Unexpected Rearrangement and Expression of the Immunoglobulin {lambda}1 Locus in Scid Mice

Norman R. Ruetscha, Gayle C. Bosmaa, and Melvin J. Bosmaa
a Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111

Correspondence to: Melvin J. Bosma, Fox Chase Cancer Center, Institute for Cancer Research, 7701 Burholme Ave., Philadelphia, PA 19111. Tel:215-728-3630 Fax:215-728-2412 E-mail:MJ_Bosma{at}fccc.edu.


  Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that V{lambda}1–J{lambda}1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH–DJH and V{kappa}–J{kappa} rearrangements. Particularly surprising, we found the level of V{lambda}1–J{lambda}1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid V{lambda}1–J{lambda}1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, ~15% of V{lambda}1–J{lambda}1 rearrangements lacked abnormal deletions. Productive {lambda}1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect {lambda}1-expressing cells by flow cytometry in the spleens of bcl-2–transgenic scid mice. Strikingly, {lambda}1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of V{lambda}1 and J{lambda}1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid {lambda}1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of V{lambda}1 and J{lambda}1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged V{lambda}1 and J{lambda}1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.

Key Words: B cell differentiation, pro-B cells, premature Ig{lambda} recombination, VJ{lambda} junctional diversity, P additions


  Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

The rearrangement of Ig genes proceeds in an ordered fashion (for review see references (1) and (2)). It begins with rearrangement of the H chain gene elements, DH and JH, followed by VH–DJH rearrangement (3). H chain gene rearrangement is initiated at the pro-B cell stage and is generally followed by L chain gene rearrangement at the pre-B cell stage (4) (5) (6), with V{lambda}–J{lambda} rearrangement occurring later (7) (8) (9) (10) and/or less frequently (11) (12) (13) than V{kappa}–J{kappa} rearrangement. The order of H and L chain gene rearrangement is not absolute, however, as studies with B lineage cell lines (14) (15) (16) and sorted pro-B cells (5) (6) indicate that rearrangement at the {kappa} or {lambda} locus may precede or occur independently of H chain gene rearrangement. Also, inactivation of the {kappa} locus by gene targeting has shown that rearrangement of the {lambda} locus does not require prior rearrangement of the {kappa} locus (17) (18), consistent with earlier evidence for independent rearrangement of {lambda} and {kappa} loci in various cell lines (16) (19) (20) (21) and in {kappa}-transgenic mice (22). Independent rearrangement of {lambda} and {kappa} loci is in agreement with a stochastic model of L chain gene rearrangement (11) (23) and contrary to a strictly regulated model of L chain gene rearrangement (for review see reference (2)).

Consistent with the stochastic model of L chain gene rearrangement, we report here a low frequency of V{lambda}1–J{lambda}1 rearrangement in severe combined immunodeficient (scid) and in wild-type (wt)1 mice with a targeted deletion of the JH locus (JHT mice) (24). As JHT mice lack a functional H chain locus, V{lambda}1–J{lambda}1 rearrangement in these mice must occur independently of H chain rearrangement. Furthermore, as both scid and JHT mice lack pre-B cells, the observed V{lambda}1–J{lambda}1 rearrangement is inferred to occur at the pro-B cell stage and independently of V{kappa}–J{kappa} rearrangement.

Although a low frequency of V{lambda}1–J{lambda}1 rearrangement at the pro-B cell stage in JHT mice could have been predicted based on the stochastic model of L chain gene rearrangement, the regular occurrence of such rearrangement in scid pro-B cells would not have been predicted. In scid mice, V(D)J recombination is severely impaired as a result of a DNA repair defect (25) (26) (27). The defect is due to a nonsense mutation in the gene coding for the catalytic subunit of DNA protein kinase (DNA-PKcs) (28) (29) (30). Because of this mutation, developing scid lymphocytes cannot efficiently join V, D, and J coding ends resulting from the initiation of V(D)J recombination (31) (32) (33) (34). Consequently, most developing scid lymphocytes are thought to die prematurely with persisting DNA breaks. Therefore, given that V{lambda}1–J{lambda}1 rearrangement generally follows DH–JH and VH–DJH rearrangement and is much less frequent than V{kappa}–J{kappa} rearrangement, we would not expect to detect V{lambda}1–J{lambda}1 rearrangement in scid mice.

Nonetheless, as shown here, V{lambda}1–J{lambda}1 rearrangement is routinely detectable in fetal liver, adult bone marrow, and spleen of individual scid mice. Moreover, in scid fetal liver we found the level of V{lambda}1–J{lambda}1 rearrangement to be comparable to that in wt fetal liver. Although most scid V{lambda}1–J{lambda}1 rearrangements showed abnormal deletions of V{lambda}1 and/or J{lambda}1 nucleotides at the VJ junction, ~15% lacked abnormal deletions. Productive {lambda}1 transcripts resulting from in-frame V{lambda}1–J{lambda}1 rearrangements were clearly evident in bone marrow and spleens of individual scid mice. Most scid {lambda}1 transcripts displayed the same recurring palindromic (P) additions of three, four, or five nucleotides. P addition (35) (36) is thought to result from asymmetric nicking of a hairpin coding end (37) (38) (39) (40), followed by fill-in of the overhang and joining to another coding end.

Our findings raise several puzzling issues: (a) Why is the level of V{lambda}1–J{lambda}1 rearrangement comparable in fetal liver of scid and wt mice; (b) Why do most {lambda}1 transcripts of individual scid mice show the same recurring P additions; and (c) How do {lambda}1 expressing scid cells survive the deleterious effect of scid and apparently emigrate from bone marrow to spleen? These three issues are discussed.


  Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Mice.
C.B-17 mice homozygous and heterozygous for the scid mutation (41) are here denoted as scid and scid/+ mice, respectively. C.B-17 scid mice hemizygous for the bcl-2-36 transgene (bcl-2 scid mice; reference (42)) were obtained from S. Cory (The Walter and Eliza Hall of Medical Research, Melbourne, Australia). Genotyping of bcl-2 mice was done by PCR using DNA from tail snips (43) and oligonucleotide primers for the SV40 sequence included in the transgene (44). Mice with both of their recombination activation gene (RAG)1 loci inactivated by gene targeting (RAG-/- mice; reference (45)) and mice with their JH elements deleted by gene targeting (24) were provided by R. Hardy (Fox Chase Cancer Center). The targeted JHT allele was backcrossed onto C.B-17 mice for three backcross generations (N3). N3F1 mice were intercrossed to generate N3F2 mice homozygous for the JHT allele (JHT mice). JHT mice were crossed with scid mice to obtain JHT/+, scid/+ mice; these were then intercrossed to obtain JHT scid mice. Genotyping for the wt and inactivated JH allele was done by PCR using tail DNA and primers specific for the wt and inactivated JH locus (see JH1 and JHT oligonucleotides below). All of the above mice were bred and maintained at the Fox Chase Cancer Center and were analyzed between the ages of 6 and 12 wk.

Flow Cytometric Analysis.
Flow cytometry was used to test for the presence of cells with surface Ig{lambda}1 ({lambda}1+ cells) in scid and bcl-2 scid mice. In brief, spleen cells of individual scid, scid/+, bcl-2 scid, bcl-2 scid/+, and RAG1-/- mice were stained with biotin-conjugated anti-CD8 (53.6), allophycocyanin (PharMingen)-conjugated anti-CD45 (B220), and FITC-conjugated anti-{lambda}1 (R11-153–FITC; PharMingen) in the manner previously described (46). Cells were analyzed by three-color flow cytometry using a dual laser FACStarPLUSTM (Becton Dickinson). Binding of biotinylated antibodies was revealed by Texas Red–conjugated streptavidin (Southern Biotechnology). Dead cells were identified by propidium iodide staining and excluded from analysis. Gates were set to score {lambda}1+ cells based on the distribution of {lambda}1 staining of spleen cells in the scid/+ positive controls. Due to the paucity of {lambda}1+ cells in scid and bcl-2 scid mice, between 0.5 and 1.0 x 107 spleen cells were analyzed per mouse. Cells were simultaneously stained for the B and T specific markers B220 and CD8, respectively, to ensure that cells scored as {lambda}1+ were indeed B lineage cells (i.e., {lambda}1+B220+CD8-). Spleen cells from RAG1-/- mice served as negative controls for background staining of {lambda}1.

Oligonucleotides.
Oligonucleotides were synthesized by an Applied Biosystems 394 DNA/RNA Synthesizer. Oligonucleotides used as primers for PCR or reverse transcriptase (RT)-PCR were as follows: VH (#91), 5'-GCCGGATCCGTGCAGCTGGTGGAGTCTGG-3'; DH (#285), 5'-ACTGCTACCTCTGGCCCCACCAG-3'; JH4 (#361R), 5'-AGATAATCTGTCCTAAAGGCTC-3'; Cµ (#289), 5'-ATGCAGATCTCTGTTTTTGCC-TCC-3'; V{kappa} (#68), 5'-GGCTGCAGGACATTGTGCTGACCCAATCTCCAGCTTCT-3'; J{kappa}2 (#367), 5'-GGTAGACAATTATCCCTCTTCCCCTAGT-3'; C{kappa} (#130), 5'-ATGGATCCAGTTGGTGCAGCATC-3'; V{lambda}1ext (#282), 5'-TCTCCT-GGCTCTCAGCTCAG-3'; V{lambda}1int (#294), 5'-AGGAATCTGCACTCACCACATC-3'; J{lambda}1 (#271), 5'-GCACCTCAAGTC-TTGGAGAG-3'; C{lambda}1 (#283), 5'-GAGGAAGGTGGAAACAG-GGTG-3'; ß2ML (#229), 5'-GAATGGGAAGCCGAACATAC-TGAACTG-3'; ß2MR (#230), 5'-TGCTGATCACATGTCTC-GATCC-3'; SV40L (#355), 5'-GGAACTGATGAATGGGAGC-AGTGG-3'; SV40R (#356), 5'-GCAGACACTCTATGCCTGTG-3'; JHTL (#370), 5'-CCTTGCGCAGCTGTGCTCGACGTTG-3'; JHTR (#371), 5'-GCCGCATTGCATCAGCCATGATGGA-3'; JH1L (#368), 5'-GGACCAGGGGGCTCAGGTC-ACTCAGG-3'; and JH1R (#369), 5'-GAGGAGACGGTGACCGTGGTGCCTGC-3'.

Genomic PCR.
~5 x 106 bone marrow or spleen cells were used to prepare genomic DNA by PureGene Kit (Gentra Systems). PCR was carried out in 50 µl with ~106 cell genome equivalents of DNA. Controls for nonspecific amplification of PCR products included the use of RAG-/- DNA and no DNA template in the reaction. Reactants included oligonucleotide primers for DH and JH, VH and JH, V{kappa} and J{kappa}, V{lambda}1 and J{lambda}1 elements or for the ß2 microglobulin (ß2M) locus along with 220 µM each of dATP, dGTP, dCTP, and dTTP, 0.4 µM primers, 20 µM Tris-HCl, pH 8.4, 50 µM KCl, 1.5 µM MgCl2, and 2.5 U of AmpliTaq DNA polymerase. The cycling reaction consisted of an initial denaturation for 4 min at 95°C, with 23 cycles of 1 min at 94°C, 45 s at 68°C, and 1 min at 72°C, and a final elongation step for 5 min at 72°C. 1/10 of each PCR reaction (1/20 for ß2M control) was electrophoresed through 1.5% LE agarose (FMC Bioproducts) in 1x Tris-acetate-EDTA buffer, turboblotted by alkaline transfer onto maximum strength Nytran Plus membranes (Schleicher & Schuell) and hybridized in Denhardt's solution with the appropriate probes. Radioactive {alpha}-[32P]dCTP labeling was done by random priming using the Prime-It II Kit (Stratagene). Hybridization probes included pJH6.3 (47), pEC{kappa} (11), and a PCR-amplified and gel-purified (QiaexII; Qiagen) VJ{lambda}1 gene fragment to score for DH–JH (or VH–DJH), V{kappa}–J{kappa}, and V{lambda}1–J{lambda}1 rearrangements, respectively. As a control for the amount of input DNA, a portion of the nonrearranging ß2M gene was PCR amplified and hybridized to a ß2M-specific probe. Blots were exposed to X-Omat (Eastman Kodak Co.) autoradiographic film and also to a PhosphorImaging plate for quantitation by a BAS1000Mac Bio-Imaging Analyzer (Fuji Photo Film Co.).

RT-PCR.
Total RNA from ~5 x 106 bone marrow or spleen cells was obtained by using RNEasy (Qiagen) as prescribed by the manufacturer. RNA was eluted into DEPC-treated H2O and stored at -73°C. RNA from the equivalent of ~1.5 x 106 bone marrow cells or ~3.0 x 106 spleen cells was used to synthesize first strand cDNA using SuperscriptII RT and 100 ng of random hexamers (Amersham Pharmacia Biotech) as directed by the manufacturer (GIBCO BRL). A portion of this cDNA (equivalent to ~3 x 105 bone marrow cells or ~6 x 105 spleen cells) was amplified by PCR using 220 µM each of dATP, dGTP, dCTP, and dTTP, 0.4 µM primers, 20 µM Tris-HCl, pH 8.4, 50 µM KCl, 1.5 µM MgCl2, and 2.5 U of AmpliTaq DNA polymerase (PerkinElmer) in a reaction volume of 50 µl. Controls for nonspecific amplification of PCR products included RAG-/- cDNA and no cDNA template in the reaction. Semiquantitative PCR using a PTC-100 Thermal Controller (MJ Research) was carried out after an initial denaturation for 4 min at 95°C, with 23 cycles of 1 min at 94°C, 45 s at 65°C, and 1 min at 72°C, and a final elongation step for 5 min at 72°C. Southern blotting and hybridization was carried out as described above. The hybridization probes included pCµ3741 (48), pEC{kappa}, and gel-purified VJ{lambda}1 and ß2M PCR-amplified gene fragments.

Quantitation.
Conditions for semiquantitative PCR were determined by varying cycle number and the amount of input DNA (or cDNA). Filters were exposed to a Fuji imaging plate to quantify the amount of {alpha}-32P–hybridized probe in experimental samples relative to that in control (reference) samples using a BAS1000Mac Bio-Imaging Analyzer (Fuji Photo Film Co.). We found that with 23 cycles of amplification, the amount of PCR product was proportional to the amount of input DNA (from 106 cells) at several different dilutions. Similarly, at 23 cycles, the amount of RT-PCR product was found to be proportional to the amount of input cDNA (from 3–6 x 105 cells) at several different dilutions.

Sequence Analysis.
To ensure sufficient PCR product for cloning, one microliter from the primary PCR or RT-PCR reaction was subjected to an additional 15 cycles of PCR using conditions as above. The V{lambda}lint primer was used with J{lambda}1 or C{lambda}1 for recovery of junctional sequences from genomic DNA or cDNA, respectively. PCR products were electrophoresed through 1.5% LE agarose, purified using QiaexII, and cloned into pCR2.1 for transformation of INV{alpha}F' bacteria (Invitrogen). Recombinant colonies were randomly chosen for plasmid recovery by Perfect Prep (5Prime-3Prime, Inc.). Plasmids were submitted for cycle sequencing using the ABI Prism Dye Terminator Reaction Kit and an ABI 377 DNA Sequencer (PerkinElmer).


  Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Evidence for V{lambda}1–J{lambda}1 Rearrangement before the Pre-B cell Stage.
To test whether the {lambda}1 locus can rearrange early in B cell differentiation, we assayed for the presence of nongermline {lambda}1 transcripts in scid mice and also in bcl-2 scid mice. As shown in Fig 1 A, {lambda}1 transcripts resulting from V{lambda}1–J{lambda}1 rearrangement ({lambda}1 transcripts) were clearly evident in the bone marrow of scid mice and more so in the bone marrow of bcl-2 scid mice. The higher abundance of {lambda}1 transcripts in the latter mice presumably reflects the greater longevity of B lineage cells in bcl-2 scid mice than in scid mice (42). Fig 1 A also illustrates that transcripts resulting from V{kappa}–J{kappa} rearrangement ({kappa} transcripts) were routinely detectable in bone marrow of bcl-2 scid but not scid mice, whereas transcripts resulting from VH–DJH rearrangement (µ transcripts) were barely detectable in some bcl-2 scid mice and not at all in scid mice (Fig 1 A). Consistent with previous reports of detectable DH–JH rearrangement in scid mice (49) (50), Dµ transcripts resulting from DH–JH rearrangement were readily detectable in scid and bcl-2 scid bone marrow.



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Figure 1. (A) Detection of {lambda}1 transcripts in bone marrow of individual scid (s/s) and bcl-2 s/s mice and (B) in pooled bone marrow of JHT s/s and JHT non-scid (+/+) mice. Transcripts were detected by RT-PCR using locus-specific primers (see Materials and Methods). Transcripts corresponding to DH–JH, VH–DJH, V{kappa}–J{kappa}, and V{lambda}1–J{lambda}1 rearrangements are denoted Dµ, µ, {kappa}, and {lambda}1, respectively. Amplification of ß2M transcripts served as a control for the amount of input cDNA. Results obtained with wt (+/+) bone marrow are provided for comparison.

As differentiation of scid B lineage cells does not generally progress beyond the pro-B cell stage, our detection of {lambda}1 transcripts in scid mice suggests that V{lambda}1–J{lambda}1 rearrangement may occur before the pre-B cell stage. To test whether this is indeed true and whether the {lambda}1 locus can rearrange independently of the H chain locus, we assayed for {lambda}1 transcripts in bone marrow of JHT mice. In these mice, B cell differentiation is completely arrested at the pro-B cell stage as a result of gene-targeted inactivation of the JH locus (24). Fig 1 B shows that {lambda}1 transcripts were readily detectable in JHT bone marrow. The abundance of {lambda}1 transcripts in JHT mice was ~40-fold less than in wt mice but ~25-fold greater than in JHT scid mice (Table 1). We conclude that V{lambda}1–J{lambda}1 rearrangement can occur at the pro-B cell stage and independently of H chain gene rearrangement.


 
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Table 1. Level of {lambda}1 Transcripts in scid Fetal Liver and JHT scid Bone Marrow Relative to wt Controls

Developmental Onset of V{lambda}1–J{lambda}1 Rearrangement in scid and wt Embryos.
To compare the developmental onset of V{lambda}1–J{lambda}1 rearrangement with that at other Ig loci, we tested genomic DNA from pooled livers of scid and wt embryos for DH–JH, VH–DJH, V{kappa}–J{kappa}, and V{lambda}1–J{lambda}1 rearrangement (Fig 2). In wt embryos, we found that DH–JH rearrangement could be detected as early as day 12, whereas VH–DJH and V{kappa}–J{kappa} rearrangements were not detectable until day 14. These results are in general agreement with earlier reports on the time course of H and {kappa} chain gene rearrangement in fetal mice (51) (52). In scid embryos, DH–JH rearrangement was not clearly evident until day 14, and VH–DJH and V{kappa}–J{kappa} rearrangements were not detected except for a VH–DJH rearrangement of aberrant size in the day 15 sample. In contrast, V{lambda}1–J{lambda}1 rearrangement was evident as early as day 12 in both scid and wt embryos. These results indicate that V{lambda}1–J{lambda}1 rearrangement is initiated early in development and may developmentally precede VH–DJH and V{kappa}–J{kappa} rearrangement.



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Figure 2. Developmental onset of V{lambda}1–J{lambda}1 rearrangement relative to that of DH–JH, VH–DJH, and V{kappa}–J{kappa} rearrangement in fetal liver of scid (s) and wt (w) mice. (A) Genomic DNA from pooled fetal liver at day (d) 12, 13, 14, and 15 of gestation was subjected to PCR using locus-specific primers to amplify DH–JH1-3, VH–DJH1-4, V{kappa}–J{kappa}1,2, and V{lambda}1–J{lambda}1 rearrangements. Amplification of the nonrearranging ß2M gene served as a control for input DNA. (B) Amplification of {lambda}1 and ß2M transcripts was done by RT-PCR.

It is important to note in Fig 2A and Fig B, that the level of V{lambda}1–J{lambda}1 rearrangement and {lambda}1 transcript in day 13–15 scid fetal liver remains relatively constant and appears comparable to that in the day 13 and 14 wt fetal liver. Indeed, quantitation of the amount of {lambda}1 hybridizing signal for V{lambda}1–J{lambda}1 rearrangement and {lambda}1 transcript in the day 13 and 14 scid samples showed this to be ~80% of that in the corresponding wt samples (Table 1). It should be noted that the observed level of V{lambda}1–J{lambda}1 rearrangement in DNA from 106 fetal liver cells was about two orders of magnitude less than in control DNA samples from 106 adult bone marrow cells of wt mice (data not shown). This is not surprising, as day 13–14 fetal liver lacks detectable pre-B cells and contains <=1% pro-B cells (reference (53) and our unpublished results).

V{lambda}1–J{lambda}1 Rearrangements from scid Mice Contain Abnormal Deletions at the VJ Junction.
PCR-amplified V{lambda}1–J{lambda}1 rearrangements were detectable not only in scid fetal liver and adult bone marrow, but also in scid adult spleen. In most of these rearrangements, the V{lambda}1 and/or J{lambda}1 coding segments were abnormally truncated by >20 nucleotides. Deletions of this magnitude were not observed in V{lambda}1–J{lambda}1 rearrangements from wt mice. Representative results are illustrated in Fig 3 and Fig 4 for adult bone marrow and spleen, respectively. More than 70% of scid V{lambda}1–J{lambda}1 rearrangements (121/147 distinct sequences analyzed) contained abnormal deletions; those lacking such deletions often showed unusually long P additions, as illustrated in Fig 4.



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Figure 3. Representative V{lambda}1–J{lambda}1 junctional sequences in genomic DNA from pooled fetal liver (FL) of scid (s/s) embryos (day 13) and from pooled bone marrow (BM) of s/s, wt (+/+), and JHT adult mice. Germline nucleotides (nt) for the 3' and 5' ends of the V{lambda}1 and J{lambda}1 gene are shown at the top (the germline V{lambda}1 and J{lambda}1 coding regions comprise 297 and 38 nt, respectively). Upper- and lowercase letters under the N/P column denote N and P nucleotide additions, respectively. The number of V or J nucleotides deleted from the V{lambda}1 or J{lambda}1 coding end is indicated in parentheses; the asterisk denotes an in-frame rearrangement.



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Figure 4. Representative V{lambda}1–J{lambda}1 junctional sequences in genomic DNA of pooled spleen (SPL) from scid (s/s) and wt (+/+) adult mice. Format is as in Fig 3.

V{lambda}1–J{lambda}1 rearrangements from bone marrow of JHT mice, in contrast to those from bone marrow of scid mice, showed nontemplated (N) additions and comparatively small deletions at the VJ junction (Fig 3). N addition is dependent on terminal deoxynucleotidyl transferase (TdT) (54) (55). This enzyme is expressed at the pro-B cell stage (56), the stage at which B cell differentiation is arrested in JHT mice (24). The absence of N additions in V{lambda}1–J{lambda}1 junctions from wt mice (Fig 3 and Fig 4) is in agreement with earlier reports (57) (58) and consistent with the occurrence of most L chain rearrangement at the late pre-B cell stage (5) (6), when TdT expression is dramatically downregulated (56). As scid and JHT mice both show an arrest of B cell differentiation at the pro-B stage, the abnormal loss of nucleotides in scid V{lambda}1–J{lambda}1 junctions must reflect the effect of the scid mutation and not a peculiarity of premature V{lambda}1–J{lambda}1 rearrangement.

{lambda}1 Transcripts in scid Adult Bone Marrow and Spleen Lack Abnormal Deletions at Their VJ Junction.
Most {lambda}1 transcripts from scid adult bone marrow and spleen corresponded to in-frame V{lambda}1–J{lambda}1 rearrangements with frequent P additions (illustrated in Fig 5). The P additions consisted of three to five nucleotides (cag, ccag, and gaaat) and were found repeatedly in individual mice. The most common recurring sequence consisted of two P additions separated by an AT dinucleotide (gaaat-AT-ccag). Interestingly, the AT dinucleotide is palindromic to the last two nucleotides of the (gaaat) P addition. Similar restricted VJ junctional sequences and recurring P additions were also observed in {lambda}1 transcripts recovered from bcl-2 scid spleen (data not shown). It is important to note that each PCR amplification of bone marrow and splenic cDNA from scid mice was done in parallel with PCR amplification of splenic cDNA from wt and RAG1-/- mice. We found that {lambda}1 transcripts from three individual wt mice lacked N/P additions (illustrated in Fig 5). No {lambda}1 transcripts were recovered from cDNA of RAG1-/- mice. Thus, the observed recurring P additions appear to be a unique property of {lambda}1 transcripts in the bone marrow and spleens of scid mice.



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Figure 5. Representative VJ junctional sequences in {lambda}1 transcripts from individual scid (s/s), wt (+/+), and JHT scid mice. Both bone marrow (BM) and spleen (SPL) of three s/s and three +/+ mice was analyzed; the results for two individuals are shown. Format is as in Fig 3.

We analyzed a total of 188 cloned sequences from {lambda}1 transcripts in bone marrow and/or spleens of five individual scid mice. We found that 115 clones contained P additions. In Fig 6, each distinct junctional sequence (denoted A–I) among the 115 clones is listed according to its representation in individual mice and overall frequency (see histogram). Sequences B and C are treated as one in the histogram, as are sequences F and G, because each of these pairs is identical except for the substitution of T (underlined) for C in the V{lambda}1 germline codon, AGC. Note that (a) sequences A–G, comprising most of the clones (110/115), corresponded to in-frame V{lambda}1–J{lambda}1 rearrangements; (b) sequences A, B, and C accounted for ~70% (83/115) of the V{lambda}1–J{lambda}1 rearrangements; and (c), the gaaat-AT-ccag P addition was present in all mice analyzed and represented nearly 40% of the clones (45/115).



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Figure 6. Recurring P additions in VJ junctions of {lambda}1 transcripts from scid adult mice. Bone marrow and/or spleens of five individual scid mice were analyzed. We obtained VJ junctional sequences from 188 clones and found that 115 of these contained P additions. 9 distinct VJ junctional sequences were found among the 115 clones; these are denoted (A–I) below the underlined germline sequence for the V{lambda}1 and J{lambda}1 coding ends. The number of mice that contained a given VJ junctional sequence is indicated at left. The asterisk denotes that the rearrangements were in frame. The overall frequency of each distinct junctional sequence (A–I) is shown in the histogram.

In contrast to scid mice, {lambda}1 transcripts from JHT scid mice lacked P additions and showed abnormal deletions at their VJ junctions similar to scid genomic V{lambda}1–J{lambda}1 rearrangements; furthermore, most corresponded to out-of-frame V{lambda}1–J{lambda}1 rearrangements (illustrated in Fig 5). These results suggest, as discussed later, that survival of {lambda}1-expressing scid cells could depend on the coexpression of a Dµ (or µ) chain. Consistent with this possibility, Dµ and {lambda}1 transcripts were the only Ig gene transcripts routinely detectable in scid bone marrow (illustrated in Fig 1). Moreover, most scid Dµ transcripts (10/16 analyzed) corresponded to DH–JH rearrangements in reading frame 2 (data not shown), which would be expected to result in the expression of a Dµ chain.

Cell Surface Expression of {lambda}1 Chains Is Detectable in Spleens of bcl-2 scid Mice.
The presence of {lambda}1-expressing scid cells in the spleen prompted us to test for possible cell surface expression of {lambda}1 chains ({lambda}1+ cells) in the spleens of scid and bcl-2 scid mice. The latter mice were included because survival of scid B lineage cells is known to be enhanced in the presence of the bcl-2 transgene (42). Large numbers (5–10 x 106) of cells were analyzed by three-color flow cytometry for expression of cell surface {lambda}1 and the B and T cell markers CD45 (B220) and CD8, respectively. Mice with an inactivated RAG1 locus (RAG1-/- mice; reference (45)) served as a negative control for background staining of {lambda}1. As illustrated in Fig 7, there were no detectable {lambda}1+B220+CD8- cells in scid and RAG1-/- mice. However, in bcl-2 scid mice, {lambda}1+B220+CD8- cells were detectable at a frequency of 0.02–0.05% versus 0.5–1.5% in the wt controls. The distribution of B220 staining for {lambda}1 gated cells is shown in the histograms on the right side of Fig 7. Note that bcl-2 scid spleen cells stained less bright for B220 and {lambda}1 than scid/+ or bcl-2 scid/+ spleen cells. These results indicate that a low frequency of scid cells express {lambda}1 chains on their cell surfaces and can be detected in the spleen of bcl-2 scid mice.



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Figure 7. Detection of {lambda}1-expressing cells in spleens of bcl-2 scid (s/s) mice by three-color flow cytometry. Gate settings for scoring {lambda}1+ cells in bcl-2 s/s mice were based on the distribution of {lambda}1 staining in positive and negative controls, bcl-2 s/+ and RAG-/- mice, respectively. {lambda}1+ cells in bcl-2 s/s and s/+ control mice are denoted in the shaded areas of the histograms for relative cell number versus {lambda}1 staining. The {lambda}1+ cells in the shaded areas were analyzed for CD8 and B220 expression; most of these cells (>85%) displayed a B cell phenotype ({lambda}1+B220+CD8-) and fell within the boxed areas. The {lambda}1+ cells in bcl-2 s/s mice represented ~0.02–0.05% of the spleen cells analyzed and showed a {lambda}1dullB220dull phenotype. In contrast, {lambda}1+ cells in s/+ and bcl-2 s/+ mice were {lambda}1brightB220bright and represented ~0.5–1.5% of the spleen cells analyzed. The overlay of B220 histograms on the far right shows the distribution of B220 staining for cells in the s/s and RAG-/- mice and for {lambda}1+ cells in the bcl-2 s/s, s/+, and bcl-2 s/+ mice.

Three-color flow cytometry was used also to analyze bcl-2 scid mice for expression of surface µ chains (µ+ cells). No µ+ cells were detected (data not shown), consistent with previous reports showing a lack of µ+ cells in bcl-2 scid spleen (42) (59).


  Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

The preceding results support earlier evidence, cited in the Introduction, that initiation of rearrangement at the {lambda} locus does not require prior rearrangement at the H or {kappa} chain locus. Our detection of {lambda}1 transcripts in bone marrow of scid and JHT mice indicates that V{lambda}1–J{lambda}1 rearrangement can occur before the pre-B cell stage and independently of H chain gene rearrangement. Moreover, the detection of V{lambda}1–J{lambda}1 rearrangement as early as day 12 in wt fetal liver, in which there is no genetic impairment of Ig gene rearrangement, suggests that the onset of V{lambda}1–J{lambda}1 rearrangement may developmentally precede VH–DJH and V{kappa}–J{kappa} rearrangement. The latter rearrangements were not detectable before day 14.

The most novel aspect of our findings is the regular detection of V{lambda}1–J{lambda}1 rearrangement in scid mice. This would not have been predicted, particularly the comparable level of V{lambda}1–J{lambda}1 rearrangement in scid and wt fetal liver. Also unexpected are the recurring P additions in scid {lambda}1 transcripts and the presence of scid cells with in-frame V{lambda}1–J{lambda}1 rearrangements in the spleen. We discuss the implications of these findings below.

Levels of V{lambda}1–J{lambda}1 Rearrangement and {lambda}1 Transcript in scid Mice.
We were surprised to find that the level of V{lambda}1–J{lambda}1 rearrangement in scid fetal liver was comparable to that in wt fetal liver (Table 1), despite the abnormal loss of nucleotides in the VJ junction of most scid V{lambda}1–J{lambda}1 rearrangements. Thus, in scid fetal liver, abnormally truncated V{lambda}1 and J{lambda}1 coding ends appear to be joined as efficiently as V{lambda}1 and J{lambda}1 coding ends in wt fetal liver. This is unexpected because previous studies have clearly shown that V(D)J rearrangement is severely impaired in B and T lineage cells of adult scid mice (31) (32) (33) (34). Indeed, as discussed below, V{lambda}1–J{lambda}1 rearrangement in scid adult bone marrow cells appears to be much less efficient than in wt bone marrow cells. The basis for the comparable level of V{lambda}1–J{lambda}1 rearrangement in scid and wt fetal liver is unexplained. Possibly, the machinery available for nonhomologous end joining in fetal liver differs from that in adult bone marrow and is able to compensate for the scid deficiency in DNA-PKcs.

Whereas the level of {lambda}1 transcripts was comparable in scid and wt fetal liver, in scid adult bone marrow (from JHT scid mice), {lambda}1 transcript levels were ~25-fold less than in control adult bone marrow of JHT mice (Table 1). The latter comparison is valid because B cell differentiation is arrested at the same stage (pro-B cell stage) in both JHT and JHT scid mice. Moreover, neither JHT nor JHT scid mice can make Dµ or µ chains, which could potentially affect the selection (or survival) of cells with productive {lambda}1 transcripts.

Two explanations can be considered for the 25-fold difference in levels of {lambda}1 transcript in JHT and JHT scid adult bone marrow. The first postulates that the scid {lambda}1 transcripts are much less stable than those generated in wt bone marrow, possibly owing to the abnormal nucleotide deletions accompanying scid V{lambda}1–J{lambda}1 rearrangement. We would expect such instability to be manifest in scid fetal liver as well, and yet in this tissue the level of {lambda}1 transcript was comparable to that in wt fetal liver. A second explanation is that the 25-fold difference in abundance of {lambda}1 transcripts in JHT and JHT scid mice primarily reflects a lower efficiency of V{lambda}1–J{lambda}1 rearrangement in scid adult pro-B cells than wt adult pro-B cells. This explanation is consistent with previous studies showing a 10–20-fold lower level of DH–JH rearrangement in scid than in wt adult bone marrow (49) (50).

The question arises as to why we did not obtain evidence of premature V{kappa}–J{kappa} rearrangement in scid bone marrow, given that a low level of such rearrangement has been previously observed in the pro-B cell fraction of wt mice (5) (6). One possible reason is that initiation of V{kappa}–J{kappa} rearrangement invariably results in cell death. Consistent with this possibility, {kappa} transcripts were readily detectable in the bone marrow of bcl-2 scid mice. The bcl-2 transgene is known to promote the longevity of scid B lineage cells (42) and their differentiation beyond the pro-B cell stage (42) (59). As reasoned elsewhere (60), primary and secondary initiation of V{kappa}–J{kappa} rearrangement in scid cells lacking the bcl-2 transgene would be expected to result in persisting chromosomal breaks and cell death.

VJ Junctions of scid {lambda}1 Transcripts Show Recurring P Additions.
Most {lambda}1 transcripts recovered from scid adult mice corresponded to in-frame V{lambda}1–J{lambda}1 rearrangements, and >60% of these transcripts contained recurring P additions. Whereas in wt mice most P additions are one or two nucleotides (61), in scid mice they are often longer (62) (63). In the present case, all of the scid P additions at V{lambda}1 were five nucleotides in length (gaaat), and those at J{lambda}1 were either three or four nucleotides in length (cag or ccag). This may reflect a strong bias in the resolution of hairpin coding ends imposed by the scid DNA-PKcs deficiency, such that J{lambda}1 and V{lambda}1 coding ends are frequently nicked three to four and five nucleotides from the hairpin tip, respectively, and then joined without further modification. In addition, cells expressing {lambda}1 transcripts with these junctions may be strongly selected, as discussed later.

Of particular interest is the recurring V{lambda}1–J{lambda}1 junctional sequence of two P additions separated by an AT dinucleotide (gaaat-AT-ccag), which was present in ~40% of the {lambda}1 transcripts with P additions (Fig 6). The basis for the AT dinucleotide is unclear. Given that scid V{lambda}1–J{lambda}1 rearrangements occur at the pro-B cell stage in the presence of high TdT levels, the AT dinucleotide could represent a nontemplated addition mediated by TdT. Another possibility is that the AT dinucleotide, which is palindromic to the last two nucleotides of the gaaat P addition, corresponds to a secondary P addition. In this scenario, one could postulate two successive recombination events. The generation of the gaaat P addition would result from an open and shut recombination (64) at V{lambda}1. This would be followed by secondary cleavage at the V{lambda}1 signal/coding border, asymmetric nicking of the V{lambda}1 hairpin coding end, and joining to a J{lambda}1 coding end with a ccag overhang. Regardless of how the AT dinucleotide is generated, we suggest that cells with the gaaat-AT-ccag junctional sequence are strongly selected to account for the repeated occurrence of this sequence in {lambda}1 transcripts of individual scid mice.

Evidence for {lambda}1-Expressing scid Cells.
Scid {lambda}1 transcripts were not only detected in bone marrow but also in spleen. Moreover, {lambda}1 transcripts with the same VJ junctional sequences were often found to recur in both of these tissues. This implies a strong selection for cells with in-frame V{lambda}1–J{lambda}1 rearrangements containing particular VJ junctional sequences. Such selection was not evident in JHT scid mice. {lambda}1 transcripts from JHT scid bone marrow contained abnormal deletions at the VJ junction, lacked P additions, and in most cases corresponded to out-of-frame V{lambda}1–J{lambda}1 rearrangements. These findings suggest that survival and selection of {lambda}1-expressing cells requires a functional H chain locus.

How might {lambda}1-expressing scid cells be dependent on a functional H chain locus? One possibility is that Dµ and {lambda}1 chains, resulting from expression of in-frame Dµ and {lambda}1 transcripts in scid bone marrow, pair and associate with the Ig {alpha} and ß signal-transducing chains (65) (66) to form a B cell–like receptor (BCRDµ/{lambda}1). Although Dµ chains cannot pair efficiently with {kappa} chains (67), they can pair with the surrogate L chain (68) (69) and might be expected to pair with {lambda}1 chains as well because the latter share some homology with the surrogate L chain (for review see reference (70)). Expression of BCRDµ/{lambda}1 in this proposed scenario would signal rapid (or direct) progression of scid pro-B cells to the recombinase-inactive B cell stage and allow these cells to survive and migrate to the periphery. But as BCRDµ/{lambda}1 would lack a VH region, we would not expect cells bearing this receptor to persist or expand in response to naturally occurring antigens. This could in part explain the very low frequency of cells with surface {lambda}1 in bcl-2 scid spleen (<=0.05% of the cells examined). What is experimentally missing in support of the above scenario, however, is evidence for bcl-2 scid cells with surface {lambda}1 and µ chains. Despite reported evidence for intracellular expression of µ (or Dµ) chains in B220+CD22+ spleen cells of bcl-2 scid mice (59), cells with surface µ chains have not been detected in bcl-2 scid mice (42) (59).

In conclusion, we suggest that (a) joining of V{lambda}1 and J{lambda}1 coding ends after the initiation of V{lambda}1–J{lambda}1 rearrangement may not be impaired in B lineage cells of scid fetal liver because of the developmental time at which such rearrangement occurs; (b) recurring P additions in scid {lambda}1 transcripts may reflect a strong bias in the resolution of V{lambda}1 and J{lambda}1 hairpin coding ends imposed by the scid defect as well as possible strong selection for cells expressing these {lambda}1 transcripts; and (c) pro-B cells with in-frame V{lambda}1–J{lambda}1 rearrangements may express a pre-BCR–like receptor and differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.


  Footnotes

1 Abbreviations used in this paper: DNA-PKcs, DNA protein kinase catalytic subunit; JHT, JH targeted deletion; P, palindromic; RAG, recombination activation gene; RT, reverse transcriptase; TdT, terminal deoxynucleotidyl transferase; wt, wild-type.


  Acknowledgements
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

We thank Kerry Campbell, Randy Hardy, Dietmar Kappes, Pam Nakajima, Gillian Wu, and Martin Weigert for review of the manuscript and Samuel Litwin for helpful discussion. We also thank Roseanne Diehl for assistance in typing the manuscript.

Grants from the National Institutes of Health (CA06927 and CA04946) and an appropriation from the Commonwealth of Pennsylvania supported this work.

Submitted: 4 February 2000
Revised: 31 March 2000
Accepted: 7 April 2000


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Introduction
Materials and Methods
Results
Discussion
Acknowledgements
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