Altered splenic B cell subset development in mice lacking phosphoinositide 3-kinase p85{alpha}

Amber C. Donahue, Kristen L. Hess, Kwan L. Ng and David A. Fruman

Center for Immunology and Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA

Correspondence to: D. Fruman; E-mail: dfruman{at}uci.edu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The signaling enzyme phosphoinositide 3-kinase (PI3K) is activated following B cell receptor (BCR) engagement and by many other receptors on B lymphocytes. Mice lacking p85{alpha}, the predominant PI3K regulatory isoform, exhibit defects in B cell development and activation that are grossly similar to those found in mice lacking Bruton's tyrosine kinase (Btk) and other critical signaling molecules. However, a detailed analysis of splenic B cell subsets in p85{alpha}-deficient mice has not been reported. Here we show that these mice are deficient in four major B cell subsets: transitional-1, transitional-2, follicular and marginal zone. These defects are distinct from those observed in Xid mice that express a mutant Btk unable to interact with PI3K lipid products. Moreover, mice with both genetic lesions exhibit even greater impairment in B cell development. Finally, we show that transgenic expression of the anti-apoptotic protein Bcl-2 in p85{alpha}-deficient mice restores the transitional B cell subsets but not the marginal zone subset, and produces a follicular population with an aberrant phenotype. These findings establish a role for PI3K-p85{alpha} in differentiation of both follicular and marginal zone B cells, and suggest that these functions are required not solely for the propagation of anti-apoptotic signals.

Keywords: B lymphocytes, cellular differentiation, signal transduction, spleen, transgenic/knockout


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Developing B cells exit the bone marrow and migrate to the spleen as immature B cells (1). Further development occurs in the spleen, with cells subject to negative and positive selection during transitional stages before becoming mature follicular (FO) or marginal zone (MZ) B cells (2,3). Advances in phenotypic discrimination have allowed a greater understanding of the developmental relationships and functional properties of splenic B cell subsets. Transitional-1 and -2 (T1 and T2) cells represent sequential stages in immature B cell development that are phenotypically, anatomically and functionally distinct (4,5). Mature FO cells circulate throughout the secondary lymphoid organs, and proliferate in lymphoid follicles and germinal centers following recognition of antigen. MZ cells are also mature and can respond to antigen, but reside permanently in the marginal zone surrounding splenic follicles, where they may have specialized functions, including a role as the first line of defense against bacterial pathogens (3).

Signaling through the B cell receptor (BCR) is essential for many steps in B cell development and for the activation of mature cells by a specific antigen. Optimal BCR signaling requires the concerted action of a diverse array of kinases, phosphatases, small GTPases, exchange factors and adaptor molecules. Together this assembly of signaling components has been termed the BCR signalosome [reviewed in (69)]. The signalosome components must interact at the membrane, and cooperate to achieve maximal activation of phospholipase C-gamma (PLC{gamma}) and efficient triggering of calcium (Ca2+) mobilization and other downstream signaling pathways. Although some critical contributors to the signalosome are physically associated with the BCR and/or are integral membrane proteins (e.g. Ig{alpha}/Igß, CD19, Src family kinases), others are recruited from the cytoplasm by reversible protein–protein and protein–lipid interactions. PI3K is thought to play an integral role in membrane recruitment of signalosome components, in particular Btk and PLC{gamma}.

In both B and T cells, PI3K is required for proliferation and survival in response to most mitogens and enhanced PI3K pathway activity is associated with lymphoproliferation, autoimmunity and leukemia (10,11). A lipid kinase, PI3K phosphorylates phosphatidylinositol (PtdIns) and its derivatives (phosphoinositides) on the 3-hydroxyl of the inositol head group (12,13). PI3K enzymes belong to a multi-gene family and the catalytic isoforms have been divided into four subclasses. Only class IA and class IB PI3Ks can phosphorylate PtdIns(4,5)P2 to generate PtdIns(3,4,5)P3, a second messenger that binds to and recruits signaling proteins containing pleckstrin homology (PH) domains (12,13). Class IA PI3K exists as a heterodimer of one of five regulatory isoforms (p85{alpha}, p55{alpha}, p50{alpha}, p85ß and p55{gamma}) bound stably to one of three catalytic isoforms (p110{alpha}, p110ß and p110{delta}) (12,13). The regulatory isoforms do not possess enzyme activity but are crucial for targeting the heterodimer to signaling complexes at cellular membranes and for activation of the catalytic subunits. Class IA PI3K members are activated downstream of the BCR via association of regulatory subunits with CD19 and various adaptor proteins (9).

Targeted inactivation of the mouse genes for either p85{alpha} or p110{delta} yields similar B cell phenotypes, including reduced numbers of mature B cells and failure to proliferate following BCR crosslinking (1418). These phenotypes show overall similarity to defects observed in mice lacking Btk or various other signalosome proteins; indeed, one criterion for identification of signalosome components has been the demonstration of developmental defects in genetically deficient mice (6,9,19). However, detailed analysis of subset development has revealed separable functions of some of these components. For example, MZ B cell development requires a subset of signaling proteins including CD19 and p110{delta}, but is not impaired in Xid mice lacking functional Btk (4,16,18,20). These findings illustrate the complexities in signaling networks that control subset development. Here we present the first detailed study of B cell subset development in p85{alpha}–/– mice, and compare these patterns to Xid mice. In addition, we determine which aspects of PI3K function in B cell development can be complemented by expression of the anti-apoptotic protein Bcl-2. Our results define a role for PI3K-p85{alpha} in development of all major splenic B cell subsets, with only T1 and T2 fully restored by Bcl-2.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Balb/c mice purchased from The Jackson Laboratory (Bar Harbor, ME), Balb.Xid mice (provided by O. Witte, UCLA, Los Angeles, CA) and Eµ-Bcl-2 transgenic mice (provided by S. Korsmeyer, Dana-Farber Cancer Institute, Boston, MA) were bred in our colony. p85{alpha}–/– mice in the Balb/c genetic background were purchased from Taconic (Germantown, NY), and bred with Balb.Xid and Bcl-2 transgenic mice under a research crossbreeding agreement to generate the new strains p85{alpha}–/–/Xid and p85{alpha}–/–/Bcl-2 Tg. p85{alpha}–/–/Xid were in the Balb/c background and p85{alpha}–/–/Bcl-2 Tg mice in a mixed genetic background containing C57Bl/6 and Balb/c. CBA/CaJ and CBA/CaN mice and CD19–/– mice (21) were purchased from Jackson. Animal use was approved by the institutional animal care and use committee.

Flow cytometric analysis of developmental subsets
Single cell suspensions were obtained from the spleens of 6–10-week-old mice and depleted of RBCs by hypotonic lysis. Splenocytes were stained with combinations of anti-CD21, anti-CD24 and anti-CD23; or anti-IgD, anti-CD24, anti-IgM and anti-CD21 (anti-CD24 antibody from eBioscience, San Diego, CA; all others including streptavidin from BD Biosciences, Mountain View, CA). Purified rat anti-mouse CD21 monoclonal antibody (BD Biosciences) was conjugated to Alexa Fluor 647 using a fluorophore-conjugation kit (Molecular Probes, Eugene, OR) and Micro Bio-Spin 30 chromatography columns (Bio-Rad Laboratories, Hercules, CA). Data from at least 30 000 total events were acquired and analyzed (FACSCalibur and CellQuest software, BD Biosciences; FlowJo software, TreeStar, San Carlos, CA).

Immunohistochemistry
Mouse spleens were harvested, embedded in OCT medium (Sakura, Torrance, CA) and frozen over dry ice. Eight-micrometer sections were cut and mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Slides were cleared with CitriSolv (Fisher), fixed with 100, 95 and 75% ethanol, and blocked with 10% goat serum (Vector Laboratories, Burlingame, CA) in PBS for 30 min at room temperature (25°C). Sections were stained serially with MOMA-1 (Serotec, Oxford, UK), goat anti-rat IgG–Alexa 488 (Molecular Probes), anti-B220–biotin (BD Biosciences) and streptavidin–Cy3 (Zymed Laboratories, San Francisco, CA), each diluted in PBS, for 1 h at room temperature. Slides were washed with PBS. Coverslips were mounted with Gelmount (Biomeda, Foster City, CA). All images shown were acquired at 10x magnification using a LSM 510 Meta microscope (Zeiss, Thornwood, NY).

Proliferation assays
B cells were purified as described previously (14) via negative magnetic selection using CD43-specific beads (Miltenyi Biotec, Auburn, CA). B cell purity was determined to be >95% by FACS analysis (FACSCalibur and CellQuest software, BD Biosciences; FlowJo software) using anti-B220 and anti-Thy1.2 antibodies (BD Biosciences). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-ME. Purified B cells (5 x 104/well) were stimulated in triplicate in 100 µl of total volume in 96-well flat bottom dishes, using mitogens at the following final concentrations: goat anti-mouse IgM [F(ab')2, 10 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA], LPS (serotype 0127:B8, 10 µg/ml; Sigma-Aldrich, St Louis, MO), hamster anti-mouse CD40 (1 µg/ml; BD Biosciences) and recombinant murine IL-4 (2 ng/ml; Endogen, Woburn, MA). Cells were pulsed from 48–64 h with [3H]thymidine (1 µCi in 50 µl of medium/well), harvested (MachII-96; TomTec, Hamden, CT) onto filters and counted with a BetaPlate scintillation counter (Wallac, Gaithersburg, MD).

Calcium flux assays
Single cell suspensions were obtained from the spleens of 6–10-week-old mice and depleted of RBCs by hypotonic lysis. Splenocytes were stained with anti-B220–FITC (BD Biosciences) in 5% FCS in PBS, then resuspended to <107/ml in sterile Ca2+ buffer (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES and 0.1% BSA, pH 7.5) plus 1 µM indo-1 AM and 0.04% Pluronic (Molecular Probes). Cells were incubated for 45 min to 1 h at 37°C, washed with Ca2+ buffer and resuspended to 2.5 x 106/ml in Ca2+ buffer. Cells were warmed for 15 min in a 37°C water bath before data acquisition on a MoFlo flow cytometer (Cytomation, Fort Collins, CO). Cells were acquired for 1 min to establish a baseline of intracellular Ca2+ levels, and then stimulated with 20 µg/ml of anti-IgM F(ab')2 followed by further data collection for 12 min. The ratio of Ca2+-bound to Ca2+-free indo-1, which fluoresce at different wavelengths, was determined for live, B220+ cells.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
p85{alpha}-deficient and Xid mice exhibit different ratios and numbers of splenic B cell subsets
Btk is a Tec family tyrosine kinase with a PH domain that binds with high affinity to PtdIns(3,4,5)P3. A point mutation that abrogates selective binding to PtdIns(3,4,5)P3 is the genetic lesion in Xid mice. Several other lines of evidence are consistent with the model that Btk is a critical PI3K effector in B cells, including the finding that mice lacking p85{alpha} or p110{delta} exhibit Xid-like defects in B cell development and function (6,9). However, this genetic correlation is based on relatively superficial analyses of B cell development and function, and other data suggest that PI3K and Btk have separable functions (22,23). To determine whether PI3K and Btk have distinct roles in development, we compared splenic B cell subsets in p85{alpha}-deficient and Xid mice. Previous studies have shown that mice lacking p85{alpha} have a reduction in both total splenic B220+/IgM+ cells and the B220+/IgMlo/IgDhi mature recirculating population (15,23). We reported a similar B cell phenotype when we targeted the gene encoding p85{alpha} in a way that also eliminated expression of the alternative splice products p55{alpha} and p50{alpha} (14). Those experiments were carried out primarily using chimeric mice generated from RAG2-deficient blastocysts, because germline inheritance of a homozygous p85{alpha}/p55{alpha}/p50{alpha} mutation causes perinatal lethality (24). In the present work we analyzed in more detail the splenic B cell deficits in mice lacking only p85{alpha}, because this strain is viable and fertile and interpretation of developmental defects is not potentially confounded by variable chimerism.

FACS analysis is commonly used to distinguish T1, T2, FO and MZ cells based upon different patterns of surface expression of CD21, CD24 (also known as heat-stable antigen; HSA) and CD23 (5). In a plot of CD21 vs CD24 (Fig. 1A), the T1 subset is identified by expression of low levels of CD21 (CD21lo) and high levels of CD24 (CD24hi). FO cells express intermediate levels of both CD21 and CD24 (CD21int/CD24int). Cells with high expression of both CD21 and CD24 include both the T2 and MZ subsets (CD21hi/CD24hi). The developmental marker CD23 allows a further distinction to be made between MZ cells, which express lower levels of CD23 (CD23lo), and T2 cells, which express high levels of the marker (CD23hi) (Fig. 1B) (5). An alternate method of determining MZ numbers, that of comparing CD21 and CD23 expression (25), yielded percentages similar to those shown in the analyses below, and provided a guide for drawing the histogram gates shown.



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Fig. 1. p85{alpha}–/– spleens exhibit broader B cell defects than Xid, and p85{alpha}–/–/Xid defects are different than either single lesion alone. Splenocytes were harvested from mice of each genotype and stained with CD21/CD24/CD23 (A and B) or IgD/IgM (C), and live cells were analyzed by flow cytometry. (A) CD21 vs CD24. FO (CD21int/CD24int), T1 (CD21lo/CD24hi) and T2+MZ (CD21hi/CD24hi) gates were drawn according to previous reports (5). The population of CD21lo/CD24lo cells that accumulates in p85{alpha}–/– spleens is mostly non-B cells (B220/IgM/IgD) and was not investigated further (data not shown). (B) expression of CD23 on cells in the T2+MZ gate (A, top left panel) allows for differentiation between T2 (CD23hi) and MZ (CD23lo) cells. Comparison of CD21 vs CD23 expression provided a guide for the T2 and MZ gates for each genotype (not shown). (C) Comparison of IgD vs IgM differentiated the most mature subset (IgDhi/IgMlo) from the intermediate (IgDhi/IgMhi) and least mature (IgDlo/IgMhi) populations in the spleen (the latter also containing MZ B cells). Representative dot plots and histograms are shown for each genotype; Xid (Balb.Xid). See Table 1 for numerical summary of replicate data.

 

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Table 1. Splenic B cell subsetsa

 
In parallel to CD21/CD24/CD23 stains, we examined the surface expression of IgD and IgM (Fig. 1C). Immature B cells that enter the spleen express high levels of IgM and very little IgD (IgDlo/IgMhi), and progress to expression of high levels of both isotypes (IgDhi/IgMhi). Mature recirculating B cells downregulate IgM and are often described as the IgDhi/IgMlo population. Mature MZ B cells retain high levels of IgM and downregulate IgD, and in this analysis are therefore present in the same quadrant as the most immature cells. It has been reported that the IgDhi/IgMlo population is equivalent to the FO B cell subset identified as CD21int/CD24int (5). However, in four-color staining experiments we found that the CD21int/CD24int FO subset included cells in both the IgDhi/IgMlo and IgDhi/IgMhi quadrants (Fig. 2).



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Fig. 2. p85{alpha}–/– and Xid ‘follicular’ B cells express higher levels of surface IgM than Balb/c, and p85{alpha}–/–/Xid express higher levels than FO B cells containing either single lesion alone. Splenocytes were harvested from mice of each genotype and stained with CD21/CD24/IgM/IgD, and live cells were analyzed by four-color flow cytometry. FO, T1 and T2+MZ gates were drawn for live cells as in Fig. 1 (left panel). The IgM/IgD profile of cells within the FO gate are shown (right panel). Numbers (upper right corner, right panel) represent the mean fluorescence intensity (MFI) of IgM ± SD, for IgD+ cells in the FO gate. Numbers of mice per group: Balb/c n = 5; Xid (Balb.Xid) n = 5; p85{alpha}–/– n = 3; p85{alpha}–/–/Xid n = 3.

 
Figure 1(A) shows representative dot plots comparing levels of CD21 and CD24, and the gates used for analysis (T1, T2+MZ, FO), for each of the strains analyzed in this study. The percentages and total numbers of subsets in replicate mice were averaged and these data are presented in Table 1. Initially we compared mice in the Balb/c background that were either wild-type (WT), Xid or p85{alpha}–/–. The total number of spleen cells was reduced in both p85{alpha}–/– and Xid mice, but this difference was only statistically significant in Xid. Both Xid and p85{alpha}–/– mice had reduced percentages and numbers of FO cells compared to WT, consistent with impaired maturation into the recirculating B cell pool. p85{alpha}–/– mice, similar to mice lacking the PI3K catalytic p110{delta} isoform (16,18), had significantly fewer MZ B cells than either WT or Xid (Fig. 1B and Table 1). In contrast, the percentages and numbers of MZ B cells in Xid mice were not reduced compared to WT, as reported previously (4). p85{alpha}–/– spleens also showed statistically significant decreases in the percentage of cells in the T1 and T2 subsets when compared to both WT and Xid, whereas Xid mice showed no such defects. Note that the unusual population of CD21lo/CD24lo cells that accumulates in p85{alpha}–/– spleens is mostly non-B cells (B220/IgM/IgD) and was not investigated further (data not shown). Thus, deletion of p85{alpha} suppresses development of all splenic B cell subsets whereas the Xid mutation selectively affects the appearance of the mature follicular subset. Consistent with previous work, we noted a significant increase in the percentage (but not numbers) of T2 cells in Xid spleens compared to WT (Table 1), which along with the decrease in FO cells establishes a developmental block in Xid mice at the T2 stage (4,5).

The broader splenic B cells defects in p85{alpha}–/– mice were supported by analysis of IgM and IgD levels (Fig. 1C and Table 1). Levels of the mature, recirculating cells (IgDhi/IgMlo) were significantly decreased in the Xid mice, whereas both IgDhi/IgMhi and IgDlo/IgMhi cells were significantly increased. Similar to Xid, p85{alpha}–/– mice had a significant reduction in the levels of IgDhi/IgMlo cells compared to WT. In contrast to Xid, p85{alpha}–/– mice also showed diminished percentages and numbers of the IgDhi/IgMhi and IgDlo/IgMhi subsets compared to WT. The decrease in IgDlo/IgMhi cells (predominantly T1 and MZ) was significant compared to WT (Table 1), and there was a significant reduction in both IgDhi/IgMhi and IgDlo/IgMhi subsets in p85{alpha}–/– compared to Xid.

Taking into consideration the possibility that some of the differences we observed might be strain-specific, we examined B cell developmental subsets in the spleens of the mice in which the Xid mutation was originally described, CBA/CaN, and in their WT counterparts, CBA/CaJ. Representative surface marker plots are shown for both genotypes in Figure 1, and the replicate percentages and numbers of cells are listed in Table 1. As with the Xid vs WT comparison in the Balb/c background, CBA/CaN spleens showed significantly decreased numbers of recirculating follicular cells as judged by either the CD21int/CD24int or IgDhi/IgMlo phenotypes. Similarly, CBA/CaN mice showed increases in the percentages of IgDhi/IgMhi and IgDlo/IgMhi subsets. Although there was a significant reduction in the absolute numbers of MZ B cells in CBA/CaN compared to CBA/CaJ, the percentages were essentially the same; the lower numbers may be a result of smaller spleen cellularity overall. These data are in agreement with another study that compared splenic subsets in CBA/CaJ and CBA/CaN mice (4). In summary, the Xid mutation was associated with similar defects in two strain backgrounds, suggesting that the less severe defects compared to p85{alpha}–/– was not due to Balb/c-specific compensation for lost Btk function.

Notably, when measuring absolute levels of surface marker expression using mean fluorescence intensity (MFI), we observed differences in the genetically altered mice as compared with Balb/c. These differences yielded noticeably shifted, though still recognizable, population distributions. Particularly evident were the differences in CD23 expression from one strain to another; T2 cells from Xid mice expressed considerably higher levels of CD23 than did T2 cells from WT (Fig. 1). We also noted that when cells were stained with the combination of antibodies to CD21, CD24, IgM and IgD, different strains displayed different surface expression of IgM among cells in the FO gate (CD21int/CD24int). As reported previously (4,26), FO cells in Xid mice had higher expression of IgM than WT (Fig. 2). We also observed a significant increase in IgM expression on FO cells from p85{alpha}–/– mice compared to WT (Fig. 2), although the difference was less pronounced than in Xid. These findings emphasize that cells identified as the FO subset based on CD21 vs CD24 plots may be developmentally skewed in mice lacking critical signaling proteins, which could partly explain reduced functional responses.

Immunohistochemical confirmation of MZ B cell defects
As a means of verifying MZ defects suggested by FACS data, we examined splenic architecture in situ by staining sections with antibodies directed against B cells (B220+) and against the metallophilic macrophages (MOMA-1+) that mark the spatial division between FO and MZ cells in the spleen (Fig. 3). p85{alpha}–/– mice showed a clear decrease in the population of MZ B cells surrounding the MOMA-1+ macrophages, whereas there was little or no visible difference between WT and Xid in the Balb/c or CBA/Ca backgrounds (Fig. 3). As a control, we stained sections from a CD19–/– mouse, which has been shown previously to lack MZ cells (20).



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Fig. 3. Immunofluorescent staining demonstrates MZ defect in p85{alpha}–/– and p85{alpha}–/–/Xid spleens. Frozen spleen sections were stained for MOMA-1+ (green) marginal zone macrophages and B220+ (red) FO and MZ B cells. The ring of MZ B cells (red) surrounding the splenic follicle is indicated by an arrow.

 
Mice deficient for p85{alpha} and carrying the Xid mutation exhibit more severe developmental defects than mice with either genetic lesion alone
If PI3K and Btk have independent functions in B cell signaling, the phenotype of double mutant mice should be more profound than that observed in mice with either mutation alone. To test this possibility, p85{alpha}–/– and Xid mice in the Balb/c background were bred to create p85{alpha}–/–/Xid mice. Splenocytes from the resulting double mutant animals were stained for surface developmental markers. Representative dot plots and histograms of the p85{alpha}–/–/Xid mice are shown in Figure 1, and the percentages and absolute numbers in Table 1. Double mutant mice had B cell developmental defects that were more severe in some aspects than in mice with either genetic lesion alone. The percentages and numbers of FO and T2 subsets in p85{alpha}–/–/Xid spleens decreased when compared to either single lesion (Table 1). Likewise, p85{alpha}–/–/Xid mice showed a significant decrease in the percentage of IgDhi/IgMlo cells compared to both p85{alpha}–/– and Xid. Of note, the cells found in the FO subset (CD21int/CD24int) of p85{alpha}–/–/Xid spleens had even greater skewing towards high IgM expression than either p85{alpha}–/– or Xid alone (Fig. 2).

Other aspects of the developmental phenotype of double mutant mice were similar to p85{alpha}–/– or intermediate between p85{alpha}–/– and Xid. p85{alpha}–/–/Xid mice retained a defect in the MZ subset similar to that seen in p85{alpha}–/– mice (Table 1), and staining of spleen sections of p85{alpha}–/–/Xid mice revealed a pattern resembling that of p85{alpha}–/– mice with respect to the striking paucity of MZ B cells (Fig. 3). Unexpectedly, the percentages and numbers of T1 cells, and the IgDlo/IgMhi population that contains T1 cells and MZ cells, were greater in p85{alpha}–/–/Xid spleens compared to p85{alpha}–/– and were not significantly different than WT. However, these populations were still less abundant than in Xid (Table 1). Further analysis will be required to determine the mechanism by which T1 cell numbers are restored in double mutant mice.

Expression of a Bcl-2 transgene in p85{alpha}-deficient mice increases B cell numbers but does not restore the MZ subset, and produces an aberrant FO population
Impaired development of PI3K-deficient B cells could be the result of increased susceptibility to apoptosis, and/or blockade of specific differentiation signals. To determine the extent to which enhanced survival signaling could restore B cell development and function in the absence of p85{alpha}, we introduced a Bcl-2 transgene into p85{alpha}–/– mice. Bcl-2 is the prototypical member of a family of anti-apoptotic proteins; in wild-type mice, transgenic expression of Bcl-2 augments the mature B cell pool and renders B cells resistant to spontaneous apoptosis (27,28). In Xid mice, expression of Bcl-2 in the B lineage restores aspects of development but does not rescue function (28).

We compared splenic B cell subset development in the absence and presence of a Bcl-2 transgene, in WT and p85{alpha}–/– mice. Representative FACS plots are shown in Fig. 4; quantitation of subset data is presented in Table 2. Bcl-2 expression led to changes in the developmental phenotypes of both WT and p85{alpha}–/– B cells. Splenocyte numbers were augmented in both WT and p85{alpha}–/– mice, though the increase was significant only for WT (Table 2). A shared effect was an increase in the percentages and numbers of FO B cells, compared to non-transgenic age-matched controls in the mixed background. However, the increase in FO cell number in p85{alpha}–/–/Bcl-2 mice was of less magnitude (~2-fold) than in WT mice ± the Bcl-2 transgene (~3-fold) (Table 2). Furthermore, although the Bcl-2 transgene increased numbers of IgDhi/IgMlo cells in both WT and p85{alpha}–/– mice, the percentage of cells with this phenotype remained significantly lower in p85{alpha}–/–/Bcl-2 mice compared to WT. Bcl-2 overexpression caused a marked increase in the IgDhi/IgMhi numbers in p85{alpha}–/– mice, but this was associated with only a small and not statistically significant increase in the T2 subset. These findings suggest that much of the increase in FO-like cells in p85{alpha}–/–/Bcl-2 mice is likely due to an accumulation of IgDhi/IgMhi cells that have downregulated CD21 and CD24. Consistent with this, we observed an increase in MFI of IgM expression on FO cells of p85{alpha}–/–/Bcl-2 mice relative to non-transgenic p85{alpha}–/– (21 ± 3 vs 15 ± 0.2, n = 2). The MFI of IgM on FO cells of WT and WT/Bcl-2 mice (7 ± 3, n = 2 and 10 ± 3, n = 2, respectively) were comparable to Balb/c (Fig. 2).



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Fig. 4. Expression of a Bcl-2 transgene restores some aspects of B cell development in p85{alpha}–/– spleens. Splenocytes were harvested from mice of each genotype and stained with CD21/CD24/CD23 (A and B) or IgD/IgM (C), and live cells were analyzed by flow cytometry as in Fig. 1. (A) CD21 vs CD24. (B) Expression of CD23 on cells in the T2+MZ gate. (C) IgD vs IgM. Representative dot plots and histograms are shown for each genotype.

 

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Table 2. Splenic B cell subsets in Bcl-2 transgenic micea

 
Expression of the Bcl-2 transgene did not restore MZ B cell development. In fact, the percentages of MZ cells in p85{alpha}–/–/Bcl-2 mice were actually lower than in non-transgenic p85{alpha}–/– mice (Fig. 4B; Table 2). The Bcl-2 transgene also did not restore development of the peritoneal B1 mature B cell subset (B220+/IgM+/CD5+), previously shown to be greatly diminished in mice lacking p85{alpha} (14,15) (Table 3). We conclude that Bcl-2 expression in p85{alpha}–/– mice increases the number of B220+ cells but does not rescue the MZ or B1 defects; moreover, the restored population of cells with a FO phenotype (CD21int/CD24int) retains high levels of IgM expression. Thus, the p85{alpha} isoform of PI3K appears to regulate B cell development both at the level of survival and by transducing specific differentiation signals. Of note, expression of Bcl-2 in WT mice favored the development of cells with a FO phenotype over MZ, with percentages and numbers of MZ B cells significantly reduced in WT/Bcl-2 mice (Table 2). However, there was no demonstrable loss of B220+ cells in the marginal zone of spleens from WT/Bcl-2 mice (data not shown), suggesting that some of the increased FO population in these mice might be mislocalized.


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Table 3. Expression of Bcl-2 does not restore the peritoneal B1 subset of p85{alpha}–/– micea

 
Expression of a Bcl-2 transgene in p85{alpha}-deficient mice does not restore B cell proliferation or signaling
We next determined whether the partial restoration of FO B cells in the p85{alpha}–/–/Bcl-2 mice was accompanied by increased B cell function, as assessed in a standard proliferation assay. Purified splenic B cells from WT, WT/Bcl-2, p85{alpha}–/– and p85{alpha}–/–/Bcl-2 mice were stimulated with various mitogens (Fig. 5A). As has been shown in Xid cells carrying the Bcl-2 transgene (28), proliferation in p85{alpha}–/– B cells in response to anti-IgM was not restored by expression of Bcl-2. In addition, the response of these cells to LPS was weak and comparable to that of non-transgenic p85{alpha}–/– cells. The response to anti-IgM plus IL-4 was partially restored in p85{alpha}–/–/Bcl-2 cells. All mutant and transgenic cells responded to stimulation with anti-CD40 plus IL-4, a combination that induces B cell proliferation in a PI3K-independent manner (14,29). Thus, loss of PI3K function impairs B cell activation in ways that cannot be overcome simply by overexpressing the pro-survival protein Bcl-2.



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Fig. 5. Expression of the Bcl-2 transgene is unable to restore proliferation or sustained Ca2+ flux in p85{alpha}–/– B cells. (A) Purified B cells were activated in triplicate with the indicated mitogens or media alone, and proliferation was measured by thymidine incorporation from 48–64 h. The experiment shown included B cell samples from three independent p85{alpha}–/–/Bcl-2 mice (gray bars); similar results were observed in three additional experiments. (B) Ca2+ mobilization was analyzed by FACS in B cells from WT and p85{alpha}–/– mice with and without the Bcl-2 transgene. Shown is one representative experiment of two.

 
Sustained Ca mobilization is a hallmark response to BCR crosslinking and is essential for productive B cell activation. We have previously shown that p85{alpha}-deficient B cells are defective in the sustained phase of the Ca2+ mobilization response following anti-IgM treatment (30), as observed in B cells derived from mice lacking other signalosome components including Btk and p110{delta} (1618,31). To identify a potential molecular explanation for the inability of Bcl-2 to restore proliferation in p85{alpha}-deficient B cells, we assessed whether expression of Bcl-2 affected BCR-mediated Ca2+ mobilization. p85{alpha}–/–/Bcl-2 B cells, despite a clear increase in the amplitude of the initial peak relative to non-transgenic counterparts, were also unable to maintain a sustained phase (Fig. 5B). The increased peak response in the p85{alpha}–/–/Bcl-2 B cells may be attributable to the abundance of relatively immature cells, which express higher levels of surface IgM and have been reported to produce greater Ca2+ responses than mature cells (32). The failure to maintain sustained increases in intracellular Ca2+ shows that p85{alpha}-deficient B cells have a cell-autonomous defect in early signaling that is not corrected by Bcl-2 expression.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have demonstrated that mice lacking the p85{alpha} regulatory subunit of PI3K exhibit impaired development of both major mature B cell subsets in the spleen (FO and MZ). In contrast, in the same genetic background, the Xid mutation in Btk causes a decrease only in the FO population. Compared to Xid, p85{alpha}-deficient mice also exhibit significant decreases in the immature T1 and T2 subsets. Mice that both lack p85{alpha} and carry the Xid mutation (p85{alpha}–/–/Xid) show an additive effect of the two mutations, in that the FO and T2 defects are more severe than in either single lesion alone. The defect in the MZ subset resembles that seen in p85{alpha}-deficient mice. Our findings are consistent with a recent analysis of double mutant p85{alpha}–/–/Btk–/– mice that showed more severe loss of mature B cells compared to single mutant mice; however, detailed comparison of splenic B cell subsets of p85{alpha}–/–, Btk–/– and p85{alpha}–/–/Btk–/– mice was not presented in that report (23).

The striking differences in phenotype between p85{alpha}–/– and Xid mice, and the partially additive effects seen in PI3K-Btk double mutant mice analyzed here and elsewhere (23), suggest that PI3K and Btk have both shared and distinct roles in signaling pathways that control B cell development. It is perhaps not surprising that PI3K mutant mice have more severe defects than Btk mutant mice, since Btk is just one of multiple downstream PI3K effectors recruited to the membrane by PtdIns(3,4,5)P3. PI3K products can promote PLC{gamma} activation and subsequent Ca2+ mobilization not only by recruiting Btk but also by recruiting PLC{gamma} via PH and/or SH2 domains and directly enhancing PLC{gamma} activity (9). Other PI3K effectors, such as Akt (23) and Bam32 (33,34), may have additional non-redundant roles in B cells.

Both PI3K and Btk are activated not only by BCR engagement but also by many other receptors that impinge upon B cell development and activation (9). The role of PI3K in Btk activation downstream of distinct receptors has not been fully investigated, and it has been suggested that Btk can be activated by G protein-coupled receptors via direct interactions with heterotrimeric G proteins (35). An involvement of Btk but not PI3K downstream of receptors distinct from the BCR could explain the augmented defects in double mutant p85{alpha}–/–/Xid analyzed here and the p85{alpha}–/–/Btk–/– mice reported elsewhere (23). Even in studies of BCR signaling, evidence has accumulated that aspects of Btk function are PI3K-independent. We found a small number of genes whose expression in BCR-stimulated B cells requires the function of Btk but not PI3K (22). Although PtdIns(3,4,5)P3 enhances Btk activity in vitro (36), it appears that BCR-mediated Btk kinase activation in cells is independent of PI3K and its product (23). What then is the role of the Btk PH domain interaction with PtdIns(3,4,5)P3 in BCR signaling? Delivery of Btk-associated PtdIns(4)P-5-kinase (PIP5K) to the membrane appears to be dependent on binding of the Btk PH domain to PtdIns(3,4,5)P3 (37). Targeting of a molecular complex containing Btk, PIP5K, PLC{gamma} and the adapter BLNK to membrane sites where PI3K is active may therefore help maintain local levels of the PtdIns(4,5)P2, the substrate for both PLC{gamma} and PI3K (37,38).

The greater developmental defects in double mutant mice studied could also be explained by the fact that partial loss of function in different components of a shared signaling pathway can cause additive defects (39). Indeed, the individual lesions probably do not result in complete loss of function of PI3K or Tec family kinases. The Tec tyrosine kinase is expressed in B cells and mice lacking both Btk and Tec have considerably more severe defects in B cell development compared to single mutant mice (40). Likewise, p85{alpha} is not the only class IA PI3K regulatory isoform expressed in B cells, and others may partially compensate for its absence. Consistent with this idea, we have found that some of the signaling defects in p85{alpha}–/– B cells are less severe than in WT cells treated with global inhibitors of PI3K catalytic function (30).

Impaired PI3K signaling sensitizes many cell types to apoptosis, including murine B cells (14). If the developmental defects in p85{alpha}-deficient B cells were simply the result of increased apoptosis, overexpression of a survival factor might be expected to compensate. However, we have shown that while expression of a Bcl-2 transgene in p85{alpha}-deficient cells (p85{alpha}–/–/Bcl-2) increases the number of splenic B cells, Bcl-2 does not restore the MZ or B1 subsets or fully reconstitute the FO subset. There is a significant increase in CD21int/CD24int cells and IgDhi/IgMhi cells in p85{alpha}–/–/Bcl-2, but the percentage of cells with a classical recirculating phenotype (IgDhi/IgMlo) is not fully restored. In contrast, the Bcl-2 transgene in WT mice augments the recirculating pool as judged by both the CD21int/CD24int and IgDhi/IgMlo populations. Thus, aspects of the developmental defect cannot be corrected by expression of a survival protein. These data, and the noted increase in mean IgM expression in the CD21int/CD24int populations from different mutant strains, also suggest that downregulation of CD21 and CD24 occurs before downregulation of IgM, and that cells in the FO gate based on CD21/CD24 staining include significant numbers of immature B cells, especially in mutant mice. Use of the AA4.1/CD93 marker has allowed the discrimination of a third immature transitional population (T3) that is partly present within the classical FO gate (41,42); further analysis using this marker will be necessary to determine if T3 cells accumulate in p85{alpha}–/– and p85{alpha}–/–/Bcl-2 mice.

It is possible that p85{alpha}–/– mice have greater developmental defects than Xid mice because p85{alpha} is more widely expressed, including in stromal cells and other cell types that influence B cell development. However, our finding that overexpressing Bcl-2 selectively in B cells restores numbers of transitional B cells supports the idea that diminished accumulation of these cells is the result of a cell-autonomous defect in survival signaling. While it is possible that the impairment in MZ B cell development is not cell-autonomous, a reduced MZ compartment is also observed in mice lacking CD19 (20,43) (Fig. 3), a B cell-specific protein whose signaling function depends on association with class IA PI3K regulatory isoforms (44).

Bcl-2 does not fully restore mature B cell function in the absence of p85{alpha}, as the resulting cells still display a proliferative defect in response to anti-IgM or LPS stimulation. At the molecular level, the proliferation defect in response to anti-IgM could be explained by our finding that the cells cannot maintain an elevated level of intracellular Ca2+. The continued impairment in LPS response could reflect the relative absence of MZ B cells that are especially responsive to this mitogen.

Previous work has demonstrated that transgenic expression of Bcl-XL, a Bcl-2 family member, expands the peripheral IgMlo B cell pool in both Xid mice and p85{alpha}–/– mice (23,26). Detailed analysis of splenic B cell subsets was not reported in the Bcl-XL transgene experiments, so it is difficult to compare the effects of Bcl-2 and Bcl-XL on B cell development in signaling-deficient mice. Expression of Bcl-XL in p85{alpha}–/– cells restored the proliferative response to anti-IgM plus IL-4, but the response to other mitogens was not reported (23). Direct comparison of the effects of Bcl-2 and Bcl-XL transgenes in signaling-deficient B cells will be required to determine if these survival proteins have differing abilities to compensate for developmental and functional defects.

It has become evident that B cells in the spleen are quite heterogeneous in signaling and functional responses to various stimuli (5,25,32,45,46). Furthermore, as we and others (4) have demonstrated, subsets defined by two or three surface markers can be phenotypically different when other markers are analyzed. These findings demonstrate an important caveat that should be kept in mind when studying B cell function in genetically altered mice. Namely, differences seen in bulk B220+ populations do not necessarily establish functional distinctions in mature B cells, but can also reflect differences in the maturity and/or surface receptor expression of the cells analyzed. Ultimately, assays capable of distinguishing responses in different subsets, and correlation with receptor expression, will provide the most useful information when comparing B cell responses among mutant mice.


    Acknowledgements
 
We thank Stephen Hou for flow cytometric Ca2+ analyses, Travis Moore for help with immunocytochemistry, Pratibha Sareen, Cattleya Buranasombati, Jennifer Dinh and Travis Moore for mouse genotyping, Owen Witte and Stanley Korsmeyer for providing useful mouse strains, Randy Hardy for helpful discussions, and Craig Walsh and Anne Satterthwaite for critical reading of the manuscript. This work was supported by National Institutes of Health grant AI50831 to D.A.F.


    Abbreviations
 
BCR   B cell receptor
FO   follicular
MFI   mean fluorescence intensity
MZ   marginal zone
PI3K   phosphoinositide 3-kinase
PLC{gamma}   phospholipase C-gamma
PtdIns   phosphatidylinositol
T1   transitional-1 cells
T2   transitional-2 cells
Xid   X-linked immunodeficiency

    Notes
 
Transmitting editor: T. Kurosaki

Received 25 June 2004, accepted 28 September 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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