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The Journal of Immunology, 2006, 177: 5459-5470.
Copyright © 2006 by The American Association of Immunologists, Inc.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited1

John E. Butler2, Patrick Weber and Nancy Wertz

Department of Microbiology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise ~80% of the preimmune repertoire. However, so-called "hybrid" VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied >3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for >95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adecade ago, we reported that the preimmune VH repertoire of swine was composed of ~20 VH3 family genes (1). In fetal piglets, 4 of these VH genes (VHA, VHB, VHC, and VHE) account for ~80% of the total usage of rearranged VH genes in DNA (2, 3, 4) and this preferential pattern of VH gene usage is also seen in transcripts from various tissues of young pigs (5). These observations are inconsistent with the multigenic paradigm established from studies in mice and humans. Rather, they are more reminiscent of rabbits in which the most 3' VH gene (VH1) is used 90% of the time (6), of sheep in which five V{lambda} genes account for >70% of the repertoire (7), and chickens in which single 3' VH and 3' V{lambda} genes account for the entire repertoire (8, 9). In rabbits, age and antigenic exposure can result in usage of genes other than VH1 (10, 11). Although both chickens and the rabbits use gene conversion (templated somatic mutation) to compensate for the restricted VH usage (12, 13), gene conversion has not been convincingly demonstrated in swine. However, so-called hybrid VH genes are frequently encountered in swine. Hybrid VH genes are chimeras containing the CDR1 region of one major porcine VH gene and the CDR2 of another. [The major porcine VH are the four genes that form >80% of the preimmune repertoire (2) plus VHF, VHY, and VHZ that when combined, comprise >95% of the preimmune repertoire (see Table I). Other VH genes shown in Table I that have been recovered in full-length from DNA and/or bacterial artificial chromosomes (BACs)3 or repeatedly in transcripts, are called minor VH genes. The term hybrid and "chimera" are used interchangeably.] In vitro studies demonstrated that presumably because of the low processivity of thermostable polymerases (14, 15), up to 38% of such hybrids could be generated as PCR artifacts when a 1:1 ratio of two different porcine VH genes were used as templates (16). However, the proportion was very small when the template ratio was large so VDJ chimeras generated as PCR hybrids from most cDNA preparations should be rare. Moreover, the 15–20% of VHA/VHB hybrids predicted from our in vitro studies has never been observed in cDNAs even though VHA and VHB transcripts together account for 40–60% of the preimmune cDNA (Refs. 2, 4, 5 ; also, this report). In contrast, hybrids of VHE/VHC approaching such frequencies have been seen but not the reciprocal VHC/VHE hybrid (this report). [VHE/VHC means CDR1 is VHE and CDR2 is from VHC. This nomenclature is further abbreviated to simply E/C (see Table I).] We reasoned that both forms should be equally abundant if the hybrids are due to the low processivity of Taq polymerase. These observations warranted the need to quantify the frequency of porcine VH gene hybrids so the constituency and diversification of the preimmune repertoire could be properly assessed. The piglet model is of great value in developmental studies because fetal piglets are not exposed to environmental Ag and maternal regulatory factors, and because postnatal exposure to such factors as normal gut flora, diet, pathogens, and maternal factors can be controlled postnatally by the experimenter.


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  		Table I. Porcine VH repertoire

 
In most of our previous studies on VH usage in swine using clonal hybridization (2, 3, 5) and those in humans using the same method (17), only single VH gene-specific probes were used. However, in limited studies using dual probes for CDR1 and CDR2, we identified hybrid clones, but because of the low frequency of their occurrence, they were included into a category labeled "other" (5). This policy was adopted not only because of their infrequent use but out of concern that such VDJ clones could be PCR artifacts (16). The "other" category also includes VH genes that are no longer able to hybridize with CDR-specific probes because of somatic hypermutation (SHM). In this study, we report on a systematic study to tabulate and characterize hybrid and other VH gene usage in transcripts from the mesenteric lymph nodes (MLN) of late-term fetal piglets, 5-wk-old colonized isolator piglets and conventional piglets heavily antigenized for a Th2 response by parasitic infection. The MLN was chosen because it is influenced by both the normal flora that colonizes the gut and infection with intestinal helminthes. We addressed the issue of hybrid/chimeric VH genes by 1) quantifying their occurrence in >3000 VDJ clones and 2) determining whether they occur in the genome by examining several porcine VH-containing BACs. We also sequenced >100 hybridizing and nonhybridizing VDJ clones to assess the role of SHM in generating genes of the other category. By comparing the repertoire in IgM and IgG transcripts, we were able to test the affect of class switch recombination (CSR) on the expressed VH Ab repertoire. A comparison of VH usage in the preimmune repertoire with that in colonized piglets or those exposed to intestinal parasites, allowed an index for quantifying the degree of repertoire diversification to be devised. This index has proven valuable in studies on viral and bacterial infections of gnotobiotic isolator piglets. The study extends our 1998 report by providing information on 29 VH genes and by showing that inclusion of three additional VH genes means that seven major genes account for >95% of the preimmune repertoire. Evidence presented here indicate that chimeric/hybrid VH genes are present in the genome, apparently as the consequence of gene duplication and recombination during evolution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Source of animal tissue

MLN were collected after euthanization of uninfected 110–114 fetal piglets and after 6 wk from young pigs infected with Ascaris suum or Trichuris suis. Samples collected were by-products of colonization studies with neonatal isolator piglets at South Dakota State University (Brookings, SD) and helminthic studies at the Agricultural Research Center (Beltsville, MD). Isolator piglets were colonized by monoassociation with Escherichia coli G58-1 (18). Animal studies were approved by the Animal Care and Use Committees of the respective research institutes. Samples were frozen in liquid nitrogen and transported on dry ice to the University of Iowa. All animals were selectively outbred making them less heterozygous than humans when typed for MHC (D. Smith, unpublished observation).

Preparation of cDNA

Frozen tissues were pulverized with a mortar and pestle and then solubilized in TRI-reagent (MRC). Total RNA was prepared according to the manufacturer’s recommendations and first strain cDNA was prepared as previously described (5, 19). Random hexamers as well as isotype-specific primers were used, the latter to enrich the recovery of isotype-associated VDJ transcripts (Fig. 1).


Figure 1
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  		FIGURE 1. Major porcine VH genes and polynucleotide probes and primers. Top, Nucleotide sequences of the seven major VH genes of the porcine preimmune repertoire. CDR probe binding regions are boxed as well as the re gion recognized by the pan-specific FR2 probe. Dots indicate identical nucleotides using VHA as the reference VH gene. VHY and VHZ are chimeric genes detected with the probes for CDR1 of VHC and CDR2 of VHA (VHY) and CDR1 of VHE and CDR2 of VH (VHZ). Bottom, Polynucleotide primers used for the preparation of cDNA and for first and second round PCR.

 
Cloning and identification by hybridization

IgM- and IgG-associated VDJs were recovered by two stage PCR. Using a primer pair consisting of antisense IgM and the common 5' framework (FR)1 primer (Fig. 1) the first round IgM-associated VDJ products were amplified. This PCR product was then separated on a 1.5% agarose gel and the polynucleotide band corresponding to the correct size for the IgM product, recovered from the gel using a Costar Spin-x spin column and subjected to a second round PCR using 3' FR1 primer and antisense JH (Fig. 1). IgG-bearing transcripts were recovered in a similar manner but using an antisense C{gamma}1 primer in the first round (Fig. 1).

The second round PCR product was cloned into pCR TOPO2.1 and grown in TOP TEN cells as described previously (5, 20). Briefly, positive colonies were selected and transferred to the wells of 96-well microtiter plates (Costar; 3799). Colonies were grown overnight at 37°C in Luria-Bertani (LB) ampicillin broth (LB-Amp), the plasmid DNA was recovered by alkaline lysis and transferred to nylon membranes (Schleicher & Schuell Microscience).

The membranes were then sequentially hybridized with 32P-labeled probes specific for the major VH genes of swine and finally a common FR2 probe to determine the total number of VDJ-containing clones (Fig. 1; Refs. 2, 3 and 5). The definition of "other" VH genes (Figs. 2 and 3) refers to all clones that hybridize with the FR2 probe but that fail to hybridize with both probes for CDR1 and CDR2 of the major VH genes (Fig. 1; Refs. 2 and 3 ; see Table I). The "other" category therefore potentially contains four different categories: 1) major genes that do not hybridize at all because of SHM in the CDR1 and CDR2 probe binding regions; 2) clones hybridizing with only one CDR probe because the probe binding region of the other CDR is mutated; 3) clones hybridizing with only one CDR probe because the other CDR is not found in a major porcine VH genes; and 4) clones that only hybridize with FR2 because they are minor VH genes for which CDR probes were not prepared (see Table I). Distinguishing among these categories was based on clonal hybridization and sequence analysis. Hybrid genes or others with unique sequences that were repeatedly recovered were considered putative germline VH genes. Those infrequently encountered that could not be readily identified as a mutant of a frequently encountered VH gene or a minor VH gene were considered to be the consequence of SHM or a PCR artifact.


Figure 2
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  		FIGURE 2. VH gene usage by newborn and E. coli-colonized piglets identified using single VH gene probes ({square}) or dual VH gene probes ({blacksquare}). The x-axis identifies the various VH gene based on their CDR regions, e.g., A/A or A/F. Complete VH gene names can be obtained by reference to Table I. The very shortest bars represent the recovery of one clone. *, Statistically different between single- and dual-probe assays.

 

Figure 3
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  		FIGURE 3. Individual and group variation in proportional VH usage by IgM and IgG transcripts. Data are presented for the five most frequently used VH genes and "other" in the MLN of newborns, mono-associated isolator piglets, and parasite-infected piglets. Each point represents a single animal and is based on 250–350 VDJ clones per point. The horizontal bar depicts the mean usage. Brackets connect groups that did not differ significantly. VHE and VHZ were grouped together in interest of space and because usage is often complementary, suggesting they may be allelic VH genes. Note that the scale of the y-axis differs among panels.

 
Sequence analysis

One hundred five VDJ clones were sequenced and analyzed, including 81 clones that hybridized with the FR2-specific probe but with only one or no probes for CDR1 or CDR2. These clones were transferred to 17 x 100 Falcon tubes and grown overnight in LB-Amp. Cultures were then processed using the Fast Plasmid Mini procedure (Eppendorf). Plasmid DNA concentration was determined by A260/280 spectrophotometry and samples containing 400 ng of DNA were provided to the University of Iowa Core Facility for analysis. Sequence analysis was done using the Applied Biosystems capillary sequencer (ABI Prism) and data were analyzed using the Omiga program (Accelyr) as previously described (20).

VH genes and mutated VH genes were identified by reference to the known sequence of repeatedly recovered porcine VH genes (Fig. 1; see Table I). Sequence data were used to distinguish between major and minor VH genes (see Table I) and hybrid combination that had not previously been described. Sequence data were also used to determine the number of mutations in probe binding regions needed to prevent hybridization. The number of transition and transversion changes was recorded as the number of R and S mutations. DH segments and CDR3 length was determined as previously described (4).

In some cases, VDJ clones hybridized with three CDR probes. Eight of these were sequenced and directly rehybridized or subcloned and rehybridized. These studies were undertaken to determine the basis of occasional "triple probe hybridization."

VH-containing BAC clones

BAC clones containing porcine VH genes were obtained in collaboration with Drs. P. Chardon and F. Piumi (Institut National de la Recherche Agronomique, Joey-en-Josas, France). These were recovered by PCR using primers for 5' FR1 and 3' FR3. The various BAC clones were grown up in LB chloramphenical medium and processed using Qiagen reagents. The process was done as a Maxiprep but using two times the normal amount of reagents as specified by the supplier. Samples were then processed through an isopropanol precipitation step, dissolved in water, and ethanol precipitated. BAC clones were digested with restriction enzymes and hybridized in Southern blots with a pan-specific (FR2) probe, certain VH gene-specific probes and a probe for the common leader sequence. The VH gene content of BAC clones was further analyzed by PCR amplification using a primer set comprised of the 5' FR1 primer and the antisense primer for the conserved region of FR3. The resultant products were then cloned into pCR TOPO2.1, plated, and the plasmid DNA was transferred to nylon membranes as described above. Clone-bearing membranes were hybridized with various VH-specific probes as described above to determine the VH content of the original BAC clone. A number of these were selected at random for sequence analysis. Because of concern for hybrid PCR products, special attention was given to the identification of VH genes in the same BAC clone that could provide donor sequence for possible PCR-induced hybrid VDJs.

Statistical analyses

Advice was provided by Dr. K. Chaloner (Department of Biostatistics, University of Iowa, Iowa City, IA). Simple mean differences were compared by two-tailed Student’s t analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dual VH gene probes reveal hybrid VDJ clones

Fig. 1 gives the complete sequences of the major porcine VH genes that comprise the preimmune repertoire. Also shown are the sites where the 10 CDR1- and CDR2-specific probes and the pan-specific FR2 probe used for clonal hybridization bind their targets. Fig. 1 reviews that differences among porcine VH genes are almost exclusively confined to CDR regions which explains why only CDR sequences are reported in Table I (see later). Fig. 2 compares the VH usage by IgM and IgG transcripts recovered from the MLN of newborn and colonized isolator piglets. The colonized piglets were monoassociated with E. coli G58-1, a benign commensal. Blots containing up to 96 clones were sequentially hybridized with one probe each for VHA, VHB, VHC, VHE, VHF, and FR2 (single-probe system) or sequentially with all 10 VH-specific probes (double probe system) and the control FR2 probe (Fig. 1). These comparisons show that while the overall profile of VH usage is similar in single- and dual-probe assays, statistically significant differences are observed. Although the overwhelming use of VHA by newborn IgG transcripts was seen with both assays, the single-probe assay most frequently overestimated the usage of VHC and VHE. This occurred because VHE/VHC chimeras account for >10% of total VH usage by IgG transcripts. However, the reciprocal chimera (VHC/VHE) was absent. Among colonized piglets, twice as many hybrids were detected among IgM transcripts than among IgG transcripts, whereas more IgG transcripts are accounted for by "other" (see later).

In summary, Fig. 2 shows that 1) up to 15% of total VH usage in the MLN of newborn and colonized piglets is comprised of chimeras of the major five VH genes (VHF is now included as a major VH gene), 2) the chimeras VHE/VHC (VHZ) and VHC/VHA (VHY) alone account for up to 10% of the repertoire, and 3) the overall profile of VH usage is the same for both dual- and single-probe assay systems.

Fidelity and reliability of clonal hybridization

VDJ transcripts should hybridize with one CDR1 and one CDR2 probe if they contain a major, nonmutated germline VH gene. On occasion, a clone may hybridize with three different CDR probes. Tables IIA summarizes data on such clones. These data show that of 80 triple hybridizers, the occurrence is lower in clones from parasite-infected pigs. Because ~5% or less of all clones belong to the triple hybridizer category, their presence can be ignored unless this form of nonspecific hybridization was VH gene specific and might therefore skew the results. Thus, nine such clones were sequenced and each was shown to be a single VDJ sequence and not a double clone (Table IIB). Six dual hybridizers were sequenced to serve as controls (Table IIC). Triple hybridizing clones were grown up, replated, and rehybridized. Half of these continued to hybridize with three probes. The sequences of VDJs that continued to hybridize with three probes were analyzed in Omiga to determine whether a spurious binding site could be found outside the CDRs, but none could be found.


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  		Table II. Analysis of triple hybridizing VDJ clones

 
The reliability of clonal hybridization as a measure of the degree of SHM was also tested using the sequences of 89 CDR1 regions and 94 CDR2 regions (Table III). These data show that CDR-specific probes differ in their ability to tolerate mutations in their CDRs. The CDR2 probe for VHB was most demanding in that only 1 of 10 clones allowed even a single mutation in the probe binding region and the average mutation frequency for nonhybridizing VHB-2 clones was only 1.6, whereas the average for all nonhybridizing CDRs was 3.3. By contrast, the CDR2 probes for VHA, VHC, and the CDR1 probe for VHE tolerated up to three mutations in some clones and could still hybridize. Although stringency differed, the overall data show that the average number of mutations in CDR regions of hybridizing clones was 0.7, whereas for nonhybridizing clones, it was 5-fold higher.


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  		Table III. Mutation analysis of hybridizing/nonhybridizing VDJ clones

 
Proportional VH usage is similar among piglets in different treatment groups

A great deal of immunological research is based on the use of genetically defined strains of mice which raises the question of whether individual genetic differences in outbred populations such as humans and swine, substantially effect conclusions drawn from studies of the type we describe. Fig. 3 shows the proportional usage of the most frequently used major VH genes/categories and other, among three different treatment groups of piglets. The results indicate that 1) the usage profile is similar in nearly every case for both IgG and IgM transcripts, 2) all piglets use the same major VH genes in forming their preimmune repertoire, and 3) differences in proportional VH usage are the consequence of different environmental exposure. VHB, VHC, and VHE/VHF usage did not differ between colonized isolator piglets and newborns. Rather, colonization affected usage of VHA and "other." The greatest individual variation in proportional usage was seen in VHA and VHB expressed with IgM among newborn piglets and VHE/VHF usage by IgG transcripts of newborns.

Parasite infection results in diversification of the preimmune IgM and IgG repertoires

Conventionally reared piglets are subjected to greater environmental exposure than are isolator piglets monoassociated with a single colonizer. In addition to a more complex gut flora, we used conventional piglets that were parasitized by intestinal helminthic parasites that favor Th2 responses. Figs. 3 and 4 compare the VH repertoire of IgM and IgG transcripts from the MLN of newborn, colonized isolator piglets and parasite-infected, conventional piglets using dual probe hybridization. Fig. 4 show that >95% of the newborn preimmune IgM repertoire is formed from VHA, VHB, VHC, VHE, VHF, VHZ, and VHY. [We define the preimmune repertoire as the one seen in fetal and newborn piglets that have never experienced maternal Abs or environmental Ag including those of colonizing bacteria.] By contrast, hybridizing forms of these genes account for only 12% of the IgM repertoire of parasite-infected animals. Rather, >80% of the repertoire of parasite-infected animals is accounted for by "other" VH genes which is significantly greater than for all other groups studied (Figs. 3 and 4). A similar difference between the preimmune IgG repertoire and that of young parasite-infected pigs was seen except that 17% of the repertoire in parasite-infected animals was still composed of hybridizing VHA. The overall IgM repertoire in colonized piglets was not statistically different from newborns (Fig. 4) but differences in VHA and other usage were significant (Fig. 3). Twenty percent of the IgM repertoire and 38% of the IgG repertoire in E. coli colonized piglets is classified as "other" as opposed to >80% for both IgM and IgG repertoires in parasite-infected piglets. The mean value for "other" in parasite-infected animals was significantly higher (0.001 level) than "other" in all other groups (Figs. 3 and 4). Age alone had almost no significant effect on the VH repertoire of IgM as indicated by comparing 5-wk-old germfree (GF) animals with newborns. The IgG repertoire was significantly diversified from that of newborns which may contain an age component although animal variation (SE = 5.3) renders this effect insignificant. The values in brackets are the repertoire diversification index (RDI). These values are explained in Discussion.


Figure 4
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  		FIGURE 4. Trends in VH gene usage in the MLN of newborn piglets, 5-wk-old GF piglets, E. coli-mono-associated (colonized) isolator piglets, and parasite-infected conventional piglets using dual probe hybridization. Data are based on the number of VDJ clones examined for each group (in parentheses). "Other" includes all VDJ clones that did not hybridize with either CDR1 and CDR2 probes for the major VH genes, but did hybridize with a pan-specific probe for FR2 (Figs. 1 and 5 and Table IV). Hyb includes all clones that hybridized with dual probes, but for different CDRs than those that identify VHA, VHB, VHC, VHE, VHF, VHZ, and VHY. The value in brackets is the calculated value for the RDI (see Discussion). To reduce clutter, error bars are only included for data on newborn piglets and statistical outcomes are described rather than indicated. The mean values for VHA, VHB, and VHE/VHF usage in IgG transcripts were statistically lower in parasite-infected animals while the value for "other" was statistically higher than for other groups. In the case of IgM transcripts, VHC usage was also significantly lower than in parasite-infected pigs.

 
The nature of nonhybridizing VDJ clones

The method of clonal hybridization results in four categories of VDJ clones: 1) clones hybridizing with two probes (CDR1 and CDR2); 2) clones hybridizing with only one CDR probe; 3) clones hybridizing with no CDR probe; and 4) clones hybridizing with three CDR probes (triple hybridizers). To qualify as a VDJ clone, all must hybridize with the pan-specific FR2 probe (Fig. 1). The nature of clones from category 4) was presented in Table II and discussed above. These triple hybridizers appear to be the result of spurious binding of a third CDR probe. Because they constitute 5% or less of all clones (Table IIA) it has a very small impact on studies of VH usage and VH repertoire diversification.

Of greater importance is information about nonhybridizing or partial hybridizing (only one CDR probe) VDJ clones. This category was formerly placed in the "other" category (Ref. 5 ; Figs. 2–4). Table IV summarizes sequence data on 105 VDJ clones including 81 that fit into categories 2) and 3) described above. Table IV shows that consistent with Table III, partial hybridizing or nonhybridizing VDJs have 2-fold more mutations than double hybridizers. Furthermore, there is a nonsignificant trend for usage of DHB to decrease and for other DH usage (27%) to increase in partial or nonhybridizing VDJ clones. Among those VDJ clones that do not completely hybridize, 90% of VH genes expressed with IgM and 80% of those expressed with IgG use major or minor VH genes and not rare hybrids. Partial hybridizing or nonhybridizing VDJs use mutated major VH genes twice as frequently as minor VH. Use of hybrids not regarded as either major or minor genes (Table I) comprise a minority of "other" for both isotypes. Noteworthy from Table IV is that there is no difference between IgM- and IgG-associated VDJs, suggesting that CSR and repertoire diversification are not linked.


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  		Table IV. Characterization of VDJ clones classified as "other"

 
Nonhybridizing transcripts from antigenized piglets have mutations that alter the Ab binding site

Fig. 5 summarizes sequence data on 105 VDJ clones. Data presented show the frequency of SHM to be 5-fold lower in hybridizing than nonhybridizing VDJ clones. In the former, there is no difference between the frequency of SHM in CDRs and FR regions and no significant difference in mutation frequency in FR regions between the two groups (Fig. 5). Thus, the higher mutation frequency in nonhybridizing clones is confined to CDR1 and CDR2. The terminal nine nucleotides of FR3 are known to be influenced by combinatorial joining, so the frequency of mutation in the terminal three codons of FR3 or in CDR3 cannot be distinguished from nucleotide changes that result from junctional diversity. Evidence suggesting that changes in 3' FR3 are the result of junctional diversity is based on evidence that codon sequences in this region do not differ between hybridizing and nonhybridizing clones while major changes in CDR regions are observed. The frequency of transitions and transversions in FR regions of both hybridizing and nonhybridizing clones and CDRs of nonhybridizing clones ranged from 6.5–14.7/kb and differences were not significant. The ratio of transitions (44.5 ± 33.6) to transversions (50.8 ± 38.7) in CDRs of nonhybridizing clones and in hybridizing clones (14.2 ± 19.6 to 10.2 ± 14.6) was the same although the frequency was, of course, 5-fold higher in nonhybridizing clones (Fig. 5).


Figure 5
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  		FIGURE 5. Mutation frequency (Mut/Kb) in VH gene segments of nonhybridizing (top) and hybridizing (bottom) VDJ clones. The number in parentheses is the number of VDJ sequences analyzed. The difference in mutation frequency between clones hybridizing with no probe or only one was small so that the results were averaged together. The shaded portion of the stacked bar graph shows the proportion of mutations that result in changes in the amino acid sequence (R mut). The terminal portion of CDR3 most likely includes changes due to combinatorial joining as well as SHM. The scattergram on the extreme right conveys the degree of individual variation that constitutes the mean (horizontal bar). The percent value above (81 or 71%) is the proportion of R mutations. The values above the bracketed bars indicate the frequency of transition (Ts) and transversions (Tv) per kilobase in the appropriately bracketed CDR and FR regions. All sequences have been submitted to GenBank.

 
Notable in the data presented in Fig. 5 is that FR2 is essentially nonmutated in all sequences (statistics considered). This validates our routine use of FR2 as a pan-specific marker for porcine VH genes (Fig. 1; Refs. 2, 3, 4, 5).

The porcine VH genome contains hybrid VH genes, suggesting a mechanism for genomic diversification

Early studies in our laboratory revealed the presence of porcine VH genes containing the CDR1 region of a well-known VH gene and a CDR2 region of another. Because these chimeras were usually recovered by using PCR somewhere in the process, the possibility that they could be PCR-generated artifacts was examined in vitro studies (16). These studies revealed that to recover a significant frequency of hybrids, e.g., 38%, a high concentration of donor templates, e.g., a 1:1 ratio of two templates, was needed. Because such a contrived situation is an unlikely in vivo event, we considered the alternative that some hybrid VH genes were already present in the germline. The ultimate answer of course rests on a complete characterization of the porcine VH genome. Because the swine is not a priority species for basic research, ultimate resolution may require decades. In lieu of such evidence, we analyzed several VH-containing BAC clones. Table V lists the occurrence of VH genes in four different VH-containing BAC clones. Noteworthy is that the VHC/VHA combination was recovered from BAC 352, but VHC (C/C; a potential donor of CDR1 of VHC) was absent. Similarly, a VHG/VHE hybrid was recovered from BAC 663 and 1084 but a donor sequence that could be a CDR1 of VHG was not recovered. Furthermore, VHG/VHE could be recovered from BAC 747 but neither potential parent (VHG or VHE) was recovered. A similar situation occurred for VHE/VHF in BAC 747 that contained neither VHE nor VHF that could serve as a donor for a PCR artifact. These data indicate that VHG/VHE (VHG, Table I), VHE/VHF (VHT, Table I), and VHC/VHA (VHY, Table I) are present as germline genes so their expression is most likely not a PCR artifact.


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  		Table V. Recovery of hybrid VH genes from BAC clonesa

 
The porcine VH repertoire

Table I lists all the VH gene sequences that have been reported from our own laboratory and those by Kim. The investigator column of Table I indicate which research group (B or K) has identified the sequence. It is presumed that Kim also observed the major VH genes (indicated by an asterisk) but did not report them because they were already in GenBank. Noteworthy is that many reported porcine VH genes are chimeras or hybrids as regard the sequences of their CDR1 and CDR2 regions. Because the FR regions of the porcine VH3 family genes are virtually identical (Fig. 1), only their CDR regions, designated by Arabic capitals are indicated. The actual sequences of these CDRs are given at the bottom of Table I. The data of Table I suggest that the porcine VH repertoire developed by duplication and exchanges of CDR segments from a small number of precursors. For example there are only nine unique CDR1 sequences and 15 unique CDR2 sequences among 29 reported VH genes. Based on their occurrence in the preimmune repertoire (Figs. 2 and 4), major VH genes are designated with an asterisk.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The isolator piglet model has provided insight into in vivo aspects of developmental immunology during the critical window of postnatal life (21, 22). The model has demonstrated its value in understanding the role of colonization and pathogen-associated molecular patterns in stimulating the development of adaptive immunity (18, 23) and the role of neonatal viruses on development of immune responses (24, 25). This has been in part possible because of a user-friendly feature of swine in that they use only a few, well-defined VH genes to form their preimmune VH repertoire. Because this repertoire changes with age and exposure to environmental factors (Figs. 3 and 4), it provides a means for potentially quantifying the degree of Ab repertoire development that occurs following different environmental stimuli. In this revisited study of the porcine preimmune repertoire, we have addressed several issues associated porcine VH genes and the methods used for studying their usage. These include the following: 1) individual variation in VH usage; 2) the origin of chimeric VH genes; 3) the fidelity of clonal hybridization; 4) the impact of SHM on hybridization; 5) the characterization of the other category of porcine VH genes; 6) establishment of a useful index of repertoire diversification; and 7) estimation of repertoire size.

A danger in the use of outbred animals such as humans or swine is that universal patterns of gene expressed may be masked by individual variation or differential treatment. Data presented in Fig. 3 should belay such concerns for the piglet system as regards VH usage. These show that all piglets in each treatment group use the same major VH genes and in similar proportions and that changes in proportional usage are related to environmental influences. This observation is not new. Sun et al. (2) showed that 89% of the VDJ rearrangements in fetal piglets used VHA, VHB, VHC, and VHE. Each animal used the same VH genes according to the following statistics: VHA (39.2 ± 7.1), VHB (7.4 ± 0.8), VHC (19.6 ± 2.5), VHE (22.2 ± 5.1). This pattern of VH usage differs from the pattern shown for transcripts in newborns only in the greater usage of VHB and the lower usage of VHE/VHZ. Accepting these differences in VH usage in DNA rearrangements vs transcription, the constancy of usage of a few major VH genes in all animals would be difficult to explain by a somatic gene rearrangement process or a somatic gene conversion progress. However, it may be influenced by the more limited genetic diversity of swine compared with humans (D. Smith, Baylor University, Waco, TX, unpublished observation).

Our argument that VHZ, VHY, and certain other chimeric genes are not somatic or PCR recombinants, but are present in the germline, is based on several criteria. First, Table V shows that chimeric genes can be recovered by PCR from BAC clones that appear to lack donor genes that could produce artifactual hybrids because of the low processivity of Taq polymerase. Second, VHZ (E/C hybrid) and VHY (C/A hybrid) were repeatedly found in the preimmune repertoire but not their reciprocal combination even though the donor genes, i.e., VHA, VHC, and VHE, are major components of the preimmune repertoire (Fig. 2). Assuming that hybrids arising as PCR artifacts are random, the lack or unequal recovery of reciprocal combination argues against the PCR artifact theory. Therefore, hybrids like VHZ and VHY are most likely germline genes. Whether all of the various chimeric combination identified (Fig. 2) and listed in Table I represents germline chimeras, cannot be determined at this time.

Species like rabbit (10, 12) that also use only a few VH genes or chickens that use one VH gene (9, 13) use templated mutations (gene conversion) to form their Ab repertoire. The chimeras we report do not resemble the mosaic gene conversion products reported by others (8, 26, 27). Rather, entire CDR1 regions are replaced, not segments of them as in the case of converted VH genes. Because all newborn piglets use the same seven VH genes in approximately the same proportions, it is unlikely that the swine preimmune repertoire develops by some somatic gene conversion process.

The clonal hybridization system we used in the analysis of >3000 cloned VDJs appears to be reliable in determining VH gene usage. A small number of clones hybridized with three CDR probes (Table IIA) although all were single clones (Table IIB). Because these represent <5% all clones, this nonspecific hybridization could not impact the overall conclusions.

We knew from limited sequence analysis that clones we identified as "other" were either 1) mutated variants of the major four VH genes that comprised the VH repertoire or 2) less frequently used VH genes (2, 3). Table III shows that VDJ clones of major VH genes that have on average >0.7 mutations in their CDR probe binding regions are unlikely to hybridize and would therefore be classified as "other." Although there were differences among probes in the number of mutations tolerated, the mean number of mutations in the probe binding region of major, nonhybridization VH genes was ~3.3, i.e., 5-fold higher than among those that hybridize. Sequence analysis also showed that the majority of nonhybridizing "other" VH genes were mutated major VH genes, not unknown hybrid VH genes (Table IV). The analyses of the 105 VDJ sequences revealed that sequence differences between nonhybridizing and hybridizing clones were concentrated in CDR regions and in the 3' FR3 region. Because the frequency of change in 3' FR3 is not statistically different between hybridizing and nonhybridizing clones (Fig. 5) and because this portion of FR3 is subject to change through junctional diversity, variation in FR3 3' is most likely not the result of SHM. As generally reported for other species, nucleotide changes that were unequivocally the result of SHM were more frequent in CDRs than FRs (Fig. 5). Although the frequency has been reported to be higher in CDR1 than CDR2 (28), we observed no significant difference. Most codon changes, even in hybridizing clones, resulted in changes in CDR protein sequences so they may potentially alter the Ab binding site. We found no significant differences in the number of codon changes resulting from transversions vs transitions among any comparable region as had been reported by Betz et al. (28). However, they studied a transgene that was not under selective pressure, whereas most of the highly mutated sequences we studied came from animals responding to a strong Th2 stimulus. The higher proportion of R:S substitutions especially in CDRs, is generally well-known (29, 30, 31) and may not exclusively result from selection because it is seen in fetal/neonatal lambs never exposed to environmental Ag (32).

Although SHM can relegate a clone to the category "other," nonhybridizers also include minor VH genes, a trend toward use of unidentified DH segments and a decreased use of the most JH proximal DH (DHB; Table IV). However, progressive nonhybridization with probes specific for the major VH genes primarily results from SHM of major VH genes, but also occurs by use of minor VH genes but least of all by use of previously unknown chimeras.

Data presented confirm our earlier observation that H chain repertoire diversity was due to CDR3 diversity not combinatorial diversity (4). Approximately 80% of all DH segments were either DHA or DHB, even among VDJ that used minor porcine VH genes (Table IV). This is no longer surprising because transgenic mice with only a single DH segment can generate a diverse repertoire (33) as can nurse sharks using only two segments like swine (34).

In an effort to assess the impact of viral infections, probiotics, colonization, diet, and maternal factors on the neonatal Ab repertoire, we constructed a first generation RDI (Ref. 5). The original equation has now been modified using data from >3000 cloned VDJs that use the seven major VH genes comprising >95% of the preimmune repertoire. The equation is derived by weighting the effect of VH gene expression based on the proportional usage of the major VH genes in the preimmune repertoire and considering how the usage of these genes is increased (numerator) or decreased (denominator) after environmental exposure (see Fig. 4).

Formula

We tested this RDI equation on data collected from the MLN. Helminthic parasites stimulate strong Th2 responses, commensal bacteria stimulate Th1 responses (35) and both should impact the MLN (36, 37, 38). Using the RDI index, the preimmune IgM repertoire of newborn gives an RDI value of 2.1, whereas a value of 73 is generated in parasite-infected pigs. An RDI of only 4.4 (marginally significant) for 5-wk-old piglets monoassociated with E. coli G58-1 vs 2.3 for 5-wk-old GF controls was calculated. Thus, the preimmune IgM repertoire scarcely diversifies with time or with monoassociation of isolator piglets in E. coli. When values for the RDI were generated for the IgG repertoire, there is more evidence for age-dependent (2.6–7.0) and colonization-dependent (2.6–9.2) diversification than in the IgM repertoire but differences were not significant. The small change in Ab repertoire in monoassociated isolator pigs compared parasite-infected piglets probably reflects not only parasitic infection but also conventional rearing in which newborn piglets encounter ~30 members of the normal gut flora. However, the high level of repertoire diversification in IgM transcripts from parasite-infected piglets was surprising because current dogma suggests that most Ags are T dependent and should drive responses that involves switch recombination and the preferential appearance of transcripts with SHM VDJ expressed with IgG or other switched isotypes (39). This suggests that in parasite-infected pigs, diversification, and SHM (Table IV, top) are not coupled to CSR. Mutated IgM sequences have also been reported in humans (30, 40, 41).

Accepting that many if not all hybrid VH genes in swine are actually germline genes, it is now possible to update information on the porcine VH repertoire. We adopted a system using Arabic letters with the anticipation that these will be eventually changed to the more standardized system of IGHV3-1, IGHV3-2, etc. (42), when their location in the genome has been established. During the interim, the A-Z system has been used (Table I). All are members of a single VH3 family (clan; i.e., IGHV3) and with the exception of VHB (Fig. 1), share identical FR regions that allow use of a pan-specific FR2 probe for total VH detection (Refs. 1 and 43 ; Figs. 1 and 5). Therefore, only sequence information for CDR regions is given in Table I. This conservation of FR regions is somewhat greater than human VH3 genes (92 vs 96%). According to this system, the VHE/VHC hybrid gene discussed in this report is designated porcine VHZ, hybrid VHA/VHB becomes VHV, and hybrid VHE/VHF becomes VHT. Assuming that the majority of the 29 VH genes (or alleles) listed in Table I are not artifacts of PCR, it suggests that the porcine VH locus arose from duplications and rearrangements of a small number of prototypic genes that differ only in their CDR regions. This pattern is similar to the VH3 family in humans in which numerous genes share CDR1 or CDR2 segments (42).

The data provided resolve numerous biological and methodological issues regarding the porcine VH repertoire. In the process, these studies provide an equation for quantifying the diversification of this repertoire in piglets exposed to the various environmental and maternal factors that impact the developing neonatal mammal. Such quantitative data has already been valuable in gaining a better understanding of the lymphoproliferative disorder caused by the porcine respiratory and reproductive syndrome virus and should be valuable in future studies with other neonatal pathogens that may collectively serve as models for events occurring in human infants.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Pork Board Grants 05-143 and 05-174 and by Cooperative Agreement 58-3625-4-155 with the U.S. Department of Agriculture-Agricultural Research Service (Ames, IA). Back

2 Address correspondence and reprint requests to Dr. John E. Butler, University of Iowa, Department of Microbiology, Interdisciplinary Immunology Program, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-butler{at}uiowa.edu Back

3 Abbreviations used in this paper: SHM, somatic hypermutation; MLN, mesenteric lymph node; BAC, bacterial artificial chromosome; CSR, class switch recombination; FR, framework; LB, Luria-Bertani; GF, germfree; RDI, repertoire diversification index. Back

Received for publication March 20, 2006. Accepted for publication June 29, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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