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Keywords:

  • B-cell lymphogenesis;
  • fetal;
  • RAG-1;
  • signal joint circles;
  • Tdt;
  • VpreB

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

VDJ and VJ rearrangements, expression of RAG-1, Tdt and VpreB, and the presence of signal joint circles (SJC) were used to identify sites of B-cell lymphogenesis. VDJ, VλJλ but not VκJκ rearrangements or SJC were recovered from yolk sac (YS) at 20 days of gestation (DG) along with strong expression of VpreB and RAG-1 but weak Tdt expression. VλJλ rearrangements but not VκJκ rearrangements were recovered from fetal liver at 30–50 DG. SJC were pronounced in bone marrow at 95 DG where VκJκ rearrangements were first recovered. The VλJλ rearrangements recovered at 20–50 DG used some of the same Vλ and Jλ segments seen in older fetuses and adult animals. Hence the textbook paradigm for the order of light-chain rearrangement does not apply to swine. Consistent with weak Tdt expression in early sites of lymphogenesis, N-region additions in VDJ rearrangements were more frequent at 95 DG. Junctional diversity in VλJλ rearrangement was limited at all stages of development. There was little evidence for B-cell lymphogenesis in the ileal Peyer's patches. The widespread recovery of VpreB transcripts in whole, non-lymphoid tissue was unexpected as was its recovery from bone marrow and peripheral blood monocytes. Based on recovery of SJC, B-cell lymphogenesis continues for at least 5 weeks postpartum.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

B-cell lymphogenesis take place in the fetal liver (FL) and then in bone marrow (BM) of mice and humans; in both species the process is continuous throughout life.[1, 2] In other species the site, duration and features of this process have been less-well studied. In the few cases in which other homeothermic species have been studied, the pattern differs from that in mice and humans. In both hen and rabbit the process is determinant. In rabbit the process essentially terminates after 4 months.[3, 4] In the Bovidae the process involves the spleen.[5-7] In some species, full development of the B-cell compartment depends on repertoire diversification in hindgut lymphoid tissues, e.g. the hen bursa, rabbit appendix and the ileal Peyer's patches (IPP) of sheep, leading to the view that higher vertebrates belong to either the BM or ‘gut-associated lymphoid tissues’ group as regards B-cell development.[8] However, surgical resection of the IPP of piglets does not affect B-cell levels or maintenance or repertoire diversification,[9, 10] indicating that swine fit best to the BM group. This report focuses on features at early sites of B-cell lymphogenesis in this BM group mammal.

The use of swine in biomedical research,[11] especially where antibody responses are involved,[12-15] is one method to better understand B-cell lymphogenesis in this species. It was previously shown that VDJ rearrangements first appear in yolk sac (YS) at 20 days of gestation (DG) and later in FL at 30 DG and thereafter in many lymphoid tissues of fetal piglets.[16, 17] This criterion for iden-tification of the B-cell lineage is consistent with the detection of putative B-cell precursors in fetal liver and BM.[18] However these studies did not provide convincing evidence that pre-B cells or B cells were actually developed at these sites. The VH gene usage is identical at all of the sites in fetal piglets,[17, 19] which could mean that: (i) B cells are derived from one common site and then disseminated, or (ii) the process follows the same programme at many different sites. In mice, different pathways have been described at both the molecular level[20-22] and the phenotype level.[23] Regarding the latter, two B-cell sub-populations develop at different sites; B-1 cells develop in early fetal life perhaps in the peritoneum whereas B-2 cells develop in late term BM and continuously thereafter in BM. In swine, two populations were only predicted on the basis of the occurrence of in-frame and out-of-frame VDJ rearrangements.[16] B cell lymphogenesis in swine is of interest because this species was originally placed in the “gut-associated lymphoid tissues” category because the continuous IPP of swine are homologous to those in sheep, the latter regarded as primary lymphoid tissue for sheep.[8, 24-28]

B-cell lymphogenesis is reviewed in the immunology textbooks. Briefly, the process is recognized by rearrangement of genes coding for the immunoglobulin heavy and light chains that are assembled from germline-encoded V, D and J segments by a series of site-specific recombination events.[29, 30] These rearrangements excise circles of intervening DNA that accumulate in the nucleus as signal joint circles (SJC). V(D)J recombination is initiated by the recombination activation gene products, RAG1 and RAG2.[31-34] In the B lymphoid lineage, RAG expression is primarily restricted to developing B cells in the BM.[30, 35] There are two distinct waves of RAG expression in BM. The first corresponding to immunoglobulin heavy-chain gene rearrangement at the pro-B-cell stage. The RAG genes are then down-regulated upon expression of a μ heavy chain, and in mice, humans and rabbits these assemble with the surrogate light chain, comprising proteins λ5 and VpreB to form the pre-B-cell receptor.[36, 37] λ5 is homologous to a JλCλ product that associates with VpreB to produce a highly charged junction that lacks a conventional CDR3.[38-40] VpreB, which is expressed selectively at the early pro-B and early pre-B cell stages, is also designated CD179, and encodes the immunoglobulin iota chain.[41] Later, the RAG genes are re-expressed in pre-B cells during V-J light-chain rearrangements. In mice and humans, rearrangement in the κ light-chain locus precedes that for λ.[2, 42] However, low-level RAG expression is also found in B cells in peripheral lymphoid organs, especially after immunization.[43-46] This re-expression is believed to be the result of receptor editing, which has been most frequently identified with secondary rearrangements in the light-chain loci.[47] In addition, Chun et al.[48] reported the detection of low levels of the RAG-1 transcript in the murine central nervous system by PCR, in situ hybridization, and Northern blot analyses. However, an authentic RAG-2 transcript could not be reproducibly detected in the central nervous system.

Terminal deoxynucleotidyl transferase (Tdt) is a nuclear enzyme that catalyses the addition of non-templated (N) nucleotides to the free 3′-OH ends of fragmented or nicked DNA.[49] So far, the only known physiological function of Tdt is the random addition of nucleotides to the V(D)J junctions of immunoglobulin heavy-chain and T-cell receptor gene rearrangements[50-53] and rarely at junctions during immunoglobulin light-chain rearrangements.[54-56] The N additions effectively increase diversity of the repertoire of the antigen receptors on B and T cells.

Data presented summarize the expression of RAG, Tdt, VpreB and the presence of SJC in DNA and VJ rearrangements in light-chain loci. Using semi-quantitative PCR, we show that only λ rearrangements are present in YS and FL meaning that VλJλ rearrangement precedes that for VκJκ in this species by at least 30 days. Furthermore we show that especially VpreB can be widely recovered including from non-lymphoid tissues and monocytes. Robust B-cell lymphogenesis in BM appears to be limited to early postnatal life.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Experimental animals

Animals used in the study included: (i) pregnant gilts procured from certified suppliers as previously described;[57] (ii) Minnesota miniature/Vietnam-Asian-Malaysian crossbred piglets bred in Novy Hradek;[58] (iii) isolator piglets reared as previously described[59, 60] and (iv) conventionally reared young and adult pigs.[19] All pigs were healthy and normal at necropsy. All animal experiments were approved by the National Animal Disease Center Institutional Animal Care and Use Committee (NADC-IACUC) and the Ethical Committee of the Institute of Microbiology, Czech Academy of Science, according to guidelines in the Animal Protection Act and housed according to NADC IACUC Guidelines.

Collection of animal tissues for transcript studies

At 20, 30, 50 and 95 DG, pregnant gilts were killed, and tissues from at least five fetuses were recovered. These included YS at 20 DG, FL at 30 and 50 DG and BM, spleen and IPP at 95 DG. BM was recovered from long bones of fetal, isolator and older conventional pigs after removal of cartilaginous ends, extrusion with saline and preservation in TriZol or DNAZol (Invitrogen, Carlsbad, CA). Placenta and uterus were obtained from gilts as negative control tissues.

Preparation of cell suspensions for flow cytometry

Cell suspensions for flow cytometry were prepared as previously described.[61-63] Briefly, heparinized (20 U/ml) blood was obtained by intracardial puncture. Leucocytes from the BM were isolated by washing femur contents with PBS. Erythrocytes from all suspensions were removed using hypotonic lysis and washed twice in cold PBS. All cell suspensions were finally washed twice in cold PBS containing 0·1% sodium azide and 0·2% gelatin from Cold Water Fish Skin (PBS-GEL, all chemicals Sigma-Aldrich, St Louis, MO), filtered through a 70-μm mesh nylon membranes and cell numbers were determined by haemacytometer. Recovery of leucocytes sufficient for flow cytometry at 20–50 DG was not possible using current technology.

Flow cytometry and cell sorting

A variety of mouse anti-pig monoclonal antibodies were used (see Supplementary material, Table S1). Goat polyclonal antibodies specific for mouse immunoglobulin sub-classes labelled with fluorescein isothiocyante (FITC), phycoerythrin (PE) or allophycocyanin were used as secondary immunoreagents (Southern Biotechnologies Associates, Inc., Birmingham, AL). Staining of cells for flow cytometry was performed as described previously by indirect sub-isotype staining.[62, 63] Briefly, multi-colour staining was performed using cells that had been incubated with a combination of three primary mouse monoclonal antibodies of different sub-isotypes. Cells were incubated for 15 min and subsequently washed twice in PBS-GEL. Mixtures of goat secondary polyclonal antibodies specific for mouse immunoglobulin sub-classes that had been labeled with FITC, PE and allophycocyanin conjugates were then added to the cell pellets in appropriate combinations. After 15 min, cells were washed three times in PBS-GEL and analysed by flow cytometry.

Samples were measured or sorted on a FACS Calibur or a FACS AriaIII flow cytometer (BDIS, Mountain View, CA). In each measurement, 300–700 thousand events were collected. Electronic compensation was used to eliminate residual spectral overlaps between individual fluorochromes. The PCLysis software (BDIS, Mountain View, CA) was used for data processing.

Preparation of DNA, cDNA and gene-specific PCR

Tissues that had been stored in liquid nitrogen were pulverized and their DNA was extracted into DNAZol as previously described.[17] RNA was extracted from the same tissues and treated with DNase for two rounds for 30 min at 37° and then converted to cDNA.[17, 59] The effectiveness of DNase treatment in removing contaminating DNA from RNA was tested. Briefly, we tested whether VDJ rearrangements could be recovered by PCR from RNA before and after DNase treatment (see Supplementary material, Fig. S1). Recovery of VDJ from DNase-treated RNA would indicate DNA contamination.

Selection of gene-specific PCR primers

Sequences were recovered by PCR amplification using the primers listed in the Supplementary material (Fig. S2). Primers used to recover VDJ, VκJκ and VλJλ rearrangements have been previously described.[64-66] Primers for amplification of RAG-1 were specific for the unique core sequence[67] that were found in nine different species; five are compared in the Supplementary material (Fig. S3a). The region amplified shares 85% homology among these species. Primers used for recovery of porcine VpreB were based on the sequence alignment of porcine VpreB with that of seven other species including mouse and human.[60] The per cent homology referenced to pig is given. Similar criteria were used for the generation of primers for porcine Tdt. Primers for SJC generated in D-J and V-DJ rearrangements were based on recent genomics map data.[68] Primer sequences and the size of the expected product are summarized in the Supplementary material (Fig. S2) and the latter are confirmed by agarose electrophoresis.

Rationale for semi-quantitative PCR

Using the primers described in the Supplementary material (Fig. S2), products of the expected size were recovered (Figs 1,2 and 3). In the case of SJC, multiple products are possible because swine have two functional DH segments (DHA and DHB) and one functional JH.[68, 69] The V-DHA product of 309 bp and the DHB-JH product (370 bp) were most pronounced (Fig. 3). Distinguishing the products is best seen in agarose gels, not by quantitative PCR. Concatemers of VDJ are also commonly seen at higher concentrations (Fig. 1). The doublet produced by VλJλ amplification results from using a mixture of plasmid DNA that represents the two major porcine Vλ families, each of which uses a different leader sequence producing products that differ by 60 nucleotides (Fig. 2). Again we consider it valuable to observe the nature of the products.

image

Figure 1. Comparative titration of products after two rounds of PCR for VκJκ and VλJλ in 30-days of gestation (DG) fetal liver (FL), 20-DG yolk sac (YS), 95-DG spleen (SP) and 95-DG bone marrow (BM). Note that there is no product in YS and FL but it is recovered at 95 DG.

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image

Figure 2. The specificity and sensitivity of the PCR amplification of rearranged VκJκ and VλJλ. Top: Specificity of primers for λ and κ when applied to plasmids containing λ and κ or a mixture (κ + λ). Lanes labelled A, B etc are explained below Fig. 1(a). Bottom: Sensitivity of PCR assays for VκJκ (left) and VλJλ (right) determined by titration. Right lanes on each gel (marked with –) are reagent blanks.

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image

Figure 3. Example of the titration analysis of PCR products from four different lymphoid tissues. The titration of the VDJ product from the same tissue was performed in parallel with that for RAG-1 and TdT. The secondary (larger) PCR product seen when the VDJ product is used at low dilutions is the result of concatemers. YS, yolk sack; FL, fetal liver; BM, bone marrow at 95 days of gestation (DG); IPP, ileal Peyer's patches at 95 DG.

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The PCR assays were conducted using a five-fold dilution sequence of the target DNA using an intial DNA concentration of 50 ng. Titrations were directly visualized on agarose gels (Figs 1, 2 and 3). We preferred this approach for its direct value, because there are no established quantitative PCR protocols for the genes or transcripts studied. In the case of VDJ and SJC recovered from DNA, titration end-points were normalized to those for VDJ recovery in the same sample based on the premise that the amount of VDJ product reflects the number of B or pre-B cells (Table 1A). In the case of transcripts we provide only titration end-points because differences in copy number cannot be normalized.

  • display math
Table 1. PCR titration end-point. (A) PCR titration end-pointsa and end-point ratio for signal joint circles and VDJ rearrangement. (B) PCR titration end-pointsa from transcripts
(A)AgeEnd-pointEnd-point ratiob
VDJVDDJVDDJ
BM95DG6251251252020
5W-BM5 week6251255200.8
IPP95DG625100.160
5W-IPP5 week625500.80
Spleen95DG3125500.80
Thymus95DG625550.80.8
FL50DG156251256.4e−30.16
YS20DG1562502500.16
(B)AgeVDJRAG-1TdtVpreBβ-actincVκJκVλJλ
  1. a

    The end-point values based on two or three replicates.

  2. b

    End-point ratios compared with VDJ; see Materials and methods.

  3. c

    Used as evidence for successful preparation of cDNA, because the β-actin segment amplified crosses an intron (see Supplementary material, Fig. S2).

  4. d

    Collected from sows during Caesarean delivery.

Fetal BM95DG156256256256251562562515625
5-week-BM5 week625252562515625125625
Fetal IPP95DG156252516256251253125
5-week –IPP5 week125625125251251255
Fetal spleen95DG625255156251562525125
Placenta112d1251131256252525
Fetal thymus95DG6251562562562531252525
Uterus112d2525125100
YS20DG31252513125156250625
FL50DG31255256256250625

β-actin is a relatively stable cytoskeletal protein generally thought to be present at some level in most cells, although there may be differences among cell types.[70] Our data are derived from tissues comprised of many cell types, so β-actin was used only as an internal control for positive transcriptional activity.[71-73]

Assays for VκJκ and VλJλ rearrangements

To validate the specificity and sensitivity in the recovery of rearrangements in the κ and λ loci, we conducted studies using plasmid DNA that contained VκJκ and VλJλ rearrangements. Assays were performed using equal amounts of κ and λ plasmid DNAs. Results were obtained using the semi-quantitative PCR system described and are given in Figs 1,2 and 3 and in Table 1).

Analysis of junctional diversity in VDJ and VλJλ rearrrangements

Junctional diversity for VDJ rearrangements was established by assigning CDR3 to the region between the arginine codon at the 3′ end of VH and the tryptophan (W) codon in JΗ. The gap determined the length, and changes within were recorded as 5′ and 3′ additions and DHA and DHB usage. In the case of VλJλ rearrangement, CDR3 was defined as the region between the ultimate cysteine of Vλ and the phenylalanine of Jλ.[74] Assignment of mutations was based on reference to the consensus sequence of the VH or Vλ gene used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

λ locus rearrangement precedes κ in swine

In an earlier report, we presented data showing that λ transcripts were detected before κ transcripts in fetal piglets.[66] At that time we suggested the possibility that recovery of λ represented expression of λ5 because in mice and humans rearrangements in the κ locus occur before those in the λ locus.[42] Here we show that in YS and FL, only VλJλ rearrangements were recovered whereas VκJκ rearrangements were first seen at 95 DG (Fig. 1, Table 1). Periodic samples between 50 and 95 DG were not collected or examined so the actual time when the VκJκ rearrangement appears was not pinpointed. This surprising early appearence of λ before κ of course raised a question about the specificity and sensi-tivity of the PCR assays used to recover VλJλ and VκJκ. We addressed this in an experiment shown in Fig. 2 by showing that both primers sets are specific and that after one or two rounds of PCR, sensitivity is equal. Even the small difference in sensitivity (if it exists) cannot explain the complete absence of VκJκ in YS and FL whereas the end-point titre for VλJλ is 625 (Table 1B). To suggest this could rest on whether plasmids versus cDNA were used does not explain the ease of recovering VκJκ rearrangements in 95-DG BM or IPP (Table 1B). Given these observations and controls, we believe that our data confirm that somatic rearrangements in the λ locus precede those in the κ locus; this is the opposite of what is seen in mice and humans.

As the primers used to recover the λ product used anti-sense Jλ, the products recovered in YS and FL are authentic rearrangement products from the λ locus, not conventional λ5. To confirm this we cloned and sequenced various VλJλ rearrangement from early fetuses and found them to be diverse like those in our panel of ~ 300 VλJλ rearrangements obtained from various tissues and animals of different age (Fig. 4; J. Vasquez, K.L. Wells, N. Wertz, J. E. Butler, unpublished). In a sample of seven VλJλ sequences from 20-DG YS, four different Vλ genes and two different Jλ genes were used. We highlighted (using a box) an example recovered from 20-DG YS containing a Vλ gene frequently found in older fetuses and adults (Fig. 4). Data show that there are no unique features of the VλJλ sequences recovered from 20–50-DG samples, such as CDR3 length, additions or deletions. However all early VλJλ rearrangements used the Vλ8 family (IGLV8; J. Vasquez, K.L. Wells, N. Wertz, J.E. Butler, unpublished).

image

Figure 4. The sequence of VλJλ rearrangements recovered from 20-days of gestation (DG) yolk sac (YS), 50-DG fetal liver (FL) and adult peripheral blood mononuclear cells (PMBC). The boxed sequence is an example of a rearrangement that is found in all three groups. Changes in 3′ Vλ in CDR3 from the consensus for each Vλ gene are underlined. There were no deletions from Jλ. The Vλ genes are designated according to those used in a submitted manuscript (J. Vasquez, K.L. Wells, N. Wertz, J. E. Butler, submitted for publication). Also shown is the λ5-like sequence reported in GenBank accession NM_001243319.

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High levels of SJC were only recovered from the BM of fetal and young pigs

The SJC are indicators of ongoing B-cell development. These were only recovered in large amounts from the BM of fetuses and young pigs (Table 1A). When normalized to the level of VDJ rearrangements in the same tissue, values of 20 were obtained. VD-SJC were recovered in small amounts from all lymphoid tissues except YS but their relative levels were < 1 (Table 1A). DJ-SJC were also recovered from fetal thymus, YS and FL. Although VDJ rearrangements were readily recovered from YS and FL, recovery of SJC was relatively poor. The failure to recover VD-SJC from YS was notable; SJC could also not be recovered from the BM of adult swine (data not shown).

B-cell lymphogenesis in the IPP is weak or absent during fetal and neonatal life

In spite of the observation that RAG-1, Tdt and VpreB are transcribed in the IPP, when results are normalized to SJC : VDJ ratios, there is little evidence to support the role of this tissue as a site of B-cell lymphogenesis. This is in agreement with data reported elsewhere that involved surgical resection of the IPP of newborn piglets to show that this tissue has no effect on B-cell development or maintenance of B-cell levels.[9, 10]

Expression of RAG-1 and VpreB transcripts does not fit expectations

Recovery of transcripts for RAG-1 and VpreB was unexpected from non-lymphoid tissues. RAG-1 was expressed in nearly all tissues including uterus but not placenta (Table 1B). The same was true for VpreB, but high levels were also recovered from placenta. The recovery of RAG-1 transcripts from fetal thymus was expected because it is also required for T-cell development. Recovery of β-actin confirmed successful preparation of cDNA. These unexpected results prompted the sorting of leucocytes followed by testing for VpreB, Tdt and RAG (Fig. 5a,b). The transcripts for VpreB, Tdt and RAG-1 were found in sorted cells that are putative precursors of B cells in the BM (Fig. 5c). These progenitors express low levels of VDJ rearrangements (Fig. 5c) compared with mature BM B cells (Fig. 5d) but no κ light-chain rearrangement (Fig. 5c). Interestingly, we found that VpreB transcripts could be found in sorted monocytes (Fig. 5b). As expected, there was no VDJ or VJ rearrangement in these monocytes (Fig. 5b). Weak expression of VpreB and RAG was also detected in mature BM B cells (Fig. 5d) but polymorphonuclear cells did not express any VDJ or VJ rearrangements (Fig. 5b). These results indicate that in sorted BM sub-populations, VpreB, Tdt and RAG transcripts are present in the developing B-cell lineage and in monocytes.

image

Figure 5. Analysis of transcripts from flow cytometry sorted cells prepared from newborn bone marrow (a–d) and adult blood (e–h). The bone marrow cells (dot plot a) were sorted according to scatter characteristic and expression of IgM, CD172a and MHC-II into polymorphonuclear IgM CD172a+ MHC-II cells [polymophonuclear cells (PMN) in dot plot b], monocytes (Mo in dot plot b), IgM CD172a+ MHC-II+ B-cell progenitors (P in dot plot c), and IgM+ CD172a MHC-II+ mature B cells (B in dot plot d). A similar approach was used for sorting of blood stained by monoclonal antibodies against CD3, TCRγδ, IgM, CD21, CD8, CD14 and SWC8. In this case, γδ T cells (γδ in dot plot e), αβ T cells (αβ in dot plot e), mature B cells (B in dot plot f), natural killer cells (NK in dot plot g), polymorphonuclear cells (PMN in dot plot h), and monocytes (Mo in dotplot h) were sorted. All sorted populations were lysed in TRIzol and their RNA/cDNA preparations were inspected by reverse transcription-PCR for presence of transcripts (see below each dot plot) for VDJ heavy-chain rearrangement (H), VJ light-chain rearrangement κ (L), VpreB (V), RAG-1 (R) and TdT (T). Note that amplification of VJ rearrangement for κ gave the same output as for λ. Results are representative of three independent experiments.

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Because we detected VpreB and RAG transcripts in non-lymphoid tissues like the uterus and placenta (Table 1B), we wondered whether transcripts for these could be recovered from peripheral blood leucocytes. Subpopulation of adult blood cells were sorted by flow cytometry and inspected for the presence of transcripts. Figure 5(e–h) shows that VpreB cannot be detected in sorted mature B cells although there is weak expression of RAG in the cells (Fig. 5f). Like BM, sorted monocytes contain VpreB transcript but no detectable VDJ and VJ (Fig. 5h). Other cell populations like αβ and γδ T cells (Fig. 5e), natural killer cells (Fig. 5g) or polymorphonuclear cells (Fig. 5h) do not contain any transcripts characteristic of developing or mature B cells.

N-region additions in heavy-chain VDJ rearrangements are more frequent at DG 95

The low expression of Tdt in 20-DG YS, but higher expression in fetal BM (Table 1A), prompted us to ask whether the number of N-region additions in 71 CDR3 sequences from VDJ rearrangements from 20-DG YS, 30- and 50-DG fetal liver was different from that in BM, IPP and spleen from 95-DG fetuses. The 33 sequences from 95-DG animals were treated as one group (called mix) because differences between tissues were not significant. Data show that total N region additions were significantly greater at 95 DG than in FL and YS, especially in terms of 5′ additions (Table 2). However, the usage of DHA and DHB was similar at all time-points.

Table 2. The number of N-region additions in 71 CDR3 sequences from 20 days of gestation (DG) yolk sac (YS), 30 DG and 50 DG fetal liver (FL) and from bone marrow, spleen and ileal Peyer's patches from 95-DG piglets (Mix)
Tissue n 5′AddDHA/DHB3′Add5′Add + 3′Add
  1. a

    Significantly higher than 20 DG YS.

  2. b

    Significantly higher than 20 DG YS, 30 DG FL and 50 DG FL.

20 DG YS113.0 ± 2.06/56.6 ± 5.29.6 ± 5.2
30 DG FL124.0 ± 2.06/69.8 ± 6.8a9.8 ± 6.01
50 DG FL153.0 ± 3.06/98.7 ± 6.48.7 ± 7.64
95 DG (Mix)337.0 ± 8.0b19/1412.0 ± 8.3a18.6 ± 11.0b

CDR3 diversity in VλJλ rearrangements

Figure 4 reveals that CDR3 diversity in VλJλ rearrangements is small and N-region additions are seldom seen. The frequency of N-region additions in VλJλ rearrangements pales in comparison to events in the heavy chain. This same difference between junctional diversity in heavy- and light-chain rearrangements has been reported by others.[74-77]

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

B-cell lymphogenesis in mice and humans is continuous throughout life in BM,[1, 2] which differs from the determinant process described for hen and rabbit; the process ceasing at birth or shortly thereafter.[3, 4] Knowing whether the process is continuous or determinant in swine may have implications for animal health because artiodactyls comprise 50% of all mammals and are an important food and agricultural species. This is the first study in which the identification of B-cell lymphogenesis was based on using markers that are standard in mice, humans and rabbits. In addition to factors required for somatic rearrangement, we used a product dilution assay to quantify rearranged VDJ, VκJκ and VλJλ at different times during development. As the study involved tedious technology based on PCR and flow cytometry, we have supplied some of the technical details in the Supplementary material.

Titration results for SJC were expressed relative to the recovery of VDJ, the latter was chosen as a measure of the total B cells or pre-B cells in the sample. In the case of cDNA, such normalization could not be justified because of differences in the rate of transcription of different genes that can result in large differences in copy number. Hence, only PCR titration end-points are of use.

Our findings confirm an earlier report that VDJ rearrangements are present in YS at 20 DG.[16] However, when compared with BM, recovery of VD SJC is conspicuously low and this observation extends to FL. This might suggest that active rearrangement primarily involves DJ at this stage of B-cell lymphogenesis. However this interpretation leaves unexplained why full VDJ rearrangements are recovered from this tissue. Perhaps the rate of rearrangement is very low and as DJ rearrangements have been reported to occur simultaneously on both alleles,[29, 78] V-DJ SJC would be present at lower levels than DJ SJC. As all SJC are rapidly degraded, a slow rate of B-cell lymphogenesis in YS might also explain our results and also our inability to recover SJC from adult BM. Therefore, B-cell lymphogenesis in these tissues may not be absent, but occurs at a frequency too low to be detected by the methods we have employed.

Although Tdt is expressed in YS, its expression is considerably lower than in fetal BM at 95 DG. To address whether this might be the result of differences in transcript copy number versus function, we looked for the effect of Tdt on N-region additions. We show a progressive increase in N-region additions in VDJ rearrangements with fetal age (Table 2) consistent with the pattern reported for mice and humans.[79] Hence the ease of recovery of Tdt transcripts from fetal liver may reflect the presence of alternative transcripts that do not have transferase activity. However, we found little evidence for N-region additions in VλJλ rearrangement regardless of age and consistent with other reports.[75-77] The preferential appearance of VλJλ rearrangements as early as 20 DG in YS and its continuance in 30 and 50-DG FL before the appearance of VκJκ rearrangements in BM at DG 95 (Fig. 1) clearly differs from the pattern seen in mice and humans.[42] It should be remembered that in many ungulates like cattle and the horse, > 90% of all immunoglobulins use λ light chains even though they possess a functional κ locus.[80] Early appearence of VλJλ rearrangement in the spleen of fetal sheep has also been reported.[7] Furthermore, vertebrates like hen, use only λ. We show that the VλJλ rearrangements recovered at 20 DG contained known V and J elements, making them authentic rearrangements, and contained the same spectrum of Vλ and Jλ segments that is seen in older animals (Fig. 4). In fact, four different Vλ and two different Jλ segments are represented in just seven sequences. The control studies we conducted eliminated the possibility that our results reflect differences in the specificity of primers used or the sensitivity of the respective PCRs (Fig. 2). Interestingly, when we submitted our sequence data for the VλJλ product recovered from 20-DG YS to a BLAST search, it was identified as a λ5-like sequence in GenBank (http://www.ncbi.nlm.nih.gov/projects/genome/guide/pig/). This reported “λ5” was in fact a VλJλCλ transcript and was identical to a common member of the porcine Vλ genes in our panel of ~300 Vλ genes (Fig. 4; Vasquez et al. unpublished). The annotated sequence was simply a rearranged λ light chain and not conventional λ5. No conventional λ5 sequence has yet been reported for swine.

One heretical explanation for our findings is that a conventional surrogate light chain is absent in swine. Rather, swine and perhaps other ungulates that use a predominance of λ light chains, may proceed directly to the use of authentic VλJλ rearrangements. That said, we offer no evidence for the absence of a surrogate light chain in swine. If it exists, it clearly does not involve the λ5-like element annotated in GenBank unless λ5 in swine is different from that in mouse.

Our most recent studies have shown that swine (and sheep) do not belong to the “gut-associated lymphoid tissues” category of higher vertebrates as has been proposed.[8] Hence, the increase in RAG-1 and Tdt expression (Table 1B) in the IPP of 5-week-old piglets may suggest that RAG-1 and Tdt are being re-expressed perhaps as part of receptor editing. We addressed this issue by normalizing titrations for SJC to the titration of rearranged VDJ (Table 1A). DJ-SJC were not recovered in IPP and VD-SJC were recovered 125-fold less frequently than in BM. This finding favours the interpretation that expression of RAG-1 in the IPP of 5-week-old piglets is associated with receptor editing/revision not B-cell lymphogenesis.

The recovery of VDJ rearrangements in thymus was previously observed.[81] The thymus of newborn calves and piglets contains B cells and synthesizes immunoglobulin.[82-84] Whereas the number of B cells in the medullary and cortex areas is quite small[85] the subcapsular region is rich in cells believed to be of the B-cell lineage. It is therefore likely that some lymphocyte stem cells that populate the thymus start in the direction of B-cell lymphogenesis and these could explain the recovery of thymic SJC.

The expression of RAG-1 and especially VpreB in non-lymphoid tissues was unexpected based on paradigms of B-cell lymphogenesis that were primarily established using laboratory rodents.[2] A careful analysis of the core sequence for RAG-1 that was amplified in the PCR assay we employed was highly conserved among the five species compared (see Supplementary material, Fig. S3A). The sequence amplified for VpreB was not found in any genes submitted to GenBank other than those for VpreB (see Supplementary material, Fig. S3B). Hence, recovery of transcripts for VpreB and RAG-1 should reflect transcripts of authentic RAG-1 and VpreB and not other genes. After two successive rounds of DNase treatment before the preparation of cDNA, data showed that VDJ-IgM or VκJκ could not be amplified, indicating that contaminating DNA was absent (see Supplementary material, Fig. S1). Furthermore we tested whether these markers of active B-cell lymphogenesis were transcribed in isolated leucocytes from BM and peripheral blood. For these reasons we sorted different subpopulations to test whether VpreB, RAG and Tdt are expressed in other than developing B cells. Results indicate that VpreB can be expressed at least in monocytes and probably in their macrophage progenies. Transcripts may also be found in other non-B cells that were not included in analyses. Studies show that macrophages may contain partial DJ rearrangement.[86] This is probably because they share a common lymphoid precursor.[87, 88] On the other hand, Tdt and RAG can be expressed not only as a consequence of T-cell and B-cell development in primary lymphoid organs but also because of receptor editing, which can take place anywhere in the periphery.[47, 89] This might explain our findings of weak expression of RAG in mature B cells sorted from the blood.

The enigma surrounding VpreB is more complicated. Based on sequence homologies (see Supplementary material, Fig. S3B) it is difficult to explain our results as some spurious PCR artifact. Given the heretical possibility that there is no Pre-B-cell receptor in swine, the tissue-wide expression of VpreB may have more global implications, which suggests that VpreB expression is not restricted to B-cell development. As we have been unable to identify previous studies that have employed PCR to recover transcripts for these genes from solid tissues, we believe that it remains an issue that needs further investigation in all species.

Our results suggest that expression of VpreB, Tdt and RAG transcripts are unreliable indicators of B-cell lymphogenesis in swine. We bypassed criticism of quantitative PCR by providing raw, visual titration data (Figs 1, 2 and 3). Nevertheless we encountered unexpected results for VpreB, RAG-1 or Tdt in many situations. By contrast, data that we report on SJC or light-chain rearrangement is not subject to the same criticism because it comes from measuring actual rearrangement events that occur in the DNA and cannot be skewed by concerns over transcript copy number or whether these transcripts are functional or whether quantitative PCR is reliable.

We conclude that in swine B-cell lymphogenesis begins in the YS, and in agreement with mouse and human studies, heavy VDJ rearrangements show fewer N-region additions in the early stages of development than later in fetal life and that light-chain rearangement shows little junctional diversity. By contrast, light-chain rearrangement begins in the λ locus in this species, continues in FL, meaning 75 days earlier than when κ rearrangements appear in late fetal life. The pattern of B-cell lymphogenesis in late fetal BM, which is associated with pronounced recovery of SJC, continues postnatally at least for 5 weeks after birth but is questionable in older animals. Our findings suggest that swine may resemble the rabbit in the lack of continuous B-cell lymphogenesis, do not belong to the gut-associated lymphoid tissues group[8] but perhaps belong to a group of mammals such as artiodactyls in which λ light-chain rearrangement precedes that for κ.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Research was supported by subcontract NBCHC080090 from Biological Mimetic, Fredrick, MD, USA; a grant P502/10/0038 from the Czech Science Foundation; and a grant ME09089 from the Ministry of Education, Youth and Sports of the Czech Republic.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
imm3615-sup-0001-FigS1.docWord document218KFigure S1. Effectiveness of double DNase treatment of RNA.
imm3615-sup-0002-FigS2.docWord document82KFigure S2. Primer sets used in the PCRs for the study.
imm3615-sup-0003-FigS3a.docWord document33KFigure S3. Sequence alignment of amplified and flanking sequences of porcine Rag-1 (A) and for VpreB (B) compared with sequences for other species in GenBank.
imm3615-sup-0004-FigS3b.docWord document37K 
imm3615-sup-0005-TableS1.docWord document35KTable S1. Monoclonal antibodies used for flow cytometry.

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