The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Will, W. M.
Right arrow Articles by Farrar, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Will, W. M.
Right arrow Articles by Farrar, M. A.
The Journal of Immunology, 2006, 176: 3350-3355.
Copyright © 2006 by The American Association of Immunologists

Attenuation of IL-7 Receptor Signaling Is Not Required for Allelic Exclusion1

Wynette M. Will*, Joshua D. Aaker*, Matthew A. Burchill*, Ian R. Harmon*, Jennifer J. O’Neil*, Christine A. Goetz*, Keli L. Hippen{dagger} and Michael A. Farrar2,*

* Department of Laboratory Medicine and Pathology and {dagger} Department of Medicine, Center for Immunology, The Cancer Center, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Allelic exclusion prevents pre-B cells from generating more than one functional H chain, thereby ensuring the formation of a unique pre-BCR. The signaling processes underlying allelic exclusion are not clearly understood. IL-7R-dependent signals have been clearly shown to regulate the accessibility of the Ig H chain locus. More recent work has suggested that pre-BCR-dependent attenuation of IL-7R signaling returns the H chain loci to an inaccessible state; this process has been proposed to underlie allelic exclusion. Importantly, this model predicts that preventing pre-BCR-dependent down-regulation of IL-7R signaling should interfere with allelic exclusion. To test this hypothesis, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA). STAT5b-CA expression restores V(D)J recombination in IL-7R–/– B cells, demonstrating that IL-7 regulates H chain locus accessibility and V(D)J recombination via STAT5 activation. To examine the effects of constitutively active STAT5b on allelic exclusion, we crossed STAT5b-CA mice (which express the IgMb allotype) to IgMa allotype congenic mice. We found no difference in the percentage of IgMa/IgMb-coexpressing B cells in STAT5b-CA vs littermate control mice; identical results were observed when crossing STAT5b-CA mice with hen egg lysozyme (HEL) H chain transgenic mice. The HEL transgene enforces allelic exclusion, preventing rearrangement of endogenous H chain genes; importantly, rearrangement of endogenous H chain genes was suppressed to a similar degree in STAT5b-CA vs HEL mice. Thus, attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B lymphocytes display a diverse repertoire of Ig receptors that allows them to recognize a large variety of Ags. This diversity is accomplished through V(D)J recombination, which involves rearrangement of the distinct V, D, and J gene segments (1). First, Igh D and J gene segments recombine on both homologous alleles (2). This is followed by recombination of V to DJ segments. If recombination is productive, a pre-BCR (µ H chain along with {lambda}5 and VpreB surrogate L chains) is expressed on the cell surface. The pre-BCR associates with the signaling components Ig{alpha} and Igbeta; this complex couples to downstream signaling pathways and ultimately prevents further Igh gene rearrangement. This process, in which only one productively rearranged H chain gene is expressed on a single pre-B cell, is called allelic exclusion (3).

The molecular mechanisms underlying allelic exclusion remain unclear. Several groups have proposed that allelic exclusion of H and L chain loci is controlled through the accessibility of these loci (4, 5). Importantly, IL-7R signaling plays a key role in regulating H chain gene accessibility. For example, recombination of distal VH gene segments has been found to be severely impaired in IL-7R{alpha}–/– mice compared with recombination of VH-proximal gene segments (4). In addition, Chowdhury and Sen (6, 7) have shown that IL-7R signaling is able to alter the chromatin accessibility of DJ-distal VH genes, such as those of the VHJ558 family. Furthermore, they observed that chromatin accessibility was reversed in pre-B cells, and this correlated with a decline in the ability of these cells to respond to IL-7. Based on these findings, they proposed that IL-7R down-regulation/desensitization reverses the histone acetylation and chromatin accessibility of VH genes and thus provides the key mechanism that drives allelic exclusion.

The IL-7R activates at least two major signaling pathways: the JAK/STAT5 and PI3K/Akt signaling pathways. We have previously shown that STAT5 is the key signaling molecule downstream of the IL-7R for B cell development, because activated STAT5 restores B cell differentiation and VH gene rearrangement in IL-7R–/– mice (8). More recent work by Stanton and Brodeur (9) (using pro-B cell lines) demonstrated that STAT5 is recruited to D-distal VH genes (such as A1) in response to IL-7 signaling and thereby induces VH gene acetylation and promotes VH locus accessibility. Similar results were obtained by Singh et al. (10) using pro-B cells; specifically, they demonstrated that STAT5–/– mice are defective in VH gene acetylation and Igh gene rearrangement. Thus, the IL-7R regulates VH gene accessibility via STAT5 activation. In the studies reported in this paper, we examined the effects of constitutively active STAT5b on allelic exclusion by crossing mice expressing a constitutively activated form of STAT5b (STAT5b-CA) (11) to IgMa congenic mice. STAT5b-CA mice are on the C57BL/6 genetic background and thus express the IgMb allotype. We found the percentage of B cells coexpressing IgMa and IgMb to be no different in STAT5b-CA and littermate control mice. Similar results were obtained when we crossed STAT5b-CA mice with transgenic mice that express a rearrangedIgh (MD2, hen egg lysozyme (HEL)3 transgene (12). The HEL transgenic system also allowed us to analyze allelic exclusion at the level of gene rearrangement as opposed to protein expression. Importantly, preventing IL-7R desensitization did not alter gene rearrangement in this model system. Thus, we conclude that down-regulation of IL-7R signaling is not sufficient for allelic exclusion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

STAT5b-CA mice were generated as previously described (11). MD2 HEL-Igh (HEL) mice, which express a rearranged H chain Ig specific for HEL, were obtained from Dr. T. Behrens (University of Minnesota, Minneapolis, MN). IgMa congenic mice were obtained from The Jackson Laboratory. All mice were backcrossed to the C57BL/6 genetic background for 10+ generations. Mice were housed in specific pathogen-free facilities at University of Minnesota, and all experiments were approved by the University of Minnesota institutional animal care and use committee. Mice used were between 5 and 10 wk of age.

Flow cytometry

Bone marrow, spleen, and lymph nodes were isolated and processed as previously described (11). RBC were removed from bone marrow and spleen samples by ammonium chloride lysis. Cells were stained with IgMa-FITC (DS-1) and IgMb-PE (AF6-78) from BD Pharmingen and B220-allophycocyanin (RA3-6B2) from eBioscience and were analyzed on either a FACSCalibur or an LSR II flow cytometer (BD Biosciences). Data was analyzed using FlowJo software (Tree Star). Intracellular staining for IgMa and IgMb was conducted using the same Abs as those used for cell surface staining; cells were permeabilized and stained using reagents from a BD Biosciences BrdU labeling kit.

Intracellular phospho-STAT5 staining

Bone marrow samples were stained with B220-allophycocyanin (RA3-6B2), IgM-FITC (1B4B1; eBioscience), and CD43-bio (BD Biosciences) plus streptavidin-Cascade Blue (Invitrogen Life Technologies) and were stimulated with IL-7 for 20 min. Samples were then stained for phospho-STAT5 as previously described (13), prepared for intracellular staining with a Fix and Perm Kit (Caltag Laboratories), and stained with phospho-STAT5-PE (14) (BD Biosciences) or PE-conjugated mouse IgG1 isotype control (eBioscience).

Isolation of CD19+ B cells

Bone marrow samples were stained with CD19-bio (MB19–1; eBioscience), followed by positive selection on an AutoMACS using streptavidin-conjugated magnetic microbeads (Miltenyi Biotec). Cell purity was typically >90%.

Analysis of VHJ558 gene usage

CD19+ B cells from littermate control, STAT5b-CA, HEL, and STAT5b-CA x HEL mice were purified as described above and genomic DNA was isolated using a DNeasy tissue kit (Qiagen). PCR and Southern blotting of the littermate control, STAT5b-CA, HEL, and STAT5b-CA x HEL genomic DNA was conducted as previously described (8), except the second-step PCR consisted of 17 cycles. Genomic DNA was normalized relative to the hypoxanthine phosphoribosyltransferase (HPRT) gene. All oligonucleotides and TaqMan probes were obtained from IDT. The primers used were described by Goetz et al. (8).

Calculations

We calculated the expected percentage of IgMa/IgMb-coexpressing cells in the absence of allelic exclusion as follows. First, we assume that four of nine of all pro-B cells will fail to productively rearrange either of their H chain alleles (two of three fail on the first allele x two of three fail on the second allele). The remaining five of nine have at least one productive rearrangement; in the absence of allelic exclusion, we expect that one of nine will have two productive rearrangements (one of three on the first allele x one of three on the second allele). The four of nine pro-B cells that do not have a productive rearrangement die by apoptosis and are not scored in our assay. This leaves 1/9 ÷ 5/9 = 1/5 or 20% as the expected proportion of IgMa/IgMb double-expressing cells in the absence of allelic exclusion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phospho-STAT5 expression in STAT5b-CA transgenic mice

Chowdhury and Sen (7) have proposed an attractive model in which attenuation of IL-7R signaling returns the H chain locus to an inaccessible state, thereby initiating allelic exclusion. To investigate the role of IL-7R/STAT5 signaling in allelic exclusion, we used mice that express a constitutively active form of STAT5b (STAT5b-CA) (11). The STAT5b-CA transgene was constructed by introducing point mutations at His298 and Ser715, thus rendering STAT5b constitutively active (Fig. 1A). The STAT5b-CA transgene was expressed using a transgene expression vector containing the Eµ enhancer and the lck proximal promoter (Fig. 1B). We have previously shown that this vector drives expression throughout B and T lymphocyte development (8, 11, 14). To examine activation of STAT5 in our STAT5b-CA transgenic mice, we isolated bone marrow from STAT5b-CA and littermate control mice. The bone marrow cells were stimulated with or without IL-7 for 20 min, then assayed for phospho-STAT5 expression by flow cytometry (Fig. 1, C and D). B cell populations were electronically gated using the marker B220; pro-B, pre-B, and immature-B cells subsets were identified using markers for CD43 and IgM (Fig. 1C). Pro-B cells (CD43+IgM–) from littermate control and STAT5b-CA mice responded to IL-7, as shown by high levels of phospho-STAT5 expression. Similar results were seen in unstimulated cells, indicating that in the bone marrow microenvironment, pro-B cells from both littermate control and STAT5b-CA mice are exposed to and can respond to endogenously produced IL-7 (Fig. 1D, left panels). Conversely, pre-B cells (CD43–IgM–) and immature B cells (CD43–IgM+) from littermate control mice showed minimal STAT5 activation, even after IL-7 stimulation in vitro (Fig. 1D, first and second rows, middle and right panels). These results are consistent with previous reports documenting attenuation of IL-7R signaling in pre-B cells (7, 15). In contrast, pre-B cells in STAT5b-CA mice showed robust STAT5 phosphorylation after IL-7 stimulation (Fig. 1D, fourth row, middle panel). Similar results were seen in immature B cells from STAT5b-CA mice (Fig. 1D, fourth row, right panel). Importantly, even in the absence of in vitro IL-7 stimulation, STAT5b-CA pre-B and immature B cells exhibited levels of phospho-STAT5 expression that paralleled those seen in pro-B cells from littermate control mice (compare Fig. 1D, first row, left panel, with third row, middle and right panels). Specifically, in this experiment we observed a mean channel of shift of 182 ± 5 for unstimulated pre-B cells from STAT5b-CA mice vs 214 ± 32 for unstimulated pro-B cells from littermate control mice. Thus, the STAT5b-CA transgene prevents the typically observed down-regulation of IL-7R signaling in pre-B cells.


Figure 1
View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 1. Phospho-STAT5 expression in pro-B, pre-B, and immature-B cell subsets. A, Diagram of the STAT5b-CA transgene, including locations of point mutations contributing to constitutive activation of STAT5b. B, Diagram of STAT5b-CA expression vector. C, Bone marrow from 5- to 10-wk old STAT5b-CA and littermate control (LMC) mice was harvested, stimulated with or without IL-7, and stained with Abs for CD43, IgM, B220, and phospho-STAT5 as described in Materials and Methods. B220+ cells were gated and CD43 and IgM markers were used to distinguish pro-B (CD43+IgM–), pre-B (CD43–IgM–), and immature B (CD43–IgM+) cell subsets. D, Phospho-STAT5 expression was then examined in the histograms to the right. Shaded gray histograms represent isotype controls, whereas black outlined histograms represent staining with phospho-STAT5 specific Abs. These results are representative of five separate experiments (16 STAT5b-CA and 10 LMC mice).

 
STAT5b-CA activation does not alter allelic exclusion

To investigate the role of IL-7R/STAT5 signaling in allelic exclusion, we crossed STAT5b-CA (IgMb) mice with IgMa congenic mice. We examined the expression of IgMa and IgMb in B220+ bone marrow, spleen, and lymph node cells (Fig. 2). If loss of IL-7R/STAT5 signaling is required for allelic exclusion, we would expect an increase in the percentage of cells coexpressing IgMa and IgMb in STAT5b-CA vs littermate control mice. However, we found similar percentages of IgMa/IgMb-coexpressing cells in STAT5b-CA and littermate control mice. Specifically, the percentages of IgMa/IgMb-coexpressing cells in the bone marrow from STAT5b-CA and littermate control mice were 0.4 ± 0.2 and 0.3 ± 0.2% (p = 0.6), respectively. Similar results were seen in the spleen (2.0 ± 1.3 vs 1.1 ± 0.7%, respectively; p = 0.15) and lymph nodes (2.1 ± 1.5 vs 1.1 ± 0.6%, respectively; p = 0.2). Thus, preventing the normal down-regulation of IL-7R/STAT5 signaling during B lymphocyte development does not alter allelic exclusion in this model.


Figure 2
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 2. STAT5b-CA activation does not alter allelic exclusion. Bone marrow (BM), spleen, and lymph nodes (LN) were isolated from 5- to 6-wk old IgMa/IgMb STAT5b-CA and littermate control (LMC) mice. Cells were stained with Abs to B220, IgMa, and IgMb and analyzed by flow cytometry. Cell populations were electronically gated on B220+ cells, and IgMa/IgMb expression is shown. Similar results were found in 10 STAT5b-CA and five LMC mice.

 
Previous studies have demonstrated that allelic exclusion of the H chain gene locus is not absolute, and that ~2–4% of pre-B cells have two productively rearranged H chain genes. However, in those studies it was observed that although allelic exclusion was broken at the level of gene rearrangement and intracellular H chain gene expression, only one H chain was effectively expressed at the cell surface (16). Thus, it is important to rule out the possibility that expression of the STAT5b-CA transgene did, in fact, block allelic exclusion, but that this was not detected in our experiments looking at cell surface expression of H chain alleles. To examine this issue, we harvested bone marrow from littermate control and STAT5b-CA mice, permeabilized the cells, and then stained them for intracellular expression of IgMa and IgMb. As shown in Fig. 3, once again we found similar percentages of IgMa/IgMb-coexpressing cells in STAT5b-CA and littermate control mice. Specifically, the percentages of IgMa/IgMb-coexpressing cells in the bone marrow from STAT5b-CA and littermate control mice were 1.0 ± 0.3 and 1.0 ± 0.7% (p = 0.9), respectively. Similar results were seen in the spleen (3.2 ± 1.1 vs 1.7 ± 0.9%, respectively; p = 0.18) and lymph nodes (1.1 ± 0.3 vs 2.0 ± 0.8%, respectively; p = 0.08). Thus, STAT5b-CA expression does not alter the frequency of H chain allele coexpression either inside the cell or on the cell surface.


Figure 3
View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 3. STAT5b-CA activation does not alter allelic exclusion. Bone marrow, spleen, and lymph nodes were isolated from 5- to 6-wk old IgMa/IgMb STAT5b-CA and littermate control (LMC) mice. Cells were permeabilized and stained with Abs to B220, IgMa, and IgMb and analyzed by flow cytometry. Cell populations were electronically gated on B220+ cells, and IgMa/IgMb expression is shown. Similar results were found in seven STAT5b-CA and two LMC mice.

 
STAT5b-CA x HEL mice do not undergo V(D)J recombination

To examine the effect of STAT5b-CA activation on V(D)J recombination more precisely, we crossed STAT5b-CA mice to transgenic mice that contain a rearranged Igh H chain (derived from the HEL Igh receptor; referred to as MD2) (12). Expression of the rearranged HEL H chain mimics pre-BCR signals and prevents rearrangement of endogenous H chain genes. If IL-7R/STAT5 signaling mediates allelic exclusion, then STAT5b-CA x HEL mice should still undergo V(D)J recombination. To examine this, we analyzed the expression of the HEL IgM allotype (IgMa) and the endogenous IgM allotype (IgMb) in B220+ bone marrow and spleen cells from littermate control, STAT5b-CA, HEL, and STAT5b-CA x HEL mice (Fig. 4). Littermate control and STAT5b-CA mice showed only IgMb expression, whereas HEL and STAT5b-CA x HEL mice showed virtually exclusive IgMa expression. Specifically, the percentages of IgMa/IgMb-coexpressing cells in the bone marrow of HEL and STAT5b-CA x HEL mice were 0.1 ± 0.04 vs 0.1 ± 0.06% (p = 0.7), respectively; similar results were seen in the spleen (2.4 ± 1.4 vs 3.0 ± 2.3%, respectively; p = 0.6). Thus, STAT5b-CA x HEL mice did not undergo V(D)J recombination, as evidenced by coexpression of IgMa and IgMb. Similar results were found in B cells isolated from lymph nodes (data not shown).


Figure 4
View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 4. STAT5b-CA x HEL mice do not undergo V(D)J recombination. Bone marrow and spleens were isolated from 6- to 10-wk old littermate control (LMC), STAT5b-CA, HEL, and STAT5b-CA x HEL mice. Cell populations were stained with Abs to B220, IgMa, and IgMb and analyzed by flow cytometry. Cell populations were electronically gated on B220+ cells, and IgMa/IgMb expression is shown. These results are representative of two separate experiments (four LMC, five STAT5b-CA, six HEL, and five STAT5b-CA x HEL mice).

 
VHJ558 recombination does not occur in HEL or STAT5b-CA x HEL mice

Although we could not detect surface coexpression of the HEL transgene (IgMa) and endogenous H chains (IgMb), it remained possible that rearrangement of endogenous H chain genes had occurred in STAT5b-CA x HEL transgenic mice, but that dual surface expression had been suppressed. To examine this possibility, we analyzed V(D)J recombination in these mice by looking at rearrangement of VHJ558 family members. As shown in Fig. 5, bone marrow from littermate control and STAT5b-CA mice exhibited extensive rearrangement of VHJ558 family genes (Fig. 5, lanes 1–4). As previously documented (12, 17), the presence of the HEL H chain suppressed rearrangement of endogenous H chain genes (Fig. 5, lanes 5 and 6). Importantly, STAT5b-CA x HEL transgenic mice showed no evidence of endogenous VHJ558 rearrangement (Fig. 5, lanes 7 and 8). All samples were normalized relative to the HPRT gene to ensure that equal numbers of cell equivalents were used in each experiment. Similar results were observed when examining VH7183 gene rearrangement (data not shown). Thus, attenuation of IL-7R signaling is not required for allelic exclusion to occur.


Figure 5
View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 5. VHJ558 recombination does not occur in HEL or STAT5b-CA x HEL mice. CD19+ B cells from the bone marrow of littermate control (LMC), STAT5b-CA, HEL, and STAT5b-CA x HEL mice were purified, and genomic DNA was isolated as described in Materials and Methods. PCR and Southern blotting of the genomic DNA for VHJ558 and HPRT were conducted as previously described (8 ). Genomic DNA was normalized relative to HPRT. Each lane represents genomic DNA from a distinct mouse. Results shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Three events are believed to play an important role in Igh recombination: repositioning of the H chain locus from a peripheral to a central nuclear location, contraction of the Igh H chain gene locus, and increased histone acetylation at VH genes. Specifically, Kosak et al. (18) have shown that the Igh H chain loci are centrally positioned in pro-B cells, a condition associated with transcriptionally active genes. In contrast, the Igh locus is localized to the nuclear periphery in cells that do not undergo VH gene rearrangement, such as embryonic stem cells or T cells. Furthermore, Roldan et al. (19) found that contraction of the Igh H chain gene is characteristic of cells undergoing rearrangement and that pre-B cells undergo decontraction of the Igh locus after production of a functional Igh H chain. Finally, increased histone acetylation of VH genes has also been shown to influence recombination (6). Thus, regulation at the genomic level via chromatin remodeling and nuclear positioning ensures that VH gene recombination occurs efficiently.

Importantly, the IL-7R plays a key role in at least two of these processes, namely, VH gene acetylation and locus contraction. For example, Corcoran et al. (4) demonstrated that distal VH genes display severely impaired rearrangement in IL-7R–/– mice, and this correlated with decreased VH germline transcription. In addition, Chowdhury and Sen (6, 7) demonstrated that IL-7 regulates changes in VH gene histone acetylation and nuclease accessibility that are associated with Igh gene rearrangement. More recently, it has been shown that Pax5, a downstream effector of IL-7R signaling in pro-B cells, promotes VH gene locus contraction and thereby regulates distal VH gene rearrangement (20). Thus, IL-7R signaling plays an important role in governing Igh gene rearrangement.

The IL-7R acts via induction of the STAT5 and PI3K signaling pathways. We have recently shown that for B cell differentiation, STAT5 is the key signaling molecule downstream of the IL-7R, because STAT5 activation is sufficient to largely restore B cell development in IL-7R–/– mice (8). These studies also documented that the IL-7R regulates Igh gene rearrangement through STAT5 signaling. Importantly, STAT5 appears to function by governing both locus contraction and VH gene acetylation. Specifically, Hirokawa et al. (21) have shown that STAT5 can bind to the pax5 gene promoter and regulate pax5 gene transcription using B cell lines in vitro; similarly, we have also observed STAT5 binding to the pax5 gene promoter in vivo (14). Furthermore, activated STAT5 has been shown to restore Pax5 expression in IL-7R{alpha}–/– pre-pro-B cells (22). These results suggest that STAT5 regulates locus contraction indirectly via pax5 induction. In addition, STAT5 has been clearly shown to regulate VH gene acetylation. For example, Stanton and Brodeur (9) have demonstrated that STAT5 acts downstream of the IL-7R to induce D-distal gene accessibility and histone H4 acetylation. Similarly, Bertolino et al. (10) have shown that both H3 and H4 acetylation of Igh genes is defective in STAT5a/b–/– mice; furthermore, they demonstrated that these mice exhibit defects in rearrangement of DJ-distal VH genes. Thus, STAT5 is the key effector of IL-7R signaling that regulates VH gene rearrangement by promoting gene accessibility via histone acetylation and locus contraction.

The importance of the IL-7R in initiating Igh gene rearrangement has suggested that it may also play a role in halting this process. Specifically, an attractive model has been proposed by Chowdhury and Sen (7) to explain the mechanism by which this occurs. They have proposed that "allelic exclusion is the consequence of losing signals that activate VH gene recombination; for the largest proportion of VH genes comprising the J558 family, this signal is IL-7." Thus, they have suggested that IL-7R down-regulation in pre-B cells reverts the Igh H chain loci back to its hypoacetylated, inaccessible state. As evidence for their model, they showed that IL-7 stimulation increased the accessibility of VHA.1 (a 5' member of the VHJ558 gene family) using a restriction enzyme accessibility assay. In addition, they found a 6-fold increase in histone acetylation of the VHA.1 gene in response to IL-7 compared with unstimulated pro-B cells. Likewise, using restriction enzyme accessibility and chromatin immunoprecipitation assays, they determined that VHA.1 was less accessible and hypoacetylated in pre-B cells compared with pro-B cells. Importantly, they correlated these changes in VH gene accessibility with responsiveness to IL-7R signaling; thus, they inferred that loss of IL-7R signaling led to these changes in nuclease accessibility and histone acetylation that in turn promote allelic exclusion.

This model makes a strong prediction, namely, if IL-7R down-regulation and/or desensitization could be prevented, then allelic exclusion should be negatively impacted. To test this hypothesis, we crossed STAT5b-CA mice (which express the IgMb allotype) with IgMa congenic mice and looked at IgMa/IgMb expression. If IL-7R/STAT5 down-regulation is required for allelic exclusion, we would expect ~20% of the B220+ cells from the STAT5b-CA x IgMa mice to coexpress IgMa and IgMb (see calculation in Materials and Methods). Instead, we found that there were minimal numbers of cells that coexpressed IgMa and IgMb on the cell surface. Identical findings were obtained when examining the intracellular expression of IgMa/IgMb. However, IL-7R/STAT5 signaling may only be required for allelic exclusion of the D/J-distal VHJ558 gene family. The VHJ558 family is the largest of the VH gene families and contains approximately half the murine VH genes; analysis of VH gene expression has demonstrated that in C57BL/6 mice >45% of all B cells express a rearranged VHJ558 family gene (23). Thus, if IL-7R/STAT5 signaling only regulates allelic exclusion of the VHJ558 gene family, then we would expect that 9% of the B220+ cells from STAT5b-CA x IgMa mice would coexpress IgMa and IgMb. This high level of IgMa/IgMb coexpression was never observed. In addition, no differences in the percentage of IgMa/IgMb-coexpressing cells were observed in STAT5b-CA and littermate control mice, indicating that attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion of VHJ558 family members.

We also looked at IgMa/IgMb expression in littermate control, STAT5b-CA, HEL, and STAT5b-CA x HEL mice. If attenuation of IL-7R/STAT5 signaling were responsible for allelic exclusion, we would expect the STAT5b-CA x HEL mice to coexpress IgMa and IgMb, because the HEL transgene is of the IgMa allotype, whereas STAT5b-CA mice are on a genetic background expressing IgMb. Instead, we found similar percentages of coexpressing cells in our STAT5b-CA x HEL and HEL mice. We tested this using both MD4 (H and L chain rearranged; data not shown) and MD2 (only H chain rearranged) HEL transgenic mice and observed similar results. Finally, because we were unable to detect surface expression of Igh gene rearrangement, we decided to investigate gene rearrangement at the genomic level. We examined VH7183 (data not shown) and VHJ558 gene rearrangements in littermate control, STAT5b-CA, HEL, and STAT5b-CA x HEL mice to examine this possibility. We found that VHJ558 was rearranged in B220+ cells from the bone marrow of littermate control and STAT5b-CA mice as expected, but was not rearranged in these cells from HEL or STAT5b-CA x HEL mice. Therefore, activated STAT5 does not promote VH rearrangement in mice expressing a rearranged Igh transgene. Importantly, we could not detect any evidence for impaired allelic exclusion at either the protein or the genomic level. Thus, IL-7R down-regulation/desensitization is not required for allelic exclusion.

Our findings do not provide evidence that attenuation of IL-7R/STAT5 signaling is responsible for initiating allelic exclusion. It is possible that other pathways downstream of the IL-7R may be involved in this process. However, we believe that this is unlikely for the following reasons. First, several studies have demonstrated that STAT5 restores V(D)J recombination in IL-7R–/– mice and directly regulates VH gene acetylation (8, 9, 10). Second, STAT5 indirectly regulates Igh H chain locus contraction through Pax5 induction (20, 22). Third, there is no evidence that PI3K, the other major pathway induced by the IL-7R, influences VH gene rearrangement (24). Thus, all available evidence supports a primary role for STAT5 in mediating IL-7R-dependent effects on Igh gene rearrangement. In conclusion, if IL-7R signaling does play a role in allelic exclusion, it is clearly a redundant one. Thus, it will be important to identify additional IL-7R-independent mechanisms that regulate allelic exclusion.


   Acknowledgments
 
We thank Dr. Tim Behrens for providing the HEL transgenic mice and the IgMa-FITC and IgMb-PE Abs, and Brendan Schatz for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported National Institutes of Health Grant AI05737 (to M.A.F.), a Pew Scholar Award, and a Cancer Research Investigator Award. C.A.G. and M.A.B. were partially supported by a gift from the estate of Eli and Dorothy Rosen and Bernard Collins. C.A.G. and M.A.B. are also supported by National Institutes of Health Training Grant 2T32-AI07313. Back

2 Address correspondence and reprint requests to Dr. Michael A. Farrar, Center for Immunology, University of Minnesota, 312 Church Street SE, 6-116 Hasselmo Hall, Minneapolis, MN 55455. E-mail address: farra005{at}tc.umn.edu Back

3 Abbreviations used in this paper: HEL, hen egg lysozyme; CA, constitutively activated; HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication August 17, 2005. Accepted for publication January 4, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Corcoran, A. E.. 2005. Immunoglobulin locus silencing and allelic exclusion. Semin. Immunol. 17: 141-154. [Medline]
  2. Bergman, Y.. 1999. Allelic exclusion in B and T lymphopoiesis. Semin. Immunol. 11: 319-328. [Medline]
  3. Schlissel, M.. 2002. Allelic exclusion of immunoglobulin gene rearrangement and expression: why and how?. Semin. Immunol. 14: 207-212. [Medline]
  4. Corcoran, A. E., A. Riddell, D. Krooshoop, A. R. Venkitaraman. 1998. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391: 904-907. [Medline]
  5. Haines, B. B., P. H. Brodeur. 1998. Accessibility changes across the mouse Igh-V locus during B cell development. Eur. J. Immunol. 28: 4228-4235. [Medline]
  6. Chowdhury, D., R. Sen. 2001. Stepwise activation of the immunoglobulin µ heavy chain gene locus. EMBO J. 20: 6394-6403. [Medline]
  7. Chowdhury, D., R. Sen. 2003. Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 18: 229-241. [Medline]
  8. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, M. A. Farrar. 2004. STAT5 activation underlies IL-7 receptor-dependent B cell development. J. Immunol. 172: 4770-4778. [Abstract/Free Full Text]
  9. Stanton, M. L., P. H. Brodeur. 2005. Cutting edge: Stat5 mediates the IL-7-induced accessibility of a representative D-distal VH gene. J. Immunol. 174: 3164-3168. [Abstract/Free Full Text]
  10. Bertolino, E., K. Reddy, K. L. Medina, E. Parganas, J. Ihle, H. Singh. 2005. Regulation of interleukin 7-dependent immunoglobulin heavy-chain variable gene rearrangements by transcription factor STAT5. Nat. Immunol. 6: 836-843. [Medline]
  11. Burchill, M. A., C. A. Goetz, M. Prlic, J. J. O’Neil, I. R. Harmon, S. J. Bensinger, L. A. Turka, P. Brennan, S. C. Jameson, M. A. Farrar. 2003. Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+CD25+ regulatory T cells versus CD8+ memory T cells. J. Immunol. 171: 5853-5864. [Abstract/Free Full Text]
  12. Hartley, S. B., C. C. Goodnow. 1994. Censoring of self-reactive B cells with a range of receptor affinities in transgenic mice expressing heavy chains for a lysozyme-specific antibody. Int. Immunol. 6: 1417-1425. [Abstract/Free Full Text]
  13. Van De Wiele, C. J., J. H. Marino, B. W. Murray, S. S. Vo, M. E. Whetsell, T. K. Teague. 2004. Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation. J. Immunol. 172: 4235-4244. [Abstract/Free Full Text]
  14. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, T. M. Johanns, M. A. Farrar. 2005. Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment. J. Immunol. 174: 7753-7763. [Abstract/Free Full Text]
  15. Smart, F. M., A. R. Venkitaraman. 2000. Inhibition of interleukin 7 receptor signaling by antigen receptor assembly. J. Exp. Med. 191: 737-742. [Abstract/Free Full Text]
  16. ten Boekel, E., F. Melchers, A. G. Rolink. 1998. Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity 8: 199-207. [Medline]
  17. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682. [Medline]
  18. Kosak, S. T., J. A. Skok, K. L. Medina, R. Riblet, M. M. Le Beau, A. G. Fisher, H. Singh. 2002. Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296: 158-162. [Abstract/Free Full Text]
  19. Roldan, E., M. Fuxa, W. Chong, D. Martinez, M. Novatchkova, M. Busslinger, J. A. Skok. 2005. Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6: 31-41. [Medline]
  20. Fuxa, M., J. Skok, A. Souabni, G. Salvagiotto, E. Roldan, M. Busslinger. 2004. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18: 411-422. [Abstract/Free Full Text]
  21. Hirokawa, S., H. Sato, I. Kato, A. Kudo. 2003. EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells. Eur. J. Immunol. 33: 1824-1829. [Medline]
  22. Kikuchi, K., A. Y. Lai, C. L. Hsu, M. Kondo. 2005. IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF. J. Exp. Med. 201: 1197-1203. [Abstract/Free Full Text]
  23. Viale, A. C., A. Coutinho, A. A. Freitas. 1992. Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice. J. Exp. Med. 175: 1449-1456. [Abstract/Free Full Text]
  24. Corcoran, A. E., F. M. Smart, R. J. Cowling, T. Crompton, M. J. Owen, A. R. Venkitaraman. 1996. The interleukin-7 receptor {alpha} chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. EMBO J. 15: 1924-1932. [Medline]



This article has been cited by other articles:


Home page
 J. Exp. Med.Home page
J. G. Bates, D. Cado, H. Nolla, and M. S. Schlissel
Chromosomal position of a VH gene segment determines its activation and inactivation as a substrate for V(D)J recombination
J. Exp. Med., December 24, 2007; 204(13): 3247 - 3256.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. A. Burchill, J. Yang, C. Vogtenhuber, B. R. Blazar, and M. A. Farrar
IL-2 Receptor beta-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells
J. Immunol., January 1, 2007; 178(1): 280 - 290.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Will, W. M.
Right arrow Articles by Farrar, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Will, W. M.
Right arrow Articles by Farrar, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS