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*
Department of Microbiology and Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242;
Roman L. Hruska Agricultural Research Service, U.S. Meat Animal Research Center, U.S. Department of Agriculture, Clay Center, NE 68933; and
Department of Animal Science, Iowa State University, Ames, IA 50011
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In fetal and neonatal mice, B cell subset distribution differs from that seen in adults, as does the use of VH, DH, and JH segments (5). In the fetus, a significant portion of the B cells are CD5+ (6); in addition, at least in mice and humans, the preimmune repertoire is characterized as having low affinity, connectivity, and multiple reactivity (7). The bias toward the use of certain VH genes in the formation of the preimmune repertoire could be the consequence of the proximity of rearranged components, the advantageous accessibility of VH genes to recombinases, common recombination signal motifs, the presence of VH gene-specific promoter or enhancer sequences, or the selection of B cells expressing certain VDJ gene products by self Ags or B cell superantigens. Whatever the mechanism that determines the selective usage of certain VH genes (or VDJs), it may have evolved because such VDJs encode binding sites that recognize pathogenic bacteria; such bacteria threaten all members of the species at birth or encode nonconventional Fv epitopes (8) that are recognized by stromal ligands. In any case, these VDJs encode the "natural Abs" of the preimmune repertoire and might even be considered part of innate immunity (9).
Another distinctive feature of fetal VDJ rearrangements that distinguishes them from those in adults is DH usage and complementarity-determining region-3 (CDR3)4 length. Fetal mice preferentially use their most 5' DFL 16.1 and their most 3' DQ52, and initial studies indicated a paucity of N-region additions. The latter was believed to result from the absence or low activity of deoxynucleotidyl transferase (10, 11, 12), although more recent studies indicate that N-region additions commonly occur during fetal life (13, 14). Fetal humans use DQ52 more frequently than other DH segments, and the average length of their CDR3 is significantly shorter than the CDR3 of their neonatal or adult counterparts (5, 15, 16). N-region diversity appears early and steadily increases with age (17). Although fetal rabbits do not preferentially use their DQ52 homologue, which lies 800 bp upstream of the most 5' JH, they do use Df (located 32 kb upstream) in 40% of the VDJ rearrangements (18).
The pig is being used to study the development of the Ab repertoire
because, unlike humans, mice, and rabbits, there is no transfer of
maternal Abs or regulatory proteins via the placenta in utero. This
means that the fetal Ab repertoire develops in isolation from the
impact of maternal regulatory factors that have been shown to affect
immunoontogeny at least during the last third of gestation (reviewed in
Refs. 19–21). Such studies are considered to be particularly relevant,
since there are now extensive maternal vaccination schemes designed to
influence the perinatal immune repertoire (reviewed in Refs. 22 and
23). The swine system offers several practical advantages for studying
fetal/neonatal repertoire development. First, pigs have 
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VH genes, all of which appear to share nearly identical
leader, adjacent 5' untranslated region (UTR), and framework 1
(FR1) sequences (Ref. 24 and Fig. 1
) and
possess only one JH (25), so that a single primer set (FR1
and antisense JH) amplifies all the VDJs. Second,
gene-specific oligonucleotide probes are available for the major
fetal/neonatal VH genes. Third, fetal material from many
outbred animals is abundant. Finally, neonatal piglets can be reared
gnotobiotically (26) or in "autosows" (27), so that the influence
of maternal factors, gut flora, and dietary factors on Ab repertoire
development in the neonate can be examined.
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Our previous observations (33) on the expression of VH genes in a neonatal piglet indicated that only five VH genes and two DH segments were used; this is a much more restricted pattern than is observed in neonatal mice and humans. However, these data were generated from the mesenteric lymph node mRNA of primarily one animal, so such preferential VH and DH use may not authentically reflect the general pattern in all piglets or reflect VH and DH usage in the DNA. The present study reports on the preimmune VH repertoire in various lymphoid tissues in 35 outbred fetuses ranging in age from 23 to 110 days. Data indicate that VDJ rearrangement is first seen on day 30, that four nonmutated VH genes account for 80% of VH usage, and that there is little evidence for individual or tissue variation in VH usage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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White cross-bred gilts (1/4 Yorkshire, 1/4 Large White, 1/4 Chester White, and 1/4 Landrace) from the Roman L. Hruska U.S. Meat Animal Research Center and Yorkshire x Meishan F1 crosses from Iowa State University were used in the study. Animals were hand-mated and scheduled for the slaughter and collection of 24-, 27-, 30-, 40-, 60-, 70-, and 110-day-old fetuses. Gestation in swine is 114 days. All gilts were healthy and normal at slaughter, and fetuses were immediately removed from the gravid uterus; fetal liver samples were collected at all slaughter dates. Fetal spleens were collected from 40-, 60-, 70-, and 110-day-old fetuses. Mesenteric lymph nodes, ileal Peyer’s patches, and bone marrow were obtained from 110-day-old fetuses only.
Synthesis of first-strand cDNA and cloning of the amplified VDJ cDNAs
Total RNA was purified using Trizol according to the
manufacturer’s instructions (Life Technologies, Gaithersburg, MD).
First-strand cDNA was synthesized as described previously (21) using an
antisense Cµ1 primer: (5'-tcacagagggtaggagca-3'). Next, 2 µl of the
first-strand cDNA product was used for the initial round of PCR. The
30-µl PCR mixture contained 3 µl of 10x buffer, 0.1 mM
deoxynucleoside triphosphate, 1 U of Pfu polymerase (Stratagene,
La Jolla, CA), 10 pmol of a primer for the 5' UTR (Fig. 1
), and 10 pmol
of an antisense JH primer (5'-tgaggacacgacgacttcaa-3').
After 2 min of initial denaturation at 94°C, the samples were
subjected to 30 cycles of amplification (1 s of denaturing at 94°C,
10 s of annealing at 63°C, and 10 s of extension at
72°C). PCR products of the predicted length were then excised from
the ethidium bromide-stained gel. The gel blocks were transferred to
1.5-ml tubes and an approximately equal volume of water was added to
each. The tubes were incubated at 65°C for 2 h. The aqueous
phase, containing the DNA that had diffused out of the gel, was then
used for the second round of PCR. The second round of PCR was conducted
under the same conditions as the first round except that: 1) the 5' UTR
primer was replaced by an internal FR1 primer (Fig. 1), and 2) the
templates were replaced by 2 µl of the aqueous solution obtained from
the first round of PCR (see above). In all cases, the second-round PCR
products were purified using a PCR purification kit (Promega, Madison
WI) and were then directly ligated into EcoRV-digested
pBluescript phagemids. The ligation mixture was used to transform XL-1
Blue competent cells.
Amplification and cloning of VDJs from DNA
DNA was purified using DNAzol (Life Technologies) and VDJ
rearrangements amplified in the same manner as for cDNA (see above)
except that: 1) 200 ng of DNA was used, and 2) both rounds of PCR were
conducted using the sense FR1 primer (Fig. 1) instead of the 5' UTR
primer. To validate the genomic DNA amplification, a 250-bp fragment of
porcine C
was used as a positive control for the PCR (see Fig. 2). The second-round PCR products from
genomic DNA were ligated into EcoRV-digested pBluescript as
described above for VDJ cDNAs.
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The first round of screening was performed with a pan VH probe as described previously (24). Positive clones were selected and grown overnight in individual wells of 96-well microtiter plates in 200 µl of Luria-Bertani-ampicillin medium. Half of the culture volume in each well was then transferred to a corresponding well in a new microtiter plate for plasmid preparation, while the remainder was stored at -70°C for further analysis. The bacterial cultures for plasmid preparation were pelleted and resuspended in 50 µl of resuspension solution. The cells were then lysed with 70 µl of lysis solution, and the mixture was neutralized with 70 µl of neutralization buffer. The composition of these solutions is described in the instructions provided with the Promega Miniprep kit. Cell lysates were then pelleted, and 100 µl of each supernatant was transferred to the corresponding well of a new microtiter plate. Subsequently, 100 µl of 20x SSC solution was added to each well. Finally, the plasmid-containing solutions were transferred to a nylon membrane using a 96-well membrane manifold (Pierce, Rockford, IL) connected to a vacuum pump. The membranes were dried, and the plasmid DNA was immobilized by cross-linking with UV radiation.
The porcine VH genes have been named in order of their
abundance in cDNA from newborn piglets
(VHA
VHE; 33 and/or in order of their
discovery (VHFVHO); these genes were not
named according to chromosomal location. The porcine VH
genes were originally cloned using anchored PCR and were shown to share
nearly identical leader and FR1 sequences (24, 33). A total of 42
additional sequences were recovered as cDNAs expressed by the newborn
piglet (33), and seven germline VH genes were recovered
from cosmid clones (Fig. 1). Eight additional germline VH
genes have been cloned from VH-containing polynucleotides
that were separated by electrophoresis from sperm DNA (34). All of
these swine VH genes share the sequence characteristics of
those originally recovered by anchored PCR (24). Thus far, we have only
identified two DH segments in the mapped genome; these are
located 15 kb downstream from VH1, a pseudogene that is the
most 3' VH gene in the VH locus (33).
Since the CDR1 and CDR2 regions of the porcine VH that have
been characterized have unique sequences, oligonucleotide probes can be
prepared that, when used at proper stringency, can specifically
identify each of these genes provided that no mutation had occurred in
their CDRs (Fig. 2). Thus, a nylon membrane containing the plasmid DNA
from individual clones can be sequentially hybridized with
32P-end-labeled gene-specific oligos (VHA:
5'-cagtagtacctacattaat-3'; VHB: 5'-gacaacgctttcagctgg-3';
VHC: 5'-ctaccactactataaatacc-3'; VHD:
5'-taccaccactacaaatacct-3'; and VHE:
5'-tcagtagttatgcagtgagc-3') to identify the VH gene usage
in cloned VDJ. Subsequently, a pan-specific VH probe can be
used in the last screen to confirm that all clones contain a
VH gene (Fig. 3, top right). The hybridization
temperature was 53°C for the VHB, VHD, and
VHE probes and 50°C for the VHA and
VHC probes. The hybridization time was usually 4 h or
overnight (no obvious differences were noted), and the membranes were
washed once in a low-stringency solution (2x SSC, 0.1% SDS, and 0.1%
sodium pyrophosphate) at room temperature for 20 min and twice in
medium-stringency solution (1.25x SSC, 0.1% SDS, and 0.1% sodium
pyrophosphate) at 55°C for VHB, VHD, and
VHE and 45°C for VHA and VHC. The
radioactive signals were imaged and enumerated from scans made using a
Packard Instantimager (Meriden, CT).
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VHE gene usage by
differential PCR product hybridization
Because pigs are outbred animals, conclusions regarding fetal or
neonatal VH gene usage are best obtained by sampling
several unrelated animals and their lymphoid tissues. However, the
screening procedure described above is cumbersome and labor intensive
when many different samples must be examined. Thus, we developed a
method that depends upon the direct hybridization of PCR products with
VH-specific oligonucleotide probes. The validity and
accuracy of this method for VH genes has been published
elsewhere (35). Briefly, PCR products from unknown samples and
standards, in which the composition of VH genes was
predetermined, are directly adsorbed on the same membrane. Each
VH gene standard curve is linear when signal intensity and
the amount present are compared. In the study reported here, five genes
(VHAVHE) were quantitatively analyzed so
that five sets of standards were applied to the same filter on which
the various test sample DNA had been immobilized. Since all of the
standards and samples were on the same filter, system variations caused
by membrane-related hybridization and imaging differences were canceled
out. Using two rounds of PCR minimized heterogeneity of DNA quantity.
The amount of the amplified VDJs that had been adsorbed to the membrane
in each sample was measured by quantifying the radioactive signal
generated when a FR2 probe was hybridized with the same immobilized
VDJs. This FR2 probe hybridized with all VH genes to the
same degree. This method was used so that the data obtained with
VH-gene specific oligos could be normalized to the total
VDJ content. The validity of the direct method has been verified in two
ways (35). First, when empirical data obtained with known mixtures of
VH gene DNAs (observed) were compared with the actual
content of these mixtures (expected), the deviation between the
observed and expected results was <10%. Second, the results obtained
using differential PCR product hybridization were in agreement with
results obtained by conventional cloning and hybridizing procedures.
Sequencing and Southern blot analysis
Conventional Southern blot and PCR-sequencing protocols were applied as described previously (24). Swine VH gene-specific primers (FR1: 5'-gaggagaagctggtggagt-3') and vector primers (T3: 5'-attaaccctcactaaag-3' and T7: 5'-aatacgactcactatag-3') were used for sequencing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although rearranged VDJs could not be amplified from the DNA of
24- and 27-day fetal livers, they could be amplified from some of the
30-day liver DNAs (Fig. 2). Failure to amplify VDJ was not due to the
loss of DNA or to technical problems, since the C segment could be
amplified in all samples. Failure to identify VDJ rearrangements in
most 30-day samples suggested that rearrangements might be infrequent
at this timepoint.
Specificity of CDR-specific oligonucleotide probes
The left panels of Figure 3 demonstrate the specificity of
representative CDR-specific oligos. Plasmid DNA containing
VHA, VHB, VHC, VHD, and
VHE was transferred to nylon membranes as described in
Materials and Methods. These clones, as well as several
representing hybrids of VHAVHE, have been
sequenced and described previously (33). When oligonucleotide probes
specific for the unique CDR1 or CDR2 regions of these VH
genes or a pan VH probe were sequentially hybridized with
the membrane, no cross-hybridization was observed, except when plasmids
containing hybrid VH genes were used. The latter result
further confirms the CDR specificity of the oligos used. This same
specificity was obtained with all of the remaining CDR-specific oligos.
The right panels of Figure 3 illustrate that 11 of 64 clones
(17%) use VHE.
Proportional usage of VH genes in VDJs from DNA and cDNA
Previous results obtained with a newborn piglet indicated that
VH gene usage in cDNA from the mesenteric lymph nodes
followed the order
VHA>VHB>VHC>VHD>VHE,
and that VHA accounted for half of the total sequenced
clones. Since these data came from cDNA clones, we wondered whether
there was any discrepancy between VH gene usage in genomic
DNA and VH gene transcripts. We also questioned whether the
preferential usage of VHA in the cDNA of the newborn piglet
was characteristic of VH usage in DNA at any time during
development. These questions were addressed by preparing DNA and cDNA
from the same spleen samples collected at 40, 60, and 110 days. Since
the prediction of an exact farrowing date can be in error by 2 days,
collecting fetuses at 110 days assures that they will be collected
before the physiologic events associated with parturition occur. Figure 4 indicates that VH
expression in the cDNA from 40- and 60-day samples is in close
agreement with VH usage in DNA obtained from the same
tissue samples. Similar results were obtained with 110-day-old fetuses
(data not shown).
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), which suggested that the pool of VDJ
rearrangements was large enough to prevent bias in the VH
gene frequency studies. To rule out the possibility of contamination of
genomic DNA in cDNA samples, a 5' primer from the 5' UTR was chosen for
the first round of PCR. If the cDNA product was contaminated by DNA,
the PCR product should be larger, because the genomic DNA has an intron
between the leader and the FR1 region. Using this criterion, no
evidence of DNA contamination was found. Furthermore, the results of
this experiment suggest that VDJ amplification from DNA is unlikely to
be a consequence of differences in the efficiency of amplification due
to gene location.
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Since it was convenient to use outbred animals in our research, we
were especially concerned with VH polymorphism. In work
reported elsewhere (34), the frequently cutting restriction enzyme
TaqI was chosen to study RFLP in unrelated animals, and a
surprising lack of animal variation was observed. To determine whether
this lack of polymorphism in genomic blots would be reflected in
variations in VH usage during fetal development, eight
fetuses from three different sows were selected, and the variation in
VHAVHE usage was determined. Since we were
concerned only with the variations among fetuses and not with the
absolute content of VHAVHE, comparisons were
expressed as the ratio of the radioactive intensity obtained with a
particular VH-specific probe to that obtained with a
pan-specific FR2 probe. These comparisons revealed that the variation
in VH gene usage among animals was generally <20% for all
of VH genes studied, except for one animal (S27-1; Fig. 5). This animal behaved differently when
tested using probes for VHA and VHE. It is
noteworthy that another animal, S27-2, which was a littermate of S27-1,
did not show a similar deviation in VH gene usage.
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Usage of VHAVHE in the spleens of 40-,
60-, 70-, and 110-day-fetal piglets was compared (Fig. 6). Except for VHD, which was
seen only in one 40-day sample, all of the remaining genes
(VHA, VHB, VHC, and
VHE) were detected in each sample. There appeared to be no
obvious trends in the usage of VHA and VHC,
although VHB usage progressively increased during days 40
to 70 and VHB was not among the VH genes cloned
from 30-day-old fetuses (Table I); VHB is the most 3'
functional VH gene in the swine VH locus
(33). The sum of VHA, VHB,
VHC, and VHE accounted for 80% of total
VH gene usage at all four timepoints studied.
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Lymphoid microenvironments have been shown to influence B cell
development. To determine whether fetal VH gene usage
differed among lymphoid tissues, late-term fetuses were examined with
the belief that differences in organ-specific microenvironments would
be maximal at that time. At day 110 of gestation, the major lymphoid
tissues are well developed and can be easily and clearly removed for
study. Four fetuses from the same sow were chosen for this purpose, and
the results are shown in Figure 7. There
was no notable variation in the pattern of VH usage among
the tissues tested.
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The use of CDR1- and CDR2-specific oligos to study VH
gene usage does not permit differences in CDR3 regions to be accessed
or explain why 20% of the total VDJs failed to hybridize with the
five VH-specific probes. These 20% could represent the
VHAVHE genes with mutated CDR1 or CDR2
regions or may indicate that other VH genes had been
used. Therefore, 35 nonhybridizing clones were sequenced (Table I).
Sequence data revealed that all but one of the nonhybridizing clones
used other VH genes, of which the most frequently
encountered were germline VHF (8), VHO (4), and
VHB2 (7). There was only one instance in which a
VHC was found among the nonhybridizing clones that could
represent somatic mutation, genetic polymorphism, or a Taq
polymerase error.
Among the 39 sequenced VDJs, 36 used either DHA or DHB. Two of the three unknown DH sequences were from 30-day-old fetuses. All six of the VDJs, which showed no evidence of N-region diversity, were found in DNAs from 30-day-old fetuses. Although the sample size was too small for statistical analyses, there was a tendency for junctional diversity to increase with age. Finally, unlike the data obtained using cDNA from a neonatal pig (which showed that all of the rearrangements were productive), a significant proportion of the VDJ rearrangements amplified from DNA were nonproductive (out of frame).
Table I also provides information on the four hybridizing
VH genes cloned from 30-day-old fetuses. Three of the four
clones were VHC, and only one showed any evidence of
N-region addition. All four of the hybridizing 30-day VDJs used
DHA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial reason for undertaking the current study was to determine
whether the very restricted VH and DH gene
segment usage seen in transcripts of the newborn piglet was a
consequence of a selective expansion/activation of B cell clones
bearing these rearrangements or whether such restriction was also seen
at the DNA level. We observed that, throughout fetal life, four of the
same five VH genes (VHA, VHB,
VHC, and VHE) predominated in DNA
rearrangements (
Figs. 4–7). Moreover, when VH usage in
cDNA and DNA from the same tissue was compared, only small differences
were observed at days 40, 60 (Fig. 4), and 110 (data not shown),
indicating that it was unlikely that B cells expressing certain
VH genes were being selectively stimulated to differentiate
to plasma cells in fetal life. Thus, our previous cDNA data from the
newborn (33) most likely reflected what was occurring at the DNA level.
In addition, as reported for the newborn piglet, >90% of all VDJs
used during fetal and neonatal life use DHA or
DHB.
Since single nucleotide changes in CDR1 and CDR2 result in
nonhybridization with our VH-specific probes, the
nonhybridizing VDJs encountered (Figs. 6 and 7) must represent either
mutations in CDR1 or CDR2 or the usage of VH genes for
which gene-specific oligonucleotides were not available. This was
clarified by sequence analyses of 35 nonhybridizing clones (Table I);
only one of these sequences could be identified as a
VHAVHE gene. The sequence in question was a
VHC from a 110-day spleen that might represent a
polymorphism, a mutation, or a Taq polymerase error.
Otherwise, all of the nonhybridizing VH genes represented
other known germline VH genes as well as VH
genes for which no germline sequences are available for comparison
(Table I; Unk). Compared with the germline sequences available for
VHF, VHO, and VHG (J.S and J.E.B.,
manuscript in preparation), no evidence of somatic mutation could be
found. Since all VHB2 genes had identical sequences in CDR1
and CDR2, we suspect that VHB2 is an allele of
VHB and not a somatic mutation of VHB. Thus, no
evidence was found that somatic hypermutation was occurring at any time
during the 84-day fetal period in which VDJ rearrangements were
recovered.
Since the data presented were obtained from 35 outbred fetuses, it is
both surprising and noteworthy that variations in the usage of
VHA, VHB, VHC, and VHE
among so many genetically different animals were not observed (Fig. 5).
Only one animal (S27-1) deviated from an otherwise regular pattern and
from its littermate S27-2. Since all three sows were unrelated
cross-bred animals, it has been concluded that the predominant usage of
four VH genes is a developmental rather than an individual
feature of this species. Because a single nucleotide change would
prevent hybridization of the CDR1 or CDR2 gene-specific
oligonucleotides, the data also suggest that polymorphism among these
four genes is generally lacking (with the possible exception of
S27-1). Further exceptions may be VHD or
VHB2, which could be alleles of VHC and
VHB, respectively. These findings are consistent with the
idea that conserved and highly monomorphic VH genes are
used to generate the preimmune repertoire in swine; this possibility is
consistent with the overuse of monomorphic VH6 in fetal
humans (37). Although VH gene usage in fetal mice and
humans has been studied and shows a selective use of certain
VH genes, there is a paucity of information that addresses
the issue of whether fetal VH usage is consistent among
individuals or strains of mice. Perhaps studies in rabbits, another
outbred species, will provide the greatest insight into this matter.
Rabbits use their most 3' VH (VH1) 90% of the
time (38); in addition, the Alicia rabbit, which has a mutation of
VH1, uses a VH1-like upstream gene to
eventually compensate for the lack of VH1. Recently,
Pospisil et al. (39) proposed that selective VH1
usage may be a result of positive selection by some stromal cell
Ag that recognizes and causes the proliferation of B cells expressing
VH1 or a VH1-like gene or at least prevents
their apoptosis. Thus, B cells expressing
VHAVHE may represent the progeny of B cells
positively selected against apoptosis (e.g. by a B cell superantigen
such as Fv). Fv is found in fetal human livers and binds Abs with
variable regions encoded by the VH3 family with graded
specificity (40, 41). All pig VH genes belong to the
VH3 family (24), swine IgG competes equally with
VH3-encoded human IgG for human Fv, and a similar substance
has been identified in fetal pig bile (G. J. Silverman, unpublished
observations).
Early studies suggested that preferential VH gene usage
reflected the location of these genes within the heavy chain locus (42, 43), although a recent systematic comparison made by Schroeder et al.
on VH gene usage at different times during gestation
indicated that the linear proximity model only fits well in early
gestation (2). We have shown that the most 3' functional VH
gene in the pig is VHB, although VHA accounted
for 50% of the expressed VH genes in a newborn piglet
(33). We show here that VHB is not the predominant
VH gene in 30-day-old fetuses (Table I). Thus other factors
besides chromosomal location must play a role in preferential
VH usage in swine.
Apparent preferential VH gene usage need not be positional
or dependent upon positive selection by some Fv-like stromal ligand.
Rather, recombinases may favor VH genes with certain
recombination signal sequences (RSSs) or sequence motifs that flank the
RSSs. Chen found that frequently expressed human VH3 family
genes such as Humhv3005 and VH26 share a unique
32-bp, 
B enhancer-like sequence that is located 16 to 17 bp
downstream of the nonomer as well as many enhancer-like motifs in the
5' UTR (44). We aligned a few of the genomic VH gene
sequences and RSSs, including those that are frequently and
infrequently used (Fig. 8), but no clue
was found that might explain the frequent usage of
VHAVHE.
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T cells) (49). Natural
Abs with similar specificities have been reported in humans and mice
(50, 51). Recently Reid et al. have shown that natural IgM Abs from B1
cells protect newborn mice from endotoxin shock (52). Thus, the
conserved and apparently limited preimmune Ab repertoire of the piglet
may have evolved to recognize a small number of pathogens that threaten
the newborn of this species.
CDR3 is generally believed to contribute most to the diversity of the
Ab repertoire. In initial studies of early development in humans and
mice, CDR3 was characterized by few N-region additions (53, 54),
nonrandom DH-JH recombinations (55), and a bias
for a particular reading frame (56). Although N-region additions
were absent in 30-day fetal VDJs, they were routinely found on and
after day 40 (Table I); more recent reports indicate that N-region
additions are routinely observed in fetal mice and humans (13, 14, 17).
Similar to mice and humans, DH usage is also nonrandom in
fetal and newborn piglets, since two DH segments account
for DH usage in >90% of fetal VDJ rearrangements.
The data presented here provide valuable information about Ab repertoire development in yet another species. Although swine and sheep are both Group III artiodactyls (19), we found no evidence of somatic hypermutation in fetal life as has been reported for fetal sheep (29). Rather, our findings are more similar to fetal rabbits in both the lack of hypermutation during this stage of development and the use of very few VH genes (28, 38, 39). The pattern in rabbits and swine clearly differs from the pattern in rodents and primates, in which many VH genes are used (57). Nevertheless, the fetal piglet appears immunocompetent (30, 31) and displays switch recombination (30, 33), and natural fetal Abs are common (30, 45, 46). The fact that hypermutation is absent and combinatorial diversity provides only limited possibilities in the fetal piglet suggests that junctional diversity in CDR3 is the primary source of Ab diversity and specificity in this species. This finding is reminiscent of what has been described for the TCR (58) and for transgenic mice that are able to develop a fully functional Ab repertoire using only one or a few VH gene segments (Ref. 59; J. L. Xu and M. M. Davis, unpublished observations). Should this be the case in swine, it raises questions as to: 1) why four VH genes and not just one are used (as in rabbits) and 2) the role of CDR1 and CDR2 in the Ab repertoire and Ab specificity for this species.
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Footnotes |
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2 Mention of trade names is necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.
3 Address correspondence and reprint requests to Dr. J. E. Butler, Department of Microbiology, College of Medicine, University of Iowa, Iowa City, IA 52242-1109. E-mail address:
4 Abbreviations used in this paper: CDR, complementarity-determining region; FR1, framework 1; FR2, framework 2; UTR, untranslated region; RSS, recombination signal sequence.
Received for publication November 21, 1997. Accepted for publication June 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ T-cells. Curr. Opin. Immunol. 9:57.[Medline]
/ß T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
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J. E. Butler, C. D. Lemke, P. Weber, M. Sinkora, and K. M. Lager Antibody Repertoire Development in Fetal and Neonatal Piglets: XIX. Undiversified B Cells with Hydrophobic HCDR3s Preferentially Proliferate in the Porcine Reproductive and Respiratory Syndrome J. Immunol., May 15, 2007; 178(10): 6320 - 6331. [Abstract] [Full Text] [PDF]
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J. E. Butler, P. Weber, and N. Wertz Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited J. Immunol., October 15, 2006; 177(8): 5459 - 5470. [Abstract] [Full Text] [PDF]
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J. E. Butler and N. Wertz Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG3 J. Immunol., October 15, 2006; 177(8): 5480 - 5489. [Abstract] [Full Text] [PDF]
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J. E. Butler, N. Wertz, J. Sun, H. Wang, P. Chardon, F. Piumi, and K. Wells Antibody Repertoire Development in Fetal and Neonatal Pigs. VII. Characterization of the Preimmune {kappa} Light Chain Repertoire J. Immunol., December 1, 2004; 173(11): 6794 - 6805. [Abstract] [Full Text] [PDF]
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C. D. Lemke, J. S. Haynes, R. Spaete, D. Adolphson, A. Vorwald, K. Lager, and J. E. Butler Lymphoid Hyperplasia Resulting in Immune Dysregulation Is Caused by Porcine Reproductive and Respiratory Syndrome Virus Infection in Neonatal Pigs J. Immunol., February 1, 2004; 172(3): 1916 - 1925. [Abstract] [Full Text]
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M. Sinkora, J. Sun, J. Sinkorova, R. K. Christenson, S. P. Ford, and J. E. Butler Antibody Repertoire Development in Fetal and Neonatal Piglets. VI. B Cell Lymphogenesis Occurs at Multiple Sites with Differences in the Frequency of In-frame Rearrangements J. Immunol., February 15, 2003; 170(4): 1781 - 1788. [Abstract] [Full Text] [PDF]
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J. E. Butler, J. Sun, P. Weber, S. P. Ford, Z. Rehakova, J. Sinkora, and K. Lager Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification J. Immunol., September 15, 2001; 167(6): 3239 - 3249. [Abstract] [Full Text] [PDF]
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J. E. Butler, P. Weber, M. Sinkora, J. Sun, S. J. Ford, and R. K. Christenson Antibody Repertoire Development in Fetal and Neonatal Piglets. II. Characterization of Heavy Chain Complementarity-Determining Region 3 Diversity in the Developing Fetus J. Immunol., December 15, 2000; 165(12): 6999 - 7010. [Abstract] [Full Text] [PDF]
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M. Sinkora, J. Sinkora, Z. Rehakova, and J. E. Butler Early Ontogeny of Thymocytes in Pigs: Sequential Colonization of the Thymus by T Cell Progenitors J. Immunol., August 15, 2000; 165(4): 1832 - 1839. [Abstract] [Full Text] [PDF]
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D. Lanning, P. Sethupathi, K.-J. Rhee, S.-K. Zhai, and K. L. Knight Intestinal Microflora and Diversification of the Rabbit Antibody Repertoire J. Immunol., August 15, 2000; 165(4): 2012 - 2019. [Abstract] [Full Text] [PDF]
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