Address correspondence to Thomas H. Winkler, Hematopoiesis Unit, Nikolaus-Fiebiger-Center, Glueckstrasse 6, 91054 Erlangen, Germany. Phone: 49-9131-8529136; Fax: 49-9131-8529106; email: twinkler{at}molmed.uni-erlangen.de
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Abstract |
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Key Words: B cell development allelic exclusion Rag TdT pre-BCR
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Introduction |
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Furthermore, signals transduced through the pre-BCR are also implicated in maintaining allelic exclusion at the Ig HC locus by preventing further rearrangement of the second, DJH-rearranged allele. This is presumably achieved by shutting down the recombination machinery (e.g., the Rag genes; reference 5) and closing of the remaining Ig HC allele during subsequent LC rearrangement. Interestingly, Ig HC allelic inclusion is observed in mice in which the transmembrane region of µHC has been mutated (µmT), demonstrating that the membrane-form of the Ig HC is crucial for allelic exclusion to occur (6). In contrast, all mouse models in which components of the SLC have been deleted still display allelic exclusion at the level of surface Ig expression (79).
Several hypotheses have been put forward to explain this inconsistency. It was suggested that premature rearrangement and expression of LC in a small number of cells could "rescue" B cell development and Ig HC allelic exclusion (10). The observation that some µHCs can associate with VpreB alone (11) led to the hypothesis that this pre-BCRlike complex could signal allelic exclusion in the absence of
5. However, the observation that allelic exclusion is still maintained in VpreB1/VpreB2/ double-deficient and VpreB1/VpreB2/
5/ triple-deficient mice (8, 9) suggested that in the absence of
5 and/or VpreB1/2 µHC pairs with other, yet undefined partners resulting in a signaling complex could substitute for some, but not all, pre-BCRspecific functions.
To achieve a greater understanding of the mechanisms contributing to allelic exclusion, especially in SLC-deficient mice, we asked how the de novo synthesis of a tetracycline-controlled transgenic µHC affects the expression of the cellular recombination machinery (Rag1/2 and terminal deoxynucleotidyl transferase [TdT]) in the presence or absence of either 5 or VpreB1/2. The same question was also investigated in mice lacking the entire SLC (SLC/). In addition, we have analyzed the cellular location and composition of putative signaling complexes in these same mice.
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Materials and Methods |
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Sorting and Culturing of BM-derived B Lineage Cells.
ProB cells were isolated from BM single cell suspension using anti-CD19coated magnetic beads (Miltenyi Biotec) following the manufacturer's instructions or by sorting green fluorescent protein+ (GFP) cells (NG-BAC) using a MoFloTM high-speed sorter (DakoCytomation). ProB cells were cultured on -irradiated ST2 cells in medium with or without IL-7 (14). IMDM medium supplemented with 2% FCS, 1 mM glutamine, 50 µM ß-mercaptoethanol (GIBCOTM; Invitrogen), and 0.3 mg/ml Primatone® RL (Sheffield Products) was used for all cell culture experiments unless stated otherwise. Tetracycline hydrochloride was added to the culture medium at a concentration of 100 ng/ml to block transgenic HC expression.
For enzymatic amplification staining of surface pre-BCR components, MACS-sorted tet-µHC CD19+ BM cells were cultured for 2 d in the presence of Tet, splitted, and recultured for an additional 18 h in the presence or absence of Tet on -irradiated stromal cells in the presence of IL-7. For SLC/ mice, MACS-sorted CD19+ cells were cultured for 4 d on
-irradiated stromal cells in the presence of IL-7 in RPMI 1640 and supplemented with 50 µM ß-mercaptoethanol, antibiotics, and 10% FCS.
Antibodies and Flow Cytometric Analysis.
Antibodies for surface stainings include: PE-conjugated anti-CD19 mAb, FITC- or PerCPTM-conjugated anti-B220 mAbs, PE-conjugated antic-kit, and anti-CD25 mAbs (BD Biosciences).
For intracellular stainings, cells were fixed and permeabilized using the FIX & PERM® kit (Caltag Laboratories) and the following antisera: FITC- or CyTM5-conjugated polyclonal goat antimouse IgM (µH chain specific) and FITC- or CyTM5-conjugated polyclonal donkey antirabbit IgG (Dianova); FITC-conjugated antiL-chain and anti
1+
2L-chain mAbs (BD Biosciences); polyclonal rabbit anti-TdT (Supertechs); and polyclonal rabbit anti-Ku70 (DPC Biermann).
RNA Fluorescent In Situ Hybridization (FISH).
The Rag1 cDNA fragment was amplified by PCR using the following primers: 5'-AGTGAGGTCTTCTCCTAGCACCTA-3'; and 5'-ATGATTTTCTGAACCTCTCTTGG-3'. The PCR product was cloned into the pCR2.1 vector (GIBCOTM; Invitrogen). To prepare probes for RNA FISH, the plasmid was first linearized by restriction enzyme digest, either 5' or 3' of the insert for preparation of antisense and sense probes. Thereafter, RNA was synthesized using T7 or T3 RNA polymerase as instructed using the MEGAscriptTM High Yield Transcription Kit (Ambion), followed by RT-PCR amplification using p(dN)6 random primers and digoxygenin-dUTP.
RNA FISH was performed as described by Gribnau et al. (15) with the exception that the hybridization mix contained 50% formamide. Before application to the slides, the hybridization mix containing the probes was denatured at 85°C. Antibody detection of labeled probe was preceded by a 45-min incubation of the slides with TSB (0.1 M Tris, 0.15 M NaCl, and blocking reagent; Roche Diagnostics Ltd.) at room temperature. Probe was detected using the following antibodies that were diluted in TSB and each incubated with the slides for 45 min at room temperature: sheep anti-DIG (Roche Diagnostics Ltd.), FITC-labeled rabbit antisheep, and goat antirabbit (Calbiochem-Novabiochem). Images were examined by microscope under oil with a 100x objective.
Enzymatic Amplification Staining.
Membrane pre-BCR components were detected by enzyme amplification staining (EAS) using the Flow-AmpTM kit for cell surface molecules stained by biotinylated primary antibodies (Flow-Amp Systems, Ltd.). EAS staining achieves signal enhancement with enzymes that catalyze the deposition of labeled molecules onto the cell surface. Although the label binds covalently to any cell surface protein, the deposition is specific because it is proximity controlled by specific antibodydependent binding to a targeted cell surface molecule. Abs used for this method include: biotin-conjugated polyclonal goat antimouse IgM (µH chain specific; Sigma-Aldrich), biotin-conjugated rat mAbs R6-60.2 (antimouse IgM), SL156 (antimouse preB cell receptor), and LM34 (antimouse 5; BD Biosciences). Hybridomas producing rat mAbs VP245 and R5 (antimouse VpreB) were provided by F. Melchers (University of Basel, Basel, Switzerland; reference 16) and M.D. Cooper (University of Alabama, Birmingham, AL; reference 17). These two mAbs were purified from hybridoma supernatants on protein GSepharose, biotinylated using standard procedures, and tested by flow cytometry on the preB cell line NFS5. Biotin was revealed using PE-streptavidin purchased from BD Biosciences. After EAS surface staining, cells were incubated with propidium iodide (Sigma-Aldrich) to exclude dead cells from analysis. Fluorescence of cells in the lymphocyte gate was determined on a four-color FACSCaliburTM instrument (BD Biosciences), and data files were analyzed using FlowJoTM software (Tree Star, Inc.) or CellQuestTM (BD Biosciences).
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Results |
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To confirm a causal connection, we took advantage of a tetracycline-controlled µHC transgene that can be expressed in the absence of endogenous V(D)J rearrangements, called tet-µHC (4). In these mice, µHC expression is completely suppressed, and B cell development is blocked at the proB cell stage after treatment with tetracycline in the drinking water for 7 d (4). CD19+ BM proB cells from these mice were cultured on stromal cells in the presence of IL-7. Expression of the transgenic µHC was induced by omitting tetracycline. After 24 h, the cells were harvested, stained for intracellular µHC and TDT expression, and analyzed by FACS®. Fig. 1 A demonstrates that µHC positive cells (Tet) showed a marked decrease in TdT protein level compared with µHC negative cells (Tet). Surprisingly, after µHC induction (Tet), a similar, though less pronounced down-regulation of TdT was observed in cells that lack the SLC components 5 or VpreB1/2, and, hence, cannot express a complete pre-BCR complex. Parallel staining for Ku70, an ubiquitously expressed protein involved in V(D)J recombination, as well as general DNA repair (22), revealed no decrease in µHC positive cells, confirming the specificity of µHC-mediated TdT down-regulation in the absence of
5 or VpreB1/2. Comparable results were obtained with cells cultured in the absence of IL-7 (unpublished data). Thus, de novo expression of µHC is causally involved in the down-regulation of TdT and, unexpectedly, de novo expression of a µHC can signal the down-regulation of TdT in the absence of the pre-BCR.
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TdT Is Down-regulated in µHC-expressing BM PreB Cells in the Absence of SLC Components and Conventional LC.
To further investigate the unexpected down-regulation of TdT expression in the absence of the pre-BCR, we analyzed wild-type mice that express a polyclonal µHC repertoire as well as mice deficient in all SL chain components. We compared TdT levels of proB and preB cells in recombination competent wild-type, VpreB1/VpreB2/, and SLC/ mice. For this purpose, four-color flow cytometric analyses were performed on CD19+ BM cells. B lineage cells were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect cytoplasmic µHC and nuclear TdT. Antibodies specific for and
LC were also included to exclude cells expressing surface or intracellular LC.
In wild-type mice, as expected, most c-kit+µHC proB cells showed high levels of TdT expression, whereas most c-kit+µHC+ cells had down-regulated TdT (Fig. 2 A, top), presumably because they already express the pre-BCR (23). As expected, in VpreB1/ VpreB2/ and SLC/ mice, most c-kit+µHC cells were positive for TdT (Fig. 2 A, middle and bottom). However, in contrast with wild-type cells, TdT levels stay elevated in most c-kit+µHC+ cells from both VpreB1/VpreB2/ and SLC/ mice.
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Induction of µHC in ProB Cells Mediates Down-Regulation of Rag Expression in the Absence of 5.
Although TdT is dispensable for Ig rearrangements, Rag1/2 complexes are crucial for execution of the initial steps of V(D)J recombination. Rag1/2 gene expression is down-regulated in large preB cells that display the pre-BCR on the cell surface (5). Surface expression of the µHC in complex with SLC (pre-BCR) is believed to be mandatory for this down-regulation and, thereby, might be implicated in allelic exclusion by preventing further rearrangement on the DJ-rearranged allele. Because allelic exclusion is still maintained in all SLC knockout mice (79), we analyzed the effect of transgenic µHC induction on Rag1/2 expression in the presence or absence of 5.
For this purpose, we used tet-µHC and tet-µHC5/ mice carrying a bacterial artificial chromosome that contains the Rag2 gene regulatory sequences, but encodes a GFP reporter instead of Rag2 (NG-BAC). The GFP reporter was shown to reflect the documented patterns of Rag expression (13). GFP+ BM cells from tetracycline-treated tet-µHC mice (proB cells) were isolated by flow cytometry cell sorting and cultured on stromal cells in medium containing IL-7 without Tet (Fig. 3). Cells were fixed at 24, 48, and 72 h; permeabilized; stained for expression of µHC; and analyzed by FACS®. The de novo expression of µHC resulted in a more pronounced down-regulation of GFP, clearly measurable after 48 h (Fig. 3, left; mean fluorescence intensities: 78 in µ negative versus 56 in µ positive cells). Because GFP degradation was shown to lag behind Rag mRNA (GFP in vivo half-life: 52 h; reference 25), more rapid effects could not be expected. We also observed a decrease in the GFP level in µHC negative cells during the cultivation (Fig. 3, mean fluorescence intensities: 189 for 24 h to 55 for 72 h). This effect presumably is induced by IL-7 in the medium as described previously (26). Surprisingly, comparison of GFP levels in µHC positive and µHC negative cells 48 and 72 h after the conditional expression of the µHC in cells lacking
5 shows that Rag2 reporter gene expression is down-regulated to a greater extent in µHC positive cells (Fig. 3, right). This result indicates that Rag2 down-regulation might be mediated by µHC, but does not depend on formation of a complete pre-BCR.
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Transgenic µHC Detected on the Surface of PreB Cells in the Absence of LC and 5 or VpreB1/VpreB2.
Our findings that µHC mediates down-regulation of TdT and Rag1/2 in the absence of SLC components raised the question of how the responsible signals are generated. It is thought that incomplete pre-BCRs are retained in the ER through binding to the Ig HC binding protein BIP (30). Because the various SLC-deficient mice still show HC allelic exclusion (79), it has been suggested that an ER-resident µHC complex may still be able to signal. However, a pre-BCR apparently has to be released from the ER to efficiently bind Src-related protein tyrosine kinases and gain signaling capacity (31). Therefore, we favor the hypothesis that a signaling competent pre-BCRlike complex is expressed on the cell surface in the absence of 5 and/or VpreB1/2.
To allow detection of low levels of surface µHC complexes, we used biotinylated Abs in combination with EAS. We analyzed surface expression of transgenic µHC and SLC on CD19+ BM cells from tet-µHC, tet-µHC 5/, and tet-µHC VpreB1/VpreB2/ mice. Cells were isolated and expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for 18 h in the presence (Fig. 5 A, shaded) or absence (Fig. 5 A, unshaded) of Tet. Expression of the transgenic µHC in the absence of Tet was confirmed by parallel cytoplasmic staining for µHC (unpublished data).
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µHC Detected on the Surface of PreB Cells from SLC/ Mice.
To exclude the possibility that only the µHC transgene used in these experiments can give rise to surface expression and to exclude the possibility that both 5 and VpreB are able to support surface µHC expression independently, we analyzed BM preB cells from SLC/ mice for cell surface expression of µHC using EAS. BM proB cells from Rag/ mice were used as negative control. As shown in Fig. 5 B, after culturing sorted CD19+ cells for 4 d in vitro, a distinct µHC positive population was detected in cultures from SLC/ but not Rag/ mice. As expected, the SLC/-cultured cells did not express
5. Furthermore, the µHC positive cells did not express either
or
LCs. Thus, a variety of µHCs can be transported to the cell surface in the absence of the entire SLC.
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Discussion |
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It has been suggested that premature rearrangement and expression of LC in a small number of cells can "rescue" B cell development and HC allelic exclusion in the absence of SLC (10, 32). However, this mechanism would predict N-nucleotide additions in LC genes of peripheral B cells in
5/ and VpreB1/2/ mice, which is not the case (33). Herein, our results clearly demonstrate that the down-regulation of TdT and Rag1 does not depend on LC expression. First, our experiments using a tetracycline-inducible µHC transgene were performed in Rag2-deficient mice, which are unable to express conventional LCs. Second, FACS® analyses of BM cells from SLC-deficient mice revealed a readily detectable population of cells that expresses µHC and has down-regulated TdT; these cells were negative for cytoplasmic or surface conventional LC. Therefore, we conclude that at least some µHCs are able to deliver a signal for down-regulation of TdT and Rag1 gene expression in the absence of SLC and conventional LC.
TdT protein was found to be down-regulated in the majority of c-kit+/µHC+ cells from wild-type mice. In contrast, TdT was not down-regulated in these cells in SLC-deficient mice. In wild-type mice, a significant proportion of cells expressing the pre-BCR are still c-kit+ and presumably already cycling (5, 23) Therefore, the down-regulation of TdT mRNA levels (5) might rapidly lead to loss of TdT protein during proliferative expansion. However, the µHC does not induce proliferative expansion in SLC-deficient mice (4). Therefore, it is conceivable that in the absence of SLC, TdT protein remains detectable for longer periods of time. Importantly, TdT is clearly down-regulated at the CD25+ stage in SLC-deficient BM cells.
The analysis of Rag1 nuclear transcripts after the de novo expression of a µHC transgene demonstrates that down-regulation of the recombinase machinery in both wild-type and SLC-deficient cells is comparable and fairly rapid. This, together with asynchronous replication (34) and nonequivalent nuclear location (27) of the IgH alleles may provide sufficient time for a productive µHC to affect feedback inhibition of the rearrangement machinery. Such a situation could also occur in mice lacking SLC. It is assumed that, either at the time when the recombinase machinery is down-regulated or later during preB cell differentiation, the second IgH allele is rendered inaccessible, thus preventing further rearrangement (35). However, the molecular mechanisms responsible for this process are currently unknown. Thus, we can only speculate as to whether this process is affected by the absence of the SLC. As immature B cells in SLC/ mice show allelic exclusion, we propose that, as in wild-type mice, the second IgH allele is already inaccessible for the recombinase machinery at the time of LC gene rearrangement.
In the BM of SLC-deficient mice, a small population of cells has up-regulated CD25, a marker associated with differentiation into preB cells (8, 9, 24). We found that these cells, representing 510% of all CD19+ surface Ig BM cells in SLC-deficient mice, were negative for TdT as well as conventional LC and, therefore, are comparable in phenotype to preB cells from wild-type mice (5, 19, 24). Presumably, in a fraction of these cells, the Rag genes are reexpressed, and the LC gene loci are undergoing recombination. In all probability, these preB cells can give rise to the few allelically excluded immature B cells that are detected in SLC/ mice (79).
Where do the signals for the down-regulation of Rag1 and TdT and the up-regulation of CD25 originate? It has been discussed that ER-resident µHCs without obligate surface expression could induce signaling cascades (3), but it is doubtful whether elements of the appropriate signaling pathways (e.g., the Src family kinases) are efficiently recruited to the ER (31). Therefore, we investigated whether some µHCs can be transported to the surface and acquire signaling competence by forming pre-BCRlike complexes in the absence of SLC. Indeed, using an enzymatic amplification staining technique, we were able to detect µHC surface expression on preB cells from 5/, VpreB1/VpreB2/, and SLC/ mice, which may be complexed with Ig-
/-ß and, thus, mediate the effects of µHC in SLC-deficient mice. These data confirm and significantly extend recent data obtained from SLP-65/ mice (36). In SLP-65/ mice, there is increased µHC expression on the surface of preB cells and a novel pre-BCR complex was detected in the absence of
5. Importantly, our results provide evidence for the presence of this incomplete pre-BCR on SLP-65containing cells as well as on cells that lack all SLC components. We propose that this receptor, as has been shown for the receptor in SLP-65deficient cells (36), is signaling competent and responsible for the observed down-regulation of the recombinase machinery. The absence of a µHC-mediated down-regulation of TdT in
5/Ig-
deficient cells provides further evidence for signaling competence.
It can be speculated that the incomplete pre-BCRlike receptor represents an alternative, possibly rudimentary mechanism to select functional µHCs and facilitate surface expression. Reports of human µHCs that do not require association with SLC for surface transport support this scenario (37). In the absence of SLC, µHCs are believed to be retained in the ER through binding to the Ig HC binding protein BIP (30). Consequently, there is a requirement for a partner to facilitate exit from the ER. A possible candidate is VpreB3, a protein resident in the ER and able to associate with µHC (38). Alternatively, other hitherto unknown proteins may be essential for this process.
It remains unclear at present whether the incomplete pre-BCR described here has a physiological role in normal preB cell development. The SLC genes are highly expressed in proB cells and rapidly down-regulated in preB cells during clonal expansion (5, 39, 40). Therefore, the degree of proliferation might be controlled, not only by the dilution of SLC with successive cell divisions but also by the half-life of SLC proteins. It is conceivable that an incomplete pre-BCR has a physiological function in normal preB cell development. Possibly, this takes place at the stage when conventional pre-BCR is down-regulated, which is observed during clonal expansion of preB cells and before LC gene recombination (i.e., at the transition from large to small preB cells). This is supported by the observation that the tetracycline-inducible µHC transgene is stably expressed in preB cell cultures over a period of at least 4 d, whereas 5, VpreB, and the conformational epitope, recognized by the pre-BCRspecific antibody SL156, were undetectable even by surface EAS staining after only 23 d (unpublished data). Interestingly, it has been observed that prolonged survival of preB cells in µHC-transgenic, Bcl-2-transgenic Rag2/ mice allows accumulation of novel precursor B cells in the spleen that showed considerable µHC expression on the cell surface (41). Most of these cells are expected to have down-regulated SLCs (42) and, therefore, the µHC might form an incomplete pre-BCR similar to the one described in our studies.
In summary, we provide evidence that in the absence of SLC, µHC can induce down-regulation of the recombinase machinery, and that this effect is dependent on Ig-. Induction of a µHC transgene in Rag2/ proB cells can mediate down-regulation of TdT, Rag1, and probably Rag2, providing a model that could explain how the mechanisms involved in the establishment of allelic exclusion are still functional in the absence of SLC and, therefore, a conventional pre-BCR.
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Acknowledgments |
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G. Galler and T. Winkler are supported by the Deutsche Forschungsgemeinschaft (grant nos. DFG WI 1183/3 and GK592). C. Mundt, M. Parker, and I.-L. Mårtensson are supported by the Biotechnology and Biological Sciences Research Council.
Submitted: 4 September 2003
Accepted: 21 April 2004
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References |
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