Cyclin D3 is selectively required for proliferative expansion of germinal center B cells

Abstract

The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

FIG. 1.

B cells require cyclin D3 for TD Ag-induced germinal centers. WT (•) and Ccnd3^−/− (▴) animals (A and B) or sublethally irradiated (5 Gy) μMT mice reconstituted 8 weeks earlier with bone marrow from WT (bWT) (•) or Ccnd3^−/− (bCcnd3^−/−) (▴) mice (C and D) were immunized i.p. with NP₁₉-KLH in CFA. At 10 days postimmunization, GC B-cell generation, indicated by CD95 and GL7 staining, was assessed by flow cytometry. Values indicate the gated population as a percentage of B220⁺ cells. The data shown represent two independent experiments with two to four animals per group (A and B) or four independent experiments with three to five animals per group (C and D). Graphs show the means and standard errors of the mean (SEM), along with the P value, as determined by using an unpaired t test.

FIG. 2.

Cyclin D3 is required for germinal center maturation but is dispensable for germinal center initiation. (A and B) WT and Ccnd3^−/− animals were immunized i.p. with SRBCs, and GC generation, as indicated by the presence of CD95⁺ GL7⁺ B cells, was assessed by flow cytometry 7 days postimmunization. (C and D) WT and Ccnd3^−/− animals were immunized i.p. with SRBCs, and GC initiation, as indicated by the presence of PNA⁺ B cells as a percentage of total lymphocytes (filled symbols) or as percentage of B220⁺ cells (open symbols), was assessed by flow cytometry 4 days postimmunization. (E and F) WT (•) and Ccnd3^−/− (▴) animals were immunized i.p. with SRBCs, and GC maturation, as indicated by GL7 and IgD profile of PNA⁺ B cells, was assessed by flow cytometry 4, 7, and 10 days postimmunization. The data shown represent two independent experiments with three to five animals per group (A to D) or three animals per group (E and F) per time point. Graphs show means and SEM, along with the P value, as determined by using an unpaired t test.

FIG. 3.

Cyclin D3 is specifically required for the proliferative expansion of GL7⁺ IgD⁻ mature germinal center B cells. WT and Ccnd3^−/− animals were immunized i.p. with SRBCs. At 5 days postimmunization, the animals were injected i.p. with 2 mg of BrdU. (A and B) At 6 to 7 h postinjection, the in vivo proliferation and cell cycle status of GC B cells was measured by flow cytometry. B-cell proliferation, as indicated by BrdU incorporation (upper panels) as a percentage of total lymphocytes (filled symbols) and as a percentage of B220⁺ (open symbols) and the GC maturation state (GL7, IgD profile) of proliferating B cells (B220⁺ BrdU⁺) (lower panels), was assessed by flow cytometry. (C and D) Proliferation (upper panels) and cell cycle status (lower panels) of total IgD⁻ GL7⁺ GC B cells from WT and Ccnd3^−/− animals were measured. The data shown represent at least two independent experiments with two to three animals per group. Graphs show means and SEM, along with the P value, as determined by using an unpaired t test.

FIG. 4.

Cyclin D3 is required for affinity maturation but is dispensable for early Ag-specific antibody production. bWT (•) and bCcnd3^−/− (▴) animals were immunized i.p. with NP-KLH in CFA, and serum was collected at the indicated time points. Total Ag-specific IgM and IgG (A), and affinity maturation (B), determined by the ratio of high-affinity IgG to Ag-specific IgG, was measured by ELISA. The data shown is from four independent experiments with three to five mice per group. (C and D) The generation of IgG class switched B cells from GL7⁺ and GL7⁻ GC B cells (B220⁺, PNA⁺) 14 days postimmunization was measured by flow cytometry. IgG1⁺ cells are represented as a percentage of PNA⁺ B cells. The data from five animals are shown. Graphs show means and SEM, along with the P value, as determined by using an unpaired t test.

FIG. 5.

Cyclin D3 but not cyclin D2 is expressed in germinal center B cells. (A) GC and non-GC B cells were sorted from spleens pooled from 5 to 10 WT animals at 5 days after immunization with SRBCs, as indicated. Values indicate the gated population as a percentage of total cells and represent at least three experiments. Total protein or RNA was prepared from the sorted cells and subjected to Western blot (B) or semiquantitative RT-PCR (C) analysis for cyclin D2 or cyclin D3, as indicated. BCL-6 levels in GL7⁺ IgD⁻ (solid line) and GL7⁻ IgD⁺ (dashed line) PNA⁺ GC B cells were assessed by intracellular staining and flow cytometric analysis (D). The shaded histogram indicates the PNA⁻ non-GC B-cell gate. The data shown are an average derived from at least three mice.

FIG. 6.

cAMP-PKA-GSK3 signaling regulates cyclin D3 stability. GC and non-GC B cells were sorted from spleens pooled from 5 to 10 WT animals at 5 days postimmunization with SRBCs. (A) Phosphorylation of GSK3 was measured by Western blot analysis. GC B cells were cultured for 120 min in LY294002, H-89, and/or LiCl, as indicated. (B) Cyclin D3 levels were measured by Western blot analysis. (C) Non-GC B cells were cultured for 30 min with or without LiCl, and the relative levels of cyclin D3 were measured by Western blot analysis. (D) Non-GC B cells were cultured in DB-cAMP with or without LiCl for the indicated time points, and the relative cyclin D3 levels were measured by Western blot analysis. Equal loading of the protein samples was confirmed by observation of the levels of β-actin, Erk, or Akt. The data are representative of at least three independent experiments.

FIG. 7.

Overview of cyclin D2/D3 function and regulation in the germinal center response.