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The Journal of Immunology, 2003, 171: 5921-5930.
Copyright © 2003 by The American Association of Immunologists

CD19 Function in Early and Late B Cell Development. II. CD19 Facilitates the Pro-B/Pre-B Transition 1

Dennis C. Otero and Robert C. Rickert2

Division of Biology and University of California-San Diego Cancer Center, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19-/- mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19-/- and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19-/- mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton’s tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transition from the pro-B cell to the pre-B cell represents the first stage at which B cell receptor (BCR)3 selection directs B cell development. Expression of the pre-BCR is dependent upon previous developmental events that are driven by stromal cell interaction and IL-7 receptor signaling. Positive feedback from IL-7 induces up-regulation of the IL-7R {alpha}-chain (essential for the high affinity IL-7R) as well as TdT expression (1). Mice deficient in IL-7 reveal a block at the pro-B cell stage similar to blocks seen in recombinase-activating gene-/- and {lambda}5-/- mice (2), mutations that prevent the formation of a pre-B cell receptor (3). The onset of pre-BCR expression coincides with the loss of stromal cell dependency. In fact, it was shown that completion of the pre-B cell stage occurred in the absence of stromal cell support and IL-7, but required a functional pre-BCR (4). Nonetheless, IL-7 can act synergistically with the pre-BCR to promote proliferation in pre-B cell cultures in what is thought to be a mitogen-activated protein kinase (MAPK)-dependent process (5, 6). Thus, the expression of the pre-BCR at this stage is necessary for the developmental progression and proliferative expansion of B cell precursors that have functionally rearranged and expressed a H chain gene and is aided by the presence of IL-7.

The pre-BCR is comprised of a functional µ-chain and a surrogate L chain (Vpre-B and {lambda}5), and signals via the associated Ig{alpha} (CD79a)/Ig{beta} (CD79b) heterodimer (7, 8). Ig{alpha}/Ig{beta} recruit Src and Syk family kinases to propagate pre-BCR-mediated signals and drive differentiation into immature B cells. As evidence, mutations of any structural component (Ig{alpha}, Ig{beta} VpreB, {lambda}5, etc.) of the pre-BCR complex results in a developmental block at the pre-B cell stage (9, 10, 11, 12). Mice deficient for the tyrosine kinase Syk or the concomitant loss of Blk, Lyn, and Fyn also show a block at the pro- to pre-B cell transition due to defects in pre-BCR signaling (13, 14, 15). Thus, signaling through the pre-BCR is important for the transition from an IL-7-dependent pro-B cell to an IgM-expressing immature B cell.

BCR signaling in immature B cells can lead to negative selection or induced receptor editing (16, 17, 18, 19). Repertoire analysis suggests that positive selection may also occur at this stage (20). Immature B cells that leave the bone marrow (BM) express high levels of IgM and low levels of IgD on the surface, and migrate to the spleen to undergo further maturation (21). This maturation coincides with the loss of immature B cell markers, such as CD25, heat-stable Ag (HSA), and 493 Ag, and up-regulation of other markers, including IgD, CD21, and CD23 (22, 23, 24). These transitional B cells undergo further BCR-dependent selection in the spleen (25), which accounts for the high rate of turnover in this population (26). Thus, generation of the long-lived B cell pool involves both positive and negative selection that take place in BM as well as spleen. These selection steps are mediated by signals through the BCR and help to direct the formation of mature B cells capable of responding to Ag.

CD19 is a B cell-restricted signaling molecule known to transduce signals initiated through the BCR. CD19-/- mice show a decrease in peripheral B cell numbers, with some B cell subsets more affected than others (27, 28). Whether this defect is derived from an earlier impairment in B lymphopoiesis has not been investigated. Analysis of the early B cell compartment in these mice is made difficult due to the dynamic nature of B cell development and the possibility of CD19 impacting B cell development at multiple stages. It is early in ontogeny (neonate) that one sees the greatest difference in B cell numbers in the spleens of CD19-/- mice compared with wild-type (WT) littermates (29). In older mice, parameters change as B cell generation in BM diminishes and the maintenance of selected long-lived B cells becomes of key importance (30). Because of its early expression and the fact that it is involved in signaling through the BCR and pre-BCR (31), CD19 may contribute to the generation of mature B cells. Here we used BM reconstitution experiments and cell labeling studies as well as primary early B cell cultures to document a role for CD19 in early B cell development that is closely associated with the pro- to pre-B cell transition. Biochemical evidence is also provided showing that CD19 contributes significantly to the propagation of signals through the pre-BCR. We conclude that CD19 contributes to B cell generation at the early Ag-independent stages of B cell development in addition to important roles in the peripheral immune system.


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

CD19-/- mice on the BALB/c (IgHb; The Jackson Laboratory, Bar Harbor, ME) background (10-generation backcross) were maintained under pathogen-free conditions and were handled in accordance with the guidelines set forth by the Animal Subjects Program at University of California-San Diego. BALB/c (IgHb) congenic mice were used as WT controls, and BALB/c (IgHa) WT mice were used as recipients in transfers. CD19 transgenic (Y482/513F) mice maintained on the CD19null 129/Sv background were provided by R. H. Carter (University of Alabama, Birmingham, AL).

Adoptive transfer

Recipient mice (BALB/c/IgHa) were lethally irradiated (1000 rad) with a cesium source and administered antibiotics (1.0 mg/ml neomycin and 0.1 mg/ml polymixin-B; Life Technologies, Gaithersburg, MD) in the drinking water postreconstitution. BM cells from WT and CD19-/- mice were depleted of B cells using MiniMACS columns (Miltenyi Biotec, Auburn CA) and anti-B220-conjugated magnetic beads. BM cells (1 x 107 total cells) were injected into the lateral tail vein of irradiated mice. To generate chimeras, B cell-depleted BM from age-matched WT and CD19-/- mice were mixed at predetermined ratios before injection. Chimeric mice were analyzed 5 wk postirradiation. For autoreconstitution, WT BALB/c, CD19-/- mice were exposed to 500 rad of ionizing radiation and analyzed for B cell subpopulations by flow cytometry 12–14 days postirradiation.

Flow cytometry

After harvesting organs, single-cell suspensions were prepared, and RBC were lysed with ACK buffer (0.15 M NH4Cl, 1 mM KHCO2, and 0.1 mM Na2-EDTA, pH 7.4). Cells (1 x 106) were stained for 15 min on ice with diluted Ab, washed with PBS containing 1% FCS, and incubated with the streptavidin-conjugated flurochrome when necessary. Abs against the following surface markers were obtained from BD PharMingen (San Diego, CA): CD43-bio, CD24(HSA)-FITC, CD24(HSA)-bio, IgD-FITC, IgM-PE, IgM-bio, CD19-PE, B220-APC, B220-PE, 5-bromo-2'-deoxyuridine (BrdU)-FITC, and streptavidin-allophycocyanin. Anti-IgD bio was obtained from eBioscience (San Diego, CA), and anti-B220 TriC was purchased from Caltag (Burlingame, CA).

Cell cycle analysis

Cycling cells were identified from BM single-cell suspensions by flow cytometric analysis of G0-G1 vs S/G2-M peaks after propidium iodide (PI) staining in 1 mM Tris (pH 8), 0.1% Triton, 0.1% sodium citrate, 0.1 mM EDTA, and 50 µg/ml PI. For BrdU incorporation, mice were injected with 1.0 mg/ml of BrdU every 12 h, and BM cells were analyzed at 24 and 48 h. Labeled cells were fixed with 1% paraformaldehyde in PBS, permeabilized with 0.5% Tween in PBS for 15 min at 37°C, treated with DNase (50 U/ml), and stained with anti-BrdU-FITC.

Generation of IL-7-dependent pre-B cell cultures and Western blotting

BM cells from WT and CD19-/- mice were cultured in the presence of IL-7 (1/100 dilution of supernatant from a J558 line, gift from B. Kee, University of Chicago, Chicago, IL) for at least 5 days, at which time nonadherent cells were >95% B220 positive. Cultures were serum-starved and IL-7-starved for 3 h before stimulation with 20 µg/ml of biotinylated anti-Ig{beta} (HM79; BD PharMingen), followed by cross-linking with 30 µg/ml avidin (Sigma-Aldrich, St. Louis, MO); lysates from 2 x 106 cells were loaded per lane. Membranes were probed for phospho-specific extracellular signal-regulated kinase (ERK; P-p44/42) and/or phospho-specific Btk (P-223) and were reprobed for total ERK (p44/42) and/or total Btk (Cell Signaling, Beverly, MA) and developed with Western Lightning (PerkinElmer, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Early B cell development in CD19-/- mice

To determine whether reduced B cell generation contributes to the diminished representation of mature recirculating B cells in CD19-/- mice, we performed a thorough examination of the BM B cell compartment in CD19-/- mice backcrossed to BALB/cIghb (F10). Flow cytometric staining for surface expression of CD43, B220, and IgM revealed slight variations in the absolute number per femur of pro-B (IgM- CD43+, 0.3 ± 0.04 x 106 WT vs 0.5 ± 0.04 x 106 CD19-/-), pre-B (IgM- CD43-, 1.4 ± 0.2 x 106 WT vs 1.5 ± 0.2 x 106 CD19-/-), and immature B cell subpopulations (IgM+ B220low, 1.1 ± 0.2 x 106 WT vs 1.0 ± 0.04 x 106 CD19-/-) and a marked difference in recirculating mature B cells (IgM+ B220high, 2.8 ± 0.07 x 106 WT vs 2.1 ± 0.04 x 106 CD19-/-; Fig. 1a). Further resolution of the pro/pre-B compartment showed a significant increase in the percentage of CD43-/BP-1+ pre-B cells in CD19-/- mice (Fig. 1b). The onset of BP-1 expression corresponds to the stage at which the expression of the pre-BCR results in the proliferative expansion of B cell precursors that have productively rearranged a H chain gene (21), and thus is coincident with the pro-B to pre-B transition (32, 33). By contrast, only subtle differences were observed in the expression of CD25 and CD2, which represent late stage pre-B cell markers (Fig. 1b) (22, 34). Therefore, it appears that the loss of CD19 has an effect on early pre-B cell development, as seen by the altered expression of CD43 and BP-1, but little effect on the accumulation of late pre-B cells (expression of CD2 and CD25), which represent the largest fraction of BM B cells. The skewing toward pro-B cells in CD19-/- mice becomes more evident when analyzing very young mice. In 2-wk-old animals, there was an increase in the relative number of pro-B cells in CD19-/- mice compared with WT mice (15.0 ± 1.1 to 8.5 ± 0.8%, respectively), with a relative decrease in pre-B cells (56.1 ± 1.3 to 64.0 ± 0.8%, respectively; Fig. 1c). These defects in early B cell development combined with the reduced survival of peripheral B cells (29) resulted in a severe decrease in the total number of B cells in the spleens of 2-wk-old CD19-/- mice (1.2 x 106 vs 5 x 106 for WT; Fig. 1c).



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  		FIGURE 1. Altered distribution of B cell subsets in CD19-/- mice. a, BM cells from adult (>8-wk-old) WT and CD19-/- mice (n = 6 each) were stained for differential expression of B220, IgM, and CD43 (see text for details). Regions represent pro-, pre-, immature, and mature B cells. b, BM cells from adult WT and CD19-/- mice (n = 6 each) were stained for B220, IgM, CD43, BP-1, CD2, or CD25. The dot plots displayed were gated on IgM-/B220+ cells. c, BM cells from 14-day-old WT and CD19-/- mice (n = 4 each) were stained for B220, IgM, and CD43. The upper plots were gated on B220+ lymphocytes. Spleen cells from 14-day-old WT and CD19-/- mice were stained for B220, IgM, and CD23. d, BM cells from adult WT and CD19-/- mice (n = 6 each) were stained for B220, HSA, IgM, and IgD. Numbers in the inset histograms indicate the percentage of HSAhigh cells in the IgMhigh/IgDhigh gate shown in the upper dot plots. Regions in the lower plot (gated on B220+ lymphocytes) represent mature recirculating B cells (HSAlow/IgD+).

 
Upon successful L chain gene rearrangement, B cells express a functional BCR on the surface to become IgMhigh immature B cells. These immature B cells leave the BM and migrate to the spleen, where they up-regulate IgD expression and differentiate into mature B cells. Interestingly, BM cells from CD19-/- mice show an increase in the relative number of IgMhigh/IgDhigh B cells (Fig. 1d), a population that is thought to be present only in the spleen (26), but may develop in the BM in the absence of CD19. Alternatively, CD19 may directly influence the expression levels of surface IgM, resulting in the production of mature recirculating B cells with elevated IgM levels. To determine whether the latter was true, the expression of HSA (CD24) was used to determine the relative number of mature recirculating B cells in BM from WT and CD19-/- mice. HSA expression is elevated on B cell progenitors, including transitional B cells in the spleen, but is down-regulated on mature recirculating B cells (35, 36). Flow cytometry revealed that the majority of IgMhigh/IgDhigh B cells in the BM of CD19-/- mice expressed low levels of CD24, indicating that they were mature recirculating cells (Fig. 1d, inset). A reduction in the relative number of mature recirculating B cells in the BM of CD19-/- mice was also evident from the direct enumeration of HSA-/IgD+ cells (Fig. 1d). Thus, there is an altered ratio of immature to mature B cells in CD19-/- mice compared with WT counterparts, although it is not clear whether this is caused by impaired progression from the immature B cell stage or reflects the known survival defect in mature CD19-/- B cells (29).

Competitive disadvantage of CD19-/- early B cells in mixed BM chimeras

Phenotypic analysis of B cell subsets from the BM of CD19-/- mice suggests an altered developmental progression. To directly evaluate defects in early B cell development caused by the absence of CD19, mixed chimeras were generated to compare the development of CD19+ and CD19- B cells in the same animal. B220-depleted cells from the BM of WT and CD19-/- mice were mixed at equal ratios (50/50) or, alternatively, at a 20/80 ratio favoring WT or CD19-/- BM cells, and were transferred by i.v. injection into lethally irradiated mice. One set of transfers is shown in Fig. 2a, and the results of all experiments are summarized in Fig. 2b. In chimeric animals analyzed 5 wk postreconstitution, an enrichment for CD19 expressing cells was already apparent at the pro-B cell stage where mice injected with only 20% WT BM cells showed 32% of their pro-B cells as CD19+ (Fig. 2a). This result is made more striking by the fact that the delayed onset of CD19 expression dictates that only a subpopulation (70%) of B220+/CD43+ cells are CD19+. The advantage attributed to CD19 expression at the pro/pre-B cell stage is maintained, but not amplified, upon progression to the immature B cell stage (Fig. 2b). Mice reconstituted with 50 or 80% BM cells from WT mice exhibit a significant, albeit less striking, advantage for CD19-expressing cells. Together these data suggest that CD19 expression confers an advantage during the early proliferative pro/pre-B transition, which is maintained during the subsequent progression to the immature B cell stage in a step that does not require additional cell division.



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  		FIGURE 2. Competitive disadvantage of CD19-/- early B cells in mixed BM chimeras. a, Flow cytometric analysis of BM cells from one set of chimeric mice 5 wk postreconstitution. Columns are labeled with the relative composition of B220-depleted BM cells injected. Histograms show the relative percentage of CD19+ and CD19- B cell subsets. b, Results from four independent series of transfers are shown with SD and the initial percentage of transferred WT B220-depleted cells indicated.

 
Reduced B cell generation in CD19-/- mice following autoreconstitution

The BM chimera experiments revealed a reduced ability of CD19-/- B cells to compete with WT B cells in early development. The nature of this defect may be distinct from mechanisms operating in CD19-/- mice. For example, competition may exist for stromal cell interactions or homotypic interactions among B cells, which has been suggested to promote differentiation (37, 38). Therefore, to further examine the effect of CD19 deficiency on B cell maturation, we assessed synchronized differentiation of B cell precursors in autoreconstituted mice. Here, mice were sublethally irradiated to remove all peripheral hemopoietic cells and BM precursors. Stem cells in the BM are resistant to this level of irradiation and will proceed to reconstitute the hemopoietic system. This assay has been well characterized in terms of the kinetics of B cell development achieved on specific days following irradiation (39). We chose to use the 12–14 day time point when large numbers of transitional B cells are present in the spleen. Fig. 3a shows the absolute numbers of B cells present at each stage of development in WT and CD19-/- mice at 12 or 14 days postirradiation. In CD19-/- animals, we found an overall decrease in B cell numbers, reflecting a striking reduction in all subpopulations of B cells from BM and spleen. Reduced B cell generation in the absence of CD19 was particularly evident when observing transitional B cells in the spleen (Fig. 3b). This defect is also evident from the increased mature B cell formation in WT mice at 14 days, although we could not discriminate a defect in entering vs leaving the transitional B cell stage in CD19-/- mice, since B cells from CD19-/- mice are slower to mature. The increase in IgMhigh/IgDhigh cells in autoreconstituted mice primarily represents transitional-2 B cells (as indicated by HSA levels; Fig. 3b) and not mature B cells that have up-regulated IgM. Consistent with this interpretation, no IgMhigh/IgDhigh cells developing in or recirculating to the BM were found in CD19-/- mice following autoreconstitution (Fig. 3b). In addition, decreased B cell numbers found in CD19-/- mice were not caused by a general defect in hemopoiesis, since there were similar numbers of T cells in the spleen of WT and CD19-/- mice at 14 days postirradiation (Fig. 3a). Thus, CD19-/- mice are defective in the generation of B cells, first made evident at the pro- to pre-B transition that is coincident with the onset of surface Ig expression.



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  		FIGURE 3. Impaired B lymphopoiesis in CD19-/- mice after sublethal irradiation. a, Absolute numbers (n = 8 from two experiments) of B cell subsets from the BM (one femur) and spleen of WT and CD19-/- mice 12 or 14 days after sublethal irradiation obtained by analysis of flow cytometric data: pro-B cells (B220low/CD43+/IgM-/IgD-), pre-B cells (B220int/CD43-/IgM-/IgD-), immature B cells (IgM+/IgD-), and transitional B cells (IgM+/IgDlow) in the BM; T-1 B cells (IgM+/IgDlow), T-2 B cells (IgMhigh/IgDhigh), and mature B cells (IgMlow/IgDhigh) in the spleen; and B cells (B220+) and T cells (CD3+) in the spleen. b, Representative flow cytometry of BM and spleen cells from WT and CD19-/- mice 14 days postirradiation and stained for IgM, IgD, HSA (CD24), and B220. Lymphocyte gates were used for all plots.

 
Decreased pre-B cell proliferation in mice lacking CD19

Reduced pre-BCR-mediated proliferation and/or survival are plausible explanations for reduced B cell generation in CD19-/- mice. In gating on B220+ lymphoid cells and measuring forward scatter as an index of cell size, we observed a significant decrease in large B220+ cells in CD19-/- mice after sublethal irradiation (Fig. 3b). The decrease was most evident in the population of large B cells in the IgM- gate of CD19-/- mice (12 ± 2.6%) relative to WT mice (26 ± 2.8%). This population represents large pre-B cells that are undergoing proliferative expansion following the expression of the pre-BCR. These data suggest that CD19 influences proliferation at the large pre-B cell stage of development, possibly by augmenting signals through the pre-BCR.

To directly determine whether pre-B cells from CD19-/- mice have a proliferation defect, experiments were undertaken to determine both the relative number of cells in cycle and the rate of proliferation. BM cells from age-matched WT and CD19-/- mice were stained for surface expression of B220 and IgM and labeled with PI after fixation and permeabilization. CD19-/- mice consistently showed fewer pro/pre-B cells in cycle (16 ± 4.0%) than WT mice (23 ± 2.4%; Fig. 4a). B cells that have differentiated to the immature stage (IgM+) are not in cycle; thus, the majority of these cells fall within the G0-G1 gate (data not shown). The nature of the staining procedure prevents the discrimination of pro-B cells from pre-B cells. Therefore, the difference in the percentage of cycling cells between CD19-/- and WT mice includes pro-B cells whose proliferation is dependent on IL-7 and may not be sensitive to the absence of CD19.



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  		FIGURE 4. Reduced pro- and pre-B cell proliferation in CD19-/- mice. a, Histograms show DNA content after PI staining of B220+/IgM- B cells from BM of WT and CD19-/- mice (n = 6 from three experiments). Numbers represent the percentage of cells in cycle (>2n DNA content). b, Representative flow cytometric analysis of BM cells from WT and CD19-/- mice injected i.p. with 1.0 mg of BrdU every 12 h. Histograms show the percentage of BrdU-labeled BM B cells from WT and CD19-/- mice 24 h after injection. c, Graph shows the percentage (±SD) of BrdU-positive pro- and pre-B cells or immature B cells 24 or 48 h after injection (n = 6 from three experiments). d, WT mice, CD19-/-mice, and mice transgenic for mutated CD19 (Y482/513F; n = 6 from three experiments) were subjected to BrdU labeling for 24 h. The graph shows the percentage of BrdU+ cells obtained by flow cytometric analysis, with gates representing pro-B cells (B220+/CD43+) and pre-B cells (B220+/CD43-/IgM-).

 
PI staining allows for determination of the percentage of cells in cycle, but does not accurately indicate the rate at which cells are progressing through cycle. Thus, to get an accurate measure of the rate of cell turnover, short term BrdU labeling experiments were performed. Age-matched WT and CD19-/- mice were injected with BrdU at 12-h intervals and analyzed 24 and 48 h after the first injection. Mature recirculating B cells (B220high/IgMlow) are not yet labeled at these early time points, so analysis was restricted to pro/pre-B cells (B220low/IgMneg) and immature B cells (B220low/IgMpos). At 24 h, there were almost 20% more BrdU-labeled pro- and pre-B cells in WT mice compared with CD19-/- mice (Fig. 4b). Similarly, there were almost twice as many BrdU-labeled cells at the immature B cell stage in WT compared with CD19-/- mice. These BrdU-labeled immature B cells represented cells that differentiated from recently proliferating pre-B cells. At 48 h, B cells from CD19-/- and WT mice began to equalize in terms of the percentage of cells labeled with BrdU, an indication of how quickly cells move through earlier stages of development. The observation that the number of BrdU+ immature B cells began to equalize in the two sets of mice after 48 h also supports the idea that immature B cells may be accumulating in the BM of CD19-/- mice. Together, these data demonstrate an impaired ability of CD19-/- B cell precursors to proliferate.

CD19 contains nine conserved tyrosines in its cytoplasmic tail that are responsible for recruiting multiple downstream signaling molecules (40, 41). Of importance are tyrosines 482 and 513, which represent binding domains (YxxM motif) for the p85 subunit of phosphoinositide 3-kinase (PI3-K) (42). Recently, it was shown that mutation of these two tyrosines resulted in B cell defects similar to those seen in mice lacking the entire CD19 molecule (43). We investigated whether these tyrosines were also important for proliferative signals generated by CD19 in pre-B cells. In this case, incorporation of BrdU was measured in B cell populations of BM 24 h after a single injection of BrdU. Similar numbers of BrdU-positive cells could be seen in the CD43+ pro-B cell population after 24 h, yet there was a reduction in the percentage of BrdU-labeled pre-B cells from CD19-/- mice as well as Y482/513F transgenic mice compared with WT mice (Fig. 4). These data indicate that proliferative signals through the pre-BCR are modulated by tyrosines 482 and 513 of CD19.

Impaired pre-BCR signaling in the absence of CD19

To correlate the proliferation defect observed in CD19-/- mice with signaling through the pre-BCR, we cultured BM cells in the presence of IL-7 to generate primary pre-B cell cultures. These cultures have been used extensively to study properties intrinsic to pre-B cells as well as their further differentiation (44). BM from WT and CD19-/- mice were cultured in the presence of IL-7 until they reached >95% purity (~5 days). We did not observe a difference in the growth properties of pre-B cells from CD19-/- mice compared with WT pre-B cells (Fig. 5a). These cells were cultured in saturating concentrations of IL-7 in the presence of stromal cells. Therefore, to determine whether CD19 contributed to IL-7 responses in a synergistic manner, cells were exposed to titrated concentrations of IL-7, and proliferation was measured by short term BrdU incorporation. Here again we did not observe a difference in the proliferative response to suboptimal concentrations of IL-7 by CD19-/- pre-B cells relative to WT pre-B cells (Fig. 5b). In addition, upon removal of IL-7, CD19-/- pre-B cells underwent apoptosis to a similar degree as WT pre-B cells (Fig. 5c). These data suggest that CD19 does not contribute substantially to IL-7 responses by pre-B cells, nor does it appear to affect the regulation of cell death at this stage of development.



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  		FIGURE 5. Impaired Ig{beta} signaling in the absence of CD19. a, Primary pre-B cells were expanded by culturing BM cells from WT and CD19-/- mice (n = 9 from three experiments) for 5 days in the presence of IL-7. Cells (1 x 105/well) were plated in a 96-well plate and counted on consecutive days for live WT ({blacksquare}) and CD19-/- (•) cells by trypan blue exclusion (0.1% in PBS). b, Pre-B cells (1 x 106) were cultured in 24-well plates in the presence of titrated levels of IL-7. After 24 h BrdU was added at 10 µM for 30 min before analysis. Cells were then stained for BrdU incorporation. c, Pre-B cells were washed twice and then cultured with medium in the absence of IL-7. Apoptosis was followed over time using PI staining for DNA content and counting events in the subG0/G1 (apoptotic) peak. d, Pre-B cells from primary cultures were serum- and IL-7-starved for 3 h and then prebound with 20 µg/ml of anti-Ig{beta} (biotinylated HM79) for 5 min on ice. Stimulation was initiated by the addition of pre-warmed 30 µg/ml avidin in PBS for the indicated times. Immunoblots were probed for anti-phospho-specific MAPK ERK(p44/42) and anti-phospho-specific Btk and reprobed for total MAPK or Btk to show equal loading. Immunoblots are representative of three independent experiments.

 
The involvement of CD19 in pre-BCR signaling was investigated by stimulation of cultured pre-B cells. Although it was shown that CD19 can be phosphorylated by signaling through the pre-BCR as well as autocross-linking in a pro-B cell line (31, 45, 46), it has not been determined whether CD19 modulates pre-BCR-induced signals. To address these issues, pre-BCR signaling was assessed by stimulation with an agonistic anti-Ig{beta} Ab (HM79). Ig{beta} is first expressed on pro-B cells and is capable of stimulating both pro- and pre-B cells (47). IL-7-dependent pre-B cell cultures were serum- and IL-7-starved for 2 h and then stimulated with HM79. The phosphorylation status of downstream effectors of pre-BCR signaling was analyzed by Western blot following stimulation. Specifically, ERK/MAPK (p44/42) and Btk were chosen because of their involvement in CD19-dependent signaling cascades (6, 48). As shown in Fig. 5d, ERK/MAPK activation showed similar kinetics in CD19-/- and WT pre-B cells, yet the presence of activated ERK/MAPK was severely reduced in the absence of CD19. Likewise, immunoblotting for phosphorylated Btk indicated reduced activation of Btk following Ig{beta} stimulation in CD19-/- pre-B cells. Both Btk and ERK/MAPK activities have been shown to be important for the proliferation of pre-B cells (6, 49). As such, reduced activation in CD19-/- pre-B cells in vitro correlates with the reduced proliferation observed in CD19-/- pre-B cells in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we present evidence of a role for CD19 in the early Ag-independent stages of B cell development. In mixed BM chimera and autoreconstitution studies, we found that CD19-expressing B-lineage cells possessed a distinct advantage during early expansion. One possible explanation for this observation is that CD19 is necessary for inducing or sustaining proliferation at early stages of B cell development. In support of this argument, we showed that pro- and pre-B cells from CD19-/- mice had fewer cells in cycle compared with pro- and pre-B cells from WT mice. A proliferative defect in CD19-/- B cell precursors was also evident from pulse labeling experiments with BrdU. Similar findings were obtained from mice expressing a CD19 molecule bearing tyrosine to phenylalanine substitutions at positions 482 and 513 (43). Since these tyrosines are known to recruit PI3-K (42), these data suggest a role for the CD19/PI3-K signaling pathway in pre-BCR-mediated proliferation of pre-B cells. We extended these findings to show that CD19 was required for effective pre-BCR signaling in terms of activation of MAPK and Btk. Reduced ERK/MAPK signaling in CD19-/- mice is significant in light of recent evidence of the importance of ERK/MAPK in coordinate signaling through the IL-7R and the pre-BCR (6).

The PI3-K effector molecule Btk has also been shown to be important for B cell development (50, 51, 52). Using competitive BM chimeras of Xid and WT cells, Hendriks et al. (51) found that Btk promoted cell progression at the pre- to immature B cell transition in the BM and at the immature to mature B cell transition in the periphery. Defects similar in scope to those reported here for CD19-/- mice, including reduced mature B cell formation, abnormal numbers of BP-1+ pre-B cells, increased expression of surface IgM, and reduced proliferation of pre-B cells were reported in mice deficient in Btk (49). Moreover, related defects in the transition from pro- to pre-B stage have been reported in mice lacking subunits of PI3-K (53), suggesting the involvement of CD19 in these processes. We have recently shown that inactivation of the phosphoinositide phosphatase PTEN and the resultant increase in cellular phosphatidylinositol-3,4,5-triphosphate levels complement the loss of CD19 in peripheral B cell maturation and function (54), indicating the importance of the PI3-K pathway in CD19 signaling. In reconstituted myeloma cells, CD19 has been shown to be necessary for efficient BCR-mediated activation of Btk (55), most likely through the PI3-K pathway and the production of phosphatidylinositol-3,4,5-triphosphate in concert with Lyn-mediated phosphorylation of Btk (56, 57). While reduced activation of Btk was suggested to be responsible for decreased BCR-induced proliferation in CD19-/- mice caused by insufficient Ca2+ mobilization (58), Btk has also been suggested to play an additional complementary role that is independent of CD19 signaling (59).

In addition to Btk, Src family kinases such as Lyn factor prominently in CD19 function, but CD19 does not appear to be necessary for signals induced by Syk kinase activity (60, 61). Thus, a model for the modulatory role of CD19 in pre-BCR signaling would include augmented activation of Src kinases, ERK/MAPK, PI3-K, and Btk. Such a role would augment signal propagation induced through the Ig{alpha}/{beta} heterodimer to promote early B cell development. Of note, while this manuscript was under revision, Kitamura and colleagues (62) reported that a CD19-dependent signaling pathway is necessary to support the residual B cell lymphopoiesis occurring in SLP65 (BLNK, BASH)-/- mice. Thus, CD19 may also be responsible for an ancillary pathway of early B cell development.

In addition to signaling requirements for cell proliferation, a signaling-competent pre-BCR or BCR is required for B cell survival (63). Inadequate signaling through the pre-BCR may result in the apoptosis of pre-B cells that fail to enter cell cycle (64, 65). Signaling through the BCR at the immature B cell stage can also result in positive or negative selection. It was recently shown in a BCR transgenic system (3-83 mice) that the absence of CD19 resulted in reduced positive selection, as evidenced by developmental arrest as well as impaired light chain allelic exclusion and increased receptor editing (66). As in the case of positive selection, negative selection is regulated by BCR signaling thresholds to which CD19 may contribute. Correspondingly, there is a reported accumulation of immature B cells in the BM of 3-83Tg/CD19-/- mice, which may suggest altered selection by endogenous ligands in the absence of CD19. However, 3-83Tg/CD19-/- mice on the deleting MHC class I H-2b background show intact negative selection to the self-Ag H-2Kb (67). Therefore, the apparent finding of an accumulation of IgMhigh B cells in the BM of CD19-/- mice favors a defect in the differentiation or emigration of immature B cells from the BM rather than failed negative selection. Additionally, we recently have shown that B cell numbers can be partially rescued in CD19-/- mice by overexpression of the survival factor Bcl-2 in B cells (29). Yet, B cell numbers in CD19-/- mice overexpressing Bcl-2 never reach the level seen in Bcl-2 mice that express CD19, indicating that the activation-associated early developmental defects described here cannot be overcome by simply promoting survival.

Previous work from several groups has focused on CD19 function in peripheral B cells, where it associates with the complement receptor, CD21. As CD21 is only expressed on mature B cells, any role ascribed to CD19 in early B cell development is CD21 independent. Recently, it was shown that CD19 can bind heparin/heparan sulfate on follicular dendritic cells as well as stromal cells of BM origin (68). This broad binding specificity represents a complement-independent function for CD19 on B cell progenitors at the stromal cell-dependent stage of development. It was also shown that CD19 can bind to the extracellular portion of IgM (68). This function is thought to be important for recognizing immune complexes, but could also influence B cell/B cell interactions important for the development of B cell progenitors (44). It is unclear at present how or if these interactions apply to CD19 binding of surface IgM or the pre-BCR. A specific ligand for the pre-BCR has not been identified, in which case signaling may be initiated by self-aggregation of the pre-BCR, as recently suggested for {lambda}5 function (69). In contrast, it was recently shown that the pre-BCR can bind galectin-1 on stromal cells, which results in the formation of an immune synapse and the initiation of pre-BCR signaling (70). In either case, preassociated CD19 would signal downstream of the pre-BCR.

It is becoming more evident that coreceptors, in addition to modulating responses to Ag in the periphery, may be influencing the generation of B cells in the BM. In addition to CD19, recent work on Fc{gamma}RIIB (CD32) and CD22 has suggested roles for each molecule in early B cell development, before the expression of surface Ig (71, 72). Mice rendered deficient in the expression of CD32 have increased numbers of B cells in BM (71). It has also been shown that FcR signaling can block apoptosis in pre-B cell lines (73). The early expression of CD22 at the pre-B cell stage and its ability to promote cell-cell interactions would suggest a role in early B cell development as well (74, 75, 76). Interestingly, an increase in immature B cell numbers was noted in CD22-/- mice as was an increase in autoantibody production, indicating the importance of CD22 in controlling signaling thresholds that govern selection and tolerance induction in immature B cells (75). Incidentally, both these receptors, CD32 and CD22, have also been implicated in the regulation of CD19 function (77, 78, 79). These studies indicate that in addition to modulating B cell responses to Ag in the periphery, coreceptors, specifically CD19, influence the development of B cells and thus the generation of the B cell repertoire that is capable of responding to Ag.


   Acknowledgments
 
We thank members of the Rickert laboratory for critical reading of this manuscript. We also thank Dr. Robert H. Carter (University of Alabama, Birmingham, AL) for use of the Y482/513F CD19 transgenic mice, and Dr. Barbara L. Kee (University of Chicago, Chicago, IL) for providing the IL-7-producing J558 cell line.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI41649 (to R.C.R.) and a Biomedical Science Grant from the Arthritis Foundation (to R.C.R.). Back

2 Address correspondence and reprint requests to Dr. Robert C. Rickert, Division of Biology and University of California-San Diego Cancer Center, 9500 Gilman Drive, MC0322, La Jolla, CA 92093-0322. E-mail address: rrickert{at}ucsd.edu Back

3 Abbreviations used in this paper: BCR, B cell receptor; bio, biotin; BM, bone marrow; BrdU, 5-bromo-2'-deoxyuridine; Btk, Bruton’s tyrosine kinase; HSA, heat-stable Ag; PI, propidium iodide; PI3-K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; WT, wild type. Back

Received for publication April 4, 2003. Accepted for publication September 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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