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. 2006 Mar 20;203(3):675-87.
doi: 10.1084/jem.20052444. Epub 2006 Feb 27.

Lineage specification and plasticity in CD19- early B cell precursors

Lynn L Rumfelt  1 , Yan Zhou, Benjamin M Rowley, Susan A Shinton, Richard R Hardy
Affiliations

Lineage specification and plasticity in CD19- early B cell precursors

Lynn L Rumfelt et al. J Exp Med. .

Abstract

We describe here three CD19- B cell precursor populations in mouse bone marrow identified using 12-color flow cytometry. Cell transfer experiments indicate lineage potentials consistent with multilineage progenitor (MLP), common lymphoid progenitor (CLP), and B lineage-restricted pre-pro-B (Fr. A), respectively. However, single cell in vitro assays reveal lineage plasticity: lymphoid/myeloid lineage potential for CLP and B/T lineage potential for Fr. A. Despite myeloid potential, recombination activating gene 2 reporter activation is first detected at low levels in most MLP cells, with 95% of CLPs showing 10-fold increased levels. Furthermore, single cell analysis shows that half of CLP and 90% of Fr. A cells contain heavy chain DJ rearrangements. These data, together with expression profiles of lineage-specific genes, demonstrate progressive acquisition of B lineage potential and support an asynchronous view of early B cell development, in which B lineage specification initiates in the MLP/CLP stage, whereas myeloid potential is not lost until the pre-pro-B (Fr. A) stage, and B/T lymphoid plasticity persists until the CD19+ pro-B stage. Thus, MLP, CLP, and Fr. A represent progressively B lineage-specified stages in development, before the CD19+ B lineage-committed pro-B stage.

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Figures

Figure 1.
Figure 1.
Identification of MLP, CLP, pre-pro–B (Fr. A), pro-B (Fr. B and C), and early pre–B (Fr. C′) stages using a combination of fluorescent reagents and 12-color flow cytometry. The staining reagents are described in Materials and methods. Two gating approaches are used to discriminate these stages: one for the three earliest, shown on the left, and another for three later stages, shown on the right. 5 × 105 bone marrow cells were analyzed. Data shown are representative of more than 10 independent analyses using 6–12-wk-old BALB/c female mice.
Figure 2.
Figure 2.
B, T, NK, and myeloid engraftment from MLP stage cells, in contrast with predominant lymphoid repopulation using CLPs, and predominant B cell repopulation using Fr. A. Cell populations defined as in Fig. 1 were isolated by cell sorting from B6.Ly5.2 mouse bone marrow and injected i.v. together with unfractionated bone marrow from B6 wild-type (Ly5.1) mice into lethally irradiated B6 mice. After 3 wk, recipient animals were killed and indicated tissues were analyzed by flow cytometry for the presence of Ly5.1/Ly5.2 cells in B (B220+CD19+IgM+), T (CD4/C8+CD3+), NK (NK1.1/DX5+), and myeloid (CD11b/GR1+) cell populations. (A) Percent engraftment reported is frequency of Ly5.2+ cells divided by total cell frequency for the indicated population. (B) Absolute numbers of Ly5.2+ cells of the indicated cell type were determined and then compared with the number obtained with MLP stage cells (defined as 1.0 for each cell type). Error bars show standard error for analyses of 6–10 individual recipients from four separate experiments.
Figure 3.
Figure 3.
Bipotential stromal cell assay reveals unexpected myeloid capacity in CLP stage cells that is lost upon progression to the B220+ (Fr. A) stage. (A) Individual cells from the indicated population were deposited into wells of a 96-well flat-bottom plate containing preestablished S17 stromal cells in complete RPMI 1640 medium, supplemented with SCF, Flt3L, and IL-7. After 7–10 d, plates were examined for cell colonies and these were harvested and analyzed by flow cytometry. Colonies were classified as B (CD11b−CD19+), myeloid (CD11b+CD19−), or other (CD11b−CD19−). No mixed colonies were observed. Representative data from more than four separate experiments is shown. (B) A similar analysis was performed with cells sorted from the bone marrow of RAG-2–GFP reporter mice, selecting the GFP intermediate (+) or brightest (++) MLP stage cells, or the GFP-high (+++) CLP or Fr. A stage cells. Gated regions are marked on the GFP histograms in Fig. 4. Representative data from more than four separate experiments is shown.
Figure 4.
Figure 4.
(A) Detection of RAG-2 gene transcriptional activation very early in hematopoietic development. LIN− cells, gated to eliminate CD43− pre–B and later stage B lineage cells, were analyzed for expression of RAG-2–GFP and CD93. Vertical bar indicates the demarcation between positive and negative GFP levels. (B) Comparison of levels of RAG-2–GFP in early stages of B lineage development in mouse bone marrow. The level of green fluorescence detected in RAG-2–GFP transgenic− littermates is shown for each cell subset analyzed. Numbers on the plots show the mean fluorescence intensity for each population. Sort gates for the cells analyzed in B/Myeloid bipotential assays (Fig. 3 B) are indicated. Representative data of three separate analyses is shown.
Figure 5.
Figure 5.
Comparable T cell potential in CLP and Fr. A stage cells. (A) Three cells of the indicated population were deposited into wells containing a fetal thymic lobe. Plates were then maintained under high oxygen submersion FTOC conditions in the presence of SCF and IL-7. Wells were analyzed after 2 wk and evaluated for the presence of CD90/Thy1.2+CD25+ early T lineage cells or CD19+ B lineage cells. B lineage cells were not efficiently generated under the conditions used. Data from five separate experiments is shown. (B) Individual CLP and Fr. A cells were deposited by electronic cell sorting into microplate wells containing DL1-OP9 or GFP-OP9 (control) stromal cells and the type of cells generated was assayed 7–10 d later. B and T lineage cells were identified as described above. “Other” stands for not falling into either gate.
Figure 6.
Figure 6.
Real-time quantitative RT-PCR analysis of gene expression in early B lineage fractions isolated from mouse bone marrow. Results show average and standard error for three separate sorted samples. For each gene, maximum expression observed in the four fractions tested is set to 1.0. (A) B lineage–associated or –restricted genes are expressed from very early stages of hematopoietic development. (B) Gene expression in MLP stage cells compared with that in mature recirculating (Fr. F) B cells, showing readily detectable levels of mRNA for TdT and RAG-2 at this very early stage. (C) Patterns of expression of transcription factors indicate an ordered sequence of gene activation early in B cell development. (D) Genes associated with other hematopoietic lineages (Csf1R, C/EBPα, myeloid; Notch-1, GATA3, and T lineage) show diminishing expression as cells progress from CLP to Fr. A to CD19+ Fr. B.
Figure 7.
Figure 7.
Cluster analysis reveals distinct patters for 1,000 genes (out of ∼40,000 features on the slides used) whose expression varies within the four early B lineage stages analyzed in this work. Heat map display of expression shows relative level for each individual gene: green, low; black, medium; red, high. Six patterns of expression, including those present at high levels early that are down-regulated (clusters D and E), those up-regulated at the CLP and Fr. A stages (cluster A), and those up-regulated with B cell development (clusters B and C). Cluster B includes many genes considered important in B cell development, such as RAG-1, BLNK, CD19, CD79a, CD79b, EBF, and VpreB.
Figure 8.
Figure 8.
Diagram of early stages in B lineage development, indicating extent of DHJH rearrangement, expression of key transcription factors, expression of a RAG-2–GFP reporter transgene, extent of B/T/myeloid repopulating capacity, and lineage plasticity in vitro.
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