Reduced rag2 expression in pro–B cells is consistent with lower numbers of pre–B cells in aged mice. Bone marrow from young (2–3.5 mo) and aged (26–27 mo) NG mice and wild-type controls (both [FVBN × CBA]F1 background) was harvested and analyzed by flow cytometry as described in Materials and Methods. (A) Flow cytometric analysis of GFP expression (a reporter of rag2 expression in NG mice) and forward light scatter (FSC) within pro–B cells (B220^LOCD43⁺AA4.1⁺) from young and old NG mice. The numbers within the gates depict the percent of pro–B cells that express GFP. Bone marrow pro–B cells from representative wild type, young NG, aged NG with a moderate loss of GFP expression, and an aged NG with a severe loss of GFP expression are displayed. (B) Numbers of pro–B (B220^LOCD43⁺AA4.1⁺) and pre–B cells (B220^LOCD43⁻AA4.1⁺CD24²⁺) in bone marrow of young and aged NG mice. White bars represent pro–B cells, and black bars represent pre–B cells. The pre:pro ratio is calculated by dividing the number of pre–B cells by the number of pro–B cells. (C) Flow cytometric analysis of GFP in pro–B cells from young (black bars) and aged NG mice (gray bars). The percent of pro–B cells that are GFP⁺ are shown for the same mice depicted in B. (D) The percent of pro–B cells that are GFP⁺ and the pre:pro ratio are displayed for each NG mouse shown in B and C. Closed diamonds represent young mice, and open diamonds represent aged mice.

rag2 expression, V(D)J recombinase activity, and the pre:pro ratio are reduced in aged mice. CD45.1 NG × H2-SVEX double transgenic mice were generated (GFP serves as a reporter of rag2 expression and VEX serves as a reporter of V[D]J recombinase activity) and used as donors for adoptive transfer. Bone marrow was harvested from wild-type CD45.2 young (3–4 mo at time of harvest) and aged (23–29 mo at time of harvest) recipient mice 5–6 wk after adoptive transfer of bone marrow from NG × H2-SVEX CD45.1 mice. Pro–B (IgM⁻B220⁺CD43⁺) and pre–B cells (IgM⁻B220⁺CD43⁻) of donor origin were identified based on expression of CD45.1. The left panel displays the mean, standard deviation, and distribution of the percent of donor origin pro–B cells that are GFP⁺, whereas the middle panel depicts these values for VEX⁺ donor pro–B cells. The right panel displays the mean, standard deviation, and distribution of the pre:pro ratio. The pre:pro ratio was calculated by dividing the percent of bone marrow that was donor pre–B cells by the percent of bone marrow that was donor pro–B cells for each recipient mouse. Y, young mice; A, aged mice.

Magnitude, renewal rates, and production rates of B lineage progenitor pools in aged and young adults. (A) Bone marrow was harvested from either young adult (black bars) or aged mice (white bars) of the strains and F1 combinations indicated and stained as described in Materials and Methods. The proportions of pro–B (IgM⁻B220⁺CD43⁺), pre–B (IgM⁻B220⁺CD43⁻), and immature B (IgM⁺B220^LO) subsets were assessed using flow cytometry and multiplied by the marrow cell estimate of Osmond (reference 50) to obtain total numbers. Bars show mean ± SD of samples from 10 to 30 mice, depending on strain. **, statistical significance for young versus old (Student's t test; P < 0.05). (B) Young adult (diamonds) or aged (squares) C57BL/6 mice were injected with 0.6 mg BrdU at 12-h intervals. Bone marrow was harvested at various times after the onset of labeling and stained for surface phenotype and BrdU incorporation as described in Materials and Methods. The proportion of BrdU-labeled cells (top) was determined using flow cytometry, and the numbers of BrdU-labeled cells (bottom) were calculated by multiplying these proportions by the Osmond estimate of total marrow cells. Labeling among pro–B, pre–B, and immature B cell subsets is shown in the left, middle, and right plots, respectively. Each point represents an individual mouse. Solid and dashed lines (young and aged, respectively) are those determined by linear regression.

Reduced RAG2 protein levels in pro–B cells are consistent with lower numbers of pre–B cells in aged RAG2-GFP KI mice. Bone marrow from young (4–7 mo) and aged (23–28 mo) RAG2-GFP KI mice was analyzed by flow cytometry. (A) Representative flow cytometric analysis of GFP in pro–B cells (Ly6C⁻DX5⁻IgM⁻B220⁺CD43⁺) of RAG2-GFP KI mice. Bone marrow from young wild type, young RAG2-GFP KI, aged RAG2-GFP KI with a moderate loss of GFP expression, and an aged RAG2-GFP KI with severe loss of GFP expression are displayed. The numbers within the gates depict the percentage of pro–B cells that express GFP. (B) Numbers of pro–B (Ly6C⁻ DX5⁻IgM⁻B220⁺CD43⁺) and pre–B cells (Ly6C⁻DX5⁻IgM⁻B220⁺CD43⁻) in bone marrow of young and aged mice. White bars represent pro–B cells, and black bars represent pre–B cells. The pre:pro ratio was calculated by dividing the number of pre–B cells by the number of pro–B cells. (C) Analysis of GFP in pro–B cells of RAG2-GFP KI mice was conducted as in A. Black bars represent young mice, and gray bars represent aged mice. The percentage of pro–B cells that are GFP⁺ are displayed for the same mice shown in B. (D) The percentage of pro–B cells that are GFP⁺ and the ratio of pre–B to pro–B cells are displayed for each KI mouse shown in B and C. Closed diamonds represent young mice, and open diamonds represent aged mice.

Pro–B cells derived from young and aged sources display similar rag2 expression and pre:pro ratios in young hosts. Bone marrow from young (3 mo) and aged (26–27 mo) NG mice were transferred into lethally irradiated young (2 mo at time of transfer) recipient mice. (A) GFP expression in pro–B cells (left) and pre:pro ratio (right) in young and aged donor mice at time of transfer. These mice are four of the six young and all of the aged mice shown in Fig. 1. Black bars represent young donor mice, and diagonally striped bars represent aged donor mice. To aid comparisons, each young donor mouse is given a designation, Y1-Y4, and each aged donor mouse was given A1-A5, depicted below each panel. (B) Analyses of bone marrow harvested from young recipient mice 5 wk after transfer of cells from either young or aged donors. Pro–B cells were defined as IgM⁻B220⁺CD43⁺AA4.1⁺ and pre–B cells were defined as IgM⁻B220⁺CD43⁻AA4.1⁺CD24⁺. The percentage of pro–B cells that are GFP⁺ and the pre:pro ratios are shown. The pre:pro ratio of two young unmanipulated NG mice used as controls in this experiment were 1.2 and 1.3. Each bar represents values obtained from one recipient mouse. The designations Y1-Y4 and A1-A5 reflect the source of the transferred bone marrow as shown in A, and brackets show the groups of recipients according to the donor designation.