Reduced production of B-1-specified common lymphoid progenitors results in diminished potential of adult marrow to generate B-1 cells

Abstract

B-1 B cells have been proposed to be preferentially generated from fetal progenitors, but this view is challenged by studies concluding that B-1 production is sustained throughout adult life. To address this controversy, we compared the efficiency with which hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs) from neonates and adults generated B-1 cells in vivo and developed a clonal in vitro assay to quantify B-1 progenitor production from CLPs. Adult HSCs and CLPs generated fewer B-1 cells in vivo compared with their neonatal counterparts, a finding corroborated by the clonal studies that showed that the CLP compartment includes B-1- and B-2-specified subpopulations and that the former cells decrease in number after birth. Together, these data indicate that B-1 lymphopoiesis is not sustained at constant levels throughout life and define a heretofore unappreciated developmental heterogeneity within the CLP compartment.

Fig. 1.

B-1 potential of neonatal and adult HSCs. HSCs (500–1,000) isolated from the bone marrow of neonatal (2.5 wk) and adult (15 wk) mice were injected i.v. in vivo, and the generation of donor (CD45.2) sIgM^high CD11b⁺ CD5⁺ B-1a and sIgM^high CD11b⁺ CD5⁻ B-1b cells in the peritoneal cavity was evaluated 6 wk later. (A) Resolution of HSCs in bone marrow based on their Lin^– CD117^high Sca-1^high CD150⁺ phenotype. The frequency of Lin^– Sca-1⁺ c-kit⁺ cells in total bone marrow is indicated in Left. The Lin^– Sca-1⁺ c-kit⁺ cells were also gated according to the phenotype shown on the plot in Right. (B) Distribution of B-1a, B-1b, and B-2 cells in the peritoneal cavity of a young (6 wk) WT B6 mouse. The frequency of the respective populations among total sIgM⁺ cells is indicated. (C) Representative FACS plots of donor B-1 reconstitution in the peritoneal cavity of recipients of neonatal and adult HSCs. The frequency of the respective populations among total sIgM⁺ cells is indicated. (D) Frequency of donor B-1a and B-1b cells in the peritoneal cavity of recipient mice 6 wk after reconstitution with neonatal and adult HSCs. (E) Total number of donor B-1a and B-1b cells in the peritoneal cavity of recipient mice 6 wk after reconstitution with neonatal and adult HSCs. Five recipients of neonatal and six recipients of adult HSCs were analyzed. *P ≤ 0.05; **P ≤ 0.01.

Fig. 2.

B-1 potential of neonatal and adult CLPs. CLPs (5 × 10³–1 × 10⁴) isolated from the bone marrow of neonatal (2.5 wk) and adult (15 wk) mice were injected i.v. in vivo, and the generation of donor (CD45.2) sIgM^high CD11b⁺ CD5⁺ B-1a and sIgM^high CD11b⁺ CD5⁻ B-1b cells in the peritoneal cavity was evaluated 6 wk later. (A) Resolution of CLPs in bone marrow based on their Lin^– CD117^low Sca-1^low CD127 (IL-7R)⁺ phenotype. The frequency of Lin^– Sca-1^low c-kit^low cells in total bone marrow is indicated in Left. Those cells were also gated based on expression of CD127 (Right). (B) Representative FACS plots of donor B-1 reconstitution in the peritoneal cavity of recipients of neonatal and adult CLPs. The frequency of the respective populations among total sIgM⁺ cells is indicated. (C) Frequency of donor B-1a and B-1b cells in the peritoneal cavity of recipient mice 6 wk after reconstitution with neonatal and adult CLPs. (D) Total number of donor B-1a and B-1b cells in the peritoneal cavity of recipient mice 6 wk after reconstitution with neonatal and adult CLPs. Six recipients of neonatal and five recipients of adult CLPs were analyzed. *P ≤ 0.05; **P ≤ 0.01.

Fig. 3.

Development of a clonal assay to quantify B-1 progenitor production. (A) Fifty CLPs from bone marrow of 4-wk-old mice were seeded into wells, and 4 d later, cells were harvested and phenotyped. The FACS plot shows that the culture conditions support the development of both B-1 and B-2 progenitors in the same well. The CD45R^−/low CD19⁺ B-1 and CD45R⁺ CD19^low/– B-2 progenitors harvested from the in vitro cultures 5 d later were resolved based on labeling of comparable populations in fresh bone marrow samples shown in Fig. 4C. (B) Replicate wells seeded with 50 CLPs were analyzed at 9 d after initiation of the cultures. CD19⁺ CD45R^low/− B-1 progenitors matured into CD19⁺ CD45R^dim cells, and the CD19^– CD45R⁺ B-2 progenitors acquired a B220⁺ CD19⁺ phenotype. (C) Single CLPs from bone marrow of neonatal (2.5 wk) or adult (15 wk) mice were seeded into wells, and the phenotype of the cells was determined 4–6 d later. Values on the plots indicate the frequency of the different progenitor cell populations in the cultures.

Fig. 4.

Clonal analysis of CLP developmental potential. (A) Frequency of wells seeded with single neonatal or adult CLPs in which B-1 and B-2 progenitors were observed. The number of positive wells over the number of wells seeded is indicated. (B) Single CLPs from bone marrow of 2.5- and 15-wk-old mice were seeded into wells. The number of B-1 progenitors per well produced from the different aged CLPs was determined 6 d later. (C) Frequency of Lin^– CD45R^−/low CD19⁺ B-1 progenitors among Lin^– CD93⁺ cells in E15 fetal liver and bone marrow of 6-wk-old WT mice. *P ≤ 0.05; **P ≤ 0.01.

Fig. 5.

Two models to explain the attenuated potential of adult CLPs to generate B-1 progenitors. In model 1, both neonatal and adult HSCs generate CLPs that can differentiate into B-1 or B-2 progenitors, but B-1 potential declines within the first few weeks after birth. In model 2, neonatal and adult HSCs generate B-1– or B-2–specified CLPs, with the production of the B-1–specified CLPs predominating during neonatal life and declining in the adult.