Research Article

Regulation of B Versus T Lymphoid Lineage Fate Decision by the Proto-Oncogene LRF

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Science  11 May 2007:
Vol. 316, Issue 5826, pp. 860-866
DOI: 10.1126/science.1140881

Abstract

Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell-fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors in mice to develop into B lineage cells by repressing T cell–instructive signals produced by the cell-fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of the cell's determination of B versus T lineage.

All hematopoietic cells are generated from a small subset of pluripotent stem cells (HSCs) via lineage-restricted progenitors. In adult mice, HSCs reside in the bone marrow (BM) and give rise to lymphoid-restricted progenitors (1), which subsequently develop into B and T lymphocytes in the BM and thymus, respectively. This developmental process is coordinated by the expression of distinct sets of genes at specific differentiation stages. Although some of the transcriptional regulators that play key roles in early stages of lymphocyte development are known (2, 3), the precise molecular mechanisms by which lymphoid-restricted progenitors are instructed toward B or T cell fates are still undefined.

The proto-oncogene LRF (4), encoded by the Zbtb7a gene, [formerly known as Pokemon (5) and also described as FBI-1 (6) and OCZF (7)] is a transcriptional repressor that belongs to the POK (POZ/BTB and Krüppel) protein family. Promyelocytic leukemia zinc finger (PLZF) and B cell lymphoma 6 (BCL6), two members of the POK family, are involved in chromosomal translocations associated with acute promyelocytic leukemia (APL) and non-Hodgkin's lymphoma (NHL), respectively (8, 9). In a similar manner, we recently reported that LRF plays a pivotal proto-oncogenic role and is highly expressed in human NHL tissues (5). Emerging experimental evidence indicates that the POK family is indispensable for normal hematopoiesis and immune system developments (912). BCL6 has been shown to be essential for germinal center (GC) formation and for T helper type 2 (TH2) inflammatory responses (9). More recently, two independent groups reported that a close homolog of LRF (Th-POK, also known as cKrox or Zbtb7b) is a master regulator of CD4+/CD8+ (CD4/8) T cell lineage specification (11, 12).

Given that LRF is broadly expressed in multiple hematopoietic lineages (fig. S2A), especially in the GC B cells (5), forms complexes with BCL6 (4), and is highly expressed in human NHL tissues, we hypothesized that this gene could play a key role in B cell development. We therefore investigated both fetal and adult lymphopoiesis using LRF gene deletion in mice.

B cell development. Deletion of the Zbtb7a gene in mouse was carried out with a conventional gene knockout approach (fig. S1, A and B). Although we did not observe a gross defect in the heterozygous mutant, homozygous deletion of the Zbtb7a gene (Zbtb7a–/–) resulted in embryonic lethality around 16.5 days post coitum (DPC) because of severe anemia. Examination of B lymphopoiesis in 14.5 DPC fetal livers (FLs) from Zbtb7a–/– mice revealed a reduction in the total number of CD19+B220+ B cells (Fig. 1A). This was mainly due to reduction of B cells after the pro-B stage of differentiation. Absolute numbers of the earliest B cell precursors (LinAA4.1+CD19B220+) were comparable to those of wild-type (WT) littermate controls, whereas total numbers of LinAA4.1+CD19+B220+ and LinAA4.1CD19+B220+ B cells were markedly decreased in Zbtb7a–/– FLs (Fig. 1A, right). Hematopoietic stem cell (HSC) and common lymphoid progenitor (CLP) populations were intact in Zbtb7a–/– FLs (fig. S2B).

Fig. 1.

LRF is indispensable for both fetal and adult B lymphopoiesis. (A) On 14.5 DPC, FL cells were stained with fluorochrome-conjugated antibodies against B220, CD19, AA4.1, and lineage markers. (Left) Representative profiles made by fluorescence-activated cell sorting (FACS) of the Zbtb7a+/+ and Zbtb7a–/– FL cells. Total numbers of FL mononuclear cells were counted, and absolute number of B cells in each developmental stage was calculated. Average cell numbers of three independent embryos for each genotype are presented (±SD). (B) The 14.5 DPC FL-HSCs (LinSca1+c-Kit+) were cultured on OP9 stromal cell layers in the presence of IL-7 and Flt3 ligand. After 10 days of culture, cells were isolated and analyzed by FACS. (C) (Left) Schematic representations of mouse breeding strategy for conditional LRF knockout experiments. (Right) Follow-up of the PB counts after pIpC (or phosphate-buffered saline) injections over time. Four groups of mice were examined according to genotype and treatment. WBC counts in the PB were measured by a hematology analyzer, and total numbers of B and T cells were subsequently calculated based on the percent positive cells having B220 and CD4/8 expression, respectively. The average cell count of five animals was plotted on each time point with error bars (±SD). (D) BM cells were stained with fluorochrome-conjugated antibodies against B220, CD19, IgM, CD43, and lineage markers (21) 1 month after the last pIpC injection. Representative FACS profiles for each genotype are shown. (E) Absolute cell number of each population was calculated according to FACS profiles. Black horizontal bars represent mean cell counts among five animals.

Pro-B cells can be propagated in vitro on OP9 stromal cell layers in the presence of interleukin 7 (IL-7) and Flt3 ligand (13). Substantial numbers of pro-B cells could be propagated from Zbtb7a+/+ fetal liver HSCs (FL-HSCs), but Zbtb7a–/– pro-B cells were barely detectable (Fig. 1B), which indicated a cell-autonomous defect in early B cell development. In contrast, Zbtb7a–/– FL-HSCs retained their capacity for T cell development with Zbtb7a–/– FL-HSCs successfully giving rise to T cells in vitro, after culture on OP9-DL1 stromal cells overexpressing the Notch ligand Delta-like 1 (14) (fig. S2C). To further investigate the defect in early B cell development in Zbtb7a–/– FLs, we performed BM competitive repopulation assays (15). Thus, Zbtb7a+/+ and Zbtb7a–/– FL cells were transplanted separately into lethally irradiated recipient mice along with WT BM cells from a congenic strain expressing the CD45.1 antigen (fig. S3A, left). In these experiments, Zbtb7a+/+ FL cells successfully gave rise to peripheral blood (PB) B cells in the recipients; however, Zbtb7a–/– FL–derived B cells were virtually undetectable (fig. S3A, right).

The role of LRF in adult lymphopoiesis was next explored by using conditional deletion of the Zbtb7a gene (fig. S1, C and D). Mx1-Cre transgenic mice were used, in which Cre recombinase is induced in HSCs by administering polyinosinic-polycytidylic acid (pIpC) (16). A series of double-mutant mice were treated with pIpC at 3 weeks of age as previously described (Fig. 1C, left) (16), and peripheral blood was analyzed at 2-week intervals. After pIpC treatment, a significant decline of white blood cell (WBC) counts in the Zbtb7aFlox/Flox Mx1cre+ mice was observed (Fig. 1C), primarily due to a considerable reduction in circulating B220+ B cells, whereas T cell numbers remained similar to those of controls (Fig. 1C).

Like the defect seen in Zbtb7a–/– mice (Fig. 1A), B cell development in the BM of Zbtb7aFlox/– Mx1cre+ mice was severely impaired (Fig. 1D). Thus, pro-B, pre-B, and immunoglobulin IgM+ B cells were drastically reduced, whereas absolute numbers of the prepro-B cells (fig. S3B) (17) were increased in the pIpC-treated Zbtb7aFlox/– Mx1cre+ BM (Fig. 1, D and E). Of note, LRF mRNA expression in the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells was essentially undetectable, as revealed by quantitative real-time fluorescence polymerase chain reaction assays (QPCR) (fig. S3C). Furthermore, proportions of the HSCs and CLPs were not grossly affected in the pIpC-treated Zbtb7aFlox/– Mx1cre+ mice (fig. S3D), although absolute numbers of their HSCs and CLPs were slightly increased as compared with control mice (fig. S3E).

Extrathymic T cell development. Both T and B cells share their origins with a CLP (1). However, we did not observe a gross defect in the T cell compartment in pIpC-treated Zbtb7aFlox/– Mx1cre+ thymus (Fig. 2A). Although a slight decrease was observed in double-negative 3 (CD4CD8CD44CD25+ or DN3) and CD4CD8CD44CD25 (DN4) thymocyte populations, the proportions of CD4 single-positive (CD4-SP), CD8-SP, and CD4/8 double-positive (DP) T cells were comparable to those of control mice (Fig. 2A). Unexpectedly however, an accumulation of extrathymic DP T cells in the BM of pIpC-treated Zbtb7aFlox/–Mx1cre+ mice was detected (Fig. 2B) that made up nearly 30% of the BM mononuclear cells (BMMNCs) 1 month after pIpC treatment (Fig. 2B). Immunohistochemical and/or fluorescent analyses further demonstrated that CD3dim DP T cells accumulated in the BM of Zbtb7aFlox/–Mx1cre+ mice (Fig. 2C). These extrathymic BM DP T cells were polyclonal in origin, as revealed by Dβ1-to-Jβ1 rearrangement status of the T cell receptor β locus (fig. S4A). Moreover, quantitative measurement of gene dosage indicated that the Zbtb7a gene was almost undetectable in both thymic and BM DP T cells in the pIpC-treated Zbtb7aFlox/–Mx1cre+ mice (fig. S4, B and C). Notably, extrathymic T cell development appeared to be limited to the BM, as these cells were not observed in spleen or Peyer's patches (fig. S4, D and E).

Fig. 2.

Extrathymic DP T cell development in the BM after LRF loss. (A) Thymic T cells were analyzed 1 month after pIpC injection. (Left) Representative FACS profiles for each genotype. (Right) Proportions of CD4/8 DN, CD4/8 DP, CD4 single-positive (CD4SP) and CD8 single-positive (CD8SP) populations were examined, and the DN fraction was further stratified according to CD44 and CD25 expression. Three mice were analyzed for each genotype. (B) BMMNCs were analyzed for CD4/8 expression 1 month after pIpC treatment. (Left) Representative FACS profiles for each genotype. (Right) Average percentage positive of three mice for each genotype is demonstrated (±SD). (C) (Top) Immunofluorescent analysis of CD3 and PU.1 expression in BM sections. (Bottom) Immunohistochemical analysis of T cell and B cell markers (CD3 and Pax5, respectively) in BM sections 1 month after pIpC treatment. (D) Either Zbtb7aFlox/+ Mx1cre+ or Zbtb7aFlox/– Mx1cre+ donor BMMNCs (CD45.2+) were transplanted into lethally irradiated recipient mice (CD45.1+). After engraftment, recipient mice were treated with pIpC. Recipients' BMMNCs were then collected and analyzed 2 weeks after the last pIpC administration. Representative FACS profiles are presented. (E) Zbtb7aFlox/– Mx1cre+ donor BMMNCs (CD45.2+) were transplanted into lethally irradiated recipient nude mice. Two weeks after transplantation, mice were treated with either pIpC or phosphate-buffered saline. Recipients' BMMNCs were harvested and subsequently analyzed 10 days after the last pIpC administration. Representative BM FACS profiles are presented.

To investigate whether the extrathymic DP T cell accumulation seen when the LRF gene was deleted was caused by defects in a cell intrinsic mechanism, LRF inactivation was induced in BM-reconstituted recipient mice. Thus, Zbtb7aFlox/+ Mx1cre+ or Zbtb7aFlox/– Mx1cre+ BMMNCs were transplanted into lethally irradiated recipient mice. After engraftment, recipient mice were treated with pIpC to induce Cre expression (Fig. 2D, left). In Zbtb7aFlox/– Mx1cre+ reconstituted mice, an accumulation of donor-derived (CD45.2+) DP T cells was seen in the BM with a significant reduction of B cells both in BM and PB in a cell-autonomous manner (Fig. 2D and fig. S4F). To determine whether extrathymic BM DP-T cell development was thymus-independent, Zbtb7aFlox/– Mx1cre+ BMMNCs were transferred into lethally irradiated athymic nude mice, and the LRF gene was subsequently inactivated after engraftment by pIpC administration. DP T cell accumulation was observed in the BM 10 days after the last pIpC administration (Fig. 2E), indicating that in the absence of LRF, lymphoid progenitors in the BM gave rise to BM DP T cells in a thymus-independent fashion.

Aberrant lymphocyte commitment. To explore whether the early B cell developmental program takes place correctly in pIpC-treated Zbtb7aFlox/–Mx1cre+ prepro-B cells, the expression of the genes encoding pre-BCR components, terminal deoxynucleotidyl transferase (TdT), and Rag recombinases was examined. In these experiments, mRNA levels of pre-BCR components (Igα, Igβ, and Vpre-B1), Rag recombinases (Rag-1, Rag-2) and TdT in Zbtb7aFlox/–Mx1cre+ mice were markedly reduced compared with those of control mice (Fig. 3A).

Fig. 3.

pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells are defective in early B cell development and demonstrate a DN T cell signature. (A) QPCR analysis of the genes encoding pre-BCR components, TdT, the Rag recombinases, and the critical transcription factors in early B cell development. mRNA expression levels were normalized to hypoxanthine-guanine phosphoribosyl transferase (Hprt) mRNA amount and are represented by bar graphs. Each sample was analyzed in duplicate, and error bars indicate ±SD. BMMNCs were collected and flow-sorted 1 month after the last pIpC injection. (B) Western blot analysis for Ebf1 and Stat5 protein in the pIpC-treated prepro-B cells. Bar graph represents normalized protein expression level over corresponding heat shock protein Hsp90 protein level. (C) QPCR analysis of Notch and Notch target genes in the prepro-B cells. QPCR was performed as described in (A). (D) CD25 and CD44 expression in prepro-B cells was examined 10 days after the last pIpC injection. FACS profiles of normal thymic DN T cell populations are also presented. (E) Schematic representation of lymphoid lineage development in LRF conditional knockout mutants.

Early B cell development is governed by a small set of cytokines and transcription factors. Both PU.1 and Ikaros are essential for the maintenance of HSCs and CLPs (18, 19); Flt3 ligand is required for the generation of CLPs but not HSCs (20). Both IL-7 and its receptor (IL-7R) are indispensable for prepro-B to pro-B transition (21) and transcription factors Bcl11a, E2A, Stat5, and Ebf1 also play critical roles at this developmental stage (3) (fig. S5A). pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells showed a significant down-regulation of E2A, Ebf1, and Pax5 mRNA (Fig. 3A). Similarly, small amounts of Ebf1 protein were detected in pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells, whereas Stat5 protein was abundant (Fig. 3B). Because the enforced expression of Ebf1 is able to rescue B cell developmental defects in PU.1–/–, IL-7R–/–, and E2A–/– mice (3), we examined whether the overexpression of Ebf1 in Zbtb7a–/– FL-HSCs might have a similar effect. Positive control LRF-transduced Zbtb7a–/– FL cells successfully gave rise to splenic B cells in recipient mice (fig. S5B). However, neither GFP– vector nor Ebf1-transduced FL cells were able to rescue the Zbtb7a–/– B cell phenotype (fig. S5B).

Given that pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells were unable to progress further in the B cell developmental program, we speculated that they might have become aberrantly committed to the T cell lineage, thus generating the extrathymic DP T cells found in the BM. To test this, we examined mRNA expression levels of T cell–specific target genes in the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells. In these analyses, mRNA levels of Notch1, Notch3, but not Notch2, and their downstream target genes were profoundly elevated in the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells (Fig. 3C). Despite expressing the cell surface B cell marker B220 (Fig. 1D), pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells appeared aberrantly committed to the T cell rather than B cell lineage. To determine whether the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells could differentiate into more mature stages of T cell development, sorted prepro-B cells were cultured on OP9 stromal cell layers. After coculture with control OP9-GFP cells, the pIpC-treated Zbtb7aFlox/+Mx1cre+ prepro-B cells, in which one allele of LRF remained intact, efficiently differentiated into pro-B cells, while the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells did not give rise to pro-B cells (fig. S6A, left). In the case of cells expressing the Delta notch ligand (OP9-DL1), however, control prepro-B cells still differentiated into pro-B cells, even though activation of the Notch pathway drives T cell development (fig. S6A). This is likely because normal prepro-B cells express very low levels of the Notch1 receptor and thus cannot respond to the DL1 signal (see Fig. 3C for Notch1 mRNA). On the contrary, the pIpC-treated Zbtb7aFlox/– Mx1cre+ prepro-B cells immediately lost B220 expression on the cell surface and effectively differentiated to CD4/8 DP T cells (fig. S6A, right). In agreement with these findings, Zbtb7aFlox/– Mx1cre+ prepro-B cells were mostly positive for CD25 and negative for the CD44 surface markers, which is reminiscent of normal thymic DN3 T cells (Fig. 3D). Taken together, these data indicate that in the absence of LRF, lymphoid progenitors give rise to aberrant B220-positive DN T–like cells, which subsequently differentiate to DP T cells in the BM at the expense of normal B cell development (Fig. 3E).

Notch repression by LRF. Notch signaling is critical for T cell development and the perturbation of this pathway can result in cellular transformation (22). Furthermore, Notch is obligatory for correct fate determination toward B or T lineage in the lymphoid progenitors (2) and was recently reported as the most commonly mutated gene in human T cell acute lymphoblastic leukemia (T-ALL) (23). Notch1 deletion in mouse HSCs results in a marked reduction in thymic T cells and simultaneous B cell development in the thymus (24). Conversely, constitutive activation of Notch1 pathways in HSCs and/or CLPs results in blocking of B cell development and a profound expansion of extra-thymic DP T cells in the BM. Although these mice eventually develop T cell leukemia in the BM (22), thymic T cell development is not impaired, for the most part (22). Given that the phenotype of LRF conditional knockout mice is seen in mice overexpressing the intracellular domain of Notch1 that leads to constitutive Notch pathway activation (22), we hypothesized that LRF might oppose Notch1 function at HSC and/or CLP stage. To test this directly, we examined expression of Notch genes and their targets in HSCs and CLPs. After pIpC treatment, LRF mRNA was efficiently eliminated both in HSCs and CLPs (Fig. 4A) and was followed by the up-regulation of all Notch target genes in the pIpC-treated Zbtb7aFlox/– Mx1cre+ HSCs and CLPs (Fig. 4A). Corresponding Notch1 mRNA levels were comparable to those of control mice (Fig. 4A). This “Notch signature” was evident mainly at the HSC and CLP stages, as relatively low levels of Notch target genes were detected in myeloid or erythroid progenitor compartments (fig. S6B). To further elucidate whether the aberrant T cell commitment in the absence of LRF was Notch-dependent, LRF conditional KO mice were treated with a gamma secretase inhibitor (GSI). GSIs are potent inhibitor of Notch signaling that act by preventing the cleavage and release of the intracellular moiety of the Notch receptor (25). Strikingly, this almost completely rescued abnormal B or T cell commitment seen in LRF conditional KO mice (Fig. 4B). After GSI treatment, neither the DP T cells nor aberrant DN T–like prepro-B cells were observed (Fig. 4B). Furthermore, Zbtb7aFlox/–Mx1cre+ prepro-B cells could give rise to pro-B cells after GSI treatment (Fig. 4B). It was noteworthy that, in these pro-B cells, expression of VpreB1, a component of the pre-BCR, resumed after GSI treatment, whereas LRF mRNA became barely detectable, which confirmed that gene targeting was correct (Fig. 4C).

Fig. 4.

LRF opposes Notch pathways at the HSC and CLP stage. (A) BMHSCs and CLPs were flow-sorted from pIpC-treated animals 1 month after the last pIpC injection. QPCR analyses were performed as described in Fig. 3A. (B) In vivo GSI treatment rescued aberrant lymphoid development in LRF conditional KO mice. Either Zbtb7aFlox/+ Mx1cre+ or Zbtb7aFlox/– Mx1cre+ BMMNCs (CD45.2+) were transplanted into lethally irradiated recipient mice (CD45.1) as described in Fig. 2D. Mice were subsequently treated with pIpC. Either GSI or vehicle alone (as a control) was orally administered as described in (15). Recipients' BMMNCs were collected and analyzed 3 weeks after the last pIpC administration. (C) RNA was extracted from flow-sorted pro-B cells, and QPCR analysis was subsequently performed as described in Fig. 3A.

Discussion. Our findings allow us to reach two conclusions. First, we identify LRF as a master regulator in determination of B versus T lymphoid fate, with loss of LRF in HSCs and CLPs, resulting in an absence of B cell development and spontaneous extrathymic DP T cell development in the BM. Second, we demonstrate that loss of LRF results in aberrant activation of the Notch pathway, with Notch target genes becoming strongly up-regulated in HSCs and CLPs. Taken together, we propose a working model for B versus T cell lineage fate determination, in which LRF plays a pivotal role as a negative regulator of T lineage commitment by opposing Notch function (Fig. 5). In normal HSCs, LRF opposes Notch function. LRF blocks basal Notch signaling triggered from BM stromal cells, which express moderate level of Notch ligands (26). BM stromal cells also express molecules that support B cell commitment and development, such as Flt3 and SDF1 (27). Therefore, HSCs and lymphoid progenitors in the BM are committed to the B cell lineage by default and differentiate into B cells in the presence of signals, such as IL-7 (21). After homing to the developing thymus, in which Notch ligands are abundantly expressed (26), HSCs and/or lymphoid progenitors efficiently give rise to thymic T cells, because, at this point, Notch signaling would overrule the repressive role of LRF on Notch function. However, in the absence of LRF, the low levels of Notch ligands expressed by the BM stroma would now be sufficient to activate Notch target genes normally repressed by LRF in HSCs and CLPs, thus aberrantly specifying T cell fate (Fig. 5). In support of this working model, exogenous expression of LRF in HSCs was seen to result in inefficient DN T cell production, as compared with mock-infected HSCs in an OP9-DL1 culture system (fig. S6C).

Fig. 5.

Proposed model for the role of LRF in determining B versus T lineage fate. (Left) In BM, where stromal cells express moderate levels of Notch ligands, LRF expression in HSCs and lymphoid progenitors functions to repress T cell–instructive signals produced by Notch. ICN, intracellular domain of Notch; CSL, CBF-1 (RBP-Jκ,Jκ recombining binding protein)–Suppressor of Hairless–Lag1 (Notch effector). (Right) However, once progenitors home in on the thymus, where Notch ligands are more abundantly expressed, this repressive role of LRF on Notch function is overruled (top), which allows efficient production of T cell precursors.

Our data provide strong evidence that LRF can oppose the Notch signaling pathway. Given that GSI treatment, which blocks the Notch pathway upstream, was sufficient to cause resumption of normal B versus T cell commitment in mutant HSC and CLPs, LRF likely targets upstream components of the pathway rather than repressing downstream Notch target genes. As we observed high LRF expression in NHL patients (5) and Notch signaling is known to play a tumor suppressive role in human B cell malignancies (28), it is tempting to speculate that LRF can also exert its oncogenic activity by opposing Notch function in the B cell compartment. In addition, given that LRF is widely expressed in the organism and Notch has described roles in regulating the development and differentiation of multiple tissues, the extent of LRF'srepressive role on Notch-dependent processes remains to be seen.

Supporting Online Material

www.sciencemag.org/cgi/content/full/316/5826/860/DC1

Materials and Methods

Figs. S1 to S6

References

References and Notes

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