Correspondence to: Ashok R. Venkitaraman, The Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease and CRC Department of Oncology, The Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, UK. Tel:44-1223-336901 Fax:44-1223-336902 E-mail:arv22{at}cam.ac.uk.
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Abstract |
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After the productive rearrangement of immunoglobulin (Ig) heavy chain genes, precursor (pre-)B lymphocytes undergo a limited number of cell divisions in response to interleukin (IL)-7. Here, we present evidence that this phase of IL-7dependent expansion is constrained by an inhibitory signal initiated by antigen receptor assembly. A line of pre-B cells from normal murine bone marrow that expresses a µ heavy chain with a D-proximal VH7183.2 region divides continuously in IL-7. IL-7 responsiveness ceases upon differentiation to the µ1, 1 stage, despite continuing expression of the IL-7 receptor (IL-7R), suggesting that antigen receptor assembly inhibits IL-7 responsiveness. This is confirmed by introduction of a rearranged
light chain gene, which inhibits proliferative signaling through the IL-7R. Inhibition is specific to the IL-7R, because it is overcome by replacement of the IL-7R cytoplasmic domain with corresponding sequences from the closely related IL-2Rß chain. Alteration of a single tyrosine residue, Tyr410, in the IL-7R cytoplasmic domain to phenylalanine also prevents the inhibition of proliferation after antigen receptor assembly. Thus, the loss of IL-7 responsiveness after antigen receptor assembly may be mediated through the recruitment of an inhibitory molecule to this residue. Our findings identify a novel mechanism that limits cytokine-dependent proliferation during B lymphopoiesis. This mechanism may be essential for the proper regulation of peripheral B lymphocyte numbers.
Key Words: interleukin 7 receptor, B lymphocyte differentiation, signal transduction, antigen receptor, immunoglobulin
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Introduction |
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A potent stimulus for the proliferation of immature B lymphocytes is provided by IL-7 (1), a cytokine secreted by stromal cells in the bone marrow. IL-7 triggers cell division by engagement of a heterodimeric receptor (IL-7R) that contains a ligand-specific chain (2), and a common
chain shared with the receptors for IL-2, IL-4, IL-9, IL-13, and IL-15 (3) (4) (5) (6) (7). We have shown elsewhere that proliferative signaling through the IL-7R is critically dependent on the integrity of a single tyrosine residue in the cytoplasmic domain of the
chain (8). This tyrosine residue is essential for the ligand-induced recruitment of the effector enzyme phosphatidylinositol 3-kinase (PI 3-k), through the Src homology 2 (SH2) domains in its regulatory subunit, p85 (9).
In mice, the ability of immature B cells to divide in response to IL-7 changes with their developmental progression. Progenitor (pro-)B cells, which have yet to complete Ig heavy chain (IgH) gene rearrangement, require stromal cell contact as well as IL-7 to divide (10) (11). Productive IgH rearrangement triggers progression to the precursor (pre-)B cell stage, at which IL-7 alone is sufficient to provoke proliferation (1) (12) (13). Pre-B cells undergo only a limited number of cell divisions before they differentiate into IL-7unresponsive, mature B lymphocytes, which express a functional antigen receptor (14) (15). It remains unclear how IL-7dependent proliferation can be limited during these developmental transitions to regulate the number of mature cells emerging to the periphery. Here, we present evidence that the IL-7dependent proliferation of pre-B cells is constrained by an inhibitory signal initiated through antigen receptor assembly.
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Materials and Methods |
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Cells.
IL-7dependent cells derived from normal bone marrow (16) were maintained in growth medium with 10 ng/ml of recombinant IL-7 (Genzyme Diagnostics). The retroviral packaging cell lines CRE (17), AM12 GP + env (18), and nx (19) were used, and were maintained in DMEM with 10% FCS.
Immunofluorescence Staining and Flow Cytometric Analysis.
Cells were washed in ice-cold PBS. Staining for CD19 and CD43 was with FITC-conjugated rat antimouse CD19 and with a biotinylated rat antimouse CD43, followed by streptavidin-R-PE. Staining for CD2 was with PE-conjugated rat antimouse CD2, and for CD25, with an FITC-conjugated rat antimouse CD25. All of these reagents were from PharMingen. Staining for µ was with FITC-conjugated goat antimouse µ (µ chain specific; Southern Biotechnology Associates), whereas staining for light chain was with PE-conjugated goat antimouse
(
chain specific; Southern Biotechnology Associates). Staining for
1 light chain was with FITC-conjugated goat antimouse
(
chain specific; Southern Biotechnology Associates). Receptors for human IL-4R were detected with biotinylated human IL-4 (R&D Systems), followed by streptavidin-PE (PharMingen). Staining for the murine IL-7R was with rat antimouse IL-7R (A7R34; gift of S. Nishikawa, Kyoto University, Kyoto, Japan), followed by a biotinylated antirat antibody (PharMingen) and streptavidin-FITC (Jackson ImmunoResearch Labs). Negative control staining was with the matched isotype controls (PharMingen). All samples were analyzed on a FACScaliburTM flow cytometer (Becton Dickinson) by standard methods using CELLQuestTM analysis software.
Sequencing of the Productive IgH Rearrangement.
A PCR-based assay for VH rearrangements was first carried out. High-molecular-weight genomic DNA was extracted by the Proteinase K method, and subjected to two rounds of PCR. Nested 3' primers from the intron downstream of the JH4 and nested 5' primers corresponding to VH7183 and VHJ558 V region families have already been described (20). Southern blotting of PCR products using a VH7183 familyspecific probe (20) confirmed that the rearrangement utilized a member of this family. The PCR product was then gel purified and sequenced using a nested primer corresponding to the 5' end of VH7183 (20). Sequence comparisons were carried out using the BLAST search algorithms (available at http://www.ncbi.nlm.nih.gov/BLAST).
Pre-B Cell Cloning Assay.
The pre-B cell line was cloned by limiting dilution in the presence of 10 ng/ml recombinant IL-7 (Genzyme Diagnostics). The pre-B cells were plated at 0.3 cells per well in 96-well plates and then cultured in recombinant IL-7 containing medium for 2 wk. The colonies were then removed, and 100 were analyzed for the expression of light chain, as described above.
Retroviral Constructs and Their Transfection into the Ecotropic Packaging Cell Line.
The human (hu)IL-4R/IL-7R and the huIL-4R/IL-2Rß are the same constructs that were used in Corcoran et al. (8). The huIL-4R/IL-7R Tyr410Phe and the huIL-4R/IL-7R Tyr456Phe were generated with a PCR-based strategy using oligonucleotides encoding the desired point mutations. The Tyr456Phe mutation is at the extreme 3' end of the coding sequence, and so the huIL-4R/IL-7RY456 mutant was constructed simply by PCR amplification using the wild-type huIL-4R/IL-7R receptor (8) as a template (forward, 5'-AGCTGAACGCGTCCATGGGGTGGCTTTGCTCTGGGCTC-3'; reverse, 5'-AACTCTGAGTCTCACTGGTTTTGGAAGCTGGAC-3'). For the Tyr410Phe mutation, the oligonucleotide encoding the mutation (5'-CTAGACTGCAGGGAGAGTGGCAAGAATGGGCCTCAT-3') and a reverse primer from the 3' end of the coding sequence (5'-AGCTTGTCTTCACTGGTTTTGGTAGAA-3') were used in PCR to generate a cytoplasmic domain fragment encoding the desired alteration. After digestion with PstI and BglII, the fragment was cloned into the corresponding sites in the cytoplasmic domain of the huIL-4R/IL-7R wild-type receptor. The huIL-4R/IL-7R Tyr410Phe and the huIL-4R/IL-7R Tyr456Phe were then digested with MluI and BglII and ligated into the MluI and BamHI sites of a vector based on the Moloney leukemia provirus (8) rendered replication defective by deletion of the pol and env genes.
The bicistronic expression vectors containing the rearranged 1 light chain gene cDNA and the huIL-4R/IL-7R, huIL-4R/IL-2Rß, and huIL-4R/IL-7R Tyr410Phe chimeric receptors were also constructed using a PCR-based approach. The
1 light chain cDNA was used as a template for two oligonucleotides (forward, 5'-TCTACAACGCGTCCATGGCCTGGATTTCACTTATA-3'; reverse, 5'-TCTAGAGGATCCGCGGCCGCTAGGAACAGTCAGCAC-3'), which contained MluI and BamHI sites, respectively. The
1 light chain fragment was linked through an internal ribosome entry site (IRES) from the EMC virus to a cDNA encoding the huIL-4R/IL-7R, huIL-4R/IL-2Rß, or huIL-4R/IL-7R Tyr410Phe chimeric receptors. A three-way ligation was performed using:
1 fragment digested MluI, BamHI; IRES fragment digested BamHI, NcoI; and the chimeric receptor cDNAs digested NcoI, NotI. These chimeric receptor sequences were generated with the correct template using oligonucleotides containing NcoI and NotI sites at the 5' and 3' ends of the fragment, respectively. The three-way digestion products were then inserted into the MluI and NotI sites of the retroviral vector. The integrity of all constructs was confirmed by nucleotide sequencing.
Constructs were cotransfected by the calcium phosphate method into an ecotropic packaging cell line (17) with a plasmid encoding NeoR. Stable transfectants were isolated by selection in 1 mg/ml of G418 sulfate (GIBCO BRL).
Retrovirus-mediated Gene Transfer in Cultures of the Pre-B Cell Line.
Pre-B cells were incubated at 106 cells/ml, on a 20% confluent monolayer of 2,000-rad -irradiated retrovirus-secreting packaging cells, in RPMI 1640 medium containing 10% FCS, 5 x 10-5 ß-mercaptoethanol (growth medium), and 10 ng/ml recombinant IL-7 (Genzyme Diagnostics) at 37°C in a 5% CO2 atmosphere. After 2 d, cultures were washed and transferred to growth medium containing 10 ng/ml IL-4 (Genzyme Diagnostics) and 5 x 10-5 ß-mercaptoethanol (growth medium) and then fed every 34 d by removal of spent medium and replacement with new.
Proliferation Assays.
Cells (5 x 105) were washed and resuspended in 3 ml of growth medium. The cells were then incubated for 4 d at 37°C in 96-well microtiter plates in 100 µl of growth medium containing the indicated quantities of recombinant human IL-4 or recombinant mouse IL-7 (Genzyme Diagnostics). Proliferation was quantified using the MTT assay (21).
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Results and Discussion |
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A Line of Pre-B I Cells from Normal Murine Bone Marrow Divides Continuously in IL-7.
We used a line of pre-B cells isolated from normal murine bone marrow (16) to study the mechanisms that limit IL-7 responsiveness during B lymphopoiesis. These cells express markers characteristic of the pre-B I stage in development (Fig 1 A). Thus, in addition to the CD19 marker typical of the B lineage (22), the cells display the marker CD43, whose expression is lost upon differentiation to the pre-B II stage (10). They also express BP-1 (16), whose expression commences at Hardy stage C (10). Finally, the cells fail to display CD25, a marker that commences its expression at the pre-B II stage of B cell development (23) (24).
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This pre-B I cell line divides continuously in the presence of IL-7 (Fig 1 B). We have found no alteration in IL-7 responsiveness after multiple rounds of passage over a period of 3 yr (data not shown).
In these cells, a productive IgH gene rearrangement results in the synthesis of membrane-inserted µ heavy chains (Fig 1 A). We have sequenced this productive rearrangement (Fig 2 A). It involves a VH gene segment from the VH7183 family located in the D-proximal region of the murine IgH locus. Comparison of the sequence with known members of the family reveals a precise match with VH7183.2 (Fig 2 B; reference (25)). In contrast, the IgL genes remain in the germline configuration (16).
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Differentiation to the µ1, 1 Stage Results in the Loss of IL-7 Responsiveness.
A small fraction of cells in this pre-B I cell line spontaneously undergoes differentiation to the µ1, 1 stage during propagation in culture (Fig 1 A). Although
95% of the cells lack
expression, between 0.5 and 5% are consistently
1 at any given time. Interestingly, the fraction of
1 cells fails to increase even after prolonged periods in culture (data not shown), indicating that they do not grow out in an IL-7dependent manner. This suggested to us that differentiation to the µ1,
1 stage might be accompanied by the loss of IL-7 responsiveness.
To confirm that this was indeed the case, the pre-B I cells were cloned at limiting dilution in the presence of IL-7, and the resulting clones were analyzed for expression of a rearranged chain gene. As shown in Table 1, this procedure gave rise exclusively to cell clones that were
95%
2, just as are the parental cells. Thus, cells that have differentiated to the µ1,
1 stage cannot be cloned out in IL-7, indicating that IL-7 responsiveness is indeed lost.
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Loss of IL-7 Responsiveness Occurs Despite the Continuing Expression of IL-7R.
We reasoned that the loss of IL-7 responsiveness upon differentiation might result from the downregulation of IL-7R expression. To test this possibility, surface expression of the IL-7R was compared in 1 and
2 cells using an mAb specific to the
chain of the mouse IL-7R. Fig 3 A demonstrates that IL-7R expression is detected to a similar level in both the
1 and the
2 fractions. These results suggest that, contrary to expectation, the loss of IL-7 responsiveness in µ1,
1 cells cannot simply be ascribed to the lack of IL-7R expression.
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Retroviral Transfer of a Chimeric HuIL-4R/IL-7R Receptor Fails to Rescue µ1, 1 Cell Outgrowth.
To subject the validity of these observations to further test, we introduced a cDNA encoding a huIL-4R/IL-7R chimeric receptor into the pre-B I cells by retroviral gene transfer. The chimeric molecule (Fig 3 B) contains the extracellular sequences of the human IL-4R, conferring strict, species-specific responsiveness to human IL-4 (26). However, its transmembrane and cytoplasmic domains are derived from the IL-7R. Expression of the chimeric receptor is promoted by the long terminal repeat sequence of the Moloney murine leukemia virus, active in a wide range of cell types (17). Therefore, it is not subject to the same transcriptional constraints as is the endogenous IL-7R.
Transduced pre-B I cells were selected in human IL-4 for expression of the huIL-4R/IL-7R before analysis. As shown in Fig 3 B, the huIL-4R/IL-7R supports the proliferation of the 2 pre-B I cell population. However, the chimeric receptor does not rescue the outgrowth of µ1,
1 cells, further substantiating that the loss of IL-7 responsiveness at this developmental stage is not simply due to the lack of IL-7R expression. Rather, our results suggest that differentiation to the µ1,
1 stage is accompanied by the inhibition of proliferative signaling through the IL-7R.
Inhibition of Proliferative Signaling at the µ1, 1 Stage Is Specific to the IL-7R Cytoplasmic Domain.
Accordingly, we tested if replacement of the IL-7R cytoplasmic domain with sequences from the related IL-2R ß chain (27) could overcome this inhibition. Pre-B I cells transduced with a huIL-4R/IL-2Rß chimeric receptor were selected in human IL-4 before analysis for expression. As shown in Fig 3 C, the huIL-4R/IL-2Rß supports the outgrowth not only of
2 pre-B I cells but also of cells that have differentiated to the µ1,
1 stage. This is in marked contrast to the huIL-4R/IL-7R (Fig 3 B). Thus, the loss of proliferative signaling at this developmental stage is specific to the IL-7R cytoplasmic domain. Taken together with the results in Table 1, this finding confirms that the differentiation of pre-B I cells to the µ1,
1 stage triggers the inhibition of proliferative signaling through the IL-7R, despite continuing expression of the receptor.
Antigen Receptor Assembly Initiates the Inhibition of Proliferative Signaling through the IL-7R.
Our results suggest that the assembly of a functional antigen receptor initiates the inhibition of proliferative signaling through the IL-7R. To test this hypothesis directly, we created bicistronic gene constructs in which a rearranged 1 light chain gene was linked through an IRES to a cDNA encoding either the huIL-4R/IL-7R or huIL-4R/IL-2Rß chimeric receptors (Table 2). These constructs were introduced into pre-B I cells by retroviral transduction, and
1-expressing cells were selected in human IL-4.
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As shown in Table 2, 1-expressing cells were readily detected in cultures transduced with the
1IREShuIL-4R/IL-2Rß construct. These
1 cells could be maintained in IL-4dependent culture for >1 mo without loss of
expression (data not shown). In contrast,
1 cells could not be isolated from cultures transduced with the
1IREShuIL-4R/IL-7R construct in multiple independent experiments, suggesting that their outgrowth was not supported by signaling through the huIL-4R/IL-7R. Taken together, our results indicate that assembly of a µ,
1 antigen receptor results in the loss of responsiveness to proliferative signaling through the huIL-4R/IL-7R, and that this inhibition is specific to the cytoplasmic sequences of the IL-7R but not the IL-2Rß.
Inhibition of Proliferative Signaling Is Dependent on Tyr410.
To investigate the mechanism by which this inhibitory signal might act, we constructed a series of mutant huIL-4R/IL-7R constructs in which cytosolic Tyr residues were altered to Phe. The cytoplasmic domain of the IL-7R contains three Tyr residues (Tyr410, Tyr449, and Tyr456) conserved in evolution between the murine and human receptors, suggesting an important function. The Tyr410Phe and Tyr456Phe mutants were tested in these experiments because we have already shown that Tyr449, which recruits the effector enzyme PI 3-k, is essential for proliferative signaling through the IL-7R (8) (9).
Fig 4 A shows that both the huIL-4R/IL-7R Tyr 410Phe and huIL-4R/IL-7R Tyr456Phe mutants are equally proficient at supporting the proliferation of pre-B I cells. Thus, neither residue is essential for proliferative signaling. However, in contrast to both the wild-type receptor (Fig 3 B) and the Tyr456Phe mutant (Fig 4 C), the huIL-4R/IL-7R Tyr410Phe mutant alone could support the outgrowth of µ1, 1 cells (Fig 4 B). This implies that mutation of a single Tyr residue can overcome the inhibition of proliferative signaling through the IL-7R.
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Accordingly, we performed a further experiment in which a rearranged 1 light chain gene was linked in a bicistronic expression vector to the huIL-4R/IL-7R Tyr 410Phe mutant receptor (Table 2). In contrast to the huIL-4R/IL-7R, but like the huIL-4R/IL-2Rß, the Tyr410Phe mutant was competent to support the outgrowth of
1-expressing cells. Taken together, these results confirm that the inhibitory signal initiated by antigen receptor assembly is critically dependent on the integrity of Tyr410 in the cytoplasmic domain of the IL-7R
chain. Therefore, we propose that the inhibitory signal may work through the recruitment of an inhibitory molecule to this residue.
In summary then, the results we have presented in this paper identify a novel mechanism that limits the expansion of pre-B lymphocytes in the murine bone marrow. By suppressing proliferative signaling through the IL-7R cytoplasmic domain upon antigen receptor assembly, this mechanism provides a feedback loop that may regulate the throughput of cells reaching the mature B lymphocyte stage. The assembly of a functional pre-B cell receptor may itself conceivably be sufficient to trigger this growth inhibitory process (28) (29), restricting the number of divisions pre-B cells can undergo before commencing IgL rearrangement. Our findings demonstrate for the first time a homeostatic mechanism that limits cytokine-dependent cell expansion during hematopoiesis.
IL-7R Signaling Inhibited by Antigen Receptor Assembly
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Acknowledgements |
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We thank Dr. J. Sims (Immunex Corp., Seattle, WA), Prof. S.I. Nishikawa (Kyoto University, Kyoto, Japan), Dr. A. Corcoran (The Cambridge Institute for Medical Research), and Dr. G. Nolan (Stanford University, Stanford, CA) for the generous gift of reagents used in this work, Dr. A. Corcoran and Dr. K.J. Patel for much helpful discussion, and Andrew Riddell for assistance with flow cytometry.
F.M. Smart received a Ph.D. Studentship from the Medical Research Council, UK. A.R. Venkitaraman holds a Professorship generously endowed by the late Dr. F.A. Zoellner. Work in A.R. Venkitaraman's laboratory is supported by the Medical Research Council, UK.
Submitted: 21 October 1999
Accepted: 3 November 1999
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References |
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