|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 September 2005, Vol. 106, No. 6, pp. 2083-2090. Prepublished online as a Blood First Edition Paper on June 2, 2005; DOI 10.1182/blood-2005-01-0283.
IMMUNOBIOLOGY The development of functional B lymphocytes in conditional PU.1 knock-out miceFrom The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage. (Blood. 2005;106:2083-2090)
As the sole source of immunoglobulin in the body, the development of functional B cells is essential in generating an adaptive immune system. B lymphocytes are produced in a stepwise process by the hematopoietic stem cell (HSC), first in the fetal liver and subsequently in the bone marrow. The self-renewing HSC is able to do this by generating a cascade of progenitors each of which is more restricted to a B-cell fate.1 The molecular mechanisms underpinning this process have come under intense scrutiny and, as a result, the transcription factor PU.1 has emerged as an essential component. PU.1 and its close relative Spi-B are members of the Ets domain-containing transcription factor family that are expressed exclusively in the hematopoietic system. Mice carrying a targeted deletion of PU.1 lack all B cells; however, they also have no T cells or macrophages and die at E18.5.2 The importance of PU.1 in B-cell development has been confirmed by an alternative knock-out strategy which, despite having a less severe T-cell and survival phenotype, lacked immunoglobulin M-positive (IgM+) mature B cells. These mice did, however, have a population of B220+ cells that did not correspond to any known B-cell fraction and did not undergo IgH rearrangement.3 There remains some controversy regarding which of these deletion strategies best represents a PU.1 null phenotype.4 The loss of multiple lineages in PU.1-deficient mice has led to the hypothesis that it is able to influence commitment to both myeloid and lymphoid fates. Experiments in which PU.1-/- fetal liver progenitors (FLPs) were transduced with PU.1 retrovirus suggest this control is exerted by different doses of PU.1, with high levels directing myeloid differentiation and low levels supporting lymphoid commitment.5 This conclusion is, however, in stark contrast to our recent finding showing the level of PU.1 in the first definable myeloid and lymphoid progenitors is the same, implying that the level of PU.1 is not deterministic at the initial definition of myeloid versus lymphoid fate.6 The absence of normal B cells in PU.1-/- mice may be due to a failure to up-regulate the transcription factor early B-cell factor (EBF), which when knocked out blocks B-cell development prior to IgH gene rearrangement.7 PU.1 has been purported to directly activate Ebf1 expression, and transducing PU.1-/- FLPs with Ebf1 has been shown to rescue early-stage B-cell development, implying the lack of Ebf1 in PU.1-/- mice blocks B-cell development.8 In addition, PU.1 has also been shown to bind to regulatory sequences in the Il7r gene,9 a fundamental component of the receptor for the cytokines interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP), both of which support lymphoid development.10-13 The loss of signals through the IL-7R in PU.1-/- mice may, however, block the generation of lymphoid progenitors rather than commitment to a B-cell fate, because Il7r is expressed from the earliest stages of lymphoid development.14
Several studies have linked PU.1 with the expression of B cell-specific genes including B220,8 IgH,15 Ig To further investigate the role PU.1 plays in B-cell development and function, we have generated a conditional PU.1 allele, which has enabled the specific deletion of PU.1 in the B-cell lineage. Surprisingly, these mice have relatively normal numbers of B cells in all defined developmental stages and are able to respond, albeit with reduced efficiency, to T-dependent immunization. The reduced number of antigen-specific cells may be due to increased apoptosis resulting from BCR cross-linking.
Mouse strains and deletion of PU.1
The PU.1 alleles PU.1FL/FL, PU.1 Monoclonal antibodies (mAbs) and flow cytometry
Antigens were detected with fluorescent or biotinylated conjugated monoclonal antibodies (mAbs) as follows: Mac-1 (M1/70), Gr-1 (RA6-8C5), B220 (RA3-6B2), IgM (331.12), and CD19 (ID3) were purified from hybridoma supernatants and conjugated in the authors' laboratory. c-Kit (2B8), IL-7R Western blotting
Protein extracts were prepared and Western blotting carried out as described by Rosenbauer et al.28 Antibodies used were rabbit anti-mouse PU.1 (T21) and goat anti-mouse Immunizations Mice were immunized by intraperitoneal injection of 100 µg alum-precipitated nitrophenyl (NP) conjugated to keyhole limpet hemocyanin (NP-KLH) (ratio, 18:1), as described.29 Mice were boosted by a further intraperitoneal injection of 20 µg soluble NP-KLH per mouse on day 28. ELISPOTs and ELISAs
The frequency of antibody-secreting cells (ASCs) was determined by enzyme-linked immunospot (ELISPOT) as reported previously.30 Briefly, 96-well filtration plates (Millipore, Bedford, MA) were coated with 100 µL of 15 µg/mL NP2BSA or NP20BSA in phosphate-buffered saline (PBS) for 12 hours at 4°C. Plates were washed with PBS and bone marrow cells or splenocytes added at 1 x 104, 1 x 105, or 1 x 106 cells per well in RPMI, 5% fetal calf serum (FCS), and 10-4 M Immunohistochemistry Tissue samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and snap frozen in isopentane on dry ice. Six-micrometer cryostat sections were stained with a combination of purified GL7 (BD Pharmingen, San Diego, CA) and biotinylated IgD (Southern Biotech). An anti-rat HRP was used to detect GL7, with subsequent staining carried out with the addition of normal rat serum to block cross-reactivity. IgD-biotin was detected using streptavidin-alkaline phosphatase (Southern Biotech) and visualized using the Fast Blue kit (Vector Laboratories, Burlingame, CA). HRP was visualized using the 3-amino-9-ethylcarbazole substrate kit (Vector Laboratories). Slides were mounted under coverslips in Aqua polymount (Polysciences, Warrington, PA). Images were analyzed with an Axioplan 2 microscope, under a 100 x /1.3 NA objective and 10 x eyepiece (Carl Zeiss, North Ryde, NSW, Australia). Images were captured with an Axiocam digital camera (Zeiss) and processed with Axiovision software (Zeiss). RT-PCR Total RNA was isolated from cells using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT; Promega, Madison, WI) according to the manufacturer's instructions. Semiquantitative RT-PCR was performed on samples previously normalized by equilibrating the level of hypoxanthine guanine phosphoribosyltransferase (Hprt) expression. All the primer combinations, the sequences for which are available upon request, spanned introns. Isolation and stimulation of B cells
Small resting B cells were isolated by macerating total spleens, treating with red cell lysis buffer, and spinning in a 80:65:50 gradient of Percoll.31 The small dense cells were removed from the 80:65 interface and the B cells positively sorted by magnetic-activated cell separation (MACS) using anti-B220 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells were cultured in RPMI supplemented with 5% FCS and 10-4 M
Conditional deletion of PU.1 in the B lineage Of the 2 strategies previously used to delete PU.1, one resulted in the complete loss of B cells in the fetal liver2 and the other reported a population of aberrant B220+ cells in the spleen and bone marrow.3 We have generated a conditional allele, which allows for spatially or temporally controlled or complete deletion of PU.1 (Figure 1A). When PU.1 was inactivated throughout the mouse by crossing to the Deleter-Cre strain, no expression of either B220 or CD19 was observed in the fetal liver at E18.5 (Figure 1B). Furthermore, no Mac-1+Gr-1+ cells were present, and knock-out mice died between E18.5 and birth despite being present in normal mendelian frequency at E18.5 (data not shown). This phenotype most closely represents that previously reported by Scott et al.2
To determine whether the lack of B cells was a result of a requirement of PU.1 for the development of the B lineage or rather a more widespread failure of hematopoietic progenitors, we crossed the PU.1fl/fl allele to CD19-Cre mice, which provided a B cell-specific source of Cre. The mice generated by this cross are termed PU.1  /CD,19 referring to deletion of PU.1 in CD19-expressing cells. The appropriate control, which is mice harboring Cre in one CD19 locus, and both wild-type PU.1 alleles are termed PU.1+/+CD19. Surprisingly, the PU.1/CD19 embryos had normal numbers of B cells in the fetal liver at E18.5 despite having a frequency of exon-5 deletion of approximately 70% (Figure 1B), suggesting that PU.1 was not essential for the development of cells committed to the B lineage. The presence of B cells in E18.5 fetal liver led us to investigate whether they were also present in adult PU.1/CD19 mice. Table 1 shows that PU.1/CD19 mice have cells in each of the previously defined B-cell fractions in the bone marrow, spleen, lymph nodes, and peritoneum. The total number of cells present in the various fractions was, however, slightly perturbed compared with controls with enhanced numbers of pre-BII and T1/T2 cells but reduced numbers of mature recirculating/follicular B cells. In contrast to a recent publication, the B cell-specific isoform of CD45, B220, was found to be expressed at normal levels on adult B cells from PU.1/CD19 mice as well as in E18.5 embryos (data not shown and Figure 1B).8
To ensure the B cells observed in PU.1 /CD19 mice had deleted the conditional PU.1 allele, we sorted cells from each fraction and subjected them to PCR analysis. Figure 1C shows that at the pre-BII stage 86% of the PCR product was from the deleted allele, a frequency that rose to more than 90% for subsequent developmental stages, indicating that most cells in each fraction are deleted. When IgM+CD19+ cells were sorted from the spleen and subjected to Western analysis, very low levels of PU.1 protein were detected from cells of the PU.1/CD19 mice. These data confirm that relatively normal B lymphopoiesis occurs in the absence of PU.1.
Expression of EBF and IL-7R
PU.1 has been suggested to directly activate the expression of Il7r9 and Ebf1,8 both of which are required for normal B-cell development.7,10 The observation that B cells from PU.1
Resting levels of immunoglobulin isotypes and response to T-dependent immunization of PU.1
Several reports have implicated PU.1 in the function of B cells, because it is thought to be part of the mechanism controlling the expression of IgH, Ig
PU.1 has also been implicated in the maintenance of the T-dependent immune response, because crossing Spib-/- mice onto a PU.1+/- background increased apoptosis in germinal center (GC) B cells over that observed in Spib-/- mice, resulting in the failure of GCs to form.21 To test if the complete loss of PU.1 alone effected the T-dependent immune response, we assessed the ability of PU.1
Following immunization, the number of NP-specific IgG1+ ASCs in the spleen and bone marrow was assessed at day 7, 28, and 35 (following boosting on day 28). Figure 3B-C shows that NP-ASCs were present at all time points in PU.1
In vitro anti-IgM stimulation of PU.1 /CD19 B cells
PU.1 and Spi-B have been implicated in the survival of B cells during an immune reaction by directly up-regulating the expression of the NF-
The ability of the PU.1 /CD19 B cells to respond to immunization by forming GCs and producing long-lived antigen-specific cells capable of secreting high-affinity antibody suggests that the apoptosis observed in vitro upon IgM cross-linking is overcome in vivo during T-dependent response. A possible explanation for this may be that T-cell help and particularly the effects of IL-4 counteract the apoptotic effects of BCR cross-linking. To test this hypothesis, we stimulated B cells with anti-IgM in the presence of IL-4 and measured the level of apoptosis observed over 30 hours. Figure 4B shows that apoptosis is significantly reduced when anti-IgM is combined with IL-4, to the extent that the PU.1/CD19 B cells are more viable than the PU.1+/+CD19 cells stimulated with anti-IgM alone. This result conflicts with data from PU.1+/-Spib-/- B cells that are not afforded protection from apoptosis by IL-4,21 suggesting a different molecular mechanism controlling BCR signal-induced apoptosis though most likely not involving A1, Bcl2, or BclXL.
In vitro anti-CD40 and LPS stimulation of PU.1 /CD19 B cells
PU.1 has been suggested to regulate several components of the machinery required for the expression and secretion of immunoglobulin15-18,33 and directly interacts with PU.1 interacting partner (PIP)/interferon regulatory factor-4 (PIP/IRF4), a factor essential for immunoglobulin production.34 The data outlined in Figure 3 show normal ratios of the different isotypes in the serum of PU.1
The in vitro development of comparatively normal ASCs from PU.1
The absence of a severe phenotype in B cells lacking PU.1 may be interpreted as there being a functional redundancy between PU.1 and some other factor. An obvious candidate for this factor is Spi-B, because it has been shown to compensate for PU.1 in some instances23; however, we have not observed any up-regulation of Spib mRNA in the absence of PU.1. In an attempt to assess whether Spi-B substitutes for PU.1 in B cells, we made nuclear extracts from LPS-stimulated PU.1 /CD19 and PU.1+/+CD19 cells and assessed the level of PU.1-like DNA binding activity by EMSA (Figure 5D). These experiments used a binding site in the SV40 promoter, which has a high affinity for PU.116,35 and presumably for any molecule able to functionally substitute for it. Extracts from PU.1+/+CD19 cells interacted strongly with the probe, whereas no equivalent DNA binding activity was observed with extracts from PU.1/CD19 cells. The band obtained from PU.1+/+CD19 extracts was confirmed as PU.1 by the addition of an anti-PU.1 antibody, which resulted in a supershift, whereas an anti-Spi-B antibody had no effect (data not shown). Taken together the data in this study suggest that PU.1 is dispensible for the development and function of B lymphocytes from the pre-B stage onward.
B lymphopoiesis is a multistep highly regulated process beginning with the HSC and ending with the production of B lymphocytes that are competent to respond to antigen by differentiating into plasma cells capable of secreting massive amounts of high-affinity immunoglobulin. Two knock-out and numerous molecular biology studies have implicated the transcription factor PU.1 as being critical for key stages in this entire process, from the initial commitment of progenitors to the B lineage to the expression and secretion of immunoglobulin. In the work presented here, we have made use of an inducible PU.1 deletion allele to rigorously test this entrenched view. The phenotype of a true PU.1 null mouse has been the point of some conjecture because the 2 alternate deletion strategies adopted resulted in some significant differences; however, in both cases the B-cell lineage was severely perturbed.2,3 Scott et al2 reported no B lymphocytes, whereas McKercher et al3 observed a population of aberrant B220+ cells that did not correspond to any known B-cell subset. In the present study, the complete deletion of the inducible PU.1 allele resulted in the loss of B cells along with all other lymphoid and myeloid cells within the fetal liver and death between E18.5 and birth. This phenotype is similar to that of Scott et al,2 supporting the conclusion that their approach best represents a true PU.1 null.
The hematopoiesis-wide defects caused by PU.1 deletion prompted us to investigate whether there was a specific failure of B lymphopoiesis or rather if the loss of B cells was due to a more catastrophic failure in hematopoietic progenitors. Deletion of PU.1 in the B lineage using a CD19-Cre transgene had no pronounced effect on development, with B cells present in the fetal liver and the adult bone marrow, spleen, lymph nodes, and peritoneum. This implies that after the point when CD19 transcription begins, when cells are committed to the B lineage, PU.1 is not essential for further development. It is interesting that within the pre-BI fraction of PU.1
PU.1 has been suggested to regulate the expression of B2208 and, in agreement with this, no B220+ cells were observed in the fetal liver of PU.1 We propose that these apparently different effects are the result of PU.1 normally functioning to initiate or create the conditions necessary for the expression of genes such as CD45 in the earliest hematopoietic cells but subsequently playing little or no role in maintenance of their expression. This would explain the apparent paradox of the widespread hematopoietic defects caused by unconditional deletion of PU.1 versus the relatively normal differentiation when deletion of PU.1 is targeted to the B-cell lineage. Another gene that may fall into this category is Ilr7, because a report by DeKoter et al9 has shown that PU.1 directly activates its expression; however, we have shown here that deletion of PU.1 after commitment to the B lineage has no effect on Ilr7 expression.
The transcription factor EBF has also been shown to be regulated by PU.1 and can substitute efficiently for PU.1 by driving in vitro B-cell development from PU.1-/- FLPs.8 We show here a contemporaneous reduction in Ebf1 levels when PU.1 is deleted, suggesting that unlike B220 or Ilr7, the activity of PU.1 is required for EBF expression at the pre-BII stage. Although there is a slight perturbation in the makeup of the B-cell fractions in PU.1
The lethality of deleting PU.1 and the failure of mutant fetal liver cells to reconstitute adult bone marrow has precluded studying if there is a requirement for PU.1 activity during B-cell function. There have been, however, several studies intimating a key role for PU.1, because it has been shown to bind regulatory elements within genes required for the function of B cells such as IgH,15 Ig
To assess the effect of deleting PU.1 alone on an immune response, we immunized mice with the well-characterized immunogen NP-KLH, which elicits a high-affinity anti-NP IgG1
An immediate and obvious retort to the lack of phenotype in B cells lacking PU.1 is that another factor—most likely Spi-B—is functionally substituting for PU.1. Indeed, the DNA binding specificities of the 2 factors have been suggested to be virtually identical.42 The argument in favor of redundancy between PU.1 and Spib is provided some validity by the observation that Spib can partially substitute for PU.1 in myeloid development but not lymphoid development,23 a somewhat enigmatic finding given that Spi-B is normally only expressed in B and T cells and not the myeloid lineages.43 The data presented here provide 2 pieces of evidence to argue against a complete functional redundancy between Spi-B and PU.1. Firstly, the expression of Ebf1, a known PU.1 target gene, is reduced in PU.1-deficient pre-BII cells despite continued expression of Spib. Secondly, gel shift assays performed with an oligonucleotide from the SV40 promoter that has been shown to have a high affinity for PU.116,35 and presumably any factor capable of functionally substituting for it suggest that there is no PU.1-like binding activity in PU.1 The data presented in this paper support the surprising conclusion that PU.1 is not required to generate functional B cells from the pre-B stage onward and rather that the failure of B lymphocytes to form in PU.1-/- mice is a result of defects in progenitor cells rather than the commitment of cells to the B lineage. In addition, despite a substantial archive of in vitro data purporting an essential role for PU.1 in the regulation of immunoglobulin expression, switching, and secretion, we have found that the deletion of PU.1 did not substantially affect the function of ASCs.
The authors express their gratitude to Jaclyn Carneli for expert animal husbandry.
Submitted January 24, 2005; accepted May 22, 2005.
Prepublished online as Blood First Edition Paper, June 2, 2005; DOI 10.1182/blood-2005-01-0283.
Supported by the Cancer Council Victoria and The National Health and Medical Research Council of Australia. S.L.N. is the Walter and Eliza Hall Institute Metcalf Fellow.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen L. Nutt, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, Australia 3050; e-mail: nutt{at}wehi.edu.au.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
,
,
(A7R34), CD21 (7G6), CD23 (B3B4), CD25 (7D4), Ig
-actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA). 
) and a reporter cassette flanked by 2 Frt sites (
) containing both the internal ribosome entry site-green fluorescent protein (IRESGFP) and the phosphoglycerate kinase (PGK) promoter driving the selection marker Neomycin (Neo) was introduced into the PU.1 locus by homologous recombination to generate the PU.1gfp allele. When crossed to mice harboring flp recombinase, the reporter/selection cassette is lost, generating PU.1fl allele. Exon 5 of PU.1 is deleted by crossing with mice carrying Cre recombinase, creating the allele PU.1
