The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38–110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

The generation of the Ab repertoire involves two rearrangement events. The first is the recombination of the V(D)J segments that takes place in primary lymphoid tissues. This is a site-specific recombination event that is mediated by recombination-activating gene-encoded recombinases that recognize specific heptamer and nonamer signal sequences (1). The second or “switch” recombination event is less precise in that break points vary widely but these are in any case deleted during RNA splicing (2). It is switch recombination that allows the Ag-specific heavy chain variable region, which is initially associated with the μ-chain of the B cell receptor, to be expressed with downstream heavy chains such as those of IgG subclasses IgA and IgE. Because the heavy chain constant regions of the various classes and subclasses of Abs mediate different biological functions, the process is important for full development of the Ab repertoire.

There is a considerable body of evidence indicating that switch recombination is mediated through costimulation by helper T cells involving CD40 and B7-1/B7-2 (3, 4, 5, 6, 7). For example, switch recombination in mice to IgG1 and IgE requires costimulation through CD40 and either IL-4 or IL-13 (8), whereas TGF-β mediates the switch to IgA (3). Switch recombination together with somatic mutation and receptor editing are major molecular events that occur as part of the germinal center (GC)3 reaction (5, 9, 10, 11, 12). Because the GC reaction occurs in response to stimulation with T-dependent Ags, switch would not be expected to occur in an environment free of T-dependent environmental Ags. Nevertheless, there are reports of IgA present as early as the second trimester in human fetuses (13, 14, 15). IgA-containing cells have been reported to be present in 180-day fetuses (16), and transcripts can be detected on day 110 (17). In addition to IgA, Cγ2 and Cγ4 transcripts have also been detected in fetal human liver (18), and switch can even occur at the time of VDJ rearrangement (19). Because LPS alone can stimulate switch recombination (20, 21, 22) by an unknown mechanism (N. Maizels, unpublished observations) the requirements for T dependence and the GC reaction are not absolute. Although lymphotoxin-α knockout mice are unable to form GC and to somatically mutate their variable region genes, they are still capable of producing IgG Abs to some Ags (23).

These observations raise the question of whether switch recombination is exclusively Ag driven and whether it is coupled to somatic mutation in vivo. Unfortunately, the human fetus is a poor model because active transport of maternal IgG to the fetus progressively increases during the third trimester, and eventually concentrations in cord blood exceed maternal levels just before birth (24, 25, 26). Furthermore, allergen-primed T cells can be found in the human fetus suggesting that allergens also can cross the placenta after they have been encountered by the pregnant mother either alone or as part of transported IgG immune complexes (27, 28, 29, 30). Although such cells could be of maternal origin, analysis of microsatellite DNA suggests that they are indeed of fetal origin (27). Responsive cells can be found even in the first 6 mo of pregnancy (29, 30), suggesting that allergens may cross the placenta alone during this stage of pregnancy and do not require shuttle as IgG immune complexes. Thus, low-level stimulation of the human fetus by T-dependent Ags that cross the placenta alone or as immune complexes may account for any fetal switched isotypes found.

Unlike the human, it has been generally believed that maternal Igs do not enter the circulation of the fetal piglet (24, 25) although there is evidence that switched isotype Igs, i.e., IgG and IgA, are present in fetal piglet serum (31, 32). Some have maintained that these are due to contamination by maternal blood (33, 34) resulting either from placental damage or contamination during collection. Although contamination may explain the IgG and IgA in fetal sera, it cannot explain the occurrence of IgA transcripts in late-term and newborn piglets (35, 36) or the presence of IgA- and IgG-secreting cells (SC) (37). This suggests that isotype switch is spontaneous and independent of environmental Ag in this species or that it results from environmental Ags such as virus, which can cross the six-layer epitheliochorial placenta of the pig by unknown mechanisms (38, 39) just as HIV crosses the placenta in humans (40).

Here we report that IgG and sometimes IgA and IgM can be detected in fetal serum as early as 38 days in utero (114 days of gestation, DG) and that IgG concentrations are ∼10-fold higher than IgM concentrations throughout gestation. The concentration of the switched isotypes markedly increases in the last 15 DG with IgA levels equal to or exceeding IgM levels and IgG levels nearly 20-fold higher. With rare exception, the isotype profile of normal fetal piglets and those infected with the PRRS (porcine respiratory and reproductive syndrome) virus, are statistically distinct from the profile in maternal serum. These exceptions notwithstanding, fetal serum Igs do not appear to result from placental leakage or contamination by maternal blood during collection. We also show that transcription of IgA and IgG first occurs midway through gestation, is especially pronounced in the thymus, and generates a highly polyclonal repertoire of switched isotypes. This repertoire is not hypermutated like that in fetal piglets that have encountered environmental Ag though viral infection. Further evidence that this polyclonal repertoire of switch isotypes develops intrinsically is demonstrated by its development in a noninductive site of the mucosal immune system of piglets maintained in germfree isolators.

Yorkshire × Meishan F1 crosses from Iowa State University (Ames, IA) and Minnesota minipigs × Vietnam Asian crosses from Novy Hradek (Czech Republic) were the sources of all normal fetal samples. Yorkshires from the National Animal Disease Center (Ames, IA) were used for viral inoculation studies and Yorkshires from a local supplier at South Dakota State University (Brookings, SD) were used in all isolator piglet studies. Animals were hand-mated, and all mothers were healthy and normal at the time of euthanization. The fetuses were immediately removed from the gravid uterus. Blood from >130 normal fetuses was collected when fetuses were at or near 40, 50, 60, 70, 90, 95, 105, and 110 days of age. The umbilical cord was carefully washed with water and saline to remove any traces of maternal blood and samples were then collected from the umbilical vein. Blood samples were also collected from 1) 27 mother pigs, including those of the fetuses studied, 2) the jugular vein or retro-orbital sinus of 29 colostrum-deprived newborn piglets, and 3) 36 fetal piglets experimentally infected in utero with an attenuated variant of the PRRS virus. Infection with the PRRS virus should theoretically allow us to test whether VDJs expressed with switched isotypes in animals with known exposure to environmental Ag differed in diversification from those in normal fetuses. Fetal liver was collected from 30-, 40-, and 50-day fetuses. Spleen was collected from all 40-day and older fetuses, and a variety of lymphoid tissues were collected when animals were 70 days and older.

Total RNA was prepared using TRIzol reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA integrity was verified by staining with ethidium bromide after agarose electrophoresis in formaldehyde-containing gels. First-strand cDNA was prepared as follows: Five micrograms of total RNA and 1 μl of the oligonucleotide mixture (IgG anti-sense CH2 (5 pmol/μl), IgM anti-sense CH2 (5 pmol/μl), IgA anti-sense CH3 (5 pmol/μl), and a random hexamer (10 pmol/μl)) were mixed together, and water was added up to a total volume of 15 μl. The mixture was heated at 85°C for 10 min and immediately cooled on ice. The first-strand cDNA synthesis solution contained 6 μl of 5× first-strand cDNA buffer, 20 U of RNasin, 300 U of Moloney murine leukemia virus reverse transcriptase, 100 μg/ml BSA, and 1 mM DTT and water to 15 μl. The denatured RNA sample was transferred into the first-strand cDNA synthesis mixture and incubated at room temperature for 10 min, then at 37°C for 40 min, and finally at 50°C for 10 min.

First-strand cDNA and the isotype-specific anti-sense primers together with a FR1-5′ end primer (see Table I) were used to amplify segments of Cμ, Cα, and Cγ. β-actin segments were amplified from these same tissues using the β-actin primer set described in Table I. All reactions were performed in a 25-μl volume containing 2.5 μl of PCR buffer, 5 U of KlenTaq (AM Peptides, St. Louis, MO), 7.5 pmol of primers, 0.5 μl of cDNA, 2.5 mM dNTP, and ∼20 μl of dH2O. Amplification was performed in a MJ-200 DNA engine under the following conditions: Initial denaturation was for 1 min at 94°C, followed by 30 cycles of denaturing at 94°C for 40 s, and then annealing at 58°C for 30 s followed by an extension at 72°C for 20 s. All products were then examined by agarose electrophoresis and Southern blotting to verify their size and identity. Five microliters of each PCR product (Cμ, Cα, Cγ, and β-actin) from the same cDNA preparation was applied to wells in a 96-well membrane transfer apparatus (Pierce, Rockford, IL) in 200 μl of 20× SSC and then blotted onto a nylon membrane as previously described (41, 42). The transferred DNA was then cross-linked to the nylon membrane by exposure to UV radiation. After prehybridization in a buffer containing 5× Denhardts solution, 0.2% SDS, 3× SSC at 53°C for 1 h, the membrane was transferred to a tube containing the same hybridization buffer and a 32P-end-labeled probe specific for Cμ, Cα, Cγ, or β-actin. Hybridization was conducted for 4 h or overnight (no differences were observed between 4 h and overnight). The membranes were rinsed twice in medium stringency buffer (0.125× SSC, 0.1% sodium pyrophosphate, and 0.1% SDS) for 20 min each. The membranes were then wrapped in Cling Wrap (Glad, Danbury, CT), and the signals were measured in a Packard Instant Imager (Palo Alto, CA). The membranes were then stripped, monitored for residual radioactivity, and hybridized with a different 32P-end-labeled probe. Representative raw data are shown in Fig. 2 and Table II. In each case, controls containing only single isotype PCR products were used, and these served to test for any cross-hybridization (see Fig. 2).

Table I.

Oligonucleotides for cDNA synthesis, PCR, and hybridizationa

No.DescriptionSequence
IgG antisense Cγ2 5′-ccgtccacgtaccaggagaa-3′ 
IgM antisense Cμ4 5′-ccacggtcctctcggtca-3′ 
IgA antisense Cα3 5′-agcctacgcaccagcacat-3′ 
FR3 sense 5′-tgagaaccgaagacacggc-3′ 
IgM antisense Cu1-2 5′-tcacagagggtaggagca-3′ 
IgG antisense Cγ2 5′-ccaccaccacgcacgtga-3′ 
IgA antisense Cα2 5′-gagcccaggagcaggt-3′ 
Pig actin sense 5′-ggtcatcaccatcggcaa-3′ 
Pig antisense actin 5′-gatccacacggagtactt-3′ 
10 Pig actin-hybridization 5′-ggactccatgcccaggaa-3′ 
11 IgA CH1-hybridization 5′-ccgtgaacgtgccctgcaa-3′ 
12 IgM antisense CH1-hybridization 5′-ctctcaggacggacgggaagt-3′ 
13 IgG CH1-hybridization 5′-tgcctggcctcaagctactt-3′ 
No.DescriptionSequence
IgG antisense Cγ2 5′-ccgtccacgtaccaggagaa-3′ 
IgM antisense Cμ4 5′-ccacggtcctctcggtca-3′ 
IgA antisense Cα3 5′-agcctacgcaccagcacat-3′ 
FR3 sense 5′-tgagaaccgaagacacggc-3′ 
IgM antisense Cu1-2 5′-tcacagagggtaggagca-3′ 
IgG antisense Cγ2 5′-ccaccaccacgcacgtga-3′ 
IgA antisense Cα2 5′-gagcccaggagcaggt-3′ 
Pig actin sense 5′-ggtcatcaccatcggcaa-3′ 
Pig antisense actin 5′-gatccacacggagtactt-3′ 
10 Pig actin-hybridization 5′-ggactccatgcccaggaa-3′ 
11 IgA CH1-hybridization 5′-ccgtgaacgtgccctgcaa-3′ 
12 IgM antisense CH1-hybridization 5′-ctctcaggacggacgggaagt-3′ 
13 IgG CH1-hybridization 5′-tgcctggcctcaagctactt-3′ 
a

Primers 1–3 were used for first-strand cDNA synthesis. Primers 4–9 were used for PCR. Primers 10–13 were only used for hybridization.

FIGURE 2.

Representative hybridization data using mixed PCR products from four tissues at three different ages with probes specific for IgM, IgG, and IgA heavy chain genes. Spl, Spleen; Thy, thymus. Raw radioanalytical scanner values, uncorrected for background, are given next to the corresponding blots. Controls on the right test the specificity of 32P-labeled probes for IgM, IgG, and IgA to hybridize with single isotype PCR products.

FIGURE 2.

Representative hybridization data using mixed PCR products from four tissues at three different ages with probes specific for IgM, IgG, and IgA heavy chain genes. Spl, Spleen; Thy, thymus. Raw radioanalytical scanner values, uncorrected for background, are given next to the corresponding blots. Controls on the right test the specificity of 32P-labeled probes for IgM, IgG, and IgA to hybridize with single isotype PCR products.

Close modal
Table II.

Representative hybridization data obtained with 32P-isotype- and β-actin-specific probesa

Tissue and DGAnimal No.IgAC.V.IgGC.V.IgMC.V.ActinC.V.
60 DG THY 63,392 8.9 30,022 9.7 23,159 4.1 36.473 9.3 
 10,712 8.3 7,803 12.1  6,047 4.0 34,533 4.7 
 5,407 6.8 7,837 5.8  7.946 7.7 29,878 1.9 
 25,310 17.8 16,664 25.9 26,293 12.4 32,535 7.8 
Mean C.V.   10.4  13.3  7.0  4.0 
60 DG SPL 1,032 17.1 1,313 23.7 153,253 1.6 35,590 1.5 
 −505 b 87.6 53 34 174,337 2.6 31.381 35 
 −2,567 1.8 1,462 63 13,473 4.9 21,014 34 
 −862 14.7 1,329 67 6,003 9.2 28,813 28 
Mean C.V.   30.3  46  10.0  24.6 
90 DG THY 176,651 15.3 143,201 9.9 15,335 8.3 49,980 43 
 308,426 1.2 220,487 7.1 36.561 8.4 41,011 32 
 36,045 9.3 36,045 9.3 11,844 2.3 39,728 1.7 
 110,945 1.1 110,947 10.7 33,178 18.6 42,683 24 
Mean C.V.  6.7  9.3  9.4  25  
110 DG THY 109,149 1.0 207,986 1.9 17,606 17.8 29,511 7.2 
 204,516 4.9 154,069 1.8 28,564 12.1 32,204 22.8 
 104,709 16.7 166,819 14.1 16,409 12.3 33,211 6.7 
 147,717 3.9 102,690 7.2 18,647 10.3 36,001 23.8 
Mean C.V.  6.6  6.25  13.1  15.1  
Tissue and DGAnimal No.IgAC.V.IgGC.V.IgMC.V.ActinC.V.
60 DG THY 63,392 8.9 30,022 9.7 23,159 4.1 36.473 9.3 
 10,712 8.3 7,803 12.1  6,047 4.0 34,533 4.7 
 5,407 6.8 7,837 5.8  7.946 7.7 29,878 1.9 
 25,310 17.8 16,664 25.9 26,293 12.4 32,535 7.8 
Mean C.V.   10.4  13.3  7.0  4.0 
60 DG SPL 1,032 17.1 1,313 23.7 153,253 1.6 35,590 1.5 
 −505 b 87.6 53 34 174,337 2.6 31.381 35 
 −2,567 1.8 1,462 63 13,473 4.9 21,014 34 
 −862 14.7 1,329 67 6,003 9.2 28,813 28 
Mean C.V.   30.3  46  10.0  24.6 
90 DG THY 176,651 15.3 143,201 9.9 15,335 8.3 49,980 43 
 308,426 1.2 220,487 7.1 36.561 8.4 41,011 32 
 36,045 9.3 36,045 9.3 11,844 2.3 39,728 1.7 
 110,945 1.1 110,947 10.7 33,178 18.6 42,683 24 
Mean C.V.  6.7  9.3  9.4  25  
110 DG THY 109,149 1.0 207,986 1.9 17,606 17.8 29,511 7.2 
 204,516 4.9 154,069 1.8 28,564 12.1 32,204 22.8 
 104,709 16.7 166,819 14.1 16,409 12.3 33,211 6.7 
 147,717 3.9 102,690 7.2 18,647 10.3 36,001 23.8 
Mean C.V.  6.6  6.25  13.1  15.1  
a

Data given as cpm.

b

Negative values indicate that hybridization was less than mean background values.

The ratio of the signal obtained with the isotype-specific probe to that obtained using the β-actin probe was designated “relative transcription”; mean data from 4–6 animals at each time point for each tissue/organ are presented in Fig. 3. Because the measured signal depends on the sp. act. of the probe used, relative transcription is only quantitatively valid for comparing a particular isotype among tissues but not directly for comparing different isotypes. Furthermore, all assays must be conducted on samples from all animals simultaneously to avoid bias caused by differences in the sp. act. of the 32P-probes used.

FIGURE 3.

Transcription of IgA, IgG, and IgM relative to β-actin in the spleen, BM, thymus, and IPP presented as the mean and SD of data from 4–6 fetal piglets at four time points during gestation. The y-axis of some plots are in log scale so low values can be visualized.

FIGURE 3.

Transcription of IgA, IgG, and IgM relative to β-actin in the spleen, BM, thymus, and IPP presented as the mean and SD of data from 4–6 fetal piglets at four time points during gestation. The y-axis of some plots are in log scale so low values can be visualized.

Close modal

In the initial test we collected data from a broad range of lymphoid tissues, but for our final analysis we decided to focus only on a few lymphoid tissues from a larger number of animals. We chose to compare spleen, bone marrow (BM), thymus, and ileal Peyer’s patches (IPP) from 4–6 animals from different litters at four different developmental time points. Spleen and thymus can be recovered at all four time points, BM was recovered from fetuses 60 days and older, but IPP was only recovered at 90 and 110 DG. The selection of these tissues was also based on their immunobiological relevance. BM is considered a major site for B cell lymphogenesis at and after 60 days (43), the spleen is a well-recognized secondary lymphoid tissue in all species, the IPP is of special interest because it is regarded in sheep as a major site for repertoire development (44), and the thymus was chosen because of renewed interest in its role as a B cell organ (45). We also studied the parotid gland, because in contrast to the IPP, it is regarded as a noninductive site of the mucosal immune system (46).

Anti-sense Cμ, Cα, and Cγ primers were used together with a FR1 sense primer to amplify the rearranged VDJs transcribed with IgM, IgA, and IgG, respectively, from the thymus of both normal and PRRS-infected fetuses. The resultant PCR products were then cloned into pBluescript as previously described (35), and 33 IgM-, 34 IgA-, and 32 IgG-associated VDJ clones were selected at random for sequencing. Clones were sequenced using the T3 primer and the four-color automated Applied Biosystems (Foster City, CA) system. Sequences were analyzed for identification of VH usage, for mutation frequency in complementarity-determining region (CDR)1, CDR2, and CDR3, CDR3 length, DH usage and length, and N and P region additions in the manner previously described (47, 48). Eight of the clones were duplicates; Table V summarize only the data from the 91 unique clones.

Table V.

Sequence analysis of thymic VDJ transcripts

Transcript SourceNo. ExaminedMutation FrequencyaTotal MutationsaCharacteristic of CDR3(VHA + VHC + VHXc )/(VHB + VHE)
CDR1CDR2CDR3Mean length ± S.D.DHA:DHBDHA lengthDHB lengthN regionb
Normal IgM 17 0.019 0.014 0.010 0.015 44.5 ± 10.4 1.4 19.8 ± 7.5 13.4 ± 7.6 17.8 ± 6.2 0.94 
Normal IgA 15 0.067♦ 0.037ΔΔ 0.008♦ 0.034ΔΔ 43 ± 11.7 0.67 17.6 ± 3.10 13.8 ± 5.6 17.7 ± 8.9 0.92 
Normal IgG 12 0.039♦ 0.020♦ 0.000♦ 0.020Δ 34.2 ± 6.9 0.10 25 12.1 ± 5.3 10.0 ± 5.8 3.8 
PRRS IgMd 13 0.011ns 0.022** 0.084** 0.033 43.0 ± 10.8 1.0 20.3 ± 8.35 18.5 ± 7.8 9.23 ± 4.18 5.5 
PRRS IgAd 19 0.046nsΔ 0.046**ΔΔ 0.101**♦ 0.057ΔΔ 38.5 ± 9.4 2.7 18.2 ± 9.5 12.0 ± 5.3 11.9 ± 6.4 1.7 
PRRS IgGd 15 0.057nsΔΔ 0.051**ΔΔ 0.120**♦ 0.062ΔΔ 38.1 ± 12.1 3.3 18.1 ± 8.5 10.7 ± 4.0 11.8 ± 7.6 5.0 
6-wk germfreee 19 0.012ns 0.017ns 0.023ns 0.013 43 ± 13.9 0.27 17.5 ± 8.5 20.5 ± 6.0 12.5 ± 9.8 0.5 
6-wk colonizede 21 0.081** 0.076* 0.058* 0.076 37 ± 13.9 2.0 15.6 ± 8.7 19.0 ± 8.2 10.0 ± 3.9 13.0 
Adulte 34 0.144** 0.186** 0.062* 0.156 45.5 ± 14.1 1.7 10.8 ± 7.3 9.8 ± 5.6 22.4 (?) (8.5) 
Transcript SourceNo. ExaminedMutation FrequencyaTotal MutationsaCharacteristic of CDR3(VHA + VHC + VHXc )/(VHB + VHE)
CDR1CDR2CDR3Mean length ± S.D.DHA:DHBDHA lengthDHB lengthN regionb
Normal IgM 17 0.019 0.014 0.010 0.015 44.5 ± 10.4 1.4 19.8 ± 7.5 13.4 ± 7.6 17.8 ± 6.2 0.94 
Normal IgA 15 0.067♦ 0.037ΔΔ 0.008♦ 0.034ΔΔ 43 ± 11.7 0.67 17.6 ± 3.10 13.8 ± 5.6 17.7 ± 8.9 0.92 
Normal IgG 12 0.039♦ 0.020♦ 0.000♦ 0.020Δ 34.2 ± 6.9 0.10 25 12.1 ± 5.3 10.0 ± 5.8 3.8 
PRRS IgMd 13 0.011ns 0.022** 0.084** 0.033 43.0 ± 10.8 1.0 20.3 ± 8.35 18.5 ± 7.8 9.23 ± 4.18 5.5 
PRRS IgAd 19 0.046nsΔ 0.046**ΔΔ 0.101**♦ 0.057ΔΔ 38.5 ± 9.4 2.7 18.2 ± 9.5 12.0 ± 5.3 11.9 ± 6.4 1.7 
PRRS IgGd 15 0.057nsΔΔ 0.051**ΔΔ 0.120**♦ 0.062ΔΔ 38.1 ± 12.1 3.3 18.1 ± 8.5 10.7 ± 4.0 11.8 ± 7.6 5.0 
6-wk germfreee 19 0.012ns 0.017ns 0.023ns 0.013 43 ± 13.9 0.27 17.5 ± 8.5 20.5 ± 6.0 12.5 ± 9.8 0.5 
6-wk colonizede 21 0.081** 0.076* 0.058* 0.076 37 ± 13.9 2.0 15.6 ± 8.7 19.0 ± 8.2 10.0 ± 3.9 13.0 
Adulte 34 0.144** 0.186** 0.062* 0.156 45.5 ± 14.1 1.7 10.8 ± 7.3 9.8 ± 5.6 22.4 (?) (8.5) 
a

Mutations/nucleotide. In the case of CDR3, mutation/nucleotide could only be recognized in DH and the JH sequence 5′ of the invariant tryptophan codon. Student’s t test was used to test the hypothesis that the mutation frequency in transcripts from PRRS-infected, germfree, colonized, and adult mice differ from thymic transcripts in normal fetuses. **, Significantly different from normal transcript at 0.01 level; *, Significantly different at 0.05 level; ns, Nonsignificant. Statistical comparisons to previously published transcripts not separated according to isotype were performed against a pooled average of IgM + IgA + IgG transcripts. A second statistical analysis was undertaken to compare the mutation frequency in switched isotypes (IgA and IgG) compared to that in IgM transcripts from the same animal group. ♦, Not significantly different from IgM transcripts; Δ, significantly different at the 0.05 level; ΔΔ, significantly different at the 0.01 level.

b

Sum of N region addition 5′ and 3′ of DH.

c

Usage of VHC and VHA together with seldom used genes like VHL or VHK characterizes differentiation of the porcine VH repertoire, whereas usage of especially VHB (analogous to VH81 × in mice) and to a lesser extend VHE, reflects the undiversified repertoire. VHX includes VH genes such as VHL and VHK, which are periodically used in diversified repertoires. Thus the ratio of VH usage is a useful parameter of repertoire diversification.

d

Data from 95-day fetuses infected at 50 days in utero with PRRS virus.

e

Data calculated from previously published sequences obtained from various nonthymic sites (4647 ). The frequency of somatic mutation in the CDR3 region of adult sequences is underestimated because of the difficulty in distinguishing highly mutated DH segments from nucleotide additions.

The serum levels of these Igs were quantified by sandwich ELISA. In the case of IgA, the globulin fraction of an α-chain-specific rabbit anti-IgA was used (B5321) for capture, whereas mAb 1459 was used for detection (provided by Dr. K. Nielsen, Animal Disease Research Institute, Nepean, Ontario, Canada). mAb 5C9B12 was used to capture IgM (provided by Dr. P. Paul, Iowa State University, Ames, IA) while detection was accomplished using rabbit anti-swine L chain conjugated to alkaline phosphatase. IgG was captured using the globulin fraction of a γ-chain-specific polyclonal antiserum (B784 + 5) and detected using the same anti-L chain conjugate described above for measuring IgM. Methods for the preparation of all polyclonal Abs and purified IgM, IgG, and IgA reference standard sera have been previously described (36, 49, 50, 51). All sandwich assays were conducted as previously described (47, 50, 52, 53).

Recovery experiments, designed to test the effect of contaminating 500 μl of several fetal sera with 1 μl of an adult serum, were performed by quantitation of IgG, IgA, and IgM in the fetal sera before and after “spiking” and in the adult serum used for spiking. The relationship of the concentrations of IgG, IgA, and IgM was established by expressing IgA and IgM levels relative to IgG and assigning the latter a value of 100. This generates an isotype profile (see Fig. 1 B).

FIGURE 1.

The serum concentration of IgG, IgA, IgM, and their isotype profiles in the sera of >150 fetal and newborn piglets. A, Data presented as the mean Ig level (micrograms per milliliter) ± SEM in which the y-axis is a log scale. The absolute mean value for each Ig at each stage of gestation is indicated. Only trace amounts of IgA and IgM were detected in the minority of 38- and 57-day samples, so mean values for these time points are not given. B, Serum Ig profiles for adult (gilts), newborn, and fetal piglets. Error bar indicate the SEM. ∗∗, Significantly different from the Ig profile in gilts at the 0.01 level; ∗, significantly different from the profile of gilts at the 0.05 level. Spiking data are based on a test with four fetal sera in which spiker is an adult serum used to contaminate the fetal samples. Because this is a different adult serum than any of the 27 gilts tested, statistical comparison between gilt sera and spiker would be meaningless. Values for spiked samples are the mean of four fetal samples. #, Statistical comparisons were between spiker and spiked, and these were not statistically different.

FIGURE 1.

The serum concentration of IgG, IgA, IgM, and their isotype profiles in the sera of >150 fetal and newborn piglets. A, Data presented as the mean Ig level (micrograms per milliliter) ± SEM in which the y-axis is a log scale. The absolute mean value for each Ig at each stage of gestation is indicated. Only trace amounts of IgA and IgM were detected in the minority of 38- and 57-day samples, so mean values for these time points are not given. B, Serum Ig profiles for adult (gilts), newborn, and fetal piglets. Error bar indicate the SEM. ∗∗, Significantly different from the Ig profile in gilts at the 0.01 level; ∗, significantly different from the profile of gilts at the 0.05 level. Spiking data are based on a test with four fetal sera in which spiker is an adult serum used to contaminate the fetal samples. Because this is a different adult serum than any of the 27 gilts tested, statistical comparison between gilt sera and spiker would be meaningless. Values for spiked samples are the mean of four fetal samples. #, Statistical comparisons were between spiker and spiked, and these were not statistically different.

Close modal

The CDR3 segments of the isotype-specific cDNAs described above were amplified using nested FR3 and anti-sense FR4 primers, which anneal to all porcine VDJ rearrangement (35, 42). These were separated on 6% polyacrylamide gels as described above and elsewhere (48). The resultant spectratypes were scanned, and different tissues of the same individual piglets were compared according to isotype.

The number of spontaneous Ig-SCs of all isotypes was determined among thymocytes, splenocytes, BM leukocytes, and lymphocytes isolated from cord blood, liver, PBMC, Peyer’s patches, and mesenteric lymph nodes (MLN) by the ELISPOT assay using isotype-specific mAbs as previously described (37). The following murine anti-swine mAbs were used as capture Abs: anti-IgM (LIG-4; Ref. 54) and anti-IgG (23.3 1a) and anti-IgA (27.9.1; both obtained from van Zaane and Holst; Ref. 55). Detection of captured Igs was visualized using a mixture of biotinylated anti-κ and -λ mAbs 27.7.1 and 27.2.1 and the streptavidin-HRP system. Data are expressed as 1) the number of Ig-SCs (according to isotype) per 106 cells (Table III), and 2) the total number of Ig-SC in each organ examined by multiplying Ig-SC per million times the total number of leukocytes recovered from each organ (Table IV).

Table III.

Ig-SC in various fetal lymphoid tissue

Tissue and FetusIg-SC per 106 Cellsa
IgMIgGIgATotal
Thymus 1 40 120 30 190 
Thymus 2 102 120 120 340 
Thymus 3 100 100 100 300 
Spleen 1 120 120 
Spleen 2 112 112 
Spleen 3 153 153 
Tissue and FetusIg-SC per 106 Cellsa
IgMIgGIgATotal
Thymus 1 40 120 30 190 
Thymus 2 102 120 120 340 
Thymus 3 100 100 100 300 
Spleen 1 120 120 
Spleen 2 112 112 
Spleen 3 153 153 
a

Assays were performed using different numbers of plating cells for spleen vs thymus, and results were adjusted to ELISPOTs/106 plating cells.

Table IV.

Number of Ig-SCa

Animal TestedFetal LiverSpleenThymusBMMLNBlood% of Ig-SC in the Thymusb
Fetus no. 1 7200 15000 68 
Fetus no. 2 9300 32000 77 
Fetus no. 3 13000 28000 68 
Animal TestedFetal LiverSpleenThymusBMMLNBlood% of Ig-SC in the Thymusb
Fetus no. 1 7200 15000 68 
Fetus no. 2 9300 32000 77 
Fetus no. 3 13000 28000 68 
a

Total number of Ig-SC recovered from various lymphoid tissues was calculated by multiplying the sum of IgM + IgA + Ig-SC determined per 106 cells times the total number of leukocytes recovered from the various organs as determined by hemocytometer.

b

Calculated as Ig-SC in thymus/Ig-SC cell in all tissues × 100.

The Ig profiles in the serum of fetal piglets of different age (DG) and treatment (e.g., PRRS-infected vs normal fetuses) were compared by Student’s t test to the mean profile established using the sera of 27 birth mothers. We tested the null hypothesis that normalized IgA and IgM levels in the sera of fetuses and newborns was identical with that in the sera of gilts. The same statistical treatment was applied to the comparison of mutation frequencies in transcripts from the thymus of normal vs PRRS-infected fetuses.

IgG was detected in the sera of all 38-day and older fetuses, whereas IgM and IgA was only detected in 2/7 of the 38-day samples and in only 1/6 of the 57-day samples (data not shown). Thereafter, all three isotypes were detected in every specimen and increased in concentration with fetal age (Fig. 1,A). In all very young fetuses and in animals 70 days and older, IgG was present at concentrations ∼10-fold higher than IgM levels. Because this concentration relationship, i.e., isotype profile, is superficially reminiscent of that in the sera of the gilts (but at a 1000-fold lower concentration), it seemed probable that the Igs in fetal serum were of maternal origin and their age-dependent increase was the result of increasing low-level placental leakage or contamination during the collection process. The extent to which such contamination of fetal sera with maternal serum could alter fetal Ig levels was tested by adding 1 μl of maternal serum to 500 μl of four different fetal serum samples containing very low levels of Igs, i.e., 0.734 ± 0.38 μg/ml IgG. When the spiked fetal sera were measured, their Ig levels increased 43-fold. The adult serum used for spiking contained 16.98 mg/ml IgG, 487 μg/ml IgA, and 5.08 mg/ml IgM. The mean concentrations of Igs recovered from four spiked fetal sera were 31.8 ± 1.8 μg IgG/ml, 916 ± 121 ng/ml IgA, and 9.86 ± 0.47 μg/ml IgM. The expected values were 33.9 μg/ml IgG, 975 ng/ml IgA, and 10.16 μg/ml IgM. Thus, the spiked fetal sera gave exactly the same profile as the maternal serum used for spiking (Fig. 1,B). Based on the outcome of this in vitro test, we reasoned that contamination during collection could be tested by comparing the isotype profiles of fetal and maternal sera. IgM and IgA concentrations were normalized to that of IgG and the latter assigned a value of 100. Representative isotype profiles of fetuses of different age, newborn piglets, and of their birth mothers are shown in Fig. 1,B. Relative IgM levels were always significantly lower than in maternal sera, and IgA levels especially increased in late gestation, attaining values 2-fold higher than IgM in colostrum-deprived newborns. This difference is significant by Student’s t test (Fig. 1, A and B). When the isotype profiles of individual birth mothers were compared with the isotype profiles of their own offspring, there was no correlation (data not shown) similar to the lack of correlation seen with nonlitter-segregated animals (Fig. 1 B).

Because porcine Parvovirus and the arterivirus responsible for PRRS have been reported to cross the placenta by an unknown mechanism, we wondered whether especially the Igs of switch isotypes in fetal serum represented an immune response to an environmental Ag. Thus, we infected fetuses in utero at 50 DG and monitored the immune response using a PRRS-specific ELISA (data not shown). The fetal response to PRRS virus is latent, perhaps because the number of target cells for the arterivirus is limiting (56). An anti-PRRS virus response is only weakly detectable by day 95, but such fetuses show a >2-fold elevation of IgG, >10-fold increase in IgA, and >5-fold increase in IgM compared with normal piglets of nearly the same age (data not shown). At 110 days, when anti-PRRS ELISA titers were robust, IgM levels were elevated 50-fold and IgG levels 5- to 10-fold compared with age-matched normal fetuses. This yielded serum Ig levels much greater than seen in normal age-matched fetuses (data not shown) and an isotype profile that differed from that in the sera of gilts because of relatively lower levels of IgA and higher levels of IgG (Fig. 1 B).

Representative raw hybridization results are shown in Fig. 2. As shown, cross-hybridization values were equal or less than background scanner values except for the IgG probe in which 1.2% cross-reactivity with IgA was observed. Table II gives scanner results obtained when equal volumes of isotype or β-actin PCR products were hybridized with 32P isotype or β-actin probes. Representative data were selected from those with very low transcription (IgA and IgG in 60 DG spleen) to those with high levels of transcripts (90 DG and 110 DG thymus). Table II provides insight into animal variation and assay reproducibility including cases of extreme variation. The latter were typically encountered when values close to background were encountered (IgA and IgG in 60 DG spleen). Nevertheless, an overall coefficient of variation (C.V.) of <12% was obtained between replicates. Extreme animal differences were seldom seen, although animals 2 and 3 differ nearly 10-fold in IgA thymic transcription but only 3-fold in IgM transcription.

When transcription of the IgM, IgG, and IgA were expressed relative to β-actin for 4–6 animals at each time point, IgM transcription was observed in all lymphoid tissues available at 50 DG (Fig. 3). IgM relative transcription was especially pronounced in spleen and BM at and after 60 DG. Although some individual splenic samples showed trace amounts of transcripts for switch isotypes (Table II), pronounced transcription was not observed until 60 DG in thymus (Fig. 3). Noteworthy is that pronounced transcription of these switched isotypes was confined to the thymus, in sharp contrast to that seen for IgM (Fig. 3). However, hybridization of a Cμ probe with thymic cDNA from 90- and 110-day fetuses was pronounced (Table II, Fig. 2). By 110 days, transcription of both IgG and IgA was also emerging in the IPP (Fig. 3). In data not shown in Fig. 3, IgG and IgA transcription could also be detected in the MLN of late term fetuses, and IgA transcripts could be detected in the parotid gland of 110-day fetuses (Fig. 4). Measured as relative transcription, this was less than that seen in the IPP (data not shown).

FIGURE 4.

Representative CDR3 spectratypes for IgM and IgA in an inductive (IPP) vs a noninductive (parotid) site of the mucosal immune systems at 110 DG (A) vs 6 wk later in a germfree isolator piglet (B). The profile seen in a 6% polyacrylamide gel is shown above the radiographic seam.

FIGURE 4.

Representative CDR3 spectratypes for IgM and IgA in an inductive (IPP) vs a noninductive (parotid) site of the mucosal immune systems at 110 DG (A) vs 6 wk later in a germfree isolator piglet (B). The profile seen in a 6% polyacrylamide gel is shown above the radiographic seam.

Close modal

Representative CDR3 spectratypes for IgM, IgA, and IgG are presented in Fig. 5. Spectratypic analysis is CDR3 length analysis. Because CDR3 is the result of the recombination of the VH, DH, and JH segments by a mechanism that involve segment trimming, N region additions, and P nucleotide additions, it is the major source of Ab and T cell repertoire diversity. CDR3 is most important for both Ab (57) and T cell specificity (58). Because in all lymphoid tissues studied except the fetal parotid (Fig. 4) the IgM spectratype was polyclonal, only the IgM spectratype at 60, 90, and 110 days in BM is shown. Although significant relative transcription of IgG and IgA appeared at 60 days in fetal thymus (Fig. 3), the CDR3 spectratype of transcripts of both switched isotypes was very oligoclonal. In 70-day and older fetuses, the prominent IgA transcripts in thymus were as polyclonal as those for IgM, and the same is true after 90 days for IgG transcripts (Fig. 5). This observation, together with data on relative transcription (Fig. 3), suggests that switch recombination is a major event with broad clonal participation in the thymus.

FIGURE 5.

The CDR3 spectratype of IgM, IgG, and IgA transcripts recovered from various lymphoid tissues at different times during gestation. Because the IgM spectratype was polyclonal in all lymphoid tissues at the times indicated, only data for BM at three different time points are shown (top). Left, IgG spectratypes; right, IgA spectratypes. Polynucleotide size markers are provided for reference at the top of each subfigure.

FIGURE 5.

The CDR3 spectratype of IgM, IgG, and IgA transcripts recovered from various lymphoid tissues at different times during gestation. Because the IgM spectratype was polyclonal in all lymphoid tissues at the times indicated, only data for BM at three different time points are shown (top). Left, IgG spectratypes; right, IgA spectratypes. Polynucleotide size markers are provided for reference at the top of each subfigure.

Close modal

Relative transcription of IgG and IgA in spleen was low (Fig. 3), although the IgG spectratype was polyclonal at 90 and 110 days (Fig. 5). In general, the IgG spectratype in spleen, IPP, and BM tended to be more polyclonal than that for IgA transcripts, although individual variations were observed (Fig. 6). Because the fetus is free from environmental Ag, these differences are either the results of random chance or are genetically determined. We observed no tendency for CDR3 lengths in any oligoclonal pattern to favor very short (12–18 nt) or long (45–60 nt) sequences, and in 60-day thymus, both were observed in the IgG spectratype (Fig. 5). In cases where IgG and IgA transcript spectratypes were oligoclonal, seldom was the exact same spectratype observed in different fetuses of the same age (Fig. 6). Thus CDR3 length differences between tissues and individuals appear random.

FIGURE 6.

Animal variation in CDR3 spectratype of IgA (top) and IgG (bottom) transcripts from the lymphoid tissues of two 90- and two 110-day randomly selected fetuses. Lymphoid abbreviations are the same as in Fig. 2.

FIGURE 6.

Animal variation in CDR3 spectratype of IgA (top) and IgG (bottom) transcripts from the lymphoid tissues of two 90- and two 110-day randomly selected fetuses. Lymphoid abbreviations are the same as in Fig. 2.

Close modal

It is generally regarded that B cell migration to noninductive sites in the mucosal immune system is driven by antigenic stimulation and amplified if Ag is present at the site (59). Fig. 4 shows that both IgM and IgA can be recovered from the fetal parotid gland, although few IgA clones are represented. However, piglets maintained germfree for 6 wk show development of a polyclonal spectratype for both IgA and IgM, suggesting that this clonal expansion is either spontaneous or driven by food protein. Because piglets maintained germfree do not respond to fluorescein-keyhole limpet hemocyanin or trinitrophenyl-Ficoll administered without adjuvant if not colonized (J. E. Butler and D. Francis, unpublished data) and no Abs to dietary proteins can be detected (J. E. Butler, P. Weber, and D. Francis, unpublished data), expansion to a polyclonal repertoire in the parotid gland of germfree piglets does not appear to be driven by environmental Ag.

The transcription data presented above indicate that Cα and Cγ transcripts are prominently expressed in the thymus. Because both switched isotypes are also present in serum, we wondered whether the number of IgA- and IgG-SC in thymus was correlated with serum IgG and IgA levels. Table IV shows that ∼70% of all Ig-SC detected by ELISPOT in the tissues studied were recovered from the thymus. Analyzed according to isotype, IgA- and IgG-SCs were only detected in the thymus, whereas IgM-SC were found in both spleen and thymus (Table III). On day 105, no Ig-SCs were observed in fetal liver, blood, BM, omentum, MLN and Peyer’s patches. However, although serum IgG levels are >25-fold higher than serum IgA, the number of IgA and IgG-SCs in thymus are essentially equal.

A total of 44 thymic VDJ transcripts from several normal late term fetuses, representing those expressed with IgM, IgA, and IgG, and 47 transcripts from two PRRS-infected fetuses were analyzed. These transcripts were not unique to the thymus because >95% used the same four porcine “fetal VH genes” (VHA, VHB, VHC, and VHE)4 that characterize the fetal preimmune repertoire, and all but five of the 91 transcripts used either DHA or DHB. The results of this analysis are summarized in Table V. In uninfected fetuses 1) relative DH usage (DHA:DHB = 0.28 for all normal transcripts) resembled that seen in germfree piglets (0.27) rather than that of colonized and conventional animals (DHA:DHA > 1.7), and usage of DHB was especially favored in IgG transcripts; 2) total CDR3 length in IgM and IgA transcripts did not significantly differ from that reported for >220 fetal VDJs (48), although because of shorter DHB segments, more N region additions were required to achieve the same CDR3 length; 3) the CDR3s of IgG transcripts were shorter than those of IgA and IgM because of fewer N region additions; 4) the formula used for VH usage (VHA + VHC + VHX)/(VHB + VHE) in IgM and IgA transcripts yielded values (∼0.9) that resembled those in germfree animals (0.5), whereas in the case of IgG transcripts, this formula yielded notably higher values (e.g., 3.8) than in germfree piglets; and 5) the overall frequency of mutations (CDR1, CDR2, and CDR3; column 6, total mutation) was similar to that in transcripts from germfree piglets and fetal VDJ in general (48) but much lower than that in colonized piglets and adult swine.

Although the overall mutation frequency resembled that of germfree piglets, i.e., <0.03, the mutation frequency was not randomly distributed among the CDRs. The CDR3 region was virtually nonmutated in all normal fetal transcripts, whereas the frequency of mutation is significantly higher in CDR2 of normal IgA transcripts, but not in IgG transcripts, compared with CDR2 in IgM transcripts (Table V). Furthermore, when the total mutation frequency was compared, VDJs transcribed with switched isotypes were significantly more mutated than those associated with IgM in both normal and virus-infected piglets (Table V). Age-matched PRRS-infected animals displayed DH ratios and VH usage profiles indicative of repertoire diversification, and these were shifted in the direction of those from colonized piglets and conventional adult swine that received known exposure to environmental Ag. Somatic mutation in thymic transcripts from PRRS-infected fetal piglets was concentrated in CDR2 and CDR3, in contrast to normal fetuses, in which mutations in CDR3 were nearly absent. Specifically, mutation frequencies were 10-fold higher in CDR3 of PRRS-infected animals than in normal fetuses.

The placentation of group III mammals, which includes ruminants, horses, and swine, is such that transfer of Igs and other protein in utero is believed not to occur (25). Nevertheless, low concentrations of Igs have been reported in the sera of newborn piglets and calves (33, 60). The origin of these Igs has been controversial with Kim and colleagues (34), ascertaining that at least in piglets, these are due to maternal contamination presumably during sample collection or as a result of damage to the placenta. Should this be the case, the major isotypes should be represented in the same proportion as in maternal serum. Although the ∼10-fold higher level of IgG than IgM is superficially reminiscent to what is seen in the sera of the gilts, the fetal isotype profile is distinct from that of their mothers (Fig. 1,B). Nevertheless, contamination during the collection process is possible and is especially problematic when collecting <0.5 ml of blood from the umbilicus of very young fetuses. The impact of such an event was demonstrated in fetal samples experimentally contaminated with maternal serum (1 μl/500 μl). This test resulted in sera with an isotype profile identical with that of the serum used for contamination and statistically distinct from the isotype profile of all normal fetuses (Fig. 1,B). Because IgA and IgM were detected in only a minority of samples before DG70, reliable mean values were not available for calculating isotype profiles before DG70. This leaves open to speculation whether any serum Igs before DG70 are of fetal origin. On one hand, only 100–500 μl can be collected from such small fetuses, so contamination with 0.1 μl of maternal blood is conceivable. In contrast, trace levels of IgG transcripts were detected at DG50 in spleen, leaving unresolved the possibility that serum IgG detected at DG57 could also be of fetal origin. The argument against contamination during the collection from older fetuses is supported by the observation that Ig levels increase, not decrease, with fetal age (Fig. 1,A). If the 0.5-ml sample collected on day 38 was contaminated with 1 μl of maternal blood, the Ig levels should be much higher than if 1 μl of maternal blood contaminated the <10-ml samples collected from 90- and 110-day-old fetuses. Many fetal sera gave an isotype profile with significantly lower levels of IgM and higher levels of IgA than are seen in maternal serum (Fig. 1,B), and newborns had absolute IgA levels higher than those for IgM (Fig. 1 A). Thus the switched isotypes in fetal serum must result from a previously undescribed selective transport or from de novo synthesis. The latter does not fit the paradigm established using the mouse model but is supported by the transcription and ELISPOT data reported here, whereas the former does not fit the paradigm established for any group III mammal.

Although some IgG in fetal sera may result from low level selective transport, the magnitude of this transport and the methods used to study it are the subject of another study (61). Here we have presented unambiguous evidence that the switched isotypes are both transcribed and secreted by the fetus. Data on transcription (Figs. 2, 3, 5, and 6) show that IgG, IgA, and IgM are all transcribed in utero, and even those for switched isotypes are pronounced on DG60 in thymus, i.e., during the first half of gestation. Not only is the thymus a prominent site for the transcription of these switched isotypes (Figs. 2 and 3), but the resulting repertoire is polyclonal (Fig. 5), and ELISPOT data indicate that both IgA- and IgG-SCs are present in thymus but not in other tissues that were studied (Tables III and IV). The less polyclonal and/or oligoclonal CDR3 spectratype observed with switched isotypes in spleen, IPP, and BM presumably reflects the paucity of IgG and IgA cells in these organs. The surprising polyclonality seen in spleen for switched isotypes despite low relative transcription in this organ, and the high degree of polyclonality seen in thymus, may indicate that splenic IgG and IgA cells are immigrants from the thymus.

Although we have observed no hypermutation in rearranged VDJs in DNA during fetal life in piglets (46, 48) and the overall mutation frequency in all fetal transcripts was similar to that in germfree piglets (Table V), VDJs associated with IgA had a higher total frequency of mutations and mutations in CDR2 when compared with IgM transcripts from the same animals (Table V). Furthermore, VH usage in IgG transcripts was heavily skewed away from VHB usage; this phenomenon is normally seen in diversified repertoires of colonized and conventional adult animals (Table V). This trend would be anticipated for thymic transcripts from fetuses actively producing Abs to an environmental Ag. This was observed in those from PRRS-infected fetuses, where VH usage is skewed away from VHB (a VH81x analog) to preferential usage of VHA and VHC. Although somatic mutations in IgG and IgA transcripts from normal fetuses are rare in comparison to colonized isolator piglets and adults, they are more frequent than in IgM transcripts from the same animals. Thus switch recombination appears to parallel repertoire diversification even when not driven by environmental Ag. Our in vivo findings in a nongenetically manipulated mammal are consistent with evidence from CD40-deficient (62, 63), μMT/μMT heavy chain minilocus transgenic (64), and lymphotoxin-α knockout mice (23), which indicates that switch recombination can proceed in the absence of antigenic stimulation and/or GC formation. In fact, studies with 3-83 transgenic mice show that the switch from IgM to IgG2a precedes GC formation (65), whereas it has been shown that some patients with X-linked hyper IgM syndrome can somatically mutate their VDJs without GC formation (66).

The ratio of IgG- to IgA-SC in thymus is nearly equal, whereas the serum IgG/IgA ratio is ∼10:1. Thus, IgG must 1) be secreted at a greater rate, 2) preferentially accumulate in the blood vascular system, 3) be in part derived from maternal blood by selective transport, or 4) be synthesized elsewhere than the thymus. Secretion outside the thymus was not detected (Tables III and IV). In mammals with the exception of primates, most serum IgA is dimeric (67, 68), and its short half-life (6 days) apparently results from its rapid transport to exocrine body fluids (68). In contrast, most IgG is not transported extravascularly, so the higher serum IgG levels we report could result from the longer serum IgG half-life, i.e., 20 days. Although the thymus may account for most fetal serum IgA, the exact origin of all fetal IgG remains unknown and awaits further experimentation. In support of de novo synthesis of all fetal IgA, late-term fetuses and colostrum-deprived newborns have Ig profiles with relative IgA levels much higher than their mothers and exceeding those of IgM (Fig. 1, A and B). This apparent increase in IgA production just before birth may reflect increased IgA production in sites such as the IPP in the last few days before parturition (Fig. 3).

We show elsewhere (61) that some low-level IgG placental transport occurs, although its impact on protective passive immunity is likely to be minimal because even in fetuses with the highest IgG levels, these are >1000-fold lower than in day-old piglets that suckle their dam (33). However, transfer of some maternal IgG across the placenta might provide an avenue for viral transfer and other environmental Ags if they could enter the fetus as part of immune complexes. The delivery of Ag in this manner could stimulate an immune response that could subsequently result in switch recombination and explain the transcription and secretion of the switched isotypes we report here. If this scenario is correct, it occurs with minimal effect on repertoire diversification but could explain the repertoire diversification observed in thymic IgA and IgG transcripts compared with IgM transcripts in normal fetuses (Table V). Although it can be maintained that viral Ags and viral infections are not representative of all environmental Ags, the repertoire diversification observed in response to infection with PRRS virus parallels those resulting from bacterial colonization. Thus it is unlikely that the switch recombination and VDJ diversification we observed in fetal piglets results from a unique environmental Ag. Thus, switch recombination in normal swine fetuses must 1) result from stimulation with fetal Ag, 2) be driven by cytokines released in an Ag-free environment, or 3) be a stochastic event in unstimulated B cells in this species. Regardless of mechanism, data from both normal and PRRS-infected fetuses show that switch recombination is associated with repertoire diversification.

The reason why the epicenter for isotype switch recombination is the fetal thymus is unexplained. There is no precedent indicating that the thymus can act as a site of Ag presentation by B cells or as a site for the B cell GC reaction. However, evidence that B cells and Ig-SC can be found in the thymus dates back to 1965 (69). More recently, evidence has been presented indicating that B cell lymphogenesis occurs in the mouse thymus (43), and we have shown that the DNA of porcine thymus (but not the cDNA) is the only lymphoid DNA that shows a CDR3 spectratype consistent with that of an unselected pro-B cell population (48). It is not transcripts from this presumed pro-B cell population that are analyzed in Table V. Rather, we and others have also shown that isotype-switched plasma cells are present in both the bovine and porcine thymus (37, 70), with IgA production being especially pronounced in cattle (70). These IgG and IgA plasma cells are predominantly found in the medulla, and we have sampled these by micromanipulation and shown them to contain the type of VDJs described in Table V (M. Sinkora, unpublished data).

Akashi and colleagues (45) calculated that ∼3 × 104 B cells leave the mouse thymus, of which 2/3 were developed intrathymically. Thus, low level transcription of IgG and IgA in many lymphoid tissues of the fetal piglet (Fig. 3) could be due to immigrant cells derived from the thymus. Although significant relative IgG and IgA transcription occurs first in the 60-day fetal thymus, this does not rule out switch recombination in other lymphoid tissues. The appearance of both IgG and IgA transcripts in fetal IPP (Fig. 3) would suggest that if the IPP of swine functions as a primary lymphoid tissue for repertoire diversification as reported for sheep (42), this diversification also involves cells with switched isotypes. We have no evidence that porcine IPP, although anatomically similar to the IPP of sheep (71), functions as a primary lymphoid tissue. Thus, it behaves in a manner distinct from the IPP of sheep and cattle (70, 71). We have observed no significant somatic mutation in any lymphoid tissue of fetal or germfree isolator piglets (46, 47, 48) as has been reported in sheep and cattle (72, 73). Because the type of switch recombination we report here has not been observed to occur in fetal hindgut lymphoid tissues of chickens, sheep, or rabbits, neither phylogeny nor anatomical homology is a reliable predictor of repertoire diversification in higher vertebrates. Thus, the in vivo piglet model continues to offer a rich opportunity to address a variety of topics concerning repertoire development in a natural and noncontrived environment.

We acknowledge the assistance of Dr. Marek Sinkora, Department of Microbiology, University of Iowa, in the collection of fetal samples; Dr. David Francis (South Dakota State University, Brookings, SD) for providing tissue samples from germfree isolator piglets; Dr. William Boersma (Central Veterinary Institute, Lelystad, The Netherlands), for providing mAb 23.3.1a, 27.9.1, 27.2.1, and 27.7.1; Dr. Klaus Nielsen (Animal Disease Research Institute, Nepean, Ontario, Canada) for providing mAb 1459; and Dr. Prem Paul (Iowa State University, Ames, IA), for providing mAb 5C9B12. We also thank Marcia Reeve for preparation of the typescript.

1

Research was supported by National Science Foundation-Molecular and Cell Biology Grant 9723721 and National Science Foundation International Grant 99-04130 (to J.E.B.).

3

Abbreviations used in this paper: GC, germinal center; DG, days of gestation; CDR, complementarity-determining region; PRRS, porcine respiratory and reproductive syndrome; IPP, ileal Peyer’s patch; SC, secreting cell; MLN, mesenteric lymph node; BM, bone marrow; C.V., coefficient of variation.

4

Fetal and newborn piglets develop their preimmune repertoire using four different VH genes that account >90% of all VH usage (4647 ). In addition, almost all DH usage can be accounted for by DHA and DHA.

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