Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim sustains B lymphopoiesis in the absence of IL-7

Abstract

IL-7 is pivotal for B cell development. Proteins of the Bcl-2 family are essential regulators of lymphocyte survival. Particularly, the pro-apoptotic BH3-only members Bim and Puma mediate lymphocyte apoptosis provoked by cytokine deprivation. Herein, we addressed whether the absence of Bim or Puma within the hematopoietic compartment could bypass the requirement for IL-7-driven B cell development in adult mice. We found that deficiency of Bim, but not Puma, partially rescued B cell development in the absence of IL-7. The numbers of both sIgM(-) and sIgM(+) B cells were markedly increased in the bone marrow of recipients lacking IL-7 upon reconstitution with Bim-deficient hematopoietic progenitors, compared with their control or Puma-deficient counterparts. The augmentation of B cell lymphopoiesis in the absence of Bim was reflected in the mature peripheral compartment by an increase in both the number of immature and mature B cells in the spleen and in the circulating IgM levels. Bim-deficient B cells were also increased in IL-7-sufficient recipients suggesting that peripheral B cells homeostasis is governed by a Bim-dependent and IL-7-independent mechanism. Our data highlight the role of Bim as a key regulator of cell survival during B lymphocyte development in vivo.

Fig. 1.

Lack of Bim, but not lack of Puma, in hematopoietic stem cells permits B cell development in the absence of IL-7. IL-7⁻ (RAG2.IL-7 DKO)-recipient mice were analyzed 9–11 weeks following reconstitution with WT, Puma KO or Bim KO LSK cells. (A) Flow cytometric analysis of bone marrow-derived single-cell suspensions. Dot plots represent three different time points from two independent experiments. (B) Quantification of total cellularity (i), numbers of total B-lineage cells (B220⁺CD19⁺) (ii), sIgM⁻ B cell progenitors (B220⁺CD19⁺IgM⁻) (iii) and sIgM⁺ B cells (B220⁺CD19⁺IgM⁺) (iv) in the bone marrow. Error bars represent the means ± SEMs of two independent experiments (n = 5–7 mice/genotype).

Fig. 2.

B cell development in IL-7-competent recipient mice. IL-7⁺ (RAG2.γ_c DKO)-recipient mice were analyzed 9–11 weeks following reconstitution with WT, Puma KO or Bim KO LSK cells. (A) Flow cytometric analysis of bone marrow-derived single-cell suspensions. Dot plots represent three different time points from two independent experiments. (B) Quantification of total cellularity (i), numbers of total B-lineage cells (B220⁺CD19⁺) (ii), sIgM⁻ B cell progenitors (B220⁺CD19⁺IgM⁻) (iii) and sIgM⁺ B cells (B220⁺CD19⁺IgM⁺) (iv) in the bone marrow. Error bars represent the means ± SEMs of two independent experiments (n = 5–7 mice/genotype).

Fig. 3.

Accumulation of Bim-deficient B cells in the spleen of IL-7-deficient mice. Flow cytometric analysis of the spleens from IL-7⁻ (RAG2.IL-7 DKO) recipients reconstituted with WT, Puma KO or Bim KO LSK cells. (A) Dot plots represent three different time points from two independent experiments. (B) B cells (B220⁺CD19⁺) were phenotypically characterized with different B cell maturation markers: immature (IgM⁺IgD⁻) and mature B cells (IgM^lowIgD⁺) (top row); follicular B cells (CD23⁺CD21^neg/low) and marginal zone B cells (CD23^neg/lowCD21⁺) (bottom row); one representative staining is shown. (C) Quantification of total cellularity and total B cells (B220⁺CD19⁺) of the spleen. (D) Quantification of B cell subsets. Error bars represent the means ± SEMs of two independent experiments (n = 5–7 mice/genotype).

Fig. 4.

Accumulation of Bim-deficient B cells in the spleen of IL-7-sufficient mice. Flow cytometric analysis of the spleens from IL-7⁺ (RAG2.γ_c DKO) recipients reconstituted with WT, Puma KO, or Bim KO LSK cells. (A) Dot plots represent three different time points from two independent experiments. (B) B cells (B220⁺CD19⁺) were phenotypically characterized with different B cell maturation markers: immature (IgM⁺IgD⁻) and mature B cells (IgM^lowIgD⁺) (top row); follicular B cells (CD23⁺CD21^neg/low) and marginal zone B cells (CD23^neg/lowCD21⁺) (bottom row); one representative staining is shown. (C) Quantification of total cellularity and total B cells (B220⁺CD19⁺) of the spleen. (D) Quantification of B cell subsets. Error bars represent the means ± SEMs of two independent experiments (n = 5–7 mice/genotype).

Fig. 5.

IL-7-deficient mice reconstituted with hematopoietic progenitors lacking Bim have higher serum IgM levels than those reconstituted with WT or Puma counterparts. Ig titers in the sera of (A) IL-7⁻ (RAG2.IL-7 DKO) and (B) IL-7⁺ (RAG2.γ_c DKO) recipients were quantified by ELISA 9-11 weeks following reconstitution with WT, Puma KO or Bim KO LSK cells. ND, not detectable. Dilutions were predetermined to produce absorbance readings in the linear range. Values were normalized to the values obtained with IL-7⁻ and IL-7⁺ recipients reconstituted with WT. Error bars represent the means ± SEMs of two independent experiments (n = 5–7 mice/genotype). (C) In vitro B cell proliferative response. CFSE-labeled splenocytes from IL-7⁻ (upper row) and IL-7⁺ (lower row) recipients following reconstitution with WT, Puma KO or Bim KO were culture in presence of LPS and IL-4. After 5 days, cultured cells were analyzed by FACS analysis. Black line: gated on B220⁺CD19⁺ cells; gray line: gated on B220⁻CD19⁻ cells. ND, not done. Data are from a representative experiment of two performed.