Deviation of the B cell pathway in senescent mice is associated with reduced surrogate light chain expression and altered immature B cell generation, phenotype, and light chain expression

Abstract

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered Ab repertoire. This likely results, in part, from reduced surrogate L chains in senescent B cell precursors and compromised pre-BCR checkpoints. Herein, we show that aged mice maintain an ordinarily minor pool of early c-kit(+) pre-B cells, indicative of poor pre-BCR expression, even as pre-BCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, B2 B lymphopoiesis shifts from dependency on pre-BCR expansion and selection to more pre-BCR-deficient pathways. B2 c-kit(+) B cell precursors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43, and decreased kappa L chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo. We hypothesize that reduced surrogate L chain expression contributes to decreased pre-B cells in aged mice. The B2 pathway is partially blocked with limited B cell development and reduced pre-BCR expression and signaling. In old age, B2 pathways have limited surrogate L chain and increasingly generate new B cells with altered phenotype and L chain expression.

FIGURE 1. λ5 knockout mice show loss of c-kit^-, but not c-kit⁺ early pre-B cells in vivo

(A) Representative dot plots of c-kit⁺ and c-kit^- precursors in wild-type (B6) (WT) and λ5 knockout (KO) mice. B220⁺CD43⁺ are pro-B/early pre-B cells and late pre-B cells are B220⁺CD43^- with percentages out of total bone marrow shown. Pro-B/early pre-B cells were further gated as surface IgM^-CD19⁺ with percent cells present in total bone marrow indicated. Surface IgM^-CD19⁺B220⁺CD43⁺ B cell precursors were gated based on c-kit and cytoplasmic μ (cμ) with proportion indicated for c-kit⁺cμ^- pro-B, c-kit⁺cμ⁺ early pre-B, and c-kit^-cμ⁺ early pre-B subsets. (B) Analysis of c-kit⁺ pro-B, c-kit⁺ and c-kit^- early pre-B, and late pre-B cell numbers for 7 pairs of WT and λ5 KO mice. *p<0.02.

FIGURE 2. Aged mice show loss of c-kit^-, but not c-kit⁺ early pre-B cells in vivo

(A) Representative dot plots of c-kit⁺ and c-kit^- precursors in young (Yg) (2-4mo) and aged BALB/c mice (21-26mo). B220⁺CD43⁺ are pro-B/early pre-B cells and late pre-B cells are B220⁺CD43^- with percentages out of total bone marrow shown. Pro-B/early pre-B cells were further gated as surface IgM^-CD19⁺ with percent cells present in total bone marrow indicated. Surface IgM^-CD19⁺B220⁺CD43⁺ B cell precursors were gated based on c-kit and cytoplasmic μ (cμ) with proportion indicated for c-kit⁺cμ^- pro-B, c-kit⁺cμ⁺ early pre-B, and c-kit^-cμ⁺ early pre-B subsets. (B) Analysis of c-kit⁺ pro-B, c-kit⁺ and c-kit^- early pre-B, and late pre-B cell numbers for 6 pairs of young and old mice. *p<0.02.

FIGURE 3. Loss of λ5 protein in aged mice occurs at pro-B and pre-B cell stages

(A) Cytoplasmic λ5 protein was stained and analyzed by flow cytometry in bone marrow c-kit⁺cμ^- pro-B, c-kit⁺cμ⁺ early pre-B, and c-kit^-cμ⁺ early pre-B cells in young and old BALB/c mice. Data shown are representative of 3 mice. Grey line=isotype control. Percent positive cells was determined with a cut-off as <2% positives in isotype controls. The change in mean fluorescent intensity (ΔMFI) was defined as (MFI experimental-MFI isotype control) and is shown in the upper left. (B) λ5 protein expression was determined in CD2^-cμ^- pro-B cells generated from young and old BALB/c mice upon bone marrow cell culture with IL-7 for 5-7 days. CD2^- pro-B cells were separated by MACS sorting prior to lysate preparation. Proteins were assayed by Western blot at 2.5, 5, and 10 ×10⁵ cell equivalents. Data are representative of 3 experiments.

FIGURE 4. C-kit⁺ and c-kit^- precursors from λ5 knock-out and wild-type mice proliferate and differentiate into B cells in vitro

Panels A-C: Bone marrow cells from WT (B6) and λ5 KO mice were MACS sorted as IgM^-CD19⁺ B cell precursors and cultured for 4 days in the presence of IL-7 (5ng/ml) and stem cell factor (SCF) (50ng/ml). Panels E-F: Bone marrow from young BALB/c mice were pooled and IgM^-CD19⁺AA4.1⁺B220⁺CD43⁺c-kit⁺ and IgM^-CD19⁺AA4.1⁺B220⁺CD43⁺c-kit^- B cell precursors were FACS sorted and cultured for 4 days in the presence of IL-7 (5ng/ml) and SCF (50ng/ml). (A,D) Stimulation index (SI) (output CD19⁺ cells/input CD19⁺ cells) of cultures initiated with precursors from either WT or λ5 KO mice (A) or BALB/c precursor populations (D). (B,E) Representative dot plot of B cells generated from either WT or λ5 KO precursors (B) or BALB/c precursor populations (E). (C,F) Relative B cell output from precursors of WT and λ5 KO mice (C) or BALB/c precursor populations (F). Relative B cell output was determined as SI x %B cells in culture. Data are summarized for 6-8 experiments. *p<0.02.

FIGURE 5. B cells derived from young and aged c-kit⁺ precursors show increased CD43 in vitro

(A) Representative histograms of CD43 expression on B cells derived from WT (B6) and λ5 KO precursors (upper panels), as described in Fig. 4, and from young and aged BALB/c c-kit⁺ and c-kit^- precursors (middle and lower panels), on day 4 of IL-7/SCF culture (grey line=isotype control). (B) Comparison of percent CD43^hi B cells derived from WT (B6) and λ5 KO precursors and isolated young and aged BALB/c c-kit⁺ and c-kit^- precursors. CD43 “high” B cells (CD43^hi) were determined as those with fluorescence greater than isotype controls as indicated in (A) * p<0.05 for each pair.

FIGURE 6. B cells derived from c-kit⁺ precursors from young, aged, or λ5 knockout mice show altered light chain expression in vitro

(A) Representative histograms show κ light chain expressing B cells derived in culture from WT (B6) and λ5 KO sIgM^-CD19⁺ bone marrow, and young (Yg) and aged BALB/c isolated c-kit⁺ and c-kit^- precursors cultured as indicated in Fig. 4. Data are representative of 3-6 experiments. (B) Summary of light chain expression on B cells derived in culture for 3-6 mice per group. *p<0.05.

FIGURE 7. Immature B cells in aged BALB/c and λ5 knockout bone marrow exhibit altered light chain expression in vivo

(A) Representative histograms show κ light chain expressing immature (AA4.1⁺) bone marrow B cells from young and old BALB/c mice and WT (B6) and λ5 KO mice in vivo. (B) Summary of light chain expression on immature bone marrow B cells from young and aged BALB/c mice and WT (B6) and λ5 KO mice. (C) Relative incidences of light chain expression among CD43^- and CD43⁺ immature B cells in young and aged BALB/c bone marrow. (D) Bone marrow (BM) immature B cells (sIgM⁺ AA4.1⁺ CD19⁺) and splenic (SP) transitional B cells (sIgM⁺ AA4.1⁺ CD19⁺) from λ5 KO and aged mice were further evaluated for κ and λ dual light chain expression. Aged mice were chosen that had substantial increases in dual light chain isotype expressing immature B cells in their bone marrow for comparison with those in spleen. Data are cumulative for 3-8 mice. *, p<0.05.

FIGURE 8. Surrogate light chain down-regulation in aged B cell precursors alters B2 B lymphopoiesis and promotes development of new B cells with distinct surface phenotype and light chain isotypes

Young Mice: Pro-B cells undergo Vh-Dh-Jh recombination (step 1); preBCR competency is dictated by Vh-Dh-Jh sequence. PreBCR signaling results in down-regulation of c-kit and expansion and population at the late pre-B cell stage (step 2). A minor proportion of early pre-B cells retains c-kit and express μ heavy chains that assemble poorly with surrogate light chains (SL). (Step 3) Late pre-B cells differentiate into immature B cells with most derived from preBCR competent precursors. Immature B cells with increased CD43 and altered light chain expression derived from c-kit⁺ pre-B cells are a minor contributor to the immature B cell pool. Aged Mice: SL and preBCR expression is low and overall pre-B cell development is reduced (step 4). C-kit⁺ pre-B cells (step 5) generate immature B cells with increased CD43⁺ and altered light chain patterns at increased frequencies (step 6).