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The Journal of Immunology, 2004, 173: 2542-2551.
Copyright © 2004 by The American Association of Immunologists

CD45-Deficient Mice Accumulate Pro-B Cells Both In Vivo and In Vitro1

Heather E. Fleming2, Craig D. Milne and Christopher J. Paige3

Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Department of Immunology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Efficient generation of mature B lineage cells requires the participation of the BCR, the pre-BCR, accessory coreceptors, and growth factor receptors. Together these receptors integrate cell intrinsic signals with regulatory pathways initiated by surrounding cells and structures. CD45 is a receptor tyrosine phosphatase expressed at high levels on all hemopoietic cells, and has been shown to modulate many signaling cascades in both positive and negative manners. In the absence of B220, the B lineage isoform of CD45, differentiation to the mature B cell stage is incomplete. We demonstrate that CD45-deficient mice also accumulate pro-B cells in the bone marrow. In vitro differentiation is altered in that B lineage populations exhibit prolonged survival in the presence of high concentrations of IL-7. Cell lines derived from CD45-deficient animals experience prolonged JAK/STAT activation in response to IL-7 stimulation, and constitutively elevated levels of phosphorylated src kinases. Aberrant IL-7R{alpha} expression is observed in vivo, and may be responsible for the skewed development present in CD45–/– animals. Demonstrating that CD45-deficient pro-B cells are affected by the absence of B220 highlights a previously unrecognized parallel in B and T lineage precursors, and emphasizes that the presence of normal numbers of peripheral B cells does not assure that the bone marrow compartment is intact.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Developing B lymphocytes experience multiple selection checkpoints during the maturation process. Two of the most defined of these checkpoints occur at the pro-B to pre-B cell transition in the bone marrow (BM),4 and at the immature to mature B cell stage in the periphery. Both of these transitions are mediated, in large part, by signals delivered by the pre-BCR complex (pre-BCR) and the complete BCR, respectively (1). Coreceptor and growth factor responses, often delivered in the context of the surrounding microenvironment, also contribute to the selection process (2). Regulation of maturation at these checkpoints appears to consist of a combination of survival, proliferation, and differentiation events. Considerable recent effort has begun to elucidate the specific signaling pathways involved in the individual checkpoint responses. We have previously presented evidence that the relative concentration of available IL-7 influences the selective expansion of pre-BCR+ cells through an ERK/MAPK-dependent signal cascade (3). In addition to activating MAPK, the pre-BCR and BCR complexes are known to signal via activation of syk and src family tyrosine kinases (4). Binding of IL-7 to its receptor triggers src kinase activation, activates the JAK/STAT cascade, and also initiates the PI3K pathway, resulting in proliferative and survival responses (5, 6, 7, 8). It is well established that IL-7 signaling results in proliferation, but the mechanism that limits expansion in response to IL-7 is not as well defined (9). CD45 is a membrane-bound tyrosine phosphatase expressed on all hemopoietic lineage cells (10). The CD45 protein can be expressed in various isoforms that are generated by alternate splicing, and is heavily modified posttranslationally by the addition of carbohydrate moieties (11, 12). B lineage cells express the 220-kDa isoform of CD45, B220. It is one of the earliest phenotypic markers used to identify early B lineage progenitors, although it is also found on a subset of NK cell precursors (13, 14). CD45 plays an important role in regulating signals mediated by Ag receptors (15). It is responsible for dephosphorylating the regulatory tyrosine residue of src family kinases such as lck and lyn, and may also turn off kinase activity by dephosphorylating residues within the active site (16, 17, 18, 19). More recently, CD45 has also been shown to be capable of dephosphorylating JAK family kinases in both human and murine cells (20, 21). We hypothesized that CD45 may be involved in regulating the proliferation of pre-BCR+ cells in response to IL-7, via its potential to dephosphorylate lyn and/or JAK1,3. Two independent strains of CD45-deficient mice were generated several years ago (22, 23). Despite the ubiquitous expression pattern of B220, generation of B lineage cells in the absence of B220 expression appeared intact in these mice. The original characterization of the CD45 knockout animals revealed a developmental defect at the immature to mature B cell transition. The defect manifests in the spleen, and results in a decreased percentage and number of recirculating mature B cells in the BM (22). In the spleen of the exon 9-targeted mice, elevated numbers of B cells with a predominantly immature phenotype were detected, and were shown to exhibit defective proliferation in response to Ig cross-linking, but intact LPS and CD40 responsiveness (23). The initial analyses of early B lineage development in CD45–/– mice were limited to frequency assays of mutant and normal BM cell colony formation in vitro. No defect in the frequency of IL-7-responsive cells was detected in the CD45 exon 6–/– mice (22).

In the current study, we present a more thorough analysis of the B lineage populations found in the BM of CD45 exon 6–/– mice and reveal a disruption of normal B cell development in the absence of B220. CD45–/– BM contains a significantly elevated percentage and number of pro-B cells, in addition to the previously reported decrease in mature B cells relative to littermate control animals. In vitro characterization of cultured BM cells demonstrates that CD45-deficient cells exhibit prolonged survival in response to high concentrations of IL-7. The mechanism responsible for the accumulation of pro-B cells in vitro and in vivo may be attributed to the observed sustained expression of the IL-7R{alpha} chain, or to the extended phosphorylation of the JAK/STAT cascade in CD45-deficient cell lines following IL-7 stimulation. This finding suggests that CD45-dependent signals may affect the efficiency of selection at the pro-B to pre-B cell transition by extending the survival of pro-B cells when high concentrations of IL-7 are available, in addition to its known role in the development of mature B cells in the periphery.


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

C57BL/6 mice were purchased from the The Jackson Laboratory (Bar Harbor, ME). CD45 exon 6–/– mice were generated previously and have been backcrossed to the C57BL/6 background (22). All mice were bred and maintained at the Ontario Cancer Institute animal facility (Toronto, Canada) and were used between 6 and 10 wk of age. All animal procedures conform with institutional animal protocol guidelines.

Cell purification

BM cell suspensions were isolated, as described (3). B cell progenitors (CD19+, CD43+, µHC–, termed sorted pro-B cells) were isolated from BM, as described, using a MoFlo high-speed cell sorter (DakoCytomation, Fort Collins, CO) (3). Typically, 0.5–1.0% of total BM was recovered from C57BL/6 or heterozygous mice, and 0.75–1.5% of total BM was recovered from CD45-null mice. Sorted populations were routinely >98% pure. Early pro-B cells were obtained by sorting with four parameters to obtain CD19+, CD43+, CD117+, BP-1– BM cells. Typically, 0.01–0.02% of total BM was recovered from CD45+/– mice using this sort protocol, and CD45–/– BM yielded up to 1.5-fold more cells than did control BM. IL-7-dependent cell lines were established from CD45-deficient and B6 mice, as previously described (3).

Cell culture conditions

Cells were grown in OPTI-MEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% FCS, 50 µM 2-ME, 2.4 g/L NaHCO3, 100 µg/ml penicillin, 100 µg/ml streptomycin, and the indicated concentration of murine IL-7 obtained from the supernatant of the stably transfected J558 line (A. Cumano, Pasteur Institute, Paris, France). Proliferation assays were performed, as described previously (3). Cells to be assayed by FACS analysis were plated at 1.0 x 104 cells/ml with the indicated concentrations of IL-7. Sorted cells to be assayed for the frequency of IL-7-responsive cells were plated, as described (24). For clonal analysis of IL-7 reponsiveness over time, wells were seeded with five sorted cells/well, and scored at day 4 for the presence of single colonies. Wells containing individual clones were scored on days 7 and 11 for survival, or on days 4, 6, 8, and 10 for the number of live cells in the colony. Surviving clones were defined as those in which at least 5% of remaining cells appeared alive by visual inspection. Cell counts and visual estimation of clone size were performed on days 4, 6, 8, and 10 only in wells that contained five or more live cells at the previous time point. Individual plates were coded to ensure that cell counts were done in a blinded fashion. Wells were fed every 4 days with the original amount of supplemented IL-7. To assay LPS induction by IgM secretion, sorted pro-B cells were grown in 5.0 ng/ml IL-7 for 4 days and then transferred at 1000 cells/well to flat-bottom 96-well plates with 1000 irradiated S17 stromal cells, or to U-bottom 96-well plates, in the absence of IL-7 and supplemented with 15 µg/ml LPS (Sigma-Aldrich, St. Louis, MO). ELISA for secreted IgM was performed after 7 days of culture, as described previously (25).

Immunofluorescence staining and analysis

Abs used for FACS analysis were purchased from BD Pharmingen (San Diego, CA), unless otherwise noted. Abs used were directly conjugated to FITC, PE, allophycocyanin, or biotin. Clones used: CD2 (RM2-7), CD19 (MB19-1; eBioscience, San Diego, CA), CD24 (M1/69), CD25 (7D4), CD43 (S7), CD117 (2B8; eBioscience), B220 (6B2; 14.8), IL-7R{alpha} (A7R34; S. Nishikawa, Kyoto, Japan), common {gamma}-chain ({gamma}c) (4G3), IgD (SBA.1), µHC (33.60), and BP.1 (6C3). Cells were stained in PBS + 3% heat-inactivated FCS at 1–3 x 106 cells/ml by incubation with FITC-, PE-, or allophycocyanin-conjugated or biotinylated Abs for 20 min on ice. After washing, cells were incubated with a streptavidin-conjugated reagent (Quantum Red (Sigma-Aldrich) or PerCP (BD Biosciences, San Jose, CA)), as above. FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). Acquisition and analysis software used was CellQuest, version 3.3.

CFSE loading and analysis

CFSE was purchased from Molecular Probes (Eugene, OR) in the Vybrant CFDA SE cell tracer kit (V-12883). Aliquots of CFSE were solubilized in DMSO to a concentration of 10 mM. Cells to be loaded with dye were washed in serum-free OPTI-MEM, resuspended at 106 cells/ml, treated with 5 µM CFSE (1/2000), and incubated at 37°C for 10 min in the dark. The loading reaction was stopped by adding cold OPTI supplemented with 10% FCS. Cells were washed in OPTI + 10% serum and cultured at 1 x 104 cells/ml supplemented with the indicated concentration of IL-7. After an 8-h incubation, untreated cells were analyzed on a FACSCalibur to determine the fluorescence level before cell division. To express the CFSE fluorescence data as the number of divisions experienced within the population, gates were drawn to divide the population into 2-fold mean fluorescence intensity drops. The percentage of live cells recovered in each CFSE gate was plotted, or used to calculate the absolute number of cells that had undergone each number of divisions.

Cell stimulation and Western blotting

Cell stimulation and Western analysis were performed, as described (3). Briefly, cells were washed in OPTI-MEM + 0.5% FCS, starved for 2 h in a 37°C humidified incubator, and resuspended at 107 cells/ml at 37°C for stimulation by the addition of 25 ng/ml IL-7 for the times indicated. Cells were lysed, and protein samples were separated using the NuPAGE (Invitrogen Life Technologies) electrophoresis and transfer apparatus. Detection of phosphorylated JAK1 (BioSource International, Camarillo, CA), STAT5, Lyn, and Akt (New England Biolabs, Beverly, MA) was performed, according to the manufacturer’s instructions. For loading controls, membranes were stripped using the Re-Blot Plus recycling kit (Chemicon International, Temecula, CA), according to the manufacturer’s instructions. Reprobing for nonphosphorylated JAK1 and STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) was performed, as above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD45 deficiency results in altered frequency of B cell progenitors in vivo

We previously reported that IL-7-responsive colony formation was intact in CD45-deficient BM populations (22). To extend these studies, four-color FACS analysis was performed using Abs that recognize a series of cell surface proteins. The data presented in Fig. 1 compare the frequency of developing B cell populations detected in individual CD45–/– and littermate BM. The mutant mice exhibit a similar percentage of BM cells that are CD19+ lymphocytes (Fig. 1). The remaining populations examined are expressed as the percentage of CD19+ cells that are designated as pro-B, pre-B, immature B, and mature B cells, according to the expression of CD43, IgM, and IgD. Mutant mice exhibit a significant decrease in the percentage of mature B cells in the BM (Fig. 1). In addition, the pro-B population in CD45-deficient animals comprises a significantly larger proportion of the CD19+ compartment than in control mice (Fig. 1). These differences are also reflected in the absolute cell numbers of the various populations found in the BM (Fig. 1B). The FACS analysis data reveal for the first time that the early developmental program of B lineage cells is altered in the BM of CD45–/– mice. It appears as though CD45 may participate in two major checkpoints during B cell maturation. In addition to the impeded immature to mature B cell transition, CD45–/– mice also exhibit an inefficient transit through the pro-B to pre-B checkpoint, known to be controlled largely by signals delivered by the pre-BCR.



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  		FIGURE 1. In vivo analysis of B lineage populations in CD45–/– and CD45+/– mice. Four-color FACS analysis was performed on BM samples from littermate animals. The percentage (A) and absolute cell number (B) of cells corresponding to pro-B (CD19+CD43+IgM–IgD–), pre-B (CD19+CD43–IgM–IgD–), immature B (CD19+CD43–IgM+IgD–), and mature, recirculating B cells (CD19+CD43–IgM+IgD+) are indicated. In A, results are presented as the percentage of CD19+ lymphocytes that are gated according to each population. The exception is the CD19+ parameter (squares), which is presented as the percentage of live cells that are gated as CD19+ lymphocytes. Data points represent individual animals assayed from three litters of CD45–/– and control littermates. Significance was determined using Student’s unpaired, one-tailed t test.

 
CD45-deficient cells exhibit elevated proliferative responses in the presence of high concentrations of IL-7

Next, the role of CD45 in the growth of cells in vitro in the presence of IL-7 was examined. Pro-B cells were isolated from wild-type (wt) or CD45–/– BM by selecting CD19+CD43+µHC– cells. Sorted populations from both mice were assayed for proliferation in response to various concentrations of IL-7, as measured by [3H]thymidine incorporation (Fig. 2A). No difference was observed in the proliferation of wt and CD45–/– cells cultured in low, picogram concentrations of IL-7. However, a reproducible increase in the proliferation of CD45-deficient cells was detected in the presence of higher, nanogram levels of IL-7, relative to wt and heterozygous cells. To determine whether the increase in proliferation reflected a higher number of progenitors, the frequency of IL-7-responsive cells present in each starting population was determined. As shown in Table I, no significant differences were detected in the frequency of responding cells that arise in culture with high or low concentrations of IL-7 between wt and mutant mice.



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  		FIGURE 2. CD45-deficient cultures exhibit elevated proliferation and accumulate early pro-B cells in the presence of high concentrations of IL-7. Sorted pro-B cells (CD19+CD43+µHC–) were cultured with the indicated concentration of IL-7 for 4 days. A, Proliferation was assayed on day 4 by the addition of [3H]thymidine. Results shown are representative of at least four independent experiments and are the mean of triplicate wells with SD error bars. B, Cells were harvested and analyzed by FACS for the percentage and number of cells with surface marker expression corresponding to normal stages of development. Populations were gated as early pro-B (CD19+CD117+CD2–µHC–), pro-B (CD19+CD117–CD2–µHC–), pre-B (CD19+CD117–CD2+µHC–), and immature B (CD19+CD117–CD2+µHC+) cells. Results shown are the mean of three independent experiments and include SE error bars. C, Sorted pro-B cells expanded in 5.0 ng/ml IL-7 for 4 days were transferred to conditions that promote IgM secretion in the presence of LPS. After 7 days, supernatant was removed from individual wells and analyzed by ELISA for IgM secretion. Results shown are representative of four independent experiments, and are the mean of five replicate wells with SD. Significance was determined using Student’s t test: ***, p < 0.0001; **, p < 0.008; *, p < 0.02.

 

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  		Table I. CD45-deficient and control sorted pro-B cell populations contain equivalent frequencies of IL-7-responsive cellsa

 
CD45-deficient populations contain an increased number and percentage of pro-B cells when cultured in vitro in the presence of high concentrations of IL-7

Phenotypic changes occur over time in populations grown in vitro in the presence of IL-7 (26). To assess whether the increased proliferation of CD45-deficient cells observed in the presence of high concentrations of IL-7 was associated with a difference in in vitro maturation, FACS analysis of the cultures was performed. Populations corresponding to early pro-B, pro-B, pre-B, and immature B cells were designated according to the expression of CD2, µHC, and CD117. The composition of these populations in each culture condition is presented in Fig. 2B. Clearly, a larger proportion of CD45–/– cultured cells exhibits early pro-B characteristics relative to the control cultures. Specifically, mutant populations contain an elevated percentage of cells that are µHC–, CD2–, and CD117+ after 4 days in culture with 5.0 ng/ml IL-7. This trend is consistent after 6 days in culture (data not shown), and results in a larger absolute number of cells that express an early pro-B phenotype (Fig. 2B, bottom). It should be noted that the population of cells that is overrepresented in the mutant cultures corresponds to the high threshold IL-7-responsive stage, shown previously to have prolonged survival in these conditions (24). In contrast, while a lower percentage of surface Ig+ cells is present in CD45–/– cultures (Fig. 2B, top), a similar number of mature cells arise in all conditions (Fig. 2B, bottom). Furthermore, the kinetics of maturation, as determined by FACS analysis on consecutive days, appears identical in both wild-type and CD45–/– cultures (data not shown). This observation suggests that maturation during culture in high concentrations of IL-7 occurs in the absence of CD45, but that CD45–/– pro-B cells may either divide faster or survive longer in these conditions than do their wt counterparts.

To test whether in vitro maturation to the LPS-responsive stage is intact in the absence of CD45, IL-7-expanded BM cells from mutant and wt mice were transferred to culture conditions that promote IgM secretion. Fig. 2C presents the amount of IgM detected by ELISA in stromal coculture supernatants. CD45–/– cells secrete levels of IgM that are equivalent to control cells grown in these conditions. In addition, culturing the IL-7-expanded B lineage cells in conditions that promote contact can replace the requirement for stromal cells and result in IgM secretion by both wt and mutant cells (Fig. 2C) (25). A slight (2- to 3-fold) decrease in the amount of IgM secreted by CD45–/– cells is detected consistently in these experiments.

CD45-deficient cells do not divide more rapidly when cultured with IL-7 in vitro

To differentiate between the possibilities that pro-B cells present in CD45–/– cultures divide more rapidly or persist longer than wt cells in high concentrations of IL-7, FACS analysis was performed using CFSE to track cell divisions. CFSE is a fluorescent cell dye that can be used to assay cell division by virtue of its property of distributing equally into both daughter cells (27). A 2-fold drop in fluorescence is observed with each round of mitosis. By loading sorted pro-B cells with CFSE, populations from wt and mutant mice cultured in high and low concentrations of IL-7 can be followed on consecutive days. Analysis of the CFSE fluorescence allows the number of cell divisions to be tracked in each case. Fig. 3 depicts CFSE fluorescence expressed as the number of divisions that individual cells have experienced since being loaded with the dye and seeded in culture. The first panel shows the percentage of live cells recovered that fall into each division (Fig. 3A). Data obtained after 3 and 4 days in culture are included in the same panel. The overlapping histograms derived from CD45–/– and control cultures indicate that the populations present in both cases have undergone the same number of divisions. The overlap is observed on both days 3 and 4, demonstrating that the rate of mitosis is equivalent in the presence or absence of CD45. Differences are apparent, however, when the same data are expressed as the absolute number of cells in each division (Fig. 3B). In this case, although the histograms still overlap, the peak height of the histograms generated from the CD45–/– cultures is higher than those from the control population. A larger histogram indicates that a greater number of cells have accumulated in the mutant cultures. Examination of specific populations within the cultures reveals that each of the cell types are present in greater numbers in CD45-deficient cultures relative to wild-type cells (Fig. 3C). The intermediate CD2+µ– population does not accumulate to the same degree in mutant cultures as do the least mature, CD2–µ– and most mature, CD2+µ+ populations. An accumulation of cells that exhibit the same rate of division suggests that CD45-deficient cells experience prolonged survival in the presence of high concentrations of IL-7.



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  		FIGURE 3. CFSE analysis of sorted pro-B cells cultured in vitro. Sorted pro-B cells were loaded with CFSE, cultured with the indicated concentration of IL-7, and analyzed by FACS at the indicated time points to determine the level of CFSE fluorescence remaining in each population. CFSE levels are expressed as the relative number of cell divisions experienced by the percentage (A) and number (B) of recovered cells. Cells cultured in this manner were also gated on pro-B, pre-B, and immature B cell populations based on CD2 and µHC. CFSE division profiles are shown for each population on day 4 of culture in C. Results shown are representative of two independent experiments.

 
CD45-deficient clones exhibit prolonged survival in the presence of high concentrations of IL-7

The data described to date have been generated from the analysis of bulk cultures of sorted pro-B cells. From these analyses, it has been shown that CD45–/– cultures exhibit elevated proliferation and an accumulation of early pro-B cells that appear to possess a heightened capacity for survival. To extend these observations to the clonal level, individual clones derived from wt and CD45-deficient mice were assayed for survival over time. IL-7-responsive clones were established by limiting dilution culture of sorted pro-B cells and examined on days 4, 7, and 11 to track persistent clone viability. The results of this assay are shown in Fig. 4A, and are presented as the percentage of clones surviving in each condition, relative to day 4. A significant increase in the survival of CD45-deficient clones is apparent in the presence of high concentrations of IL-7 when the responses of the mutant and control cells are compared. An extension of the clonal analysis was made by determining the absolute cell number present in each well at every time point. An increased number of cells generated by a single clone would be predicted based on the results presented in Figs. 2 and 3. Indeed, individual clone size is elevated when CD45–/– cells are cultured in the presence of high concentrations of IL-7 (Fig. 4B). Therefore, while CD45-deficient pro-B populations contain the same frequency of IL-7-responsive cells, more CD45–/– clones are able to persist when the cultures are supplemented with high concentrations of IL-7.



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  		FIGURE 4. CD45-deficient IL-7-responsive clones survive longer, and accumulate more cells when grown in the presence of high concentrations of IL-7. A, IL-7-responsive colonies arising from wells seeded with five sorted pro-B cells/well were scored for live colonies on day 4, 7, and 11 following the initiation of culture. Percentage of clones surviving was calculated relative to the number of live colonies present on day 4. Results shown are the mean from six independent experiments and include SE. B, The number of cells per IL-7-responsive colony was determined on days 4, 6, 8, and 10 by visual inspection. The mean colony size of surviving colonies was calculated. Results are the mean of three independent experiments and include SE. C, Sorted pro-B cells were plated at five cells/well in 5.0 ng/ml IL-7 (left) or 0.05 ng/ml IL-7 (right). The absolute cell number present in each well was determined by visual inspection on days 4, 6, and 8 of culture. The results are presented as the percentage of wells with live cells on day 4 that produce colonies that: expand by greater than 10-fold or by 1- to 10-fold between time points; decline in size after initial scoring on day 4; or expand and then decline on days 6 and 8, respectively. Data are presented as the mean of three independent experiments and include SE. Significance was determined using Student’s t test, comparing mutant and wt cells grown in 5.0 ng/ml IL-7: **, p < 0.02.

 
It is important to acknowledge that the CD19+CD43+ population of sorted pro-B cells is heterogeneous and contains precursors of varying stages of development (28). With this in mind, a more extensive analysis of the growth dynamics of individual clones was performed. IL-7-responsive clones were generated by limiting dilution and examined on days 4, 6, and 8 after the initiation of culture. Preliminary experiments revealed that distinct, reproducible patterns of clone growth and decline were evident among the starting population. Therefore, at each time point, the number of cells present in each well was determined by visual inspection. The growth patterns exhibited by individual clones were categorized as: constant growth of 10-fold or greater, or of 1- to 10-fold; constant decline after initial identification of the clone on day 4; and clone growth followed by decline at a later time interval (Fig. 4C). In the presence of high concentrations of IL-7, a significantly higher percentage of CD45-deficient clones exhibits 10-fold or greater increases in cell number over 8 days in culture (Fig. 4C, left). In contrast, no significant differences were observed over three independent experiments in the distribution of clonal growth patterns of wt and mutant cells supplemented with low concentrations of IL-7 (Fig. 4C, right). Together, the results presented in Fig. 4 indicate that pro-B populations from CD45–/– BM are more likely to give rise to clones that accumulate a large number of cells in response to high concentrations of IL-7. The bulk analysis suggests that this is due to an enhanced survival capacity of the CD45-deficient cells. However, the pro-B population from wt and mutant animals contains a similar frequency of clones that will die with the same kinetics (Fig. 4C), indicating that not all CD45–/– cells exhibit prolonged survival.

Cultures initiated with CD45-deficient early pro-B cells also exhibit prolonged survival

The pro-B population defined by CD43 expression can be further subdivided based on the presence of BP.1 and CD117. These markers were used to dissect the in vivo pro-B population of CD45–/– and control BM by FACS analysis (Fig. 5A). It is apparent that the pro-B population present in mutant mice is enriched for very early CD43+CD117+BP.1– pro-B cells, with less consistent increases in the other pro-B populations. Similar results were obtained using CD24 and BP.1 staining to subdivide the CD43+ cells into Hardy fractions A, B, and C (28), in which fraction B cells were the most prominent (data not shown).



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  		FIGURE 5. Early pro-B cells from CD45-deficient mice also yield higher cell numbers when supplemented with high concentrations of IL-7. A, Four-color FACS analysis was performed to subdivide the CD19+CD43+ pro-B BM compartment based on expression of CD117 and BP.1. Results are presented as the percentage of CD19+ cells that correspond to each pre-pro-B population. Data points represent individual animals assayed from three litters of CD45–/– and control littermates. Significance was determined using Student’s t test. B and C, Early pro-B cells (CD19+CD43+CD117+BP.1–) were sorted and cultured in 5.0 ng/ml IL-7. Cells were harvested on days 4, 6, 8, and 10 after the initiation of culture and analyzed by FACS. Populations were assessed for the percentage (B) and number (C) of cells that resemble early pro-B (CD117+CD2–µHC–), pro-B (CD117–CD2–µHC–), pre-B (CD117–CD2+µHC–), and immature B (CD117–CD2+µHC+) cells. Results shown in B are the mean of three independent experiments with SE. Data presented in C are representative of three independent experiments.

 
The in vitro experiments presented to date have used both bulk culture and limiting dilution clonal methods to track the growth and development of cells in culture containing IL-7. Results from the FACS analysis presented above indicate that the pro-B population sorted from the BM and used to initiate these cultures is not equivalent in wt and mutant mice. Indeed, the population that is most overrepresented in vivo shares phenotypic characteristics with the abundant population found in CD45-deficient in vitro cultures supplemented with high concentrations of IL-7. To examine a more comparable population of wt and CD45–/– cells, early pro-B cells (CD19+CD43+CD117+BP1–) were sorted and cultured in the presence of high or low concentrations of IL-7. FACS analysis was performed on days 4, 6, 8, and 10 following the initiation of culture with Abs that recognize CD117, CD2, and surface µHC. Using these markers, four distinct populations that mimic in vivo differentiation were tracked: early pro-B, CD117+CD2–µ–; pro-B, CD117–CD2–µ–; pre-B, CD117–CD2+µ–; and immature B, CD117–CD2+µ+. The proportion of the populations generated by normal and mutant cells cultured in high concentrations of IL-7 corresponding to each of these four populations is shown in Fig. 5B. In contrast to the results presented in Fig. 2B, when the cultures are initiated with a more homogenous population, no significant differences are observed in the relative composition of the four developmental stages. However, the CD45-deficient cells continue to exhibit prolonged survival relative to the control cells, in that a larger absolute number of CD45–/– cells is apparent (Fig. 5C). The mutant cultures accumulate cells in all of the populations examined, and the difference in cell number tends to increase with time. Slightly elevated total CD45–/– cell numbers were also obtained in cultures supplemented with low concentrations of IL-7, but no consistent differences were observed in these conditions (data not shown). Limiting dilution cultures were also initiated with the CD19+CD43+CD117+BP1– sorted cells. The frequencies of IL-7-responsive cells present in the populations sorted from CD45–/– and control mice were higher than those reported in Table I, but were not significantly different from each other (data not shown). Assessment of the cell numbers present in each of the colonies revealed that the average CD45-deficient clone size was larger than wt in high concentrations of IL-7 (data not shown), consistent with the increased cell numbers observed in the bulk culture. Together, these results suggest that the differences observed in vitro using the more heterogeneous sorted pro-B cells cannot be attributed to the elevated percentage of CD117+BP1– cells present in vivo in the CD45–/– mice.

Activation of the JAK/STAT cascade in response to IL-7 stimulation is prolonged in the absence of CD45

To investigate potential differences in the signaling pathways activated by IL-7 in the presence and absence of CD45, clonal, IL-7-dependent pre-BCR– cell lines were isolated by limiting dilution of long-term cultures of normal and mutant mice. Cells from multiple lines were stimulated with IL-7 for the indicated times and then assayed by Western blot for the presence of phosphorylated lyn, a member of the src kinase family. Consistent with the established role of CD45 as a src family phosphatase, elevated levels of phosphorylated lyn were observed in CD45-deficient samples (Fig. 6, top panel). However, lyn activation was not altered following IL-7 stimulation, suggesting that the loss of src phosphatase activity is not responsible for the prolonged IL-7 responsiveness of CD45-deficient pro-B cells. Similar results were obtained when another src family kinase, blk, and Akt, a PI3K pathway target, were examined (data not shown). In addition to analysis of the src kinases, activation of the JAK/STAT cascade was assayed. The results presented in Fig. 6 demonstrate that phosphorylation of the JAK/STAT kinase pathway is maintained up to 3 h following IL-7 stimulation in CD45-deficient cells, in contrast to the abrogation of this signal in wild-type cells. This finding is consistent with other groups who have demonstrated that CD45 exhibits JAK as well as Src phosphatase activity (16, 20, 21), and provides a potential mechanism by which CD45 deficiency may lead to the prolonged survival of pro-B cells in the presence of high concentrations of IL-7. The direct or indirect targets of the JAK/STAT cascade that lead to the extended life span of CD45-deficient pro-B cells have yet to be established.



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  		FIGURE 6. Activation of the JAK/STAT cascade is prolonged in CD45-deficient cells in response to IL-7. IL-7-dependent cell lines were isolated by limiting dilution of long-term cultures of wild-type and CD45-deficient BM. Cells were washed of IL-7, starved for 2 h at 37°C, stimulated with 25 ng/ml IL-7, lysed, and subjected to Western blot analysis with the indicated phospho-specific Abs. Membranes were stripped and reprobed with JAK1 or STAT5 Abs to control for equal loading. Results are representative of three independent experiments with at least two cell lines per genotype.

 
CD45-deficient cells exhibit prolonged expression of the IL-7R in vivo

Substantial data have been presented to support the conclusion that CD45-deficient cells exhibit prolonged survival and responsiveness to high concentrations of IL-7 in vitro. Analysis of the relative percentage of B lineage populations present in vivo revealed a pronounced elevation of both the pro-B and pre-B compartments (Fig. 1). To examine the potential for IL-7 signaling in an in vivo setting, additional four-color FACS staining was performed on CD45–/– and littermate BM cells to assay the expression level of the IL-7R on individual developing B lineage cells. As shown in Fig. 7A, a large proportion of mutant CD43– pre-B cells maintains IL-7R{alpha} chain expression at high levels, whereas most wt CD43– cells express less IL-7R{alpha} than do CD43+ cells. In contrast, normal and CD45-deficient pro-B and pre-B cells express equivalent levels of the other IL-7R chain, {gamma}c (Fig. 7A, bottom). The elevated percentage of mutant IL-7R{alpha}+ pro- and pre-B cells is consistent when multiple littermate animals are compared, whereas a less significant difference between CD45–/– and control IL-7R{alpha}+/IgM+ populations is observed (Fig. 7B). These data are consistent with the hypothesis that in the absence of CD45, the regulation of IL-7R signaling may be impaired, resulting in prolonged IL-7R{alpha} expression and the maintenance of IL-7 responsiveness.



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  		FIGURE 7. CD45-deficient pre-B cells exhibit prolonged expression of the IL-7R{alpha} chain in vivo. FACS analysis of BM from CD45–/– and control mice was performed with Abs to distinguish IL-7R expression on pro-B, pre-B, and IgM+ B cells. A, IL-7R{alpha} (top) and {gamma}c (bottom) expression of BM cells gated on pro-B (CD19+CD43+) and pre-B (CD19+CD43–) cells. Results are representative of four independent experiments. B, Percentage of CD19+ lymphocytes that are IL-7R{alpha}+ pro-B cells, IL-7R{alpha}+ pre-B cells, and IL-7R{alpha}+ IgM+ B cells in the BM of individual CD45–/– and control littermate animals. Significance was determined by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous analysis of CD45 exon 6–/– and CD45 exon 9–/– mice revealed normal or elevated numbers of splenic B lineage cells that expressed no detectable surface CD45 protein (22, 23). Despite the normal number of peripheral B cells, maturation to the IgMlowIgDhigh stage was inhibited in the absence of CD45. This developmental defect was accompanied by incomplete responsiveness to stimulation through the BCR (23, 29). Very little analysis of the BM compartment of the CD45–/– mice was performed in either of the CD45-deficient strains. In this study, we have fully assessed the frequencies of individual B lineage subsets in the BM of CD45 exon 6–/– mice. In agreement with the original publication, a similar frequency of IL-7-responsive cells is detected in in vitro analyses. However, FACS analysis of ex vivo BM suspensions reveals a significant elevation of the frequency and number of early pro-B cells in the absence of CD45. Similar differences are not apparent at later developmental stages in the BM, with the exception of the originally identified paucity of mature, recirculating B cells. A specific increase in cells at the pro-B cell stage suggests that the efficiency of the transition to the pre-B cell stage and beyond is altered in CD45-deficient animals, or that normal regulation of death and expansion of pro-B cells is impaired.

The transition from pro-B to pre-B is a significant selection checkpoint in the generation of B lineage cells (1). Recently, we have put forward a model in which a change in the threshold of responsiveness to IL-7 participates in the selection of pre-B cells (30). Our model is based on the observation that concurrent activation of the ERK/MAPK cascade by the pre-BCR and the IL-7R permits the proliferation of pre-B cells in concentrations of IL-7 that do not support the growth of pre-BCR–, pro-B cells (3, 24). Therefore, the developmental checkpoint can be controlled by the relative availability of IL-7 as the precursors transit through the BM toward the central sinus. This IL-7 threshold model predicts that pro-B cells exist, at least temporarily, in a location with high concentrations of IL-7 to permit their expansion. The second component of the model requires that B cell precursors pass through a region of decreased IL-7 availability, resulting in the selective survival and expansion of precursors that have successfully matured to the pre-B cell stage. The in vivo and in vitro data presented in the current manuscript, when interpreted in the context of this model, are consistent with the hypothesis that CD45 is required for efficient transition at the pro-B to pre-B stage in vivo, in that pro-B cells can be maintained in an environment in which high concentrations of IL-7 are available. CD45–/– BM contains a population of pro-B cells that exhibit prolonged survival when cultured in vitro with high concentrations of IL-7. The same phenotypic markers define the pro-B population that is overrepresented in the BM of CD45–/– mice. CD45–/– in vitro cultured pro-B cells can mature and proliferate in response to low concentrations of IL-7. Similarly, pre-B cells are efficiently generated in vivo even in the absence of CD45. One difference between the in vivo and in vitro CD45–/– analyses is that only pro-B cells are found in greater numbers in the BM, while cells of all maturation stages accumulate in vitro. We propose that in CD45–/– mice, the BM niche containing high levels of IL-7 permits pro-B cell accumulation via their enhanced survival capacity, possibly via a prolonged activation of the JAK/STAT kinase cascade. However, the selection pressure favoring pre-BCR+ cells in the neighboring niche containing low IL-7 levels is intact, resulting in the generation of close to normal numbers of immature B cells. Were CD45–/– animals to be artificially supplied with supraphysiological levels of IL-7, whether by transgenic or injection methods, it is predicted that mice would produce increased numbers of B cells at all stages of development. This finding is precisely what is observed in vitro, in which the regulation provided by a three-dimensional niche structure is overcome. Of note, a recent report has implicated CD45 in participating in CXCR4-dependent T cell chemotaxis (31). Given that the ligand for CXCR4, CXCL12, is an important mediator of early B cell precursor homing and migration, it is possible that CD45 may also influence the migration of pro-B cells as they navigate from one BM niche to another (32).

Recent studies have identified that CD45 is able to dephosphorylate JAK kinases in murine and human cells, in addition to its established role as a src family phosphatase (16, 20, 21). The hypothesis that the persistent responsiveness to IL-7 observed in CD45-deficent B cells is due to a prolonged activation of the JAK kinase cascade downstream of the IL-7R was tested. Using IL-7-dependent cell lines isolated from normal and mutant mice, a consistent extension of the activation-induced phosphorylation of JAK1 and STAT5 was observed in the absence of CD45. In addition, while no IL-7-dependent changes were observed in the phosphorylation of src family kinases in CD45–/– cells, mutant cells did exhibit high levels of constitutively activated lyn and blk. It is conceivable that a combined action of these active kinases with the prolonged activation of the JAK/STAT cascade influences the survival of IL-7-responding pro-B cells in vitro and in vivo. The precise mechanism by which CD45-deficient pro-B cells exhibit prolonged survival in response to high concentrations of IL-7 is, as yet, undetermined.

Despite the similarities in the signaling cascades induced by IL-7 in ex vivo cultured wt and mutant pro-B cells, an increased proportion of pre-B cells that express IL-7R{alpha} was observed in CD45–/– BM. This chain of the IL-7R is normally expressed at lower levels following acquisition of the pre-BCR, and continues to decline as the mature BCR is expressed (24, 33). Sustained presence of the IL-7R{alpha} chain could result in maintenance of IL-7 responsiveness and lead to the prolonged survival of B cell precursors observed in CD45–/– populations in vitro. Alternatively, responsiveness to IL-7 may be controlled directly by signaling induced by the Ag receptor complex. One published report found that BCR cross-linking resulted in an inability to activate the IL-7R signal cascade, despite continued expression of the IL-7R{alpha} (34). It remains to be tested whether a signal initiated by the pre-BCR leads to the reduced expression of, or responsiveness to the IL-7R{alpha} chain. Control of IL-7 signaling by the pre-BCR to limit expansion of cells at the pro-B to pre-B transition is logical, in that cells at this stage will be required to exit the cell cycle to undergo L chain rearrangement. If this is the case, our results suggest that CD45 comprises part of the pre-BCR complex in mediating this particular downstream target. Certainly, CD45 is an integral component of the mature Ag receptor complex in both B and T lineage cells (15).

The mechanism of activation of CD45 on B lineage cells has yet to be clearly established. Previous studies have identified multiple binding partners for CD45, including CD22 and galectin-1 (35, 36). Given that CD22 is first expressed at the pre-B cell stage, and galectin-1 can bind to an artificially solubilized form of the pre-BCR, it is conceivable that CD45 binding to either of these ligands participates in the regulation of the pro-B to pre-B transition (37, 38). Several recent studies have indicated that CD45 activity may be regulated by the formation of homo- and heterodimers of different CD45 isoforms (39, 40). Certainly, in T lineage cells, CD45 cross-linking can influence multiple signaling pathways, including the Ras/MAPK/ERK cascade, and may also modulate apoptotic death induced by galectin-1 binding (41, 42). In fact, our report that pro-B cells accumulate in vivo in CD45–/– BM parallels the previously identified disruption in T lymphopoiesis in these mutant mice. Specifically, pro-T cells accumulate at the CD4, CD8 double-negative to double-positive transition (23). However, a direct comparison between B and T cell precursors with respect to CD45 expression is complicated by the fact that T lineage cells express different CD45 isoforms at different developmental stages, whereas B cells express only the B220 isoform (12). Despite this distinction, the similarity in the stage and extent of the defect observed in CD45–/– pro-B and pro-T cells merits further evaluation. It should also be noted that Ogilvy et al. (43) demonstrated recently that complementation of CD45 exon 9–/– mice with specific RB and RO isoforms of CD45 was sufficient to rescue T lymphopoiesis, but not mature B cell differentiation. It remains to be tested whether the pro-B phenotype reported in this study would be rescued in Ogilvy’s transgenic mice.

In summary, we have presented the first evidence that early B lineage development is altered in vivo in the absence of CD45, in addition to the known defect observed in the periphery of CD45–/– mice. These findings are reminiscent of the requirement of thymocytes for efficient maturation at a parallel differentiation checkpoint. Our results demonstrate that developing B cell precursors accumulate in vivo at a stage before their selection by the pre-BCR, and that aberrant maintenance of activated JAK/STAT kinases may be responsible for the prolonged survival of IL-7-stimulated pro-B cells.


   Acknowledgments
 
We are grateful to S.-I. Nishikawa for providing the IL-7R{alpha} Ab, and to C. Cantin for his expert cell sorting efforts. We thank members of the Paige and Wu laboratories for their insights into this project.


   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 has been supported by funds granted by the Canadian Institutes for Health Research and the Terry Fox Foundation through National Cancer Institute of Canada. Back

2 Current address: Center for Regenerative Medicine and Technology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. Back

3 Address correspondence and reprint requests to Dr. Christopher J. Paige, Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Department of Immunology, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. E-mail address: paige{at}uhnres.utoronto.ca Back

4 Abbreviations used in this paper: BM, bone marrow; {gamma}c, common {gamma}-chain; wt, wild type. Back

Received for publication August 29, 2003. Accepted for publication June 17, 2004.


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