Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci

Abstract

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.

Figure 1

Surface phenotype and cell cycle status of pre-B cell subpopulations as defined by surface expression of V_preB, CD10, CD34, and κL and λL chains. Bone marrow lymphoid cells from a young donor were stained with purified anti-V_preB, BIO-labeled anti-CD10, and FITC-labeled anti–κL/λL chain antibodies, followed by incubation with streptavidin TRICOLOR and PE-labeled anti–mouse IgM, as described in Materials and Methods (left panel). Cells were gated for lymphoid cells. IgM⁻ (κL⁻/λL⁻) (R1) CD10⁺ lymphoid precursors were FACS^® sorted in two fractions (R2 and R3), and based on the surface expression of V_preB, were FACS^® sorted and further analyzed for CD34 expression. A comparable separation could be achieved using CD19⁻ instead of CD10⁻ specific antibody, since we had seen that sIgM⁻ CD10⁺ CD19⁺ cells coexpressed CD34, sIgM⁻ CD34⁺ CD10⁺ cells coexpressed CD19, and sIgM⁻ CD34⁺ CD19⁺ cells coexpressed CD10. CD34⁺ CD10⁺ CD19⁻ cells were not detectable. Fraction R2 was further subdivided into CD34⁺V_preB ⁺ (R4) and CD34⁻V_preB ⁺ (R5). Cellular fractions R4, R5, and R3 were FACS^® sorted, fixed, and stained intracellularly with FITC-labeled antibodies for intracytoplasmic TdT and μH chain expression, as described in Materials and Methods. To analyze TdT and μH chain expression in cells of fraction R4, a separate staining and sorting with BIO-labeled anti-CD10 and PElabeled anti-CD34 had to be performed. V_preB expression was tested by staining with purified anti-V_preB, followed by incubation with FITC-labeled anti–mouse IgM. The fraction (R3) was also subdivided based on CD34 expression and forward and side scatter profile (R6 and R7). The subpopulations R4, R5, R6, and R7 were sorted, fixed, and treated to prepare nuclei for analyses of DNA content, as described in Materials and Methods.

Figure 2

(a) Frequencies of CD19⁺ CD10⁺ B lineage cells and their subpopulations among total lymphoid cell compartment in human bone marrow of donors of different ages (one donor = one symbol). The frequencies of these subpopulations among all CD19⁺ CD10⁺ cells are also given. (b) Frequencies of CD19⁺CD10⁻sIgM⁺ mature B cells in the lymphoid compartments of human bone marrow, and their relative contribution to all CD19⁺ cells in human bone marrow of donors of different ages.

Figure 2

(a) Frequencies of CD19⁺ CD10⁺ B lineage cells and their subpopulations among total lymphoid cell compartment in human bone marrow of donors of different ages (one donor = one symbol). The frequencies of these subpopulations among all CD19⁺ CD10⁺ cells are also given. (b) Frequencies of CD19⁺CD10⁻sIgM⁺ mature B cells in the lymphoid compartments of human bone marrow, and their relative contribution to all CD19⁺ cells in human bone marrow of donors of different ages.

Figure 3

Scheme of human B cell differentiation in bone marrow. Two alternative pathways of immature B cell generation are shown (see Discussion). The dotted arrow between small pre-B II and sIgM⁺ immature B cells in alternative II indicates that the RAG-expressing pre-B II cells continue to rearrange κL and λL chain loci (21). G, germline configuration. For a comparison to mouse B cell development, see Rolink et al. (36).