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The Journal of Immunology, 2006, 177: 5480-5489.
Copyright © 2006 by The American Association of Immunologists, Inc.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG31

John E. Butler2 and Nancy Wertz

Department of Microbiology and Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fetal piglets offer an in vivo model for determining whether Ag-independent IgG subclass transcription proceeds in a manner that differs from subclass transcription in pigs exposed to environmental Ags and TLR ligands. Our data from ~=12,000 C{gamma} clones from >60 piglets provide the first report on the relative usage of all known porcine C{gamma} genes in fetal and young pigs. Studies revealed that among the six C{gamma} genes, allelic variants of IgG1 comprised 50–80% of the repertoire, and IgG2 alleles comprised <10% in nearly all tissues. However, relative transcription of allelic variants of IgG1 randomly deviate from the 1:1 ratio expected in heterozygotes. Most surprising was the finding that IgG3 accounted for half of all C{gamma} transcripts in the ileal Peyer’s patches (IPPs) and mesenteric lymph nodes but on average only ~=5% of the clones from the thymus, tonsil, spleen, peripheral blood, and bone marrow of newborns. Lymphoid tissues from late term fetuses revealed a similar expression pattern. Except for IgG3 in the IPPs and mesenteric lymph nodes, no stochastic pattern of C{gamma} expression during development was seen in animals from mid-gestation through 5 mo. The age and tissue dependence of IgG3 transcription paralleled the developmental persistence of the IPP, and its near disappearance corresponds to the diversification of the preimmune VDJ repertoire in young piglets. We hypothesize that long-hinged porcine IgG3 may be important in preadaptive responses to T cell-independent Ags similar to those described for its murine namesake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gene duplication in various mammals has lead to multiple subclasses of IgG in rodents, humans, domesticated ruminants, horses, swine, camelids, and guinea pigs (1, 2, 3, 4). Similar duplication has resulted in 13 subclasses of IgA in rabbits (5) and two subclasses of IgA in humans (6). Four IgG subclasses occur in humans, and these possess different biological characteristics. IgG1 accounts for nearly three-fourths of total serum IgG, and both IgG1 and IgG3 decorate phagocytic cells because of their high affinity for Fc{gamma}R1. IgG3 is also a powerful activator of complement. The four IgG subclasses of laboratory rats and mice also exhibit differences in biological function and physiological distribution. The subclass called IgG1 in cattle is the predominant IgG in all exocrine fluids and comprises 90% of the IgG in colostrum but only 50% of serum IgG (7, 8). Similar physiological differences are seen with IgA subclasses in which human IgA1 is relatively more prevalent in blood than secretions (6), perhaps because it is more susceptible to IgA proteases than IgA2 (9, 10). The 13 rabbit IgA subclasses also differ in their distribution among tissues (11), suggesting anatomically related differences in function. It is likely that similar differences in biological functions and relative expression will be found among Ig subclasses in animals that have been less well studied.

Our research has focused on immunoontogeny and uses fetal and neonatal piglets to study the roles of colonizing bacteria, viral infection, and dietary protein on the development of adaptive immunity in truly naive offspring. In swine, no maternal IgG or environmental Ags are transported to the fetus in utero, and cesarean-derived fetuses are precocial and can be reared in isolator units in which maternal and environmental influences are under the control of the experimenter (8, 12, 13, 14). In the course of such studies we and others have observed IgA and IgG transcripts and low levels of circulating IgG and IgA during fetal life (15, 16). Class switch recombination (CSR)3 occurs as early as mid-gestation in the thymus of fetal piglets that have no exposure to either environmental Ag or maternal IgG that might deliver Ag-containing immune complexes (15, 17). Newborn piglets also transcribe IgE in thymus and mesenteric lymph nodes (MLNs) (16).

CSR is one of several temporally coincident events that characterize the maturation of adaptive immune responses to thymus-dependent Ags. These also include germinal center formation, expression of CD40L (CD154) on activated T cells, repertoire diversification by somatic hypermutation, and, of course, changes in relative isotype/subisotype expression. CSR requires cleavage of switch regions comprised of highly repetitive sequence motifs followed by ligation to cleaved downstream switch regions and subsequent repair mediated by activation-induced cytidine deaminase (AID) (18, 19, 20, 21). Switching increases when cells are caused to proliferate, which appears to accelerate the expression of AID (22) and is heavily influenced by activated T cells and the cytokines they secrete (23). Certain cytokine profiles favor switching to certain subclasses or isotypes (24, 25, 26, 27, 28, 29, 30). Polarization of naive CD4 T cells toward Th1 or Th2 cytokine profiles is often paralleled by the expression of certain IgG subclasses and influenced by the type of Ag or microbe-associated molecular pattern (MAMP) encountered by the APC (31).

The dependence of CSR on cytokines from T cells responding to environmental Ags raises the question of how CSR proceeds in Ag-deprived fetal piglets. Evidence for an alternative mechanism comes from several observations, including our own. For example, low levels of IgA can be found in early human fetuses (32, 33, 34) as well as IgG transcripts (35). Kearney et al. (36) showed that LPS given to young mice resulted in IgG-producing cells, and this group would later identify these as short-lived IgG3 plasma cells in the splenic marginal zone (MZ) (37). Serum IgG is also present in CD40/CD40L-deficient mice (38) and in patients with X-linked hyper-IgM syndrome (39). CSR can occur in responses to thymus-independent Ags (40) and TLR ligands (18, 41) and during B cell lymphogenesis independently of T cells (42, 43). Furthermore, both CSR and somatic hypermutation can occur outside of germinal centers (44). An alternative to the classical concept of T cell dependence is the stochastic model of subclass expression. This model is reminiscent of the preferential and sequential usage of JH-proximal VH genes that occurs during Ag-independent B cell development (45, 46) and is perhaps facilitated by proliferation (22). This has been speculated by Deenik et al. (47). B cell and Ab repertoire development can be monitored for 84 days in utero in fetal piglets, and thereafter piglets can be maintained germ-free for an additional 5–7 wk postpartum (14, 48). During this postpartum window they can be colonized, infected with virus, and/or administered Ags of choice by the experimenter (13). Thus, the fetal/neonatal piglet model can be used to address whether CSR to various IgG subclasses follows a stochastic course and/or is determined by exposure to environmental Ag or MAMPs.

Eight C{gamma} gene sequences have been described in swine to date, extending the number up from the five reported more than a decade ago (49). Our recent studies indicate these are encoded by six C{gamma} genes, with two allelic variants of IgG1 and two allelic variants of IgG2 (Fig. 1) (J. E. Butler, N. Wertz, N. Deschacht, and I. Kacskovics, unpublished observations) Because speciation preceded subclass diversification, these are not phylogenetic homologues of their namesakes in other species (3, 4, 48, 50) and were named in order of their discovery. Immunochemical reagents do not exist for the study of putative IgG subclass and allelic usage in swine, so we have used a PCR recovery, cloning, and hybridization method to address the issue of whether subclass expression is intrinsic or Ag driven. This method permits proportional transcription to be quantified while simultaneously allowing new C{gamma} sequences to be discovered. We used this approach to obtain data from ~=12,000 clones and show that the allelic variants of IgG1 account for 50–80% of the C{gamma} genes transcribed in all piglets regardless of age or treatment. Most striking among the three new sequences recovered was a long-hinged IgG (designated IgG3 in Fig. 1) that accounts for ~=50% of all IgG expression in the ileal Peyer’s patches (IPPs) and MLNs of newborns but precipitously declines in usage after colonization. Except for the behavior of IgG3, no other in vivo evidence for an age-dependent or stochastic pattern of transcription of C{gamma} genes in fetal and neonatal piglets was observed. Because the developmental expression of porcine IgG3 parallels the Ag-independent life span of the IPP and the tenure of the undiversified preimmune VH repertoire, we have speculated that porcine IgG3 may be important in the preadaptive repertoire in the manner described for mouse MZ B cells.


Figure 1
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  		FIGURE 1. Unique and conserved regions of the eight human porcine C{gamma} gene sequences showing the annealing sites for the various gene-specific and pan-specific probes used in clonal hybridization studies. Dots indicate that the nucleotide is the same as the one above. Dashes indicate missing nucleotides that were added to allow alignment. Amino acid sequences are shown where appropriate. The complete sequences will be published elsewhere (J. Butler, N. Wertz, N. Deschacht, and I. Kacskovics, unpublished observations). The exact nucleotide sequences of the gene-specific probes differ somewhat from the C{gamma} gene segments to which they anneal, so they are therefore given in Table II.

 

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

Animal tissues were a by-product of studies on the following: 1) gut colonization in isolator piglets at South Dakota State University (Brookings, SD); 2) helminthic parasitism at the Beltsville Agriculture Research Center (Beltsville, MD); 3) the effect of porcine reproductive and respiratory syndrome virus at the National Animal Disease Center (Ames, IA); and 4) fetal developmental studies at the U.S. Meat Animal Research Center (Clay Center, NE). Tissues were collected after euthanizing the animals. Protocols were approved by the Animal Use and Care Committee of the respective collaborating institutes. All solid tissue was immediately frozen in liquid nitrogen and flown to the University of Iowa (Iowa City, IA) on dry ice. Blood samples were collected in EDTA in vacutainers and processed for the recovery of PBLs as described previously (16, 49, 51, 52). The PBLs were then stored in TRI Reagent (Molecular Research Center).

Table I summarizes the animals used. The focus of the study was on piglets of 110–114 days of gestation (DG), hereafter designated newborn. Thus, a complete analysis of all subclasses in all tissues from fetal, newborn, neonatal, and young pigs was not undertaken. Rather, certain groups and certain tissues were added to address specific questions that emerged during the initial studies on fetal and newborn piglets. Analysis of others was considered not applicable (NA in Table I).


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  		Table I. Animals and tissues examined

 
Preparation of RNA and cDNA

Briefly, frozen-solid tissues were homogenized using a Potter-Elvehjem device and dissolved in TRI Reagent so that all starting materials for RNA were in TRI Reagent. Total RNA was prepared as previously described (15, 16, 53). The RNA concentration and quality were determined by OD 260/280 absorbency. First-strand cDNA was prepared as previously described (15, 16, 53), except that a primer for the 3' untranslated region shared by all known C{gamma} genes, as well as random hexamers, was used (Table II).


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  		Table II. PCR primers and oligonucleotide probes

 
Characterization of porcine IgG genes

The sequences for five different IgG transcripts from a single swine were reported by Kacskovics et al. (49). These were recovered from a cDNA library and reported to represent five different IgG subclasses. We have subsequently characterized three additional C{gamma} genes designated IgG5, IgG6, and IgG3. The IgG we have named IgG3 replaces IgG3 of the Kacskovics nomenclature, because we have shown the former IgG3 to be an allelic variant of IgG1 that we now designate IgG1a. In addition, we have shown that IgG2a and IgG2b of the Kacskovics scheme are alleles of IgG2 and are now so designated. These changes in nomenclature have been incorporated in Fig. 1 and used throughout this report. An extensive study describing the characterization, structure and genetics of the porcine C{gamma} genes will be separately published (J. Butler, N. Wertz, N. Deschacht, I. Kacskovics, unpublished observations).

Quantitation of relative subclass expression by clonal hybridization

C{gamma}-containing products were recovered by two-stage PCR from first strand cDNA. Although Taq polymerase was routinely used, we also repeated a number of studies with Vent polymerase to reduce polymerase errors, but none were found. In the first round, amplification was done using a pan-specific primer set involving framework 1 (FR1) of VH (FR1 is identical in all swine VH genes) (14, 48, 53) and an anti-sense CH3 primer that annealed to a sequence common to all known swine IgG genes (Table II). The first round product was analyzed by electrophoresis in 1.5% agarose gel, and the appropriate-sized C{gamma}-containing band was removed from the gel and purified using Costar Spin-X columns (Corning). This purified product was then used as the target for the second-round PCR, which used an internal primer set recognizing conserved sequences in the CH2 domain and the 5' region of CH1 (Table II). The product of the second round PCR was cloned into a pCR-TOPO 2.1 vector, grown in TOP TEN cells for 1 h at 37°C, and plated on Luria-Bertani agar containing 50 µg/ml kanamycin. Positive clones were then selected and transferred to the wells of round-bottom 96-well microtiter plates (Costar 3799, Corning) and grown overnight at 37°C in Luria-Bertani with kanamycin. Plasmid DNA was harvested by alkaline lysis and centrifugation. Plasmid DNA was transferred and cross-linked to nylon membranes (Schleicher & Schuell Microscience) as previously described (16, 54, 55). Membranes, each containing plasmid DNA from 96 different clones, were then hybridized with C{gamma} subclass-specific probes and eventually a pan-specific C{gamma} probe (Table II and Figs. 1 and 2) in the same manner as we have routinely used for the enumeration of VH gene usage (16, 54, 55). The annealing sites for the various probes are shown in Fig. 1. As shown, two probes used in succession were needed to identify IgG6 (Fig. 1 and Table II).


Figure 2
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  		FIGURE 2. Representative clonal hybridization results. Each panel shows the same membrane hybridized with three different gene-specific probes (B–D) and a pan-specific probe (A). The microtiter plate coordinates are given in A.

 
Statistical analysis

Statistical analyses when required were done with the assistance of Dr. K. Chaloner, Department of Biostatistics, University of Iowa (Iowa City, IA). Simple mean differences were tested in two-tailed Student t tests, and allotype expression was tested by {chi}2 analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of IgG genes by clone hybridization

Fig. 2 presents representative hybridization data. Fig. 2A allows all C{gamma} -containing clones to be identified using a pan-specific C{gamma} probe (Fig. 1 and Table II), whereas Fig. 1, B–D show the same membrane sequentially hybridized with probes specific for IgG1b, IgG3, and IgG5 respectively. After each hybridization, the labeled probes were removed by two successive 10-min incubations at 80°C in water. Complete removal was verified using a Hewlett-Packard radio analytical scanner. In some cases, hybridization with two C{gamma}-specific probes was necessary to identify a certain C{gamma} gene, e.g., IgG6 (Table II). Clones, especially those that weakly hybridized (Fig. 2B; coordinates D-12, D-8, H-2) were regularly grown out and their plasmid DNA sequenced to confirm the identity of the cloned C{gamma} gene sequence.

Reproducibility of subclass and allotype expression data

Fig. 3A compares the effect of calculating proportional use data by sampling 32, 48, or 96 clones from the thymus of the same piglet. These results show that for highly expressed C{gamma} gene sequences the coefficient of variation of proportional usage data ranged from 2.1–6.6% when data for 48 and 96 wells were compared. Because in four of five cases data from 48 and 96 clones differed only slightly, and to reduce the work load, all subsequent comparisons were made using a panel of 48 clones (see Fig. 2). Fig. 3A also shows that IgG4 and IgG6 are not expressed in all tissues of a particular piglet. The animal tested was later shown to be a IgG2b/b homozygote. Although Fig. 3A tests for the effect of clone population size on proportional usage data, it does not test for variation in measurements made from a single tissue. Fig. 3B compares the proportional recovery of different C{gamma} gene transcripts in three independent analyses from the MLN of the same piglet. In each case, a fresh RNA sample was prepared from the same tissue, and the procedures for cDNA preparation, PCR, cloning, and hybridization were followed as described in Materials and Methods. The results indicate that experimentally induced differences in nucleic acid preparation, PCR, cloning, or hybridization do not generate significantly different values for proportional usage. SD values for seldom-recovered C{gamma} genes (<3% usage) ranged only from 0.5 to 1 times the mean even when tested only three times. We repeated these studies using Vent polymerase but found no differences in the clones recovered compared with classical Taq (48). Data in Fig. 3B also show that significant differential IgG1 allotype expression occurs in certain tissues of heterozygotes.


Figure 3
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  		FIGURE 3. Precision of the clonal hybridization method. A, Influence of the number of clones (32, 48, and 96) tested upon calculation of proportional usage by the thymus of the same piglet. The encircled value is the coefficient of variation of usage data obtained by comparing 48 and 96 clones. The noncircled values are the coefficients of variation for data comparing 32, 48, and 96 clones. ND, not detected. Note the unusually high proportional expression of IgG3 in this thymus and that the animal tested is an IgG2b/b homozygote. B, Reproducibility of values on proportional usage when the same MLN sample was tested three times, each time initiated from a fresh tissue sample. The horizontal bars are the mean value and the SD (boxed) is given for each. The vertical brackets indicate that the p values were obtained when differences in IgG1 and IgG2 allotype expression were compared by Student t tests.

 
IgG1 dominates expression in bone marrow (BM) and most lymphoid tissues from late term fetuses

Table III shows that when relative IgG subclass transcription for various lymphoid tissues from newborns was examined, IgG1b and IgG1a dominate the expression profile. With the exception of tissues expressing a high proportion of IgG3 (see below), nearly 80% of transcripts were IgG1 alleles. The ratio of allotype expression in >5,000 clones over all tissues of all animals was 1:1 (Table III, row labeled "Mean"). When BM, spleen, and thymus of fetuses of all ages were examined, similar IgG1 dominance was seen (Fig. 4). However, variations among animals were large as seen by the error bars in Fig. 4, and although the overall expression ratio of IgG allotypes in heterozygotes was 1:1, this ratio varied among tissues (Table III). For example in 40- to 60-DG thymi, IgG1a accounted for >70% of clones and IgG1b for 20% (Fig. 4). In the animals randomly chosen for reproducibility testing, IgG1 allotype ratios were equally skewed and differences were significant at p < 0.001 level (Fig. 3B). The dominant occurrence of IgG1 in swine provided the opportunity to more carefully examine allotype expression in this subclass, because >90% of outbred animals are heterozygous for IgG1 (J. Butler, N. Wertz, N. Deschacht, and I. Kacskovics, unpublished observations). Table IV provides representative data for 17 animals from the >70 examined. Data show that skewed expression can be in either direction and in opposite directions among tissues from the same piglet. The same skewing was seen among tissues from the >50 other animals studied (data not shown). Expression among peripheral blood cells was least skewed while skewing was observed in seven of 10 randomly selected BM samples (a primary lymphoid tissue for swine) (4, 50, 71 ; Tables III and 4).


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  		Table III. Proportional expression of C{gamma} genes in various tissues of newborn piglets

 

Figure 4
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  		FIGURE 4. Relative transcription of allelic variants of IgG1, IgG2, and the other four IgG subclass genes in BM/fetal liver, IPP, spleen, and thymus of fetal piglets at 40–60 DG, 70–90 DG, and in newborn piglets. The number of animals used and clones tested is given in Table I. Error bars indicate SD values. *, Allotype expression differences are statistically significant. **, Significant differences in the proportion of IgG3 expression.

 

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  		Table IV. Skewing of IgG1 allotype expression in outbred swinea

 
IgG3 comprises half of IgG gene expression in IPP and MLN of late term fetuses and is age dependent

Table III shows that overall the allelic variants of IgG1 account for 65% of all transcripts and those of IgG3 for only 25%. However, proportional usage is not uniform among tissues, and IgG3 expression predominates in the MLN and the IPP (>50%) but not in the jejunal Peyer’s patch (JPP). The latter behaves as a compromise between the IPP/MLN pattern and tissues like spleen, tonsil, etc. Interestingly, IgG3 is also highly expressed in the inguinal lymph node, although this is not normally considered to be connected to the mucosal immune system in the manner of the IPP and MLN. In contrast to MLN, IPP, and the inguinal lymph node, IgG3 expression constitutes <6% of IgG expression (~10-fold less) in BM, PBL, spleen, thymus, and tonsil (Table III and Fig. 4). However, thymi from exceptional fetal animals did not follow this pattern (see below and Fig. 3A).

The IPP of sheep has been implicated as a developmentally dependent site for B cell repertoire diversification (56, 57). Thus, we wondered if relative IgG3 expression was developmentally dependent. Fig. 4 shows that IgG3 usage in IPP is lower in 70–90 DG fetuses than in newborns, whereas Fig. 5 shows that the prominent usage of IgG3 in the MLN and IPP of fetal piglets is reduced to ~=5% in parasite-infected conventionally reared young pigs. Thus, we examined proportional IgG3 expression in selected lymphoid tissues of fetal animals and especially in MLNs and IPPs of isolator piglets receiving different treatments (Table V). IgG3 expression in fetal liver or bone marrow ranged from 0 to 12% and was detected in spleen at 50 DG, although proportional expression in this lymphoid organ did not increase during fetal life. IgG3 expression was significant in IPP at the earliest appearance of this organ (20% at 90 DG; Table V). Proportional expression reached a maximum (>50%) in IPP and MLN in newborns and decreased only slightly in 3–6 wk-old germ-free isolator piglets even when virus infected. However, expression decreased to <10% in colonized isolator piglets and fell to ~=5% in 5-mo-old conventional young pigs (Table V). The difference between parasite-infected conventionally reared pigs and their age-matched control littermates was not significant, so data were pooled. Thus, helminthic infection does not uniquely influence IgG3 expression, but age and conventional rearing do. In summary, IgG3 expression increases dramatically in late gestation in IPP and MLN, declines after birth in conventional and colonized isolator piglets, but remains elevated in neonates maintained noncolonized even though virus-infected. Very little IgG3 is expressed in the spleens of isolator piglets or conventional young pigs (Table V).


Figure 5
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  		FIGURE 5. Relative transcription of six IgG subclass genes in the MLNs and IPPs of nine newborn piglets and in the MLNs and IPPs of young pigs infected with Ascaris suis and Trichuris suis. Error bars are SEM. *, Mean group difference significant at the 0.05 level. **, Significantly different at 0.01 level. Because data are expressed as a proportion of the total, the decrease in IgG3 expression at 5 mo results in a relative increase in expression of IgG1. PIC, parasite-infected conventionally reared.

 

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  		Table V. Relative expression of IgG3 in selected tissues of fetal and neonatal piglets

 
Transcript expression of other IgG subclasses and allotypes

Apart from the situation involving IgG3, there are few other examples of unusual expression of a particular C{gamma} gene or C{gamma} gene allotype. Expression of IgG5 in spleen and peripheral blood averages 8–10% vs 2.6 ± 1.4% for all other tissues examined (Table III). Expression of IgG4 and IgG6 is too low to allow any conclusions to be drawn about differential tissue expression. Whereas the dominant expression of IgG1 allowed allotype expression in heterozygotes to be examined (Table IV), IgG2 was expressed at levels too low to allow statistically useful data on differential allotype expression and, therefore, could not be addressed in the manner of IgG1 (Fig. 3, A and B, and Table III). In certain tissues, no IgG4 or IgG6 was detected (Fig. 3A).

Table III shows that although IgG2, IgG4, and IgG6 are readily recovered from IgG clones, their combined contribution in late term fetuses is ~=6%. Most notably, IgG4 and IgG6 constitute <1% of the expressed IgG repertoire in fetal piglets, although relative levels of IgG2 and IgG4 are higher in parasite-infected animals (Fig. 5). In such animals, IgG4 and IgG2 can together comprise >10% of the repertoire.

Thymus and tonsil resemble nongut lymphoid tissues

The porcine fetal thymus transcribes nearly all isotypes (16) and contains a high proportion of transcripts for IgA and, to a lesser extent, IgG (15, 17). This quasimucosal pattern might suggest that early thymus, perhaps like the IPP, is a progenitor organ for mucosal immunity in swine. Table III and Fig. 4 show that fetal thymus and tonsil are more similar to PBLs and spleen than to the IPP or MLN, which highly express IgG3. However, the pattern in thymus was not consistent among individual piglets. At DG 90, ~=25% of the clones in one animal expressed IgG3, which is >4-fold higher than the mean level of IgG3 in all thymi tested (Fig. 3A and Table III). This exceptional animal is deliberately featured in Fig. 3A.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In mice, humans, cattle, and horses the physiological distribution and effector functions of Igs, including IgG subclasses, have been partially or completely characterized (3, 58). Such characterization was made possible because of subclass-specific antisera and purified subclass proteins. The current lack of availability of such reagents for swine and the complexity of the swine IgG repertoire has delayed efforts to characterize the distribution and function of the various IgG subclasses. Rather than postpone all Ab-dependent characterization, we have applied molecular genetic technology to study the relative transcription (and presumably protein expression) of the various IgG genes, especially during late fetal development. In our previous studies, Ig transcription and secretion occurred in parallel in the same fetal tissues (15). Rather than real-time PCR, we choose a cloning/hybridization procedure so we could verify our findings by sequence analysis, which also gave us the opportunity to identify three new sequences. Consistent with the RFLP data of Kacskovics et al. (49), we have now identified eight C{gamma} gene sequences in swine. A separate study describing the genetics, structural analyses, functional motifs, and phylogenetic comparisons of the porcine C{gamma} gene sequences now defines six C{gamma} genes in swine with two alleles each for IgG1 and IgG2 (J. Butler, N. Wertz, N. Deschacht, and I. Kacskovics, unpublished observations). The C{gamma} sequence named IgG3 by Kacskovics et al. (49) has been identified as an allele of IgG1. Hence, the recently discovered long-hinged C{gamma} gene (Fig. 1) is now designated IgG3. This C{gamma} gene had apparently been overlooked in the study by Kacskovics et al. (49), perhaps because only adult pigs were studied.

The selection of animals and lymphoid tissues used in this study reflects our primary focus on the observation that IgG transcripts can be recovered very early in fetal development, especially in the thymus, and that fetal sera contain low levels of IgG, IgA, and IgM (15). The occurrence of these switched isotypes was surprising, because the fetal piglet system is closed to both maternal Igs and environmental Ags that could act as immune modulators. Therefore, it offers the possibility to test whether CSR might be stochastic in a T cell-independent manner as speculated (47). T cell-independent CSR has been reported in mice (38, 42, 43), and for >25 years murine IgG3 was associated with T cell-independent responses to carbohydrate Ags (59, 60).

The idea that IgG subclass usage might follow a developmental pattern is a projection based on studies of fetal VH gene usage in which usage of the most 3' VH genes are favored (45, 46). In swine, VHB is one of the two most frequently used genes in the preimmune IgM repertoire and is the most 3' functional VH gene (reviewed in14, 48). Furthermore, the most JH proximal CH genes that encode IgM and IgD in mammals are used to form the earliest BCRs. Thus, position favors usage of Ig gene segments both immediately 3' and 5' of JH and the H chain enhancer. Subsequent usage systematically spreads upstream in the VH locus and downstream in the CH locus by CSR. Because the organization of the porcine C{gamma} locus is unknown, our data cannot test for a positional effect but can determine whether there is progressive change in C{gamma} expression during development and when piglets are exposed to environmental Ag. Data presented in Tables III and V and Figs. 4 and 5 would argue that, with the exception of IgG3 in the IPP and MLN (see below), there is no evidence for an intrinsically programmed change in the pattern of C{gamma} transcription in the fetuses of a species that is free from Ag encounter. Rather, there appears to be an intrinsic program that determines that the allelic variants of IgG1 dominate expression in all animals ranging from early gestation fetuses to 5-mo-old conventionally reared young pigs. This intrinsic domination is not altered by exposure to environmental Ag. The significant increase in IgG3 expression is likewise Ag independent, although its decline appears dependent on Ags or MAMP derived from colonizing bacteria (Table V; see below).

The quantitative importance of IgG3 expression in fetal life and its association with the IPP and MLN suggest that its expression might be related to B cell repertoire diversification. Thus, we focused our attention on primary lymphoid tissue during development and IgG3 expression postnatally up to 5 mo. In swine, B cell lymphogenesis begins at DG 20 in yolk sac, proceeds to the fetal liver, and becomes active in BM after DG 60 (14). However, because <5% of C{gamma} transcripts from fetal liver and BM of 40 DG fetuses expressed IgG3, expression does not appear to be associated with lymphogenesis. In 50-DG fetal spleens, 9% of clones expressed IgG3, and this low level of splenic IgG3 expression was seen in postnatal piglets of up to 6 wk of age (Table V). The most striking observations was that the dominant expression of IgG3 in the IPP and MLN of newborns progressively declined, especially in colonized neonates, and comprised <5% of total IgG transcripts by 5 mo (Fig. 5 and Table V). The longevity of IgG3 expression parallels the prominence of the IPP in piglets (61, 62). We and others (63) observed that the IPP of newborns exclusively expressed IgM but that 5 wk later all major isotypes were expressed, especially in colonized piglets (64). This pattern temporally correlates with the near disappearance of IgG3 expression (Table V). JPPs are far more responsive to environmental Ag provided by gut flora than IPPs (63), although the transition of hindgut lymphoid tissues from a B cell diversification center to mucosal tissue has been previously suggested (65). We have proposed that the IPP is engaged in early B cell diversification as in lambs (14, 56, 57), and in this work we suggest that it may be involved in T cell-independent CSR to IgG3 and that IgG3 Abs to T cell-independent bacterial polysaccharides play an important role in early host defense (see below). In contrast to the IPP, the conventional mucosal immune response of the JPP is highly T cell dependent. AID is required for CSR (66), and AID is up-regulated in the IPP of sheep (57) and also in swine (67). Because Peyer’s patch cells are known to traffic to the MLN (68), the recovery of IgG3 transcripts from the MLN or newborn and neonatal piglet is not surprising.

T cell-independent responses to carbohydrate Ags in mice are known to be of the IgG3 subclass (59, 60), and this subisotype is characteristic of MZ cells in the spleen that recognize TI-2 immunogens (23). Such responses often carry the specificity of the preimmune repertoire in forming a preadaptive response against bacterial pathogens (69). (Preadaptive means a functional repertoire that generates an immediate source of antibodies that bypasses T cell-mediated events such as germinal center formation, CSR, and somatic hypermutation.) In swine, the preimmune repertoire is characterized by usage of four VH genes that comprise 80% of the repertoire or seven that can account for >95% (48, 55). This preimmune VH repertoire diversifies with age and experience so that >80% of the VH repertoire of conventionally reared animals, such as the 5-mo-old young pigs used in this study, no longer resemble the preimmune repertoire (55). As shown in Table V, IgG3 expression in these animals is reduced to ~=5%. The decreased expression of IgG3 in colonized isolator piglets also parallels the increased diversification of the preimmune VDJ repertoire in these piglets (55, 64). Thus, the decline in IgG3 expression that we report in this study seems to parallel the diversification of the preimmune repertoire (48) and the disappearance of the IPP (61, 62). Perhaps porcine IgG3 performs the same role described for its namesake in mice by responding to T cell-independent Ags with a preadaptive response to bacterial pathogens. This hypothesis of porcine IgG3 function can be tested when and if an anti-IgG3 mAb becomes available.

The hypothesized parallel function of swine and mouse IgG3 must reflect parallel evolution, because IgG subclass diversification came after speciation (3, 4, 48, 50), and the Abs designated IgG3 in humans, cattle, horses, and swine are all phylogenetically unrelated. However, it is of some interest that the IgG3s in horses, cattle, humans, and swine are all minor IgGs in blood and represent the long-hinged IgGs of their respective species, although mouse IgG3 is not long hinged.

The enigmatic observation that played a role in initiating the current study, i.e., mid-gestation CSR to IgG, still remains an enigma. Of all the tissues studied, the thymus was the most variable in regards to IgG gene transcription. Fig. 4 shows that the variation in 70- to 90-DG thymi is very large. In data not shown, one fetus expressed 80% IgG1a but little IgG1b, although skewing of IgG1 allotype expression was not uncommon (Table IV and Fig. 3B). Fig. 4 shows that 70- to 90-DG thymi expressed ~=15% IgG3, although one fetus that we chose to highlight expressed ~=25% (Fig. 3A). We previously reported that the fetal thymus contains a large population of unselected pre-B cells (51, 70) that are located in the interlobular region beneath the capsule (71). If these give rise to the B cells and plasma cells of the thymus (15, 17), they might be expected to display C{gamma} expression more similar to that of IPP than of the spleen. However, the variation among C{gamma} subclass expression by the fetal thymus adds little toward resolution of the B cell enigma of the porcine thymus. Resolution will require a dedicated developmental study, including cell trafficking studies and microdissection with the aid of B cell specific mAbs (currently unavailable), to identify different stages of B cell development in various organs of swine.

Unlike rodents and humans, the so-called inverted lymph nodes of swine results in the fact that most lymphocytes traffic in blood (72). Because PBLs can be regularly sampled, it is of practical value to know whether the subclass expression by PBLs is generally representative of secondary lymphoid tissue. In fetal piglets, the pattern of C{gamma} expression by PBLs is similar to that in spleen, tonsil, BM, and to a lesser extent in JPP but is not representative of that in IPP or MLN. Importantly, IgG1 allotype expression by PBLs of heterozygotes was most consistent with the predicted outcome of allelic exclusion (Table IV). Thus, the B cells in blood may most accurately represent all lymphoid tissues except the IPP and MLN.

Allelic exclusion predicts that among heterozygotes 50% of their B cells should express one Ig H chain constant allele, and 50% the other. In studies of the porcine IgA allotypes IgAa and IgAb, expression in heterozygotes could be as disparate as 4:1 (73). The same disparity was seen in the studies we report here for the allotypes of porcine IgG1 (Table IV). These include fetal animals never exposed to environmental Ag. Thus, allotype expression in heterozygous swine can significantly deviate from the predicted 1:1 ratio in the absence of Ag selection in any particular tissue (Table IV). However, when >5,000 clones from all tissues of up to nine newborns were examined, the ratio approximates the value predicted by allelic exclusion (Table III). Because we examined on average >70 clones for each cloning reaction and up to 80% were IgG1, the control studies presented in Fig. 3 suggest it is unlikely that the skewing we show in Table IV represents a random sampling error. This argument is emphasized by results presented in Fig. 3B and because deviations from 1:1 were often greater for tissues from which large numbers of clones were tested (e.g., no.3 JPP in Table IV).

This is the first report on the differential transcription of genes encoding the porcine C{gamma} genes and their alleles. More importantly, it is the first to address whether intrinsic CSR progressively changes the relative expression of IgG subclass usage during fetal and neonatal development in fetuses and neonates not exposed to maternal factors and environmental Ags. We show that newborns, neonates, and young animals exposed to environmental Ags and pathogen-associated molecular patterns all show a profile of C{gamma} transcription dominated by the ~50–80% usage of the allelic variants of IgG1. The only exception is the long-hinged IgG3, which can account for >50% of the transcripts in newborn IPP and MLN but then progressively declines in usage as neonates encounter colonizing gut bacteria. Perhaps the developmentally dependent expression of long-hinged porcine IgG3 and the possibility of similar developmentally dependent expression of long-hinged forms of IgG in other species (IgG3 of human, horse, swine, and cattle) might reflect examples of parallel evolution of an IgG that plays an important role in the preadaptive immune response of mammals.


   Disclosures
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 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 research was supported by U.S. Department of Agriculture-Agricultural Research Service Cooperative Agreement 58-3625-4-155 and the University of Iowa Carver Trust. Back

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

3 Abbreviations used in this paper: CSR, class switch recombination; AID, activation-induced cytidine deaminase; BM, bone marrow: DG, day of gestations; IPP, ileal Peyer’s patch; JPP, jejunal Peyer’s patch; MAMP, microbe associated molecular pattern; MLN, mesenteric lymph node; MZ, marginal zone. Back

Received for publication May 25, 2006. Accepted for publication August 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Butler, J. E.. 1997. Immunoglobulin gene organization and the mechanism of repertoire development. Scand. J. Immunol. 45: 455-462. [Medline]
  2. Butler, J. E.. 2003. Disparate mechanisms drive antibody diversity among mammals: an important addition to immunology textbooks. Curr. Trends Immunol. 5: 1-18.
  3. Butler, J. E.. 2006. Why I agreed to do this (preface): antibody repertoire development. Dev. Comp. Immunol. 30: 1-17. [Medline]
  4. Nyugen, V. K.. 2001. Generation of heavy chain antibodies in camelids. PhD thesis 109-111. Free University of Brussels, Brussels, Belgium.
  5. Burnett, R. C., W. C. Hanly, S.-K. Zhai, K. L. Knight. 1989. The IgA heavy chain gene family in rabbit: cloning and sequence analysis of 13 C{alpha} genes. EMBO J. 8: 4041-4047. [Medline]
  6. Mestecky, J., I. Moro, M. A. Kerr, J. M. Woof. 2004. Mucosal immunoglobulins. J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, and L. Mayers, eds. Mucosal Immunology 153-181. Elsevier/Academic Press, Burlington, MA.
  7. Butler, J. E.. 1969. Bovine immunoglobulins. J. Dairy Sci. 52: 1895-1909. [Abstract/Free Full Text]
  8. Butler, J. E.. 1974. Immunoglobulins of the mammary secretions. B. L. Larson, and V. Smith, eds. In Lactation, a Comprehensive Treatise Vol. III: 217-255. Academic Press, New York.
  9. Kilian, M., M. W. Russell. 2004. Microbial evasion of IgA function. J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, and L. Mayer, eds. Mucosal Immunology 291-303. Elsevier/Academic Press, Amsterdam.
  10. Plaut, A. G., J. V. Gilbert, M. A. Artenstern, J. D. Capra. 1975. Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science 193: 1103-1105.
  11. Spieker-Polet, H., P.-C. Yam, K. L. Knight. 1993. Differential expression of 13 IgA heavy chain genes in rabbit lymphoid tissues. J. Immunol. 150: 5457-5465. [Abstract]
  12. Butler, J. E., M. E. Kehrle. 2004. Immunocytes and immunoglobulins in milk. J. Mestecky, and M. E. Lamm, and W. Strober, and J. R. McGhee, and L. Mayer, and J. Bienenstock, eds. Mucosal Immunology 3rd Ed.1763-1793. Academic Press, New York.
  13. Butler, J. E., D. Francis, J. Freeling, P. Weber, J. Sun, K. L. Nielsen, A. M. Krieg. 2005. Antibody repertoire development in fetal and neonatal piglets. IX. Three pathogen-associated molecular patterns act synergistically to allow germfree piglets to respond to TI-2 and TD antigens. J. Immunol. 175: 6772-6785. [Abstract/Free Full Text]
  14. Butler, J. E., J. Sun, N. Wertz, M. Sinkora. 2006. Antibody repertoire development in swine. Dev. Comp. Immunol. 30: 199-221. [Medline]
  15. Butler, J. E., J. Sun, P. Weber, S. P. Ford, Z. Rehakova, J. Sinkora, K. Lager. 2001. Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primary in fetal thymus occurs independent of environmental antigen and is only weakly associated with repertoire diversification. J. Immunol. 167: 3239-3249. [Abstract/Free Full Text]
  16. McAleer, J., P. Weber, J. Sun, J. E. Butler. 2005. Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B cell repertoire develops independently from that in blood and mesenteric lymph nodes. Immunology 114: 171-183. [Medline]
  17. Cukowska, B., J. Sinkora, L. Mandel, I Splichal, A. T. J. Bianchi, F. Kovaru. 1996. Thymic B cells of pig fetuses and germ-free pigs spontaneously produce IgM, IgG and IgA: detection by ELISPOT method. Immunology 87: 487-492. [Medline]
  18. Stavnezer, J., G. Radcliffe, Y-C. Lin, J. Nieutupski, L. Berggren, R. Sitia, E. Seversinson. 1988. Immunoglobulin heavy chain switching may be directed by prior induction of transcripts from constant region genes. Proc. Natl. Acad. Sci. USA 85: 7704-7708. [Abstract/Free Full Text]
  19. Arudchandran, A., R. M. Bernstein, E. E. Max. 2004. Single-stranded DNA breaks to cytosine occurs during Ig gene class switch recombination. J. Immunol. 173: 3223-3229. [Abstract/Free Full Text]
  20. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 102: 565-575. [Medline]
  21. Wang, C. L., M. Wabl. 2004. DNA acrobats of the Ig class switch. J. Immunol. 172: 5815-5821. [Abstract/Free Full Text]
  22. Rush, J. S., M. Liu, V. H. Odegard, S. Unniraman, D. G. Schatz. 2005. Expression of activation-induced cytidine deaminase is regulated by cell division providing a mechanistic basis for division-like class switch recombination. Proc. Natl. Acad. Sci. USA 102: 13242-13247. [Abstract/Free Full Text]
  23. Snapper, C. M., K. B. Marcu, P. Zelazowski. 1997. The immunoglobulin class switch: beyond "accessibility.". Immunity 6: 217-223. [Medline]
  24. Stavnezer, J.. 2000. Molecular process that regulate class switching. Curr. Top. Microbiol. Immunol. 245: 127-168. [Medline]
  25. Bergstedt-Lindquist, S., H.-B. Moon, U. Persson, G. Moller, C. Heusser, E. Severinson. 1988. Interleukin 4 instructs uncommitted B lymphocytes to switch to IgG1 and IgE. Eur. J. Immunol. 18: 1073-1077. [Medline]
  26. Coffman, R. L., D. A. Lebman, P. B. Rothman. 1993. Mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol. 54: 299-270.
  27. Collins, J. T., W. A. Dunnick. 1993. Germline transcripts of the murine immunoglobulin gene: structure and induction by IFN{gamma}. Int. Immunol. 5: 885-891. [Abstract/Free Full Text]
  28. Lebman, D., F. Lee, R. Coffman. 1990. Mechanisms for transforming growth factor beta and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures. J. Immunol. 144: 952-959. [Abstract]
  29. Lin, Y., J. Stavnezer. 1992. Regulation of transcription of the germline Ig {alpha} constant region gene by an ATF-element and by novel transforming growth factor beta-1 response elements. J. Immunol. 149: 2914-2925. [Abstract]
  30. Pan, Q., L. Hammarstrom. 2000. Molecular basis of IgG subclass deficiency. Immunol. Rev. 178: 99-110. [Medline]
  31. Modlin, R. L.. 2002. Mammalian toll-like receptors. Ann. Allergy Asthma Immunol. 88: 543-547. [Medline]
  32. Gathings, W. E., H. Kubagawa, M. D. Cooper. 1981. A distinctive pattern of B cell immaturity in perinatal humans. Immunol. Rev. 57: 107-126. [Medline]
  33. Miyano, A., T. Kiyamichi, M. Nakayama, H. Kitajima, A. Shimizu. 1996. Effect of chorioamnionitis on the levels of serum proteins in the cord blood of premature infants. Arch. Pathol. Lab. Med. 120: 245-248. [Medline]
  34. Quan, C. P., S. Watanabe, F. Forestier, J. P. Bouvet. 1998. Human amniotic IgA inhibits natural IgG autoantibodies of maternal or unrelated origin. Eur. J. Immunol. 28: 4001-4009. [Medline]
  35. Milili, M., M. Fougereau, P. Guglielmi, C. Schiff. 1991. Early occurrence of immunoglobulin isotype switching in human fetal liver. Mol. Immunol. 28: 753-761. [Medline]
  36. Kearney, J. F., M. D. Cooper, A. R. Lawton. 1976. B cell differentiation induced by lipopolysaccharide. IV. Development of immunoglobulin class restriction in precursors of IgG-synthesizing cells. J. Immunol. 117: 1567-1572. [Medline]
  37. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, J. F. Kearney. 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur. J. Immunol. 27: 2366-2374. [Medline]
  38. Manis, J. P., M. Tian, F. W. Alt. 2002. Mechanism and control of class-switch recombination. Trends Immunol. 23: 31-39. [Medline]
  39. Fischer, A.. 2004. Human primary immunodeficiency diseases: a perspective. Nat. Immunol. 5: 23-30. [Medline]
  40. Fagarasan, S., T. Honjo. 2000. T-independent immune response: new aspects of B cell biology. Science 290: 89-92. [Abstract/Free Full Text]
  41. He, B., X. Qiao, A. Cerutti. 2004. CpG DNA induces IgG class switch DNA recombination by activating human B cells through an innate pathway that requires TLR9 and cooperates with IL10. J. Immunol. 173: 4479-4491. [Abstract/Free Full Text]
  42. Melamed, D., E. Miri, N. Leider, D. Nemazee. 2000. Unexpected autoantibody production in membrane Ig-µ-deficient/lpr mice. J. Immunol. 165: 4353-4358. [Abstract/Free Full Text]
  43. Seagal, J., E. Edry, Z. Keren, N. Leider, O. Benny, M. Machluf, D. Melamed. 2003. A fail-safe mechanism for negative selection of isotype-switched B cells precursors is regulated by the Fas/FasL pathway. J. Exp. Med. 198: 1609-1619. [Abstract/Free Full Text]
  44. Williams, J., C. Euler, S. Christensen, M. J. Shlomchik. 2002. Evolution of autoantibody responses via somatic hypermutation outside of germinal centers. Science 297: 2066-2070. [Abstract/Free Full Text]
  45. Schroeder, H. W., Jr, J. L. Haillson, R. M. Perlmutter. 1987. Early restriction of the human antibody repertoire. Science 238: 791-793. [Abstract/Free Full Text]
  46. Yancopoulos, G. D., S. V. Desiderio, M. Paskind, J. F. Kearney, D. Baltimore, F. W. Alt. 1984. Preferential utilization of the most JH proximal VH gene segments in pre-B cell lines. Nature 311: 727-733. [Medline]
  47. Deenick, E. K., J. Hasbold, P. D. Hodgkins. 1999. Switching to IgG3, IgG2b, and IgA is division linked and independent revealing a stochastic framework for describing differentiation. J. Immunol. 163: 4707-4717. [Abstract/Free Full Text]
  48. Butler, J. E., M. Sinkora, N. Wertz, W. Holtmeier, C. D. Lemke. 2006. Development of the neonatal B- and T-cell repertoire in swine: implications for comparative and veterinary immunology. Vet. Res. 37: 417-441. [Medline]
  49. Kacskovics, I., J. Sun, J. E. Butler. 1994. Five subclasses of swine IgG identified from the cDNA sequences of a single animal. J. Immunol. 153: 3566-3573.
  50. Kehoe, J. M., J. D. Capra. 1974. Nature and significance of immunoglobulin subclasses. NY State J. Med. 74: 489-491. [Medline]
  51. Butler, J. E., P. Weber, M. Sinkora, J. Sun, S. J. Ford, R. Christenson. 2000. Antibody repertoire development in fetal and neonatal piglets. II. Characterization of heavy chain CDR3 diversity in the developing fetus. J. Immunol. 165: 6999-7011. [Abstract/Free Full Text]
  52. Lemke, C. D., J. S. Haynes, R. Spaete, D. Adolphson, A Vorwald, K. Lager, J. E. Butler. 2004. Lymphoid hyperplasia resulting in immune dysregulation is caused by PRRSV infection in pigs. J. Immunol. 172: 1916-1925. [Abstract/Free Full Text]
  53. Sun, J., J. E. Butler. 1996. Molecular characteristics of VDJ transcripts from a newborn piglet. Immunology 88: 331-339. [Medline]
  54. Sun, J., C. Hayward, R. Shinde, R. Christenson, S. P. Ford, J. E. Butler. 1998. Antibody repertoire development in fetal and neonatal piglets. I. Four VH genes account for 80% of VH usage during 84 days of fetal life. J. Immunol. 161: 5070-5078. [Abstract/Free Full Text]
  55. Butler, J. E., P. Weber, and N. Wertz. Antibody repertoire development in fetal and neonatal pigs. XIII. "Hybrid VH genes" and the pre-immune repertoire re-visited. J. Immunol. In press.
  56. Reynaud, C. A., C. R. MacKay, R. G. Miller, J. C. Weill. 1991. Somatic generation of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyers patches. Cell 64: 995-1005. [Medline]
  57. Jenne, C. N., L. J. Kennedy, J. D. Reynolds. 2006. Antibody repertoire development in the sheep. Dev. Comp. Immunol. 30: 165-174. [Medline]
  58. Wagner, B., D. C. Miller, T. L. Lear, D. F. Antczak. 2004. The complete map of the Ig heavy chain constant region reveals evidence of seven IgG isotypes and for IgD in the horse. J. Immunol. 173: 3230-3242. [Abstract/Free Full Text]
  59. Fulton, R. J., M. H. Nahm, J. M. Davie. 1983. Monoclonal antibodies to streptococcal group A carbohydrate. II. The Vk1GAC light chain is preferentially associated with serum IgG3. J. Immunol. 131: 1326-1331. [Abstract]
  60. Perlmutter, R. M., D. Hansburg, D. E. Briles, R. A. Nicolotti, J. M. Davie. 1978. J. Immunol. 121: 566-564. [Abstract/Free Full Text]
  61. Carlens, O.. 1928. Studien über das lymphatische Gewebe des Darmkanals bei einigen Haustieren, mit besonderer Berücksichtigung der embryonalen Entwicklung, der Mengenverhältnisse und der Altersinvolution dieses Gewebes im Dünndarm des Rindes. Z. Anat. Entwicklungsgesch. 86: 393-493.
  62. Pabst, R., M. Geist, H. J. Rothkötter, F. J. Fritz. 1988. Postnatal development and lymphocyte production of jejunal and ileal Peyers patches in normal and gnotobiotic pigs. Immunology 64: 539-544. [Medline]
  63. Barman, N. N., A. T. Bianchi, R. J. Zwart, R. Pabst, H. J. Rothkotter. 1997. Jejunal and ileal Peyer’s patches in pigs differ in their postnatal development. Anat. Embryol. 195: 41-50. [Medline]
  64. Butler, J. E., P. Weber, M. Sinkora, D. Baker, A. Schoenherr, B. Mayer, D. Francis. 2002. Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens. J. Immunol. 169: 6822-6830. [Abstract/Free Full Text]
  65. Weinstein, P. D., A. O. Anderson, R. G. Mage. 1995. Rabbit IgH sequences in appendix germinal centers: VH diversification by conversion-like and hypermutation mechanisms. Immunity 1: 647-659.
  66. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, T. Honjo. 2000. Class switch recombination and hypermutation requires activation-induced cytidine deaminase (AID), a potential editing enzyme. Cell 102: 553-563. [Medline]
  67. Lemke, C. D.. 2006. PRRSV infection of neonatal piglets induces immune dysregulation and modulation. PhD thesis University of Iowa, Iowa City, IA.
  68. Kelsall, B. L., F. Leon, L. E. Smythies, P. D. Smith. 2004. Antigen handling and presentation by mucosal dendritic cells and macrophages. J. Mestecky, Jr, and M. E. Lamm, Jr, and W. Strober, Jr, and J. Bienenstock, Jr, and J. R. McGhee, Jr, and L. Mayer, Jr, eds. Mucosal Immunology 3rd Ed.451-485. Academic Press, New York.
  69. Lopez-Carvalho, T., J. Foote, J. F. Kearney. 2005. Marginal zone B cells in lymphocyte activation and regulation. Curr. Opin. Immunol. 17: 244-250. [Medline]
  70. Sinkora, M., J. Sun, J. Sinkorova, R. K. Christenson, S. P. Ford, J.E. Butler. 2003. Antibody repertoire development in fetal and neonatal piglets. VI. B cell lymphogenesis occurs in multiple sites with differences in the frequency of in-frame rearrangements. J. Immunol. 170: 1781-1788. [Abstract/Free Full Text]
  71. Sinkora, M., J. E. Butler, W. Holtmeier, J. Sinkorova. 2005. Lymphocyte development in fetal piglets: facts and surprises. Vet. Immunol. Immunopathol. 108: 177-184. [Medline]
  72. Binns, R. M., R. Pabst, S. T. License. 1985. Lymphocytes immigrating from lymph nodes by blood in the pig and efferent lymph in the sheep. Immunology 54: 105-111. [Medline]
  73. Navarro, P., R. Christenson, P. Weber, M. Rothschild, G. Ekhard, J. Lemky, J. E. Butler. 2000. Porcine IgA allotypes are not equally transcribed or expressed in heterozygous swine. Mol. Immunol. 37: 653-664. [Medline]



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