodonCode, Dedham, MA) (43,
44). Compiled sequences of the two BAC clones have been uploaded in
GenBank (AB513624 and AB513625). Restriction enzyme digestion was
performed to confirm the sequence assembly. BAC clones were digested
Table I. Probe sequences used to identify common porcine VH genes, JH, and Cm
Probe Sequence for CDR1
VH Gene
VHA
VHB
VHG
VHE
VHF
VHC
VHZ
VHY
VHA*
Pan
JH
Cm
CDR1
CDR2
A
B
G
E
F
C
E
C
A
A
B
E
E
F
C
C
A
A
39 FR1
CAGT
TTCAGT
TCAGT
GT
GT
TCAGT
GT
CDR1
AGTACCTACATTAAC
GACAACGCTTTCAGC
AGTTATAGCATGGAGC
AGTTATGCAGTGAGC
AGTTATGGCGTAGGC
AGTTATGAAATCAGC
AGTTATGCAGTGAGC
AGTTATGAAATCAGC
AGAGACAACTCCCAGAACACG
CGCCAGGCTCCAGGGAAG
TGAGGACACGACGACTTCAA
GCACATGCCGCTCGTGTA
Probe Sequence for CDR2
59 F2
T
TGG
T
TGG
TG
T
TG
39 F2
CDR2
TGGCA
GCTATTAGTACTAGT
GCCATTGCTAGTAGTGAC
GGTATTGATAGTGGTAGTTA
GGTATTGATAGTGGTAGTTA
TCTATTGGTAGTGGTA
GGTATTTATAGTAGTGGTAG
GGTATTTATAGTAGTGGTAG
GCTATTAGTACTAGT
FR3
FR2
JH
CH4
TGGCA
Columns labeled CDR1 and CDR2 list the vernacular designation used in the laboratory for the porcine VH genes (see also Table II). The upstream VHA (IGHV10) is
designated as VHAp and requires a special probe that binds in the FR3 region for identification. Detection of VHN requires a combination of the probes described in Table I. This
method of recognition was confirmed by sequence analysis.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
preimmune repertoire, but these map to the middle of the locus (20).
Alternatives to the locus position concept include: 1) negative selection for B cells utilizing VH genes that recognize certain self-Ags;
2) positive selection for B cells by stromal ligands based on features
of their BCR; and 3) the evolutionary conservation of BCRs bearing
VH genes that encode binding sites that recognize bacteria that
threaten the newborn. The basis for 2) and 3) above could be the same.
For example, human V3-23, mouse VH283 (a VH2 family gene), and
shark VH genes share .80% sequence similarity at the protein level
(7, 21) and encode BCRs that are polyreactive and recognize bacterial
polysaccharides (22). In humans and mice, such Abs are often associated with IgG3 (23, 24) and class switch that occurs in the absence of environmental Ag (25, 26) and in CD40/CD40L-deficient
mice (27). In swine, IgG3 expression accounts for .60% of the total
IgG expression in the ileal Peyer’s patches (IPP) of fetal piglets
(28). The IPP of swine is the anatomical homolog of the IPP of sheep,
in which Ab repertoire development is considered to be Ag-independent (29). In both the human and mouse genome, IgG3 is the
most Cd proximal functional Cg gene (reviewed in Refs. 30, 31).
In contrast to rabbits, mice, and humans, fetal piglets present
a different pattern of fetal/neonatal development that could affect VH
gene usages. In swine, there is no known protein transfer from
mother to fetus including Igs (32–34). Because maternal Igs can
influence fetal immune responses (35, 36) and presumably B cell
selection and development, usage of certain VDJ rearrangements is
independent of this influence in fetal piglets. Although it is known
that certain porcine viruses can cross the placenta, the mechanism
remains unknown. In any case, our studies on VDJ usage in fetal
piglets were done in animals free of all detectable viral pathogens.
Offspring of swine are precosial and routinely reared in isolators
separate from their mothers, allowing the experimenter to control
environmental factors that might influence the development of
adaptive immunity (37). In swine, VH, DH, and JH usage is highly
restricted. In the expressed preimmune repertoire, four to seven VH
genes account for 75–95% of the repertoire (9, 38) (Table I). Although the mechanism is unknown, swine use only a single JH and
two DH segments to generate their preimmune Ab repertoire (38–
40). We believed these observations might be better understood by
mapping the VH, DH, and JH loci of swine. We were especially
interested to know if the predominant use of a few VH genes in the
preimmune repertoire was confined to those at the 39 end of the VH
locus as reported in rabbit, mouse, and some human studies.
We describe in this study the characterization of a 229-kb segment
of swine chromosome 7 containing the VH-DH-JH-Cm-Cd and 59 Cg
3736
with HindIII, PvuI, or FspI, and the fragment sizes were compared with
those predicted by a Genetyx sequence editor (Genetyx, Tokyo, Japan).
The number of IGHV genes contained in the BAC clones was confirmed by
PCR methods using each BAC clone DNA as a template. Each IGHV genewas
amplified using a primer set annealing to sequences in 59 framework region
(FR) 1 (59-GAGGAGAAGCTGGTGGAGT-39) and 39 FR3 (59-GCCGTGTCTTCGGTTCTCA-39) that are common to all known porcine VH genes
except VHB. To amplify the VHB, 59 FR1 (59-GAGGAGAAGCTGGTGGAGT-39) and FR3R IGHVB (59-GCCGTGTCTTCGGTTCTGA-39) were
used. The products were cloned into the TOPO 2.1 vector (Invitrogen,
Carlsbad, CA). More than 80 clones were selected at random from each PCR
product for sequence analysis.
Analysis of mapping data
Quantitation of VH gene usage in fetal liver and late-gestation
bone marrow
DNA from fetal liver at 30 d of gestation (DG30) from four piglets each
from four unrelated gilts and RNA from the bone marrow of five fetuses at
DG95 were used to recover VDJ rearrangement from DNA and cDNA,
respectively, as previously described (9, 39, 40). The VDJs recovered by
PCR were cloned into pCRÒ4-TOPO (Invitrogen), and 632 clones from
fetal liver and 432 from bone marrow transcripts were then sequentially
hybridized with polynucleotide probes specific for the CDR1 and CDR2
regions of the most common porcine VH genes (9, 39) (Table I). These
clones were also hybridized with a pan-specific FR2 probe to identify all
clones with a plasmid containing a VDJ insert (Table I).
Statistical analysis
VH usage was complied in Excel spreadsheets (Microsoft, Redmond, WA)
and displayed as proportional usage (mean and SD) in GraphPad Prism
(GraphPad, San Diego, CA). Mean differences were compared by Student t
test routines embedded in the Prism program (GraphPad).
Results
Genomic structure of the region containing porcine IGHD,
IGHJ, and the 39 IGHV genes
WescreenedseveralBACclonescontaining IGHDandIGHJsegments
by PCR to identify clones that encompass the full locus. Clone 851A09
contained 118,088 bp with sequences from the IGHD3 segment to
IGHV15. Clone747A01 overlapped 851A09 by ∼29.6 kbp and encompassed IGHG2 through IGHV1P (Fig. 1). Together the two
clones represent 229,148 bp of genomic sequence. The two BAC
clones were subjected to shotgun sequencing as described in Materials and Methods. The sequence overlap between 747A01 and
851A09 was 100% homologous, indicating that they were derived
from the same allele. By analyzing open reading frames and using
BLASTagainst GenBank sequences, we identified 15 IGHV, 4 IGHD,
FIGURE 1. Genomic structure of the porcine Ig H chain region encompassing Cg5 to IGHV15. SP6 and T7 denote primer sites located in the vector that
flank the cloning site and assist in determining the orientation of the BAC clones. The putative coding sequence (CDS) and the switch region of IgM (Sm) are
indicated by black boxes and a gray oval, respectively. Genes in which exon 1 was collapsed or contained stop codon(s) in the V segment are regarded as
pseudogenes and are indicated by P. The enhancer region is indicated by a white circle, the position of which was estimated in a previous study (49). Restriction
sites of HindIII, PvuI, and FspI are shown in the lower part of the figure. Numbers indicate the sizes of the BAC clone fragments generated by digestion with
each enzyme. Bars below the gene map indicate the region of gene duplication. Both the IMGT and vernacular nomenclatures for VH genes are given. The
familiar names of isotypes have been used because the IMGT does not distinguish between IGHD (IgD) and IGHD (diversity segment).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Sequence similarity was examined using the BLAST2 program (45) (www.
ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi), and interspersed repetitive
sequences in the pig genome were detected by using RepeatMasker (A. Smit
and P. Green, unpublished observations; www.repeatmasker.org/) with
Repbase (46) (www.girinst.org/repbase/index.html). Homologous regions
between genomic sequences were identified by using the PipMaker program
(47). Sequence alignment and phylogenetic analysis were performed by the
CLUSTAL W (1.83) program (48). The detected IGHV genes were analyzed
by the BLAST program with public nucleotide databases (DNA Data Bank
of Japan, EMBL, and GenBank). Only sequences that showed .98%
identity to previously identified porcine IGHV sequences were adopted.
VH USAGE AND GENOME ORGANIZATION
The Journal of Immunology
3737
amplify the pig IGHV segments. The fragments obtained from genomic DNA were identical to VH genes previously reported for this
species (9, 40). This, along with our restriction analysis (Fig. 1), indicates that the assembly process had been performed correctly.
Among the VH segments detected, exon 1 and a part of exon 2 were
collapsed in 4 of the 15 IGHV genes (IGHV3, IGHV7, IGHV9, and
IGHV13), suggesting they are pseudogenes. The IGHV gene located
on the 39-most side (IGHV1) had a stop codon in exon 2, indicating
that it was also a pseudogene, confirming a previous observation (40).
VHA and VHB were highly expressed in newborn piglets and
were duplicated in the porcine genome sequence
We found a large degree of duplication in the porcine genomic sequence carrying IGHV9P to IGHV14 with that containing IGHV3P to
IGHV8 (Supplemental Fig. 1). Repetitive sequences interspersed
around IGHV4 to IGHV6 were also highly conserved with those
around IGHV10 to IGHV12 (Fig. 2). We analyzed the duplication
event in the IGHV region by using repetitive sequences. As shown in
Fig. 2B, the repetitive units include long interspersed nucleotide element (LINE) sequences such as L1_Art and L1_BT. We used the
excised sequences of the respective LINEs (commonly conserved
among the sequences) to demonstrate that L1_Art and L1_BT are
highly conserved between the two repetitive IGHV units in comparison with the others (Fig. 2C). The excised L1_BT sequences
derived from the two repetitive units [L1_BT (a) and L1_BT (c)]
(135 bp) were completely identical, which implied that the two repetitive units were established recently. We estimated the divergence
time of the repetitive units by using L1_Art because the length of
conserved regions was much longer (.800 bp) than that of L1_BT. It
revealed that L1_Art (b) and L1_Art (d) in Fig. 2B were diverged
Table II. IgH genes identified from the genome sequence determined in this study
IMGT
Vernaculara
No. of Exons
Start
End
CDS Length (bp)
IGHV15
IGHV14
IGHV13P
IGHV12
IGHV11
IGHV10
IGHV9P
IGHV8
IGHV7P
IGHV6
IGHV5
IGHV4
IGHV3P
IGHV2
IGHV1P
IGHD1
IGHD2
IGHD3
IGHD4
IGHJ1
IGHJ2
IGHJ3
IGHJ4
IGHJ5
Smb
IGHM
IGHD
IGHG1
IGHG2
VHN
VHY
JH5
2
2
1
2
2
2
1
2
1
2
2
2
1
2
2
1
1
1
1
1
1
1
1
1
IgM (Cm)
IgD (Cd)
IgG3c (Cg3)
IgG5bd (Cg5)
4
5
4
4
3032
9306
12,154
15,678
27,618
34,235
37,094
42,141
45,002
48,526
60,198
66,808
69,661
76,020
90,845
108,609
109,219
113,937
131,317
132,104
132,277
132,587
132,979
133,523
135,726
139,705
147,270
188,628
209,664
3473
9747
12,366
16,126
28,065
34,676
37,306
42,589
45,214
48,974
60,645
67,249
69,873
76,470
91,288
108,646
109,246
113,973
131,327
132,157
132,329
132,634
133,029
133,576
139,170
141,406
152,588
190,144
211,155
353
353
213
359
359
353
213
359
213
359
359
353
213
359
352
38
28
37
11
54
53
48
51
54
3445
1216
1060
1001
974
VHB*
VHF
VHA*
VH E
VHB
VH T
VHA
VHG
Psg 2
DHA
DHB
Comment
Exon 1 and part of exon 2 were collapsed
Exon 1 and part of exon 2 were collapsed
Exon 1 and part of exon 2 were collapsed
Exon 1 and part of exon 2 were collapsed
Stop codon in exon 2
a
VH gene names were classified according to CDR1 and CDR2 sequence specificities (9). The vernacular terminology is based on order of discovery,
whereas the IMGT system is based on the position in the genome as shown in Fig. 1.
b
Switch region for IgM
c
IgG nomenclature based on recent studies of Butler et al. (41).
d
IgG5b is an allele of IgG5 (41).
Psg, pseudogenes discovered in genomic DNA prior to mapping studies (see Ref. 9).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
and 5 IGHJ gene segments as well as the constant regions of IgM (Cm)
and IgD (Cd) and the two most 59 IgG genes (Cg3 and Cg5) (Fig. 1,
Tables II–IV). The sequence of the switch region and C region of IgM
had been reported previously (49); this sequence had a high level of
identity to the sequence determined in this study, showing that these
sequences were derived from the same region in the locus.
Previous studies showed that functionally rearranged VDJs used
only two IGHD segments and a single IGHJ segment (38, 40).
Although only transcripts carrying IGHD1, IGHD2, and IGHJ5 are
in the public databases, we identified four IGHD segments and five
IGHJ segments. The porcine genomic locus carrying the IGHD and
IGHJ genes was much shorter than its human counterpart (9 IGHJ
and 27 IGHD are detected in the corresponding region of the human
genome: NT_026437.11, 86930000 to 87630000). In the case of JH,
only reading frame III of IGHJ5 translates to the sequence of the
only expressed JH region of swine (38); IGHJ1, IGHJ2, and IGHJ4
have noncanonical heptamer sequences (Table IV). A sequence
encoded by IGHJ3 has yet to be found in a rearranged or expressed
VDJ, although its recombination signal sequence (RSS) seems
functional, and no stop codons are present.
Analysis of the DH shows that IGHD1 (DHA) and IGHD2 (DHB)
have canonical RSSs, whereas both the heptamer and nonamer of
IGHD3 are noncanonical (Table III). The RSS of IGHD4 should
be functional, but if expressed, the tiny segment it encodes (3 aas)
could be easily overlooked in a region where TdT-dependent
nucleotide additions are common and exonucleases are active.
The 15 IGHV genes found in the sequence had very homologous
sequences. Because of this, reads could be misassembled following
shotgun sequencing. Thus, we cloned the PCR fragments generated
from clones 851A09 and 747A01 using the primer commonly used to
CCCGGCCAG
ACTGACCAAACT
Fig. 3 summarizes VH usage in VDJ rearrangement in 632 clones
from DG30 fetal liver and 432 cloned transcripts from DG95 bone
marrow. VH genes are ordered on the x-axis according to their
position in the locus, 39 to 59 (Fig. 1, Table II). The results confirm
earlier findings that IGHV4, IGHV6, IGHV10, and IGHV12
dominate the early repertoire (∼30%) but also show that IGHV14
(VHY) and IGHV15 (VHN) play an equally dominant role at
DG30. Noteworthy is that the first functional VH gene IGHV2
(VHG) (Fig. 1) is seldom used. Thus, early usage in a primary
lymphoid tissue cannot be ascribed to the position of the VH genes
in the locus. In older fetuses (i.e., DG95), usage of IGHV14 and
IGHV15 nearly disappears, whereas VHC and both IGHV4 (VHA)
and IGHV10 (VHA*) are significantly increased. VH usage at
DG95 DNA rearrangements or transcripts from bone marrow, IPP,
and spleen does not significantly differ (J. E. Butler, X. Z. Sun, N.
Wertz, K. M. Lager, and G. Tobin, unpublished observations).
These data reject the notion that VH usage during development
follows a continuous progression of VH usage from 39 to 59. In
both DG30 liver and DG95 bone marrow, usage of unidentified VH
genes (UNK) does not change. Previous studies (9) indicate that
many VDJ clones identified as UNK in conventionally reared
young pigs use the same common genes that account for 70% of
the preimmune repertoire (Fig. 3) but have mutated CDRs so they
no longer hybridize with gene-specific probes (Table I). This low
frequency of somatic hypermutation occurs in utero in the complete absence of environmental Ag stimulation, although there is
no accumulation of mutations in CDR regions as is the case in Agstimulated piglets (S. Bratsch, N. Wertz, T. Kunz, and J.E. Butler,
unpublished observations).
As indicated above, Enh, Cm, and Cd map to the same region as
previously described (40, 51). Among the six Cg subclasses and
eleven allotypes (41), Cg3 and Cg5 are the most Cd proximal C
region genes.
CACAGTG
TGCAGAAAC
GGGCCTTGGAAC
CACAGCA
GCAAAAACC
ACAGACCCGGTG
CACAGTG
GCAAAAACC
ACAGACCCGGTG
CACAGTG
ACAAAAACC
TCACAGCGCCCT
CACAGCA
VH gene usage in primary lymphoid tissues at DG30 and DG95
I
II
III
CACAGTG
GCCTGTG
CACTGTG
I
II
III
I
II
III
I
II
III
GAATTGCTATAGCTATGGTGCTAGTTGCTATGATGACC
ELL*LWC*LL**
NCYSYGASCYDD
I A I A M V LVA M M T
TGACTATAGCGGTTGCTATAGCGGTTAC
*L*RLL*RL
DYSGCYSGY
T I AVA I AV
CGGCTGAGCGCACAGCCCTGAGGCCCCAGCTGGCTGG
RLSAQP*GPSWL
G * A H S P E A PAG W
A E RTA L R P Q L A
CTCACCGGGAC
LT G
SPG
HRD
CTCACCGGGAC
LT G
SPG
HRD
CACTGTG
I
II
III
Reading Frame
7-mer
from each other ∼1.6 million y ago. This calculation is based on the
estimated mutation rate in pseudogenes: 4.6 3 1029 (50).
Evidence for recent duplication of the IGHV region is further
supported by the observation of conserved HindIII and PvuI restriction fragments in Fig. 1. This duplication unit contained the
duplicated VHA and VHB genes, which, along with VHC and VHE,
comprise .75% of the preimmune repertoire in piglets (39). The
exon-intron structures of the detected VHA and VHB genes were
also conserved, perhaps a clue that these genes are important in
generating the Ab repertoire.
ACGGGCCTGTGG
CAAGGCCTTTCC
TGAAGCCTCCGT
CGAGCCAGGAAC
GGTTTGAGA
GCTTTTTGC
CGGCCGACC
GCTTGGGC
IGHD1
IGHD2
IGHD3
IGHD4
Canonical heptamers and nonamers are underlined.
*Stop codon.
12-mer
9-mer
Gene
Table III. IGHD genes detected in this study
Discussion
We present the first partial map of the porcine H chain locus that
covers the 59 portion of the CH region, JH region, DH region, and the
first 15 of the most 39 VH genes. These results confirm previous data
on Cm and Cd (51) and Sm and Cm (49). Data presented in this study
also explain .15 y of observations that swine express only two DH
segments, IGHD1, formally called DHA, and IGHD2, formally
called DHB (40). Our mapping data also explain why swine only
express a single JH sequence (38). Evidence that only IGHJ5 (JH5) is
functional has recently been confirmed in studies in which this
segment was mutated, resulting in B cell knockout piglets (M.
Mendicino, J. Ramsoondar, C. Phelps, T. Vaught, S. Ball, T. LeRoith, J. Moonahan, S. Chen, A. Dandro, J. Boone, P. Jobst, A. Vance,
N. Wertz, Z. Bergman, X-Z Sun, I. Polejaeva, J. Butler, Y. Dai, D.
Ayares, and K. Wells, personal communication). Collectively, the
map and sequence data we provide for DH, JH, Sm, Cm, and Cd are
consistent with earlier partial mapping studies (49, 51) and explain
why swine express only two DH segments and a single JH (38, 40).
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Sequence and Deduced Amino Acid Sequence
7-mer
9-mer
VH USAGE AND GENOME ORGANIZATION
12-mer
3738
I
II
III
Boldface text indicates only expressed J region.
a
Canonical heptamers and nonamers.
pStop codon.
CACTGTGa
TGGGGCGAGCCTGGAGATTGCAC
AGGTTTTTGa
IGHJ5
54
CAATGTG
CTGGGATCCGGGCTTAGTTGTCG
CTTTCTTAAa
IGHJ4
51
I
II
III
CACTGTGa
ACACCCCCTAATGGGGGCCACAG
GGTTTTTGGa
IGHJ3
48
I
II
III
GACTGTG
TCAAGGAGGGGCAACAGGGTGCA
GTGTATTTG
IGHJ2
53
I
II
III
GCCATGGCTACTTAGATTCGTGGGGCCAGGGCATCCTGGTCACCGTCTCCTCAG
A M AT * I R G A R A S W S P S P Q
PWLLRFVGPGHPGHRLL
H G Y L D S W G Q G I LV T V S S
CCCCTGGAAACTTGACCACTGGGGCAGGGGCGTCCTGGTCACCGTCTCCTCAG
PLET*PLGQGRPGHRLL
P W K L D H W G R G V LV T V S S
P G N LTT G AG A S W S P S P Q
ACCATCTTCACAGCTGGGGCCGAGGAGTCGAGGTCACCGTCTCCTCAG
T I F TAG A E E S R S P S P Q
PSSQLGPRSRGHRLL
HLHSWGRGVEVTVSS
ACAACGGGCTCGAAAGCTGGGGCCAGGGGACCCTAGTCTACGACGCCTCGG
TTGSKAGARGP*STTPR
QRARKLGPGDPSLRRL
N G L E S W G Q G T LV Y DA S
ATTACTATGCTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAG
ITMLWISGAQALKSSCPQ
LLCYGSLGPRR*SRRVL
Y YA M D LW G P G V E V V V S S
I
II
III
54
TGGTGTG
TGCCAGGGCCTGGGTGGGCACAT
GGTTCTGTG
IGHJ1
IGHJ (bp)
7-mer
23-mer
9-mer
Gene
IGHJ genes detected in this study
Table IV.
3739
The partial map of the VH locus identified the same major VH
genes that our previous studies had identified (9, 39, 40) (Table II).
When the location of particular genes in the locus (e.g., IGHV4,
IGHV15, and IGHV2) is compared with their proportional usage,
there is no positive correlation between position and usage (Fig. 3).
This sharply differs from the situation in rabbits, in which the most
39 VH gene is used in ∼90% of early rearrangements (18), or in mice,
in which 7183 and Q52 dominate early VH usage (12, 13), and
IGHV5 and IGHV6 are dominant in humans (17). In fact, IGHV2 is
seldom used in swine at any time in development (Fig. 3) (J. E.
Butler, X. Z. Sun, N. Wertz, L. M. Lager, and G. Tobin, unpublished
observations). Our findings are more consistent with the studies by
the Milner group that selective usage is seen at all stages of development (20).
The much higher level of control of factors that might influence
Ab repertoire development during fetal life in piglets argues that
the pattern of VH usage that we observed is intrinsic and therefore
may differ from earlier studies in mouse and human fetuses. The
mapping studies provided in this paper eliminate gene position as
an explanation for VH, DH, and JH expression. Although it is
a mistake to extrapolate data from one mammal to another, our
findings and the controversy in the literature make us skeptical of
any arguments that VH usage in early development is exclusively
positionally dependent. The studies of Pospisil et al. (33) using
the Alicia rabbit would support our skepticism.
Our study does offer several insights that may be related. First is
the evidence for recent duplication within the porcine VH locus.
IGHV4 (VHA) and IGHV10 (VHA*) share identical CDR1 and
CDR2 regions (Table I) and differ only in one nucleotide in FR3.
The latter difference allowed us to prepare a probe that could
distinguish the expression of these two genes by probe hybridization (Fig. 3, Table I). Likewise, IGHV6 (VHB) and IGHV12
(VHBp) differ by only one nucleotide in CDR1. For this reason,
gene-specific probes for VHB do not distinguish between IGHV6
and IGHV12 so that usage of both is included in VHB usage (Fig.
3). Other similarities suggest that IGHV9P to IGHV14 and
IGHV3P to IGHV8 (Fig. 1) may be recent duplicons. Similar duplicons have been reported in the human VH genome (52). Thus,
whatever factors control VH usage would be likely to affect all of
the genes in each of these duplicon cassettes. Although overall VH
usage is not positional, proportional usage for VH genes within
each cassette may be the case (e.g., IGHV3P to IGHV8 in the first
cassette and IGHV9P to IGHV14 in the second cassette).
Swine genetics, especially the fine genetics of lymphocyte
receptors, are still in infancy compared with mouse genetics. Most
of the nearly 30 VH genes described for swine were mostly recovered from outbred farm pigs so that the number very likely
contains numerous alleles (9). The BAC library used in this study
was made from the Large White breed that was interbred at Institute Nationale Recherche Agronomique, whereas the data presented in Fig. 3 were derived from United States animals, largely
Landrace, Yorkshires, and their crosses that are preferred over
inbred animals for fecundity, health, and absence of viral disease,
all of which are also important criteria for their use in fetal and
neonatal isolator studies. We know from studies on Ig genetics that
even randomly inbred swine can be homozygous in the IGHC locus
(53). This background might therefore explain one of two troubling
observations made in this study. The first concerns our failure to
localize VHC within the region of the VH locus that was mapped,
despite its prominent expression (Fig. 3) (9, 39, 40). IGHV14
(VHY) (Fig. 1, Table II) shares CDR1 with VHC and CDR2 with
IGHV4 (VHA) and IGHV10 (VHA*); all porcine VH genes have
essentially identical framework sequences. It is altogether possible
that IGHV14 (VHY) and VHC are alleles and the BAC library came
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Reading Frame
Sequence and Deduced Amino Acid Sequence
The Journal of Immunology
3740
VH USAGE AND GENOME ORGANIZATION
A
C
FIGURE 2. A, Phylogenetic tree of porcine IGHV genes detected in this study. Human b2-microglobulin (huB2M: NM_004048.2) was used as an
unrelated reference. Exon 2 sequences (or sequences corresponding to exon 2 of functional IGHV segments) were used for the alignment. Because all
porcine VH genes belong to the VH3 family of clan III, we designated the various subgroups as VH3-1, -2, and -3, respectively. Bootstrap values for 1000
replicates are indicated beside the branches. B, Genomic structure in the vicinity of the region coding IGHV. Repetitive sequences such as LINE/L1 and
PRE1 are indicated by black boxes. IGHV genes are indicated by white boxes. Areas surrounded by dashed lines are highly conserved regions. C,
Phylogenetic tree estimating the divergence of the two major duplicons within the mapped locus. Line, long interspersed nucleotide element; Pre, porcine
repeat element family belonging to short interspersed nucleotide elements.
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B
The Journal of Immunology
from a swine homozygous for IGHV14. The second observation
concerns IGHV5 (VHT) that is expressed in low frequency in outbred North American swine (9). However, based on its position in
the first duplicon (Fig. 1), high expression might be expected.
IGHV5 (VHT) shares CDR1 with IGHV8 (VHE) and CDR2 with
IGHV11 (VHF), respectively. Because all three (IGHV5, IGHV8,
and IGHV11) map to separate loci, IGHV5 cannot be an allele of
either IGHV8 or IGHV11. Recent progress with the porcine genome project might resolve these issues.
Although the gene mapping studies we present can explain the
expression of only two DH segments and one JH, they fail to explain the basis of selection for VH gene usage and expression.
Clearly, it is not position in the locus. At DG30 in liver, there is
very little transcription and no BCR expression as determined by
flow cytometry and immunohistochemistry (54). Because this is
not true at DG95, the changes in VH usage in fetal BCRs at DG95
could be due to selection by intrinsic ligands for B cells that use
certain VH genes to form their BCRs.
Theories to explain positive selection of B cells expressing
certain surface receptors date to the pre-B selection hypothesis of
Melchers (55) and the work of Pospisil et al. (56). The latter
studies used Alicia rabbits that carry a deficient VH1. These rabbits show increased expression of a VH1-like gene that was generated by gene conversion following gut colonization. As
indicated above, the authentic VH1 gene is normally used in
.90% of rearrangements in newborn rabbits (18). These observations by Melchers (55) and the Pospisil team (56) can be
interpreted to mean that B cells at certain stages of development
display BCRs that are recognized by stromal receptors that promote their survival and proliferation.
The dominant role of CDR3 in determining Ab specificity is well
accepted and was experimentally shown in studies by Padlan (57) and
Xu and Davis (58). The latter group showed that mice with a single
VH gene could make Abs to nearly all of the same Ags as control
littermates with a full germline VH repertoire as long as the DH and
JH region was intact. Because of limited VH, DH, and JH usage in
swine, we estimated that .95% of the repertoire is determined by
CDR3 (59). In the case of TCR, CDR1 and CDR2 are purported to be
mainly responsible for MHC recognition, whereas CDR3 is responsible for peptide (Ag) recognition. Because TCR and BCR are
structural and genetic homologs, what is true for the goose may also
be true for the gander. Perhaps VH usage by surviving B cells in
swine is a result of their recognition by certain stromal receptors that
recognize epitopes expressed in FRs due to the conformation imposed by the VH genes they use. Perhaps this also explains the data of
Pospisil et al. (56) in rabbits with a mutated VH1 gens.
Finally, our data reveal that porcine IgG3, regarded as the ancestral IgG of swine (41) and which comprise 60% of all IgG expression in the fetal IPP (25), is most 39 proximal to Cd. Because
speciation preceded subclass diversification, IgG3 in swine, human,
and mouse are not homologous genes (1, 41). Coincidentally, the
Cg gene called IgG3 in all three species is the most 59 Cg gene and
appears to be involved in early natural Ab responses to bacteria
such as those expressed by marginal zone B cells. In swine, the
dominant expression of IgG3 in the fetal IPP of piglets would be
consistent with this concept because the IPP is considered the site of
Ag-independent repertoire development (29).
Our data argue that there is a practical reason to understand how
the IgH locus is organized and how the Ab repertoire is developed,
and, in this case, to expose the Achilles’ heel of a species that
supplies 60% of the world’s meat supply. Although a mutation of
VH1 in the Alicia still allows rabbits a second chance, probably by
gene conversion, a mutation of JH5 in pigs destroys the entire Ab
system of this species, as has been experimentally demonstrated
(M. Mendicino et al., personal communication). Thus, JH5 of swine
represents a deleterious genetic target of spontaneous or designed
mutation.
Disclosures
The authors have no financial conflicts of interest.
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VH USAGE AND GENOME ORGANIZATION
The Journal of Immunology
Corrections
Eguchi-Ogawa, T., N. Wertz, X.-Z. Sun, F. Puimi, H. Uenishi, K. Wells, P. Chardon, G. J. Tobin, and J. E. Butler. 2010. Antibody repertoire
development in fetal and neonatal piglets. XI. The relationship of variable heavy chain gene usage and the genomic organization of the
variable heavy chain locus. J. Immunol. 184: 3734–3742.
The fourth author’s name was published incorrectly. The correct spelling is Francois Piumi.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1290085
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