Ordering human CD34+CD10-CD19+ pre/pro-B-cell and CD19- common lymphoid progenitor stages in two pro-B-cell development pathways

Abstract

Studies here respond to two long-standing questions: Are human "pre/pro-B" CD34(+)CD10(-)CD19(+) and "common lymphoid progenitor (CLP)/early-B" CD34(+)CD10(+)CD19(-) alternate precursors to "pro-B" CD34(+)CD19(+)CD10(+) cells, and do the pro-B cells that arise from these progenitors belong to the same or distinct B-cell development pathways? Using flow cytometry, gene expression profiling, and Ig V(H)-D-J(H) sequencing, we monitor the initial 10 generations of development of sorted cord blood CD34(high)Lineage(-) pluripotential progenitors growing in bone marrow S17 stroma cocultures. We show that (i) multipotent progenitors (CD34(+)CD45RA(+)CD10(-)CD19(-)) directly generate an initial wave of Pax5(+)TdT(-) "unilineage" pre/pro-B cells and a later wave of "multilineage" CLP/early-B cells and (ii) the cells generated in these successive stages act as precursors for distinct pro-B cells through two independent layered pathways. Studies by others have tracked the origin of B-lineage leukemias in elderly mice to the mouse B-1a pre/pro-B lineage, which lacks the TdT activity that diversifies the V(H)-D-J(H) Ig heavy chain joints found in the early-B or B-2 lineage. Here, we show a similar divergence in human B-cell development pathways between the Pax5(+)TdT(-) pre/pro-B differentiation pathway that gives rise to infant B-lineage leukemias and the early-B pathway.

Fig. 1.

In the first wave of development, CD34^highCD10⁻CD19⁻ precursors asymmetrically self-renew and differentiate to acquire CD19 before CD10 in S17 cocultures. CFSE^highCD34^highCD10⁻CD19⁻ precursors were purified by FACS (99.8 ± 0.1% pure, n = 8) from CB mononuclear cells loaded with CFSE; labeled with CD34-, CD10-, and CD19-specific antibodies; and cultured on S17 stroma for 7 days (D7) or 10 days (D10). The CFSE^highCD34^highCD10⁻CD19⁻ progeny was stained with CD34-, CD10-, and CD19-specific antibodies and submitted to flow cytometry analyses to quantify CFSE content (numbers on top of each peak designate the number of progeny divisions, with 0 representing no division) (A) and surface CD34 levels in each generation dot-plot analysis of CD34 expression vs. CFSE content (B), using the same samples as in A [position of undivided cells is shown (▾)]. (C) Day 10 expression of CD10 and CD19 in gated CD34⁺ cells ordered by their progeny generation, as defined by CFSE levels and indicated by numbers on top of the plot. Numbers in quadrants represent the frequency of positive cells. The input cell number was 10⁴ cells per well, and average yields were 2.76 ± 0.9 × 10⁴ cells per well at D7 and 15.04 ± 3.5 × 10⁴ cells per well at D10. Cells in parallel wells were pooled, as indicated before for FACS analyses (18). Data are representative of the results from eight independent experiments.

Fig. 2.

CD34^highCD45RA⁻Lin⁻ pluripotent precursors develop into a CD34^highCD45RA^low/intLin⁻ intermediate stage that acquires either CD10 or CD19 in a mutually exclusive manner. (A) Magnetically enriched CB CD34^high cells were labeled with antibodies against CD34, CD45RA, CD10, and CD19 antigens, and CD34^highCD45RA⁻CD10⁻CD19⁻ or CD34^highCD45RA^low/intCD10⁻CD19⁻ populations gated as indicated (Left) were sorted. (Right) Reanalysis and purity of sorted cells (99.9 ± 0.1% and 99.0 ± 0.2%, respectively; n = 4). (B) CD34, CD45RA, CD10, and CD19 expression patterns in CD34^highCD45RA⁻Lin⁻ (Upper) and CD34^highCD45RA^low/intLin⁻ (Lower) progenies, respectively, after 2 weeks of S17 cocultures. The input cell number was 10³ cells per well, and the 2-week yield was 50.78 ± 6.9 × 10³ or 304.38 ± 45 × 10³ cells per well for CD34^highCD45RA⁻CD10⁻CD19⁻ or CD34^highCD45RA^low/intCD10⁻CD19⁻ progeny, respectively. Data are representative of four independent experiments.

Fig. 3.

Precursor–product relations among CD34^highLin⁻ progeny developed in vitro delineate two distinct B lineage development pathways. Highly pure CB CD34^highCD10⁻CD19⁻ precursors were cocultured on S17 stroma for 13–16 days; labeled with antibodies to CD34, CD10, and CD19 to sort CD34⁺CD10⁻CD19⁻, CD34⁺CD10⁻CD19⁺, and CD34⁺CD10⁺CD19⁻ progenitors (>99%, >99%, and 98.5 ± 0.3% pure on reanalysis, respectively); and replated on fresh S17 for an additional 1 week. (Middle) Purity of sorted populations. (Right) CD10 and CD19 expression in each sorted subset differentiated progeny. The input cell number was 10³ cells per well, and 13-day yields were 289 ± 48 × 10³, 224 ± 59 × 10³, and 72 ± 14 × 10³ cells per well for CD34⁺CD10⁻CD19⁻, CD34⁺CD10⁻CD19⁺, and CD34⁺CD10⁺CD19⁻ progenitors, respectively. Data are representative of four independent experiments.

Fig. 4.

CB alternate B-cell progenitor HCDR3. (A) V_H-D-J_H sequences from individual cells of CD34⁺CD19⁺IgM⁻ phenotype, with each segment contribution. Thirty-six cells were sorted and analyzed; all were GAPDH⁺Pax5⁺, but only 5 of 36 expressed mRNA transcripts for V_H-D-J_H rearrangements, a frequency of pre-B-I cells in this CB B-cell progenitor compartment (~14%) that is consistent with our prior report (21). Consensus HCDR3 definition is the interval between the marked (▾) TGT (92 cysteine) and TGG (103 tryptophan) codons (22). (B) Defined contribution to the sequence of V_H segments, P junctions, TdT N-region insertions, D genes, and J_H sequences to the HCDR3 length and diversity is depicted for every single cell. Sequences were analyzed using the ImMunoGeneTics software tools, which are freely available at http://www.imgt.org. That approximately three of five rearrangements were out of frame is a feature expected in sμH⁻ B-cell precursors and confirms the progenitor and unselected nature of these cell repertoires (22).