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. 2016 Mar 1;196(5):2195-204.
doi: 10.4049/jimmunol.1501690. Epub 2016 Jan 25.

Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells

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Free PMC article

Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells

Ana V Miletic et al. J Immunol. .
Free PMC article

Abstract

Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.

Conflict of interest statement

DISCLOSURES

The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1. Survivin expression is required for B cell development in the bone marrow
(A) Survivin expression in: (1) pro- and large pre-B cells (CD43+, B220+, IgM, IgD), (2) small pre-B cells (CD43, B220+, IgM, IgD), (3) immature B cells (CD43, B220+, IgM+, IgD), (4) mature recirculating cells (CD43, B220+, IgM+, IgD+), (5) GC B cells (CD11c, CD43, IgD) and (6) non GC B cells (CD11c, CD43, GL7) determined by western blot. Actin was used as a loading control. One of two independent experiments is shown. (B) Survivin expression in (1) unstimulated B cells or treated with (2, 3) 10µg/mL anti-IgM (intact or F(ab’)2 fragment, (4) 10 µg/mL LPS, (5) 5 µg/mL CpG (6) 5 µg/mL anti-CD40, (7) 25 ng/mL BAFF or (8) 100ng/mL APRIL (C) Flow cytometric analysis of splenocytes from survivinL/Lmb1Cre, survivinL/+mb1Cre, and survivin+/+mb1Cre mice with indicated antibodies. (D) Number of total splenocytes and splenic B cells from survivinL/Lmb1Cre, survivinL/+mb1Cre, and survivin+/+mb1Cre mice. (E) Flow cytometric analysis of early B cell compartment in the BM of survivinL/Lmb1Cre and survivin+/+mb1Cre mice. (F) Absolute numbers of B lineage cells at each stage of maturation in the BM of survivinL/Lmb1Cre and survivin+/+mb1Cre mice. (G) Cell cycle analysis of pro- and pre- B cells from survivinL/Lmb1Cre and survivin+/+mb1Cre mice. Results are representative of 2 independent experiments.
Figure 2
Figure 2. Survival of mature B cells is survivin-independent
(A) Flow cytometric analysis of splenocytes from survivinL/Lcd21Cre and survivin+/+cd21Cre mice with indicated antibodies. (B) Enumeration of splenocytes and splenic B cells in survivinL/Lcd21Cre and survivin+/+cd21Cre mice. (C) Immunohistology of spleens from survivinL/Lcd21Cre and survivinL/+cd21Cre mice. Red = B220+ B cells; Blue = CD3+ T cells; Green = moma-1+ macrophages. (D) SurvivinL/Lcd21Cre and survivin+/+cd21Cre mice were continuously provided BrdU in the drinking water for a 7 week period. The graphs show the percentage of BrdU-positive cells in the indicated B cell subpopulations in the spleen and BM. (E) Deletion efficiency of survivin in LPS (10 µg/mL) stimulated B cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice after 2 days of culture.
Figure 3
Figure 3. Survivin is required for the production of natural antibodies
(A) Total serum IgM and (B) total serum IgG levels from unimmunized survivinL/Lcd21Cre and survivin+/+cd21Cre mice. (C) Flow cytometric analysis of peritoneal cavity cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice with indicated antibodies. (D) Graphs show total cell numbers in the peritoneal cavity (top panel), the relative frequency of B1a, B1b and B 2 cells in the population of lymphocytes (middle) and the absolute cell numbers of B1a, B1b and B 2 cells (bottom) in the peritoneal cavities of survivinL/Lcd21Cre and survivin+/+cd21Cre mice.
Figure 4
Figure 4. Survivin is required for TI-2 and TD antibody responses
(A) Relative concentration of TNP-specific IgM in sera from survivinL/Lcd21Cre and survivin+/+cd21Cre mice immunized with TNP-Ficoll. (B) Relative concentration of SRBC specific IgM and IgG1 in the sera from survivinL/Lcd21Cre and survivin+/+cd21Cre mice immunized with SRBC. (C) Relative frequency of GC B cells (B220+, GL7+, Fas+) within the splenic B cell compartment of survivinL/Lcd21Cre and survivin+/+cd21Cre mice 7 days post-SRBC immunization. (D) Immunohistology of spleens from survivinL/Lcd21Cre and survivin+/+cd21Cre mice 7 days post-SRBC immunization. B220 was used to detect B cells. PNA was used to detect GC B cells and CD35 was used to detect follicular dendritic cells. (E) Percentage of IgG1+ GC B cells on day 7 post-SRBC immunization. (F) Frequency of GC B cells within the B cell compartment in Peyer‘s Patches isolated from unimmunized survivinL/Lcd21Cre and survivin+/+cd21Cre mice. (G) Expression levels of phospho-H2AX in GC B cells from Peyer‘s patches isolated from unimmunized survivinL/Lcd21Cre and survivin+/+cd21Cre mice. Histograms are representative of 3 independent experiments.
Figure 5
Figure 5. Survivin is required for B cell proliferation, but not survival of activated B cells
(A) Splenic B cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice were cultured in the presence of the indicated stimuli for 3 days. Viability was determined by flow cytometry. The following concentrations were used: 10 ng/mL IL4; 10 ng/mL BAFF, 5 µg/mL anti-CD40, 13 µg/mL anti-IgM F(ab‘)2 fragments, 10 µg/mL LPS. Statistics were performed using the student’s t-test. (B) Splenic B cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice were cultured with the indicated stimuli for 3 days. Graphs show the dilution of the dye eFluro670 as a measure of proliferation. Histograms are representative for 3 independent experiments. (C) Survivin expression in B cells stimulated with LPS (10 µg/mL) plus IL-4 (10 ng/mL) for 3d. (D, E) Splenic B cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice were stimulated with anti-CD40 (5 µg/mL) plus IL-4 (10 ng/mL) (D) or LPS (10µg/mL) plus IL-4 (10ng/mL) (E). Plasma cell generation (left) and isotype switching (right) were measured by flow cytometry on day 3 of culture. Results are representative of 3 independent experiments.
Figure 6
Figure 6. Survivin-deficient B cells accumulate aberrant levels of DNA after mitogenic stimulation
(A) B cells from survivinL/Lcd21Cre and survivin+/+cd21Cre mice were cultured unstimulated or in the presence of the indicated stimuli for 3 days. Graphs show 3H-thymidine incorporation. The following concentrations were used: 100ng/mL IL4; 25ng/mL BAFF, 5µg/mL anti-CD40, 10µg/mL anti-IgM (B) Morphology and DNA content of LPS (10 µg/mL) plus IL-4 (10 ng/mL) stimulated survivinL/Lcd21Cre and survivin+/+cd21Cre B cells on day 3 of culture was analyzed using an ImageStream Imaging Flow Cytometer. Arrows indicate 2n and 4n DNA content. Results are representative for 2 independent experiments. (C) Cell growth of OCI-Ly1 (GCB-DLBCL), OCI-Ly3 (ABC-DLBCL), OCI-Ly19 (GCB-DLBCL), Daudi (Burkitt‘s lymphoma), JeKo (Mantle cell lymphoma), Raji (Burkitt‘s lymphoma) cells 1, 2 and 3 days after the beginning of cell culture in the presence of the indicated concentration of the survivin inhibitor S12 was determined using a colorimetric cell counting assay. Displayed are the obtained OD values minus the value of a blank sample. The results are representative of three independent experiments.

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