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V_H Gene Replacement in Thymocytes¹

Rachel Golub^2,*, Denise Martin^*, Fred E. Bertrand, Marilia Cascalho, Matthias Wabl and Gillian E. Wu^*

^* Department of Immunology and Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada; University of Minnesota Cancer Center, Minneapolis, MN 55455; and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143

	Abstract

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The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V_H gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V_H gene replacement. In normal mice, thymic DJ_H rearrangements are common, but VDJ_H rearrangements are not. We found intermediate products of V_H replacement in double-positive CD4⁺CD8⁺ cells of the QM thymus, demonstrating that the inserted V_H gene was accessible and ruling out the possibility that a V_H gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no µ H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V_H gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V_H-to-VDJ_H joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V_H joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.

	Introduction

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In the mouse, the primary repertoire of B and T cell Ag receptor (BCR³ and TCR) genes is generated in the bone marrow and thymus. This is an ordered, regulated process that ensures that the appropriate gene segments are arranged at the appropriate stage; functional TCR gene rearrangements do not occur in B cells, nor functional BCR gene rearrangements in T cells (1). Ag per se is thought not to play a role in this process. Although further diversification in the periphery is commonly thought to be rare for the TCR (2, 3), somatic hypermutation and isotype switching are common for the BCR (4, 5, 6).

Understanding the generation of TCR and BCR diversity has been aided by mutant mouse strain studies. To name a few, the µMT, the recombination-activating genes (RAG)1/2 ^-/-, SCID, PMS2^-/-, Eh^-/-, and the L chain and H chain transgenics (7, 8, 9, 10, 11, 12, 13, 14). The strains with knocked-in prerearranged H chain genes have been particularly informative for studies questioning which diversification mechanisms are used by mice with near-monoclonal repertoires (15, 16). One aspect of diversification exposed by these mice is the extent to which V gene replacement can be used. V gene replacement at the IgH locus is the activity wherein a new upstream V_H replaces the one in the original V_HDJ_H rearrangement, with a loss of the intervening DNA including the original V_H (17, 18, 19, 20, 21). V_H replacement is mediated by the embedded heptamer, a sequence near the 3' end of most V_H, possibly in conjunction with a nonstandard nonamer (22, 23, 24, 25). It is the only known mechanism of receptor editing at the IgH locus (18). Because V_H replacement was not detected in RAG-deficient mice (26), it is likely to be RAG mediated and hence take place only in cells that have RAG activity. Most V_H, V, V, and V gene segments contain a sequence that qualifies as an embedded heptamer (27), and any cell with a sufficient level of RAG activity might undergo V gene replacement.

Because the new incoming V_H segment replaces all but a few bases of the original V_H, it has been difficult to assess how extensive a role V_H replacement plays in generating diversity. However, an intermediate in the replacement reaction—the signal-end intermediate of the original V_H—can be assessed (28, 29, 30). The signal end, which is blunt and 5'-phosphorylated, can be detected by a ligation-mediated PCR (LM-PCR) assay (28). We have reported the existence of such intermediates in the spleen and bone marrow of quasi-monoclonal (QM) mice (31), one of the knock-in strains that bear a V_HDJ_H gene segment inserted into the natural location in the IgH locus (16, 32, 33, 34).

We have used thymocytes from a QM mouse that bears a knocked-in IgH to help answer questions about the requirements to diversify the IgH gene. Based on a number of studies including another knocked-in mouse model (22), it has been proposed that receptor editing, and hence V gene replacement, is regulated by Ag-driven stimuli through the BCR. We decided to examine the IgH locus in developing thymocytes, where, in normal mice, DJ_H rearrangements are frequent and rearrangements extending to the V_H region are not (35, 36, 37, 38, 39). Indeed, although DJ_H alleles are sometimes transcribed, Dµ protein has not been detected. The QM thymus, where by definition B lineage-specific signals cannot be, presented an ideal model system to determine whether Ag-driven stimuli are absolutely required for V_H replacement.

Here, we demonstrate V_H replacement in thymocytes. Thus, neither activation of the BCR nor of any other B cell-specific stimulus is absolutely required for V_H gene replacement. The question remains as to why in normal thymocytes the V_H locus is not open to rearrangement, whereas in the knocked-in thymocytes it is.

	Materials and Methods

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Mice

QM mice are heterozygous at the IgH locus. One homologue has a knocked-in, rearranged VDJ_H containing VH17.2.25; on the other, all J_H segments have been knocked out (34). QM mice are also homozygous for an inactive allele at the L chain locus (33).

Preparation of cells and flow cytometric analysis

Thymocytes were isolated from 4- to 8-wk-old QM mice by standard methods and stained with a combination of three Abs (PharMingen, San Diego, CA): PE-conjugated anti-B220, FITC-conjugated anti-CD4, and biotin-conjugated anti-CD8 developed with streptavidin-Quantum Red (Sigma, St. Louis, MO). Populations of stained cells were sorted with a FACStar^Plus equipped with Turbo Sort using Lysis2 software (Becton Dickinson, Mountain View, CA). Sorted samples were reanalyzed with the same machine and the same Abs to check purity. Cells were then counted and prepared for DNA extraction.

For the FACS data shown in Fig. 5, cells of QM and C57BL/6 mice were triple-stained with the following Abs: FITC-conjugated anti-CD4 (PharMingen), PE-conjugated anti-CD8 (PharMingen), and biotin-conjugated anti-µ H chain (mAb 33-60). Staining with biotinylated Abs was revealed using Quantum Red-conjugated streptavidin (Sigma). To detect surface and cytoplasmic µ protein, cells were stained with PE-conjugated anti-CD3 (PharMingen), µ H chain-specific FITC-conjugated anti-IgM (Southern Biotechnology Associates, Birmingham, AL), and biotin-conjugated anti-µ H chain (mAb 33-60). For cytoplasmic µ protein, surface proteins were stained using standard methods followed by a 1-h incubation with ice-cold 70% ethanol and intermittent vortexing. Cells were washed twice and then incubated with FITC-conjugated anti-IgM for 45 min on ice to detect intracellular µ. The data were analyzed with the CellQuest program (Becton Dickinson).

View larger version (46K): [in this window] [in a new window]   FIGURE 5. FACS analysis of thymocytes and splenocytes from QM and C57BL/6 mice: Ig µ H chain protein is not expressed in thymocytes. A, Thymocytes stained for CD4, CD8, and µ, and for CD3 and cytoplasmic µ. Staining profiles in QM and control C57BL/6 mice are comparable. No significant surface or cytoplasmic µ H chain protein was detected in thymocytes of either strain. B, Total splenocytes from QM and C57BL/6 mice stained for cytoplasmic and surface µ. There are normal numbers of cells expressing surface and cytoplasmic µ H chain protein in both strains. Specificity and sensitivity of the anti-µ reagent and the streptavidin-Quantum Red reagents are shown.

All PCR primers and probes used in this study are listed in Fig. 1.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. PCR primers and oligomer probes used in this study.

DNA from cell lysates (1.5 µg) was ligated to the BW linker (40) (8 ng) with 2 U T4 ligase (Life Technologies, Rockville, MD) for 16 h at 16°C and heated to 95°C for 15 min. The first round of PCR was performed with 300–400 ng ligated DNA, 15 ng each of primers BW-1HR (28) and V_HA, 10 mM Tris (pH 8.3), 50 mM KCl, 0.5% Triton X-100, 2 mM MgCl₂, and 2.5 U Taq polymerase (Boehringer Mannheim, Indianapolis, IN). A "touch down and hot start" PCR program was used: 15 cycles of 30 s denaturation at 94°C, 1 min annealing at 56°C, and 1 min elongation at 72°C, then another 15 cycles in which the annealing temperature is decreased to 53°C. The second round of PCR was done under the same conditions, with 1 µl of a 1/50 dilution of the first PCR and 8 ng each of BW and nested primer V_HB.

Signal joint PCR on DNA from cell lysates

Seminested PCR was performed with 100 ng DNA to amplify the signal-end deletion product from a recombination signal sequence (RSS) fusion of the embedded heptamer of VH17.2.25 to the RSS of a member of the V_HJ558 family. The first 30 cycles of amplification used the 5' primer V_HB and a 3' J558 RSS consensus primer. The primer hybridization temperature starts at 56°C and decreases to 53°C. The second round of PCR uses 1 µl of a 1/50 dilution of the first PCR as template DNA, with the same conditions except that the 5' nested primer is V_HC.

RT-PCR

RNA was purified with Tri-Reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized using standard methods with avian myeloblastosis virus-reverse transcriptase (Boehringer Mannheim). PCR conditions using the primers described were: 30 µl volume, 30 cycles of 30 s at 95°C, 30 s at 55°C, 1 min at 72°C.

Analysis and cloning of PCR products

PCR products were run on 1% agarose gels. DNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) by capillary blotting and cross-linked with standard protocols. Southern blots were probed with V_Hall (see Figs. 2 and 3) or Cµ1-5' (see Fig. 6), exposed to PhosphoImager plates for 4 h, and analyzed with a Storm PhosphoImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The TA-cloning method (Invitrogen, San Diego, CA) was used according to the manufacturer’s protocol to clone PCR products. A T7 sequencing kit (Pharmacia, Piscataway, NJ) was used to sequence clones in both directions by the dideoxy method.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. A, Strategy of the LM-PCR. The signal-end intermediates are amplified with 5' primers VHA and VHB specific for VH17.2.25 of the QM mouse and a 3' primer specific for the BW linker. B, Analysis of VH replacement intermediates in bone marrow (BM), spleen (Spl), and thymus (Thy) of QM mice by the LM-PCR assay. Ethidium bromide-stained agarose gel of amplification products of 511 bp. Below, The Southern blot probed with VHall, a primer specific for VH segments.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. Analysis of VH replacement intermediates in thymocytes of QM mice at four stages of T cell development by the LM-PCR assay. The amplification strategy for VH replacements is the same as in Fig. 2. Actin DNA was amplified as a quantification and DNA quality control. A, Ethidium bromide-stained VH amplification products. B, Southern blot of A probed with VHall. C, Ethidium bromide-stained actin amplification products. D, Amplification of transcripts by RT-PCR from sorted QM DP thymocytes, T cell marker CD3, neo, and the B cell marker CD19. The fourth lane is the control without reverse transcriptase. The PCR lane is amplification of DP thymocyte cell lysis DNA with the CD19 primers.

View larger version (61K): [in this window] [in a new window]   FIGURE 6. Detection of transcripts from unsorted thymocytes. A, RAG1 and control without reverse transcriptase; B, V-Cµ and CD3; C, Southern blot of B probed with Cµ1-5'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Using the LM-PCR assay (Fig. 2A), we searched for V_H signal-end intermediates at the IgH locus in DNA from the thymus of QM mice. For comparison, the same strategy was applied in parallel to spleen and bone marrow DNA (Fig. 2B). Southern blots were probed with V_Hall, a primer specific for V_H segments, to confirm the specificity of the 511-bp amplification product obtained from spleen, bone marrow, and thymus (Fig. 2B). Then, from each tissue, the amplification product was cloned. From the thymus samples, nine plasmid clones were sequenced and then compared with three clones from the bone marrow and seven from the spleen. Most sequences (15/19) were signal-end intermediates of V_H17.2.25, the V_H knocked into QM mice. As in the bone marrow (31), every V_H17.2.25 cleavage product in the thymus was a true V_H replacement intermediate; the product ended at the 3'-embedded heptamer at the last G of the TACTGTG motif (Table I). We conclude that V_H replacement intermediates can be generated at the IgH locus in the thymus of QM mice.

To identify the cells in which V_H replacement takes place, we sorted thymus cells into subsets based on CD4 and CD8 expression. B220⁺ cells, which comprised <0.2% of the original population, were first eliminated by sorting. From the B220^- population, double-negative CD4^-CD8^-, DP CD4⁺CD8⁺, and the two single positives CD4⁺CD8^- and CD4^-CD8⁺ were isolated. For the double-negative fraction, two sorts were pooled to obtain enough cells. Upon re-analysis, the purity of the sorted cell fractions was at least 99%.

The four fractions were analyzed by LM-PCR for the presence of linker-ligated V_H17.2.25 segments (Fig. 3A). After seminested PCR, an amplification product was evident only in the DP fraction both by ethidium bromide staining (Fig. 3A) and by Southern analysis with V_Hall (Fig. 3B). The absence of signal in the other fractions was confirmed by prolonged exposure of the blots. In parallel, the actin gene was amplified by PCR (Fig. 3C); this confirmed the quantity and quality of DNA used from the four fractions. Using a sensitive RT-PCR assay for CD19 RNA, a well-expressed marker of the B lineage, we could not detect CD19 cDNA in DP cells; as a control, CD19 DNA was easily detected in the same cells (Fig. 3D). This result ruled out the possibility that the replacement intermediates were in contaminating B cells and leads to the conclusion that V_H replacement intermediates can be generated at the IgH locus in DP thymocytes of QM mice.

Fused signal-end joints generated by V_H replacement in DP thymocytes of QM mice

To confirm that the free signal-end intermediates detected were generated in V_H replacement events, we searched for signal joints, specifically for the embedded heptamer of V_H17.2.25 fused to an RSS from V_HJ558, the largest mouse V_H gene family. Using 5' primers V_HB and V_HC, specific for V_H17.2.25, and a 3' J558 RSS primer, a band of the expected size, 473 bp, was found in the QM DP thymocyte and splenic cell lysates (Fig. 4). This band was absent in a C57BL/6 splenic lysate; that is, the primers used are specific for the product and do not nonspecifically amplify germline V_HJ558 genes present in both QM and C57BL/6. We conclude that fused signal-end products generated by V_H replacement at the IgH locus are present in DP thymocytes of QM mice.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Analysis of VH signal joint products by PCR in QM DP thymus cells. VH deletion products are present in QM thymus and spleen, but not in normal C57BL/6 spleen.

To determine whether an external signal through the BCR might promote V_H replacement in thymocytes from QM mice, we looked for evidence of µ protein in these cells. Thymocytes from QM and C57BL/6 control mice were stained with anti-CD4, anti-CD8, anti-CD3, and anti-µ (Fig. 5A). There were no detectable surface µ⁺/CD4⁺ or µ⁺/CD8⁺ thymocytes in either strain. When stained for CD3 and cytoplasmic µ, there were no double-stained thymocytes. Because T cells do not express µ^--associated proteins required for surface expression, including Ig, the absence of stable µ protein in thymocytes is not surprising. Nonetheless, the absence of µ protein suggests that H chain protein is not required for V_H replacement. The sensitivity and specificity of the FACS was assessed using splenocytes from the same animals. As would be expected, the splenocytes of the two strains have cytoplasmic and surface µ⁺ cells in comparable numbers (Fig. 5B).

µ-Chain transcripts in thymocytes of QM mice

From unsorted QM thymocytes, we monitored µ H chain mRNA by RT-PCR using primers V_Hall and C_µ-ex1, with RAG1 and CD3 assayed in parallel as controls (Fig. 6). IgH transcripts that included the knocked-in V_HDJ_H were present.

To determine whether some of these transcripts resulted from V_H replacement in DP thymocytes, we amplified and sequenced µ transcripts from the DP fraction. The 5' primer V_Hall can amplify any V_H, and the 3' primer is complementary to J_H4. Each cloned VDJC_µ rearrangement was digested with the restriction enzymes StuI, which is unique to V_H17.2.25, and EcoRV, which is unique to the vector. Clones that were not digested by this combination of enzymes were potential new V_HDJ_H rearrangements.

Of 19 VDJCµ clones from two independent experiments of two mice each, six had lost the restriction sites and were sequenced. All six were new rearrangements (Fig. 7). Five clones resulted from V_H replacement, with the new V_H gene segment being from four V_H families, including V_HJ558. The V segment of clone 5 differed from V_H17.2.25 at only 2 nt, but had been processed (3 N nt added, 6 nt deleted from D) at the V-D junction. Clone 5 could be either an open-and-shut rearrangement of the knocked-in QM VDJ_H4, which is generally a rare event (41), or a V_H gene replacement using the endogenous V_H17.2.25. The same 6 nt were deleted from the D segment of clone 3. Clones 7Q and 1Q (and another clone with identical sequence to 1Q) have coding-end processing typical of that previously observed in V_H replacements. In the two 1Q sequences, there is an additional D segment, D_SP2.2; in B lineage cells from the QM mouse, additional D_H (V-D-DJ_H4) are frequently found (16, 32). From the sequences of these transcripts, we conclude that V_H replacement occurs in DP thymocytes of the QM mouse.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Alignment of VHDJH rearrangements amplified by RT-PCR from DP thymocytes. Based on a GenBank search, each incoming VH gene segment was attributed to a VH family. QM denotes the knocked-in H chain of the QM mouse. In clones 1, 3, 1Q (found twice), and 7Q, the QM VH has been replaced. Clone 5 is either a VH replacement by the endogenous VH17.2.25 gene or an open-and-shut rearrangement. Clone 1Q possesses a new incoming DH segment, which is double-underscored.

	Discussion

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Some of the elements regulating V-to-D joining are different from those regulating D-to-J joining (45, 46, 47). We can think of two possibilities as to why V_H replacement occurs during thymic development in the QM strain but is not detectable in unmanipulated strains. First, it may well be that the V_H promoter in the proximity of its J-C intron enhancer leads to a complex that opens up the V_H region, making it accessible to recombination (48, 49). Second, silencers and barriers to recombination have been detected in a few species and loci (45, 50, 51, 52). It may well be that the insertion of knocked-in prerearranged H chain genes (16) disrupted a barrier or silencer that functioned to prevent V_H-to-D_H joining in the T lineage, thus allowing V_H-to-D_H joining in DP thymocytes.

We used three independent approaches that established the existence of V_H replacement in the thymocytes of QM mice: 1) signal-end intermediates in V_H replacement were identified with the LM-PCR assay; 2) fused signal-end joints generated by V_H replacement were amplified by normal PCR; and 3) µ transcripts of genes with V_H replacement were amplified by RT-PCR and sequenced. These results mean that V_H replacement can diversify an IgH receptor while not necessarily requiring a BCR-mediated external signal. Because no µ protein could be demonstrated in thymocytes, it would seem unlikely that a µ-mediated internal signal would be needed. Moreover, the absence of CD19 transcripts in QM thymocytes reinforces the FACS data and ensures that no contaminating B cells were present. These data do not rule out the possibility that V_H replacement can be induced through signals via the BCR. In addition, V_H replacement might occur during the normal course of early B cell development, implicating a pathway other than signaling through the mature surface BCR. In this regard, we have found evidence in QM bone marrow of intermediates of V_H replacement in the pro-B cell compartment (data not shown). Thus, we favor, as do others, the hypothesis that V_H replacement is a mechanism that can be used to diversify the B cell repertoire (19).

It would follow that receptor editing is a selective rather than an instructional event. That is, in the B lineage, V_H replacement may occur in any cell in which RAG is still expressed. The cell is not instructed to replace its V_H segment after the first VDJ-encoded protein has been tested and found wanting; instead, after that protein is found wanting, some of the cells that have undergone V_H replacement are selected.

	Acknowledgments

 
We thank Charley Steinberg for discussions and early help with the preparation of this manuscript and Queenie Lam and Stacy Hirano for excellent technical assistance.

	Footnotes

 
¹ R.G., D.M., and G.E.W. were supported by the Medical Research Council of Canada, the Terry Fox Marathon of Hope, the Leukemia Society of Canada, and the Cancer Research Society of Canada. F.E.B. is a Special Fellow of the Leukemia Society of America. M.C. and M.W. were supported by National Institutes of Health Grant R01 AI41570, a Howard Hughes Transgenic Mouse Grant, and the Junta Nacional de Investigação Científica e Tecnológica of Portugal.

² Address correspondence and reprint requests to Dr. Rachel Golub, Unité du Développement des Lymphocytes, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

³ Abbreviations used in this paper: BCR, B cell receptor; QM, quasi-monoclonal; RAG, recombination activating genes; LM-PCR, ligation-mediated PCR; DP, double- positive; RSS, recombination signal sequences.

Received for publication August 2, 2000. Accepted for publication October 19, 2000.

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