Commitment to the B lymphoid lineage occurs before DH-JH recombination

Abstract

Lineage commitment in B lymphopoiesis remains poorly understood due to the inability to clearly define newly committed B lineage progenitors and their multipotential descendants. We examined the potential of three recently described progenitor populations in adult mouse bone marrow to differentiate into each hematopoietic lineage. The earliest of these, termed fraction (Fr.) A0, exhibited myeloid, erythroid, and B and T lymphoid progenitor activity and included individual cells with myeloid/B lymphoid potential. In sharp contrast, two later populations, termed Frs. A1 and A2 and characterized by surface B220 expression and transcription of the germline immunoglobulin heavy chain (IgH) locus, lacked progenitor activity for all hematopoietic lineages except B lymphocytes. These observations, together with single cell polymerase chain reaction analysis showing a lack of DHJH rearrangements in each population and experiments showing identical precursor potentials when these populations were derived from recombination activating gene (Rag)-1(-/-) and JH-/- mice, demonstrate that commitment to the B lymphoid lineage occurs before and independently of VHDHJH recombination.

Figure 1

Early B lineage precursor cell populations in adult BM. (A) Four-color immunofluorescence analyses reveal the three indicated populations. (B) 10⁴ sorted cells from Fr. A₀, A₁, or A₂ from adult BALB/c BM were cultured for 4 or 8 d on S17 stromal cells with 100 U/ml rIL-7, and the number of B220⁺ HSA^int pro-B cells was determined by flow cytometry.

Figure 2

Surface molecule expression among defined B lineage subsets. Five-color (Frs. A₀–A₂) or four-color (Frs. B/C) immunofluorescence of adult BM cells. For Frs. A₀–A₂, cells were stained simultaneously with antibodies to CD4, B220, HSA, AA4.1, and the indicated surface molecule, and expression of the indicated surface molecule on the indicated population was determined as described in Materials and Methods. For Frs. B/C, cells were stained with antibodies to CD43, HSA, B220, and the indicated surface molecule, and expression was determined by gating on CD43^low B220^low HSA^int cells. BM cells were from BALB/c mice except for Sca-1/Ly6C, for which C57BL/6 mice were used.

Figure 3

Myeloid lineage progenitor activity in Frs. A₀, A₁, and A₂. (A) Expression of c-fms, lysozyme, and μ₀ among Frs. A₀, A₁, and A₂ was determined by RT-PCR, and expression levels were normalized to β-actin expression. Data are from three separate experiments. (B) Cultures established at limiting dilution using 1, 100, and 10 cells/well for Frs. A₀, A₁, and A₂, respectively, were stained at day 14 for CD45R/B220 and CD11b expression. The numbers of wells containing exclusively B220⁺ CD11b⁻ cells, B220⁻ CD11b⁺ cells, or both are shown.

Figure 4

T lymphocyte progenitor activity in Frs. A₀, A₁, and A₂ from C57BL/6 or J_H ^−/− mice. Intrathymic transfers of the indicated BM fraction 19 d after transfer. 10⁴ sorted cells from C57BL/6 (open symbols) or J_H ^−/− (filled symbols) (both Ly5.1⁺) mice were transferred into each irradiated B6.Ly5.2 recipient, and the number of Ly5.1⁺ thymocytes was determined.