Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: J Immunol. 2021 Jul 23;207(3):799–808. doi: 10.4049/jimmunol.2100059

Protein Kinase CK2 Regulates B-cell Development and Differentiation

Hairong Wei *, Wei Yang *, Huixian Hong *, Zhaoqi Yan *,†, Hongwei Qin *,‡, Etty N Benveniste *
PMCID: PMC8323969  NIHMSID: NIHMS1710007  PMID: 34301844

Abstract

Protein kinase CK2 (also known as Casein Kinase 2) is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α’) and two regulatory CK2β subunits. CK2 is overexpressed and overactive in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphomas, leading to inappropriate activation of the NF-κB, JAK/STAT and PI3K/AKT/mTOR signaling pathways and tumor growth. However, whether CK2 regulates normal B-cell development and differentiation is not known. We generated mice lacking CK2α specifically in B-cells (using CD19-driven Cre recombinase). These mice exhibited cell-intrinsic expansion of marginal zone B (MZB) cells at the expense of transitional B (TrB) cells, without changes in follicular B (FoB) cells. TrB cells required CK2α to maintain adequate B-cell receptor (BCR) signaling. In the absence of CK2α, reduced BCR signaling and elevated Notch2 signaling activation increased MZB cell differentiation. Our results identify a previously unrecognized function for CK2α in B-cell development and differentiation.

Introduction

B-cells are central to humoral immunity, producing pathogen-reactive antigen-specific antibodies (1, 2). Based on phenotype, developmental program and functional properties, B-cells are divided into B1 and B2 cell lineages. B1 cells derive from fetal progenitors, predominate in the pleural and peritoneal cavities, and contribute most serum immunoglobulin M (IgM), including during early phases of infection (3). B2 cells develop in the bone marrow from common lymphoid progenitor cells, then pass through pro-B, pre-B, and immature B-cell stages. Newly formed immature B-cells migrate from the bone marrow to the spleen as transitional B (TrB) cells, where they further mature into follicular B (FoB) or marginal zone B (MZB) cells (4). FoB cells circulate among lymphoid organs and give rise to germinal-center B-cells that undergo somatic hypermutation in T-cell-dependent responses (5). MZB cells localize in the splenic marginal zone, and provide a first-line-of-defense by rapidly producing antibodies in response to infection by blood-borne viruses and encapsulated bacteria (3, 6).

TrB cells instructed to become FoB or MZB cells in the spleen depend on B-cell receptor (BCR) and Notch2 signaling (4, 7). BCR signaling strength drives B-cell subset fate determination (4). Weak BCR signaling seems to induce MZB cell development whereas relatively strong BCR signaling favors FoB cell development (4). Notch2 is another crucial B-cell fate determination factor, particularly for MZB cells. MZB cell instruction requires triggering of Notch2 on developing B-cells by the Notch2 ligand Delta-like 1 (Dll1) expressed by splenic venules in the red pulp and marginal zone (8–10). Aside from BCR and Notch2 signaling, B-cell maturation requires B-cell activating factor (BAFF) and NF-κB signaling (4).

Protein kinase CK2 is a highly conserved serine-threonine kinase present in cells as a tetramer consisting of two catalytic subunits (CK2α and/or CK2α’) and two regulatory subunits (CK2β) (11, 12). The catalytic subunits CK2α and CK2α’ (encoded by different genes Csnk2a1 and Csnk2a2, respectively) are closely related and share similar enzymatic characteristics. However, functional distinctions between CK2α and CK2α’ have been reported. First, global deletion of CK2α is embryonic lethal (13), while CK2α’ knockout mice have viable offspring (14), suggesting that CK2α has the capacity to partially compensate for CK2α’. Stably transfected tumor cell lines with catalytically inactive forms of CK2α or CK2α’ also provide evidence for functional differences between CK2α and CK2α’. Cell proliferation and viability were compromised by induced expression of catalytically inactive CK2α’ (15). By comparison, induced expression of catalytically inactive CK2α in these cells was without effect on proliferation or viability, suggesting that CK2α’ may have unique functions associated with the control of proliferation or viability. Therefore, CK2α and CK2α’ share many similarities, but do have some functional differences. CK2 phosphorylates serine or threonine residues specified by acidic side chains in many proteins, including growth factor receptors, transcription factors, and cytoskeletal proteins (16, 17). CK2 has been primarily studied in cancers, with aberrant expression and high kinase activity in many cancers, promoting tumor survival and growth (18, 19). In tumor cells, CK2 sustains pro-survival and proliferative signaling cascades that depend on NF-κB, PI3K/AKT/mTOR and/or JAK/STAT signaling, which are also centrally important in lymphocyte biology (20). Our previous studies provided the first evidence that CK2 is critical for activating JAK/STAT signaling in tumor cells (21, 22). Growing evidence suggests CK2 involvement not only in cancer cells, but also in regulating both innate and adaptive immune cell functions (23). Ulges et al., and our group demonstrated that CK2 activity promotes CD4+ Th17 and Th1 cell differentiation, and inhibits Foxp3+ Treg-cell generation (24–27). CK2β is also involved in the suppressive function of CD4+ Foxp3+ Tregs against allergy-promoting Th2 cells (28). CK2 is critical for monocyte-derived dendritic cells to mature and produce cytokines necessary to polarize effector T-cells in response to allergic contact dermatitis-triggering allergens (29). Inhibiting CK2 disrupts myeloid cell differentiation in the tumor microenvironment, leading to substantial augmentation of antitumor activity by immune therapy (30). A recent study demonstrated the suppressive effects of CK2 on inflammatory myeloid cell responses, rendering host tissues more susceptible to bacterial expansion during infection (31). Whether CK2 has specific functions in B-cell development and differentiation has never been examined.

Here, we examined CK2α specifically in B-cells in vivo. We demonstrate that CK2 expression and kinase activity are induced and/or enhanced upon B-cell activation. To assess the B-cell specific CK2α function in vivo, we generated mice with B-cell specific conditional deletion of CK2α by crossing gene-floxed Csnk2a1fl/fl mice with mice expressing Cre recombinase specifically in the B-cell lineage (CD19-Cre) (32). Deleting CK2α in B-cells led to MZB cell accumulation in the spleen accompanied by decreased TrB cells, without change in FoB cells. CK2α biases TrB cell development into MZB cells through regulating the magnitude of BCR and Notch2 signaling. Our data demonstrate that CK2α is an important regulator of B-cell development and differentiation, specifically, differentiation of TrB cells into MZB cells.

Materials and Methods

Mice.

B6.PEPBOY.CD19Cre/+ mice (32) were obtained from Dr. F. Lund (University of Alabama at Birmingham, UAB). Csnk2a1fl/fl mice were obtained from Dr. H. Rebholz (33). Csnk2a1fl/flCD19Cre/+ conditional knockout (CK2α-cKO) mice were created by crossing Csnk2a1fl/fl mice to CD19Cre/+ mice (32). Csnk2a1+/+ CD19Cre/+ wild-type (WT) mice were used as controls. All animals including Rag1−/− mice and C57BL/6 mice were bred and maintained under specific pathogen-free conditions in the animal facility at UAB. Experiments were performed using male and female mice between 8- and 12-weeks of age. All experimental procedures involving animals were reviewed and approved by the UAB Institutional Animal Care and Use Committee.

Flow Cytometry.

Single-cell suspensions obtained from spleen, bone marrow (one femur and one tibia), and peritoneal cavity were counted using a hemocytometer and stained in FACS buffer (DPBS supplemented with 2% FCS) for 30 min on ice and incubated with fluorescence-labeled antibodies for 30 min at 4°C. The following antibodies were used in this study (all Biolegend except where noted otherwise): anti-CD19 BV650/APC/BV786 (clone 6D5); anti-B220 APC-Cy7/BV510 /BV605 (clone RA3-6B2); anti-CD23 PE/BV421 (clone B3B4); anti-CD21/35 PerCP-Cy5.5 (clone 7E9); anti-CD93 APC (clone AA4.1); anti-IgM PE-Cy7 (clone RMM-1); anti-IgD FITC/BV605 (clone 11-26c.2a ); anti-CD24 Pacific Blue (clone M1/69); anti-CD43 APC (clone S11); anti-CD249 PE (clone BP-1, BD Bioscience); anti-CD38 APC-Cy7 (clone 90); anti-FAS Alexa 647 (clone Jo2); anti-CD138 PE/BV421 (clone 281-2); anti-CD45.1 Alexa Fluor 488/PerCP-Cy5.5 (clone A20); anti-CD45.2 Alexa 647/APC-Cy7 (clone 104); anti-CD11b FITC (clone M1/70); anti-CD5 PerCP-Cy5.5 (clone 53-7.3); anti-ADAM10-PE (clone 139712, R&D Systems); anti-CD79a (pY182) Alexa 647 (clone D1B9, Cell Signaling Technology); anti-Syk (pY352) Alexa 647 (clone 65E4, Cell Signaling Technology); anti-BTK (pY223) BV421(clone N35-86, BD Bioscience); anti-PLC-γ2 (pY759) Alexa 647(clone K86-689.37, BD Bioscience); and anti-ERK1/2 (pT202/pY204) Alexa 488 (clone 20A, BD Bioscience). For live- versus dead-cell discrimination, the Live/Dead Fixable Blue Stain Kit (L23105; ThermoFisher Scientific) or Live/Dead Fixable Aqua Stain Kit (L34957; ThermoFisher Scientific) was utilized. Stained cells were acquired on an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Inc).

For intracellular staining, spleen B-cells were purified by negative selection using CD43 Dynabeads (11422D; ThermoFisher Scientific), routinely to 90-95% purity, washed, and suspended in 2% FCS- RPMI. Cells were warmed at 37°C for 30 min. and incubated at 37°C with 10 μg/ml anti-IgM (Jackson ImmunoResearch) for the indicated time points. Subsequent surface and intracellular staining was performed using the BD Cytofix/Cytoperm protocol.

Confocal Imaging.

Confocal imaging was performed on spleen sections. Spleens were harvested, embedded in Frozen Tissue Media and snap-frozen in dry ice and 100% ethanol bath followed by cryosectioning. Eight um thick spleen frozen sections were dried in air for 20 seconds before fixation in ice cold acetone for 15 min. and rehydration with 1% BSA for 10 min.. Tissues were blocked with 10% normal rat serum at room temperature for 30 min., stained with purified rat anti-mouse CD169 (MOMA-1, Bio-Rad) plus biotin-anti-B220 (RA3-6B2, BD Biosciences), and then with Alexa Fluor 488-conjugated goat polyclonal anti-rat IgG secondary antibody plus Alexa Fluor 555-streptavidin (Invitrogen). Images were taken using a Nikon A1 confocal microscope at the UAB High Resolution Imaging Facility.

In Vitro B-cell Stimulation.

Purified spleen B-cells were cultured in B-cell medium (RPMI 1640 with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES and 50 μM β-mercaptoethanol) followed by stimulation with 10 μg/ml LPS (Sigma-Aldrich), 100 ng/ml CD40L (R&D) plus 10 ng/ml IL-4 (Biolegend), or 10 μg/ml anti-IgM plus 10 ng/ml IL-4 for 1-3 days.

Immunoblotting.

B-cells were lysed in RIPA buffer, protein lysates were separated by electrophoresis, transferred to a nitrocellulose membrane, and blotted with anti-CK2α (abcam, Cambridge, MA), CK2β (abcam, Cambridge, MA), CK2α’ (Santa Cruz Biotechnology) and β-Actin (Sigma-Aldrich) Abs, as previously described (24, 25).

CK2 Kinase Assay.

The CycLex CK2 Assay/Inhibitor Screening Kit (MBL International Corporation) was used to assess CK2 kinase activity. Cells were lysed, and both catalytic subunits (CK2α and CK2α’) were immune-precipitated. Resulting lysates were assayed for CK2 kinase activity according to manufacturer’s instructions.

TrB Cell Stimulation on OP9-Dll1 Cells.

OP9-Dll1 cells originally from Dr. Juan Carlos Zuniga-Pflucker (34) were obtained from Dr. Robert Welner (UAB). Four × 104 OP9-Dll1 cells were seeded in 24-well plates in 1-ml medium (α-MEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin) to reach 80% confluence. CD93+ transitional B-cells were enriched from splenocytes by first staining with CD93-APC, then using magnetic-bead enrichment with anti-APC beads and LS columns (both from Miltenyi) according to manufacturer’s instructions. Enriched CD93+ TrB cells (5 × 105), routinely 85% pure, were resuspended in B-cell media and added to the OP9-Dll1 cells. Non-adherent cells were harvested and analyzed for MZB cell phenotype by flow cytometry 3 days later. To inhibit Notch2 signaling, 25-μM DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; Sigma) (35), which interferes with γ-secretase-mediated Notch protein cleavage, was added to the wells.

RNA Isolation, RNA Sequencing, and Quantitative RT-PCR.

RNA sequencing was performed as described (24). Briefly, MZB cells were sorted from WT and CK2α-cKO mice. Total RNA was extracted from FACS-sorted B cells using the miRNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to manufacturer’s protocols and submitted to GENEWIZ (South Plainfield, NJ) for RNA sequencing (RNA-seq) and bioinformatics analysis. RNA sequencing data was submitted to the Gene Expression Omnibus (GEO) Repository under accession number GSE165140 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE165140). Genes with an FDR less than 0.05 with a fold-change more than 1.5 or less than −1.5 were considered differentially expressed genes (DEG). Further pathway analysis was performed by Gene Set Enrichment Analysis (GSEA) available through the Broad Institute.

For quantitative RT-PCR (qRT-PCR) analysis, 1,000-2,000 ng RNA was reverse-transcribed into cDNA using M-MLV Reverse Transcriptase (Promega) as described (24). cDNA was subjected to qRT-PCR using TaqMan primers (Thermo Fisher Scientific). Relative gene expression was calculated according to the ΔΔ threshold-cycle (Ct) method.

Mixed Bone-Marrow Chimeric Mouse Generation.

Five-10 × 106 bone-marrow cells from CD45.1 WT or CK2α-cKO mice and CD45.2 C57BL/6 mice were mixed in a 1:1 ratio and i.v. injected into lethally irradiated CD45.2 Rag1−/− mice. Chimeric mice were euthanized 8 weeks later to analyze reconstitution of MZB, FoB, and TrB cells. Reconstitution was calculated as the ratio of CD45.1 WT or CK2α-cKO to CD45.2 WT cells.

Adoptive Transfer of TrB Cells.

TrB cells (CD93+) were sorted from CD45.1 WT or CK2α-cKO mice. One × 106 TrB cells were mixed with 10 × 106 CD45.2 C57BL/6 splenocytes and injected i.v. into CD45.2 Rag1−/− recipient mice. Recipient Rag1−/− mice were sacrificed 5 days post-transfer to evaluate splenic MZB cell differentiation.

Statistical Analysis.

Statistical differences for comparison between two groups were determined by Student’s t-test using GraphPad Prism Software. Multiple comparisons were performed by one-way ANOVA. p values are indicated as: * p<0.05, ** p<0.01, *** p<0.001. All error bars represent mean ± SD.

Results

B-cell Activation Induces CK2 Expression and Kinase Activity.

To study how CK2 may regulate B-cell development and differentiation, we first examined CK2α, CK2β, and CK2α’ expression patterns during B-cell activation. In wild-type naïve B-cells, upon stimulation in vitro with the T-cell independent stimulus LPS, and the T-cell dependent stimuli CD40L plus IL-4, or anti-IgM antibody plus IL-4, expression of CK2α, CK2β and CK2α’ was strongly induced (Fig. 1A). Consistent with immunoblot results, intracellular staining revealed increased CK2α protein expression upon stimulation by LPS in a time-dependent manner (Fig. 1B). Increased protein expression also correlated with increased Csnk2a1 (CK2α), Csnk2b1 (CK2β) and Csnk2a2 (CK2α’) mRNA levels upon LPS stimulation compared with untreated B cells (Fig. 1C). Aside from subunit expression, a significant increase in overall CK2 kinase activity occurred upon LPS activation (Fig. 1D). These results demonstrate induction of CK2 protein expression and kinase activity upon B-cell activation, and suggest potential CK2 involvement in B-cell functions.

Figure 1. B-cell Activation Induces CK2 Expression and Kinase Activity.

Figure 1.

Open in a new tab

(A) Immunoblotting analysis of CK2α, CK2β, and CK2α’ in B-cells left untreated (UN) or activated with LPS (10 μg/ml), CD40L (100 ng/ml) plus IL-4 (10 ng/ml), or αIgM (10 μg/ml) plus IL-4 (10 ng/ml) for 1-3 days. Actin serves as a loading control. (B) Intracellular staining of CK2α in B-cells left untreated (UN) or activated for 1-3 days with LPS (10 μg/ml). IC, isotype control. (C) RNA was extracted from B-cells left untreated (UN) or activated for 2 or 3 days with LPS (10 μg/ml) and qRT-PCR performed using primers for Csnk2a1, Csnk2b1 and Csnk2a2. (D) At day 2, untreated and LPS-activated B-cells were assayed for CK2 kinase activity. Values shown as the mean ± SD. ** p < 0.01, Student’s t-test. n=3.

CK2α Deficiency Promotes MZB Cell Expansion.

Globally deleting CK2α is embryonically lethal (13). To determine CK2α involvement during B-cell development, we first crossed Csnk2a1fl/fl mice with mice bearing Cre recombinase under control of the endogenous B-cell specific Cd19 locus (32). The resulting Csnk2a1fl/flCD19Cre/+ (CK2α-cKO) mice were viable and underwent normal development with no signs of systemic or organ-specific inflammation, and had specific deletion of CK2α in B-cells. In the splenic B-cell compartment, deletion of CK2α was confirmed by qRT-PCR analysis (Fig. S1A) and a significant reduction in CK2 kinase activity was observed in B-cells from CK2α-cKO mice (Fig. S1B). Detailed characterization of the bone-marrow B-lineage cells demonstrated a decreased frequency of pre-B cells [Hardy Fraction (Fr) D] (B220+IgM+CD43−) and an increased frequency of mature circulating B-cells (Fr F) (B220+IgM+IgD+CD43−) (Figs. S1C and S1D), and by absolute cell counts, we observed a reduced number of pre-B cells (Fr D) (Fig. S1E). Thus, CK2α deletion slightly impacts bone-marrow B-cell development. We next evaluated the impact of CK2α deletion on peripheral immune-cell development. CK2α-cKO mice exhibited an increase in spleen weight (Fig. 2A) and total splenocytes (Fig. 2B). CK2α-cKO mice had comparable frequencies of total CD19+B220+ B-cells (Fig. 2C) and CD3+ T-cells (including CD4+ and CD8+ T-cells) compared with WT mice (Fig. S2A). Because of the increase in total splenocytes, the number of B-cells (Fig. 2C) and CD4+ T-cells (Fig. S2B) was increased in CK2α-cKO mice. CK2α-cKO mice had a similar distribution of naïve (CD44−CD62L+) and effector T-cells (CD44+CD62L−), as detected by CD44 and CD62L expression (Fig. S2C).

Figure 2. CK2α Deficiency Promotes Marginal-Zone B (MZB) Cell Increases.

Figure 2.

Open in a new tab

(A) Spleen weights from WT and CK2α-cKO mice. (B) spleen cells from WT and CK2α-cKO mice were counted. (C) Percentage and number of B220+CD19+ B-cells in the spleens of WT and CK2α-cKO mice. (D) Representative flow-cytometry staining of spleens for CD21/35hiCD23lo MZB cells and CD21/35−CD23hi FoB cells. (E, F) Percentage and absolute number of MZB cells (E) and FoB cells (F) within splenic B220+CD19+ B-cells in WT and CK2α-cKO mice. (G) Representative flow-cytometry staining of spleens for CD93 expression on total B-cells. (H) Percentage and absolute number of CD93+ TrB cells in the spleens of WT and CK2α-cKO mice. (I) Spleen sections from WT and CK2α-cKO mice (8 weeks old) were analyzed for CD169 and B220 expression by immunofluorescent staining. Bar, 100 μm. (J) Quantification of IgM and IgD expression on MZB cells from WT and CK2α-cKO mice. n=6. (K, L) Representative flow-cytometry staining and summarized data for B1a (CD19+CD11b+CD5+), B1b (CD19+CD11b+CD5−), and B2 (CD19+CD11b−CD5−) cells in the peritoneal cavity. Values shown as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001, Student’s t-test. n=10.

Among B-cells, CK2α-cKO mice exhibited increased MZB cells and decreased TrB cells, with no effect on FoB cells (Figs. 2D–H). The localization of CK2α-cKO B-cells within the splenic microenvironment was examined. We stained spleen sections from WT and CK2α-cKO mice with B220 antibody to detect B-cells and CD169 antibody, which recognizes metallophilic macrophages located at the border between the follicle (Fo) and marginal zone (MZ) areas. In WT mice, the majority of B-cells were localized in the Fo area, whereas in CK2α-cKO mice, more B-cells were localized in the MZ area (Fig. 2I). Thus, B-cells developing in CK2α-cKO mice show an abnormal distribution within the splenic microenvironment that is skewed toward an MZ localization. Moreover, we analyzed IgM and IgD expression on MZB cells and found that expression of IgM on MZB cells was comparable between WT and CK2α-cKO mice, while expression of IgD was significantly lower in CK2α-cKO MZB cells compared to WT MZB cells (Fig. 2J). Interestingly, upon LPS stimulation, we found MZB cells from CK2α-cKO mice that differentiated into CD138+ blasts were significantly reduced compared to MZB cells from WT mice (Figs. S3A–B), which suggests that CK2α promotes MZB cell differentiation into plasma cells. Characterization of B-cell subsets in the peritoneal cavity revealed no differences in peritoneal B1a (CD19+CD11b+CD5+), B1b (CD19+CD11b+CD5−), and B2 cells (CD19+CD11b−CD5−) (Figs. 2K and 2L).

MZB Cell Increase in CK2α-cKO Mice is B-cell Intrinsic.

To determine whether increased MZB cells in CK2α-cKO mice was B-cell intrinsic or caused by changes in the stromal structures or other hematopoietic cells, we created mixed bone-marrow chimeras by lethally irradiating CD45.2 T-cell and B-cell deficient Rag1−/− recipient mice and reconstituting them with equal numbers of CD45.2 WT and CD45.1 WT or CK2α-cKO BM cells. Recipient mice were characterized 8 weeks post-transfer to investigate chimerism with B-cell development stages (Fig. 3A). Splenic CD23hiCD21/35lo FoB cells were completely chimeric, and CD21hiCD23lo MZB cells were generated mostly from CD45.1 CK2α-cKO hematopoietic cells (Figs. 3B and 3C). These findings demonstrate that enhanced MZB cell differentiation in CK2α-cKO mice is not due to micro-environmental changes, but rather is cell-intrinsic. We next evaluated whether TrB cells in the spleen might be affected. There was an underrepresentation of spleen TrB cells of the CD45.1 CK2α-cKO genotype (Fig. 3C). Collectively, increased MZB cells in CK2α-cKO mice is cell-intrinsic, and this enhancement starts from TrB cell stages, which may indicate that signals instructing TrB cells to become MZB cells are increased in the absence of CK2α.

Figure 3. Enhanced MZB Cell Differentiation in CK2α-cKO Mice is B-cell-intrinsic.

Figure 3.

Open in a new tab

(A) Schematic of experimental design. To generate mixed bone-marrow chimeric mice, 5–10 × 106 WT or CK2α-cKO (CD45.1) BM cells and 5–10 × 106 WT (CD45.2) BM cells were co-transferred into lethally irradiated Rag1−/− mice for 8 weeks before analysis. (B) Flow-cytometry staining of spleens from chimeric mice to evaluate the ratio between CD45.2 WT and CD45.1 WT or CK2α-cKO cells within the FoB cell and MZB cell populations. Numbers adjacent to gates represent percentages of cells within the gates. (C) Ratios of WT/WT and WT/CK2α-cKO frequencies of MZB, FoB and TrB cell populations per mouse. ** p<0.01, *** p<0.001, n=5.

CK2α Restricts TrB Cell Differentiation into MZB Cells.

To examine how CK2α deletion in B-cells leads to higher MZB cell numbers, we quantified cell proliferation and survival. To measure proliferation, WT and CK2α-cKO mice were injected with BrdU (a thymidine analogue incorporated into DNA during S phase) 22 h pre-sacrifice. We observed no difference in BrdU+ FoB and MZB cells in CK2α-cKO mice compared with WT mice (Fig. 4A). Because there was no evidence of altered cycling under homeostatic conditions, we next analyzed cell survival. Splenic FoB and MZB cells exhibited comparable Caspase-3 staining, a measure of apoptosis (Fig. 4B). Together, these results show that CK2α deletion in B-cells promotes MZB cell accumulation without altering proliferation or cell survival.

Figure 4. CK2α Restricts TrB Cell Differentiation into MZB Cells.

Figure 4.

Open in a new tab

(A) WT or CK2α-cKO mice were injected with 1 mg BrdU i.v. 22 h before sacrifice. Frequency of BrdU+ cells in splenic B-cell subsets in WT or CK2α-cKO mice. (B) Frequency of Caspase 3+ splenic B-cell subsets in WT or CK2α-cKO mice. (C) Flow-cytometry staining of spleens for the presence of CD21/35hiCD23lo MZB cells and CD21/35lowCD23hi FoB cells in Rag1−/− recipients (CD45.2) adoptively transferred with 1 × 106 CD93+ TrB cells (CD45.1) from WT or CK2α-cKO mice mixed with 10 × 106 CD45.2 splenocytes. Numbers adjacent to outlined areas indicate the percentage of CD21/35hiCD23lo MZB cells and CD21/35loCD23hi FoB cells after adoptive transfer of TrB cells. (D) Quantification of MZB cells and FoB cells formed in Rag1−/− recipients adoptively transferred with CD93+ TrB cells from WT or CK2α-cKO mice. Values shown as mean ± SD. *** p<0.001. n=6.

To further address whether CK2α expression limits MZB cell differentiation, we performed adoptive-transfer experiments into Rag1−/− mice. We purified CD45.1+CD93+ TrB cells, and these were injected together with a ten-fold greater number of CD45.2 wild-type splenic cells, to avoid homeostatic proliferation that might bias toward MZB development (Figs. 4C and 4D). Five days post-transfer, we studied the fate of transferred CD45.1 spleen cells by flow cytometry. WT and CK2α-cKO TrB cells gave rise to both FoB and MZB cells, whereas CK2α-cKO TrB cells gave rise to a higher frequency of MZB cells (Figs. 4C and 4D), suggesting that CK2α regulates TrB cells committed to become MZB cells. We also analyzed surface markers of TrB cells from WT and CK2α-cKO mice. We found that IgM expression was significantly lower in TrB cells from CK2α-cKO mice, while the expression levels of IgD, CD21 and CD23 are comparable between WT and in CK2α-cKO mice (Supplemental Fig. 4A). ADAM10 surface expression on TrB cells has been shown to mark commitment to the MZB cell fate (36). We analyzed ADAM10 expression in TrB cells. Interestingly, ADAM10 expression was lower in CK2α-cKO TrB cells (Supplemental Fig. 4B), which suggests that increased MZB cells in CK2α-cKO mice is independent of ADAM10 expression.

CK2α Regulates MZB Cell Differentiation Through BCR and Notch2 Signaling.

TrB cell development into FoB versus MZB cells depends, in part, on the strength of BCR signaling, and also requires Notch2 signaling (4). To address involvement of these signaling pathways relative to in vivo B-cell CK2 biology, we first examined whether BCR signaling is affected in CK2α-cKO TrB cells. When exposed to anti-IgM, CK2α-cKO TrB cells showed impaired BCR signaling as demonstrated by reduced levels of multiple phosphorylated intermediates, including CD79a, Syk, BTK, PLCγ 2, and ERK1/2 (Figs. 5A and 5B).

Figure 5. Reduced B-Cell Receptor (BCR) Signaling in CK2α-deficient TrB Cells.

Figure 5.

Open in a new tab

(A) Representative flow cytometry analyzing phosphorylation of CD79a (pY182), Syk (pY352), BTK (pY223), PLCγ2 (pY759) and Erk1/2 (pT202/pY204) in WT and CK2α-cKO TrB cells stimulated with anti-IgM (10 μg/ml) for the indicated time points. (B) Summarized data show phospho-specific antibody binding to indicated intracellular signaling substrates downstream of the BCR, after stimulation with anti-IgM. Values shown as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. n=5.

To address the mechanism of how CK2α regulates MZB differentiation, spleen MZB cells were isolated from WT and CK2α-cKO mice, and subjected to RNA sequencing. A total of 129 genes were significantly increased, and 67 genes were significantly decreased in CK2α-cKO MZB cells compared to WT MZB cells (Fig. 6A). GSEA of the data revealed that differentially expressed genes (DEG) were associated with the Notch-signaling pathway (Fig. 6B). CK2α-cKO MZB cells express higher levels of genes associated with Notch signaling pathways like Heyl, Hes5 and Dtx1 (Fig. 6C) (37). We also analyzed Notch2 target-gene expression levels in TrB cells from WT and CK2α-cKO mice by qRT-PCR, and found significantly higher expression levels of Heyl, Hes5, and Dtx1 in TrB cells from CK2α-cKO mice compared to WT mice (Fig. 6D). To assess if Notch2-mediated differentiation of MZB cells from TrB cells was enhanced in CK2α-cKO mice, CD93+ TrB cells from WT and CK2α-cKO mice were cultured on OP9 cells stably transfected with Dll1 (OP9-Dll1 cells), which stimulate Notch2 signaling through Delta-like-1 ligand expression. After 3 days of culture, a significant enhancement of CD21/35+IgM+ MZB cell differentiation was observed from TrB cells from CK2α-cKO mice compared to WT mice (32% vs 20%) (Fig. 6E). Accordingly, an inhibitor of Notch signaling (DAPT, 25 μM) mitigated in vitro MZB development (Fig. 6E). These results suggest that CK2α sustains BCR signaling, and negatively regulates Notch2 signaling, acting as a master regulator of MZB cell differentiation.

Figure 6. CK2α Deficiency Enhances Notch2 Activation in TrB Cells.

Figure 6.

Open in a new tab

(A) RNA sequencing of MZB cells from spleens of WT and CK2α-cKO mice. Summary of genes differentially regulated by CK2α using the following cutoffs are shown: p < 0.05, fold change >1.5. n=3. (B) GSEA plot shows enrichment of RNA-sequencing data compared with Reactome Notch-pathway dataset. n=3. (C) Heat map shows Notch signaling relative gene expression in MZB cells. (D) Quantification of Notch target-gene expression in CD93+ TrB cells from WT and CK2α-cKO mice. n=4. (E) Flow-cytometry analysis for induction of CD21/35 and IgM expression on TrB cells cultured on OP9-Dll1 cells for 3 days in the absence or presence of the Notch inhibitor DAPT (25 μM). The fluorescence minus one (FMO) control was used for gating. ** p<0.01, *** p<0.001. n=3.

Discussion

Herein we identify a novel biological function of protein kinase CK2 in homeostasis of mature B-cell development. We found that CK2 expression and kinase activity were strongly induced upon in vitro B-cell activation, and deletion of CK2α in B-cells led to aberrant enhanced accumulation of MZB cells. This accumulation of CK2α-deficient MZB cells was associated with reduced BCR signaling and elevated activation of Notch2 signaling in CK2α-deficient TrB cells. Our results identify the unrecognized involvement of CK2α in B-cell development and differentiation.

Previous studies demonstrated that CK2 is involved in malignant lymphocyte biology and acts as a pro-survival and signaling-boosting kinase, both in precursor and mature B-cell tumors (20, 38). CK2 is overexpressed, hyperactive, and essential for PI3K/PTEN/AKT signaling cascade activation in B-acute lymphoblastic leukemia (ALL) (39). Increased CK2 expression in B-ALL phosphorylates Ikaros and impairs Ikaros function (40). In mature B-lymphoid tumors including B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL), CK2 is overexpressed and essential for cell growth (19, 41). CK2 also positively regulates STAT3 and NF-κB-dependent signaling in MCL cells (42). We report, for the first time, that mouse CK2α is integral to normal B-cell development. Interestingly, deletion of CK2α in B-cells resulted in increased cellularity of the spleen. We found that CD4+ T-cells, but not CD8+ T-cells, were significantly increased in CK2α-cKO mice, while B-cells were dramatically increased in CK2α-cKO mice. Among B-cells, CK2α-cKO mice exhibited increased MZB cells and decreased TrB cells, with no effect on FoB cells. It has been reported that MZB cells are far superior to FoB cells in inducing CD4+ T-cell expansion (43). Although it is not known whether naive MZB cells are able to efficiently activate CD4+ T-cells, it has been shown that freshly isolated MZB cells from naive animals exhibit high levels of CD80 and CD86, indicative of previous antigenic experience (43). Therefore, more MZB cells could lead to CD4+ T-cell expansion in CK2α-cKO mice.

B-cell specific deletion of CK2α increased MZB cell differentiation, but not that of FoB cells, which raises questions about how CK2α selectively affects one cell fate over another. MZB cell accumulation in the absence of CK2α may be caused by two possible mechanisms: enhanced lineage commitment from TrB cell to MZB cells and/or high survival of MZB cells. We did not find any evidence indicating reduced apoptosis or excessive proliferation of MZB cells in the absence of CK2α. When we performed adoptive-transfer experiments, we found that CK2α-cKO TrB cells gave rise to a higher frequency of MZB cells, suggesting that CK2α regulates TrB cells to become MZB cells. Also, in mixed bone-marrow chimeras, MZB cells were generated mostly from CK2α-cKO hematopoietic cells, while there was an underrepresentation of spleen TrB cells of the CK2α-cKO genotype. Therefore, increased MZB cells in CK2α-cKO mice start from TrB cell stages. TrB cells face a decision to mature into either FoB or MZB cells. It has been reported that BCR signaling causes immature type I TrB cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10 (36). BCR signaling strength has a critical role in determining TrB cell maturation into MZB or FoB cell compartments. Wen et al. demonstrated that the promotion of MZB cell development by exposure to a defined transgenic antigen, with FoB cell generation in its absence, indicates a BCR signal strength requirement that is higher for MZB cells than for FoB cell development (44). However, Pillai et al. proposed that relatively strong BCR signals favor the development of FoB cells, while weak BCR signals induce MZB cell development (4, 45, 46). Relatively strong BCR signals render TrB cells in the follicle impervious to the presence of Dll1-mediated triggering of Notch2, whereas weak BCR signaling may enhance expression of one or more components of the Notch2-signaling pathway (4, 45, 46). Therefore, signals through BCR, Notch2, possibly BAFF, and signals involved in migration and retention of MZB cells may contribute cooperatively to the development of MZB cells. In this study, we found that deleting CK2α was associated with impaired BCR signaling in TrB cells, and decreased expression of IgM on CK2α-cKO TrB cells could be one of the reasons for impaired BCR signaling in TrB cells from CK2α-cKO mice. These data indicate CK2α is required for sustaining adequate BCR and BCR signaling to regulate B-cell differentiation. Others reported that CK2 inhibition with CX-4945, a specific CK2 inhibitor (47), impaired AKT phosphorylation and intracellular Ca2+ mobilization upon BCR engagement in DLBCL cells, and that CX-4945 acted synergistically with either SYK or BTK inhibitors to induce DLBCL cell death (48). However, the underlying molecular mechanisms by which CK2 directly or indirectly regulates BCR signaling requires further study.

Notch2 signaling is another important factor determining the differentiation fate of mature B-cells into FoB versus MZB cells (8, 10). Notch is a target of phosphorylation by CK2, and this phosphorylation negatively regulates Notch function (49, 50). Inhibiting CK2 by siRNA or by pharmacological inhibition restored Notch signaling in myeloid cells and substantially improved their differentiation (50). In our study, we demonstrate that in B-cells in vivo, CK2α negatively regulates the Notch2-signaling pathway, because in the absence of CK2α, Notch2 signaling was enhanced. This enhanced Notch2 signaling then seems to promote MZB cell differentiation, thereby linking CK2α to the signaling pathway critical for MZB cell differentiation.

In summary, our data identify a previously unrecognized function of protein kinase CK2α in B-cell development and differentiation, especially a critical function in MZB cell development. We demonstrate that CK2α is vital for sustaining adequate BCR signaling, and suppression of Notch2 signaling in B-cells, because in the absence of CK2α, weak BCR signaling and enhanced Notch2 signaling promotes MZB cell differentiation (Fig. 7). Thus, CK2α is an important regulator of MZB cell development and differentiation.

Figure 7. Model Depicting Involvement of CK2α in Mature B-cell Differentiation.

Figure 7.

Open in a new tab

(A) In the presence of CK2α, B-cell activation induces CK2 expression and kinase activity, which increases the magnitude of BCR signaling and suppresses Notch2 signaling. Strong BCR signaling and suppression of Notch2 signaling instructs B-cell development into FoB cells. (B) In the absence of CK2α, weak BCR signaling enhanced Notch2 signaling drives B-cell differentiation into MZB cells.

Supplementary Material

1

Key points:

  1. CK2 expression and kinase activity are induced upon in vitro B-cell activation.

  2. Deletion of CK2α in B-cells leads to aberrant enhanced accumulation of MZB cells.

  3. CK2α sustains BCR signaling and suppresses Notch2 signaling in B-cells.

Acknowledgments

We acknowledge assistance from The Comprehensive Flow Cytometry Core at UAB and the UAB High Resolution Imaging Facility. We thank Dr. Frances Lund (UAB) for providing B6.PEPBOY.CD19Cre/+ mice and Dr. Robert Welner (UAB) for providing OP9-Dll1 cells.

Footnote:

This work was supported by National Institutes of Health Grants R01NS057563 and R01CA194414 (to E.N.B.), and National Multiple Sclerosis Society Grant RG-1606-24794 (to H.Q.).

References

  • 1.Cooper MD, Peterson RD, and Good RA. 1965. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature 205: 143–146. [DOI] [PubMed] [Google Scholar]
  • 2.yLeBien TW, and Tedder TF. 2008. B lymphocytes: how they develop and function. Blood 112: 1570–1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Martin F, Oliver AM, and Kearney JF. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14: 617–629. [DOI] [PubMed] [Google Scholar]
  • 4.Pillai S, and Cariappa A. 2009. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 9: 767–777. [DOI] [PubMed] [Google Scholar]
  • 5.Rajewsky K 1996. Clonal selection and learning in the antibody system. Nature 381: 751–758. [DOI] [PubMed] [Google Scholar]
  • 6.Cerutti A, Cols M, and Puga I. 2013. Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes. Nat Rev Immunol 13: 118–132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wang H, Morse HC 3rd, and Bolland S. 2020. Transcriptional control of mature B cell fates. Trends Immunol 41: 601–613. [DOI] [PubMed] [Google Scholar]
  • 8.Tanigaki K, Han H, Yamamoto N, Tashiro K, Ikegawa M, Kuroda K, Suzuki A, Nakano T, and Honjo T. 2002. Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells. Nat Immunol 3: 443–450. [DOI] [PubMed] [Google Scholar]
  • 9.Hozumi K, Negishi N, Suzuki D, Abe N, Sotomaru Y, Tamaoki N, Mailhos C, Ish-Horowicz D, Habu S, and Owen MJ. 2004. Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo. Nat Immunol 5: 638–644. [DOI] [PubMed] [Google Scholar]
  • 10.Saito T, Chiba S, Ichikawa M, Kunisato A, Asai T, Shimizu K, Yamaguchi T, Yamamoto G, Seo S, Kumano K, Nakagami-Yamaguchi E, Hamada Y, Aizawa S, and Hirai H. 2003. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity 18: 675–685. [DOI] [PubMed] [Google Scholar]
  • 11.Litchfield DW 2003. Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem. J. 369: 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dominguez I, Sonenshein GE, and Seldin DC. 2009. Protein kinase CK2 in health and disease: CK2 and its role in Wnt and NF-kappaB signaling: linking development and cancer. Cell Mol Life Sci 66: 1850–1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lou DY, Dominguez I, Toselli P, Landesman-Bollag E, O’Brien C, and Seldin DC. 2008. The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development. Mol. Cell Biol. 28: 131–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Xu X, Toselli PA, Russell LD, and Seldin DC. 1999. Globozoospermia in mice lacking the casein kinase II α’ catalytic subunit. Nat. Genet. 23: 118–121. [DOI] [PubMed] [Google Scholar]
  • 15.Vilk G, Saulnier RB, St Pierre R, and Litchfield DW. 1999. Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms. J Biol Chem 274: 14406–14414. [DOI] [PubMed] [Google Scholar]
  • 16.Duncan JS, and Litchfield DW. 2008. Too much of a good thing: The role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2. Biochim Biophys Acta 1784: 33–47. [DOI] [PubMed] [Google Scholar]
  • 17.Bian Y, Ye M, Wang C, Cheng K, Song C, Dong M, Pan Y, Qin H, and Zou H. 2013. Global screening of CK2 kinase substrates by an integrated phosphoproteomics workflow. Sci Rep 3: 3460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rabalski AJ, Gyenis L, and Litchfield DW. 2016. Molecular pathways: Emergence of protein kinase CK2 (CSNK2) as a potential target to inhibit survival and DNA damage response and repair pathways in cancer cells. Clin Cancer Res 22: 2840–2847. [DOI] [PubMed] [Google Scholar]
  • 19.Pizzi M, Piazza F, Agostinelli C, Fuligni F, Benvenuti P, Mandato E, Casellato A, Rugge M, Semenzato G, and Pileri SA. 2015. Protein kinase CK2 is widely expressed in follicular, Burkitt and diffuse large B-cell lymphomas and propels malignant B-cell growth. Oncotarget 6: 6544–6552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Mandato E, Manni S, Zaffino F, Semenzato G, and Piazza F. 2016. Targeting CK2-driven non-oncogene addiction in B-cell tumors. Oncogene 35:6045–6052 [DOI] [PubMed] [Google Scholar]
  • 21.Zheng Y, Qin H, Stuart F, Deng L, Litchfield DW, Terfferi A, Pardanani A, Lin F-T, Li J, Sha B, and Benveniste EN. 2011. A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway. Blood 118: 156–166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zheng Y, McFarland BC, Drygin D, Yu H, Bellis SL, Kim H, Bredel M, and Benveniste EN. 2013. Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma. Clin Cancer Res 19: 1–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gibson SA, and Benveniste EN. 2018. Protein Kinase CK2: An emerging regulator of immunity. Trends Immunol 39: 82–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gibson SA, Yang W, Yan Z, Liu Y, Rowse AL, Weinmann AS, Qin H, and Benveniste EN. 2017. Protein kinase CK2 controls the fate between Th17 cell and regulatory T cell differentiation. J Immunol 198: 4244–4254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gibson SA, Yang W, Yan Z, Qin H, and Benveniste EN. 2018. CK2 controls Th17 and regulatory T cell differentiation through inhibition of FoxO1. J Immunol 201: 383–392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Yang W, Gibson SA, Yan Z, Wei H, Tao J, Sha B, Qin H, and Benveniste EN. 2020. Protein kinase 2 (CK2) controls CD4(+) T cell effector function in the pathogenesis of colitis. Mucosal Immunol 13:788–798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ulges A, Witsch EJ, Pramanik G, Klein M, Birkner K, Buhler U, Wasser B, Luessi F, Stergiou N, Dietzen S, Bruhl TJ, Bohn T, Bundgen G, Kunz H, Waisman A, Schild H, Schmitt E, Zipp F, and Bopp T. 2016. Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development. Proc Natl Acad Sci U S A 113:10145–10150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ulges A, Klein M, Reuter S, Gerlitzki B, Hoffmann M, Grebe N, Staudt V, Stergiou N, Bohn T, Bruhl TJ, Muth S, Yurugi H, Rajalingam K, Bellinghausen I, Tuettenberg A, Hahn S, Reissig S, Haben I, Zipp F, Waisman A, Probst HC, Beilhack A, Buchou T, Filhol-Cochet O, Boldyreff B, Breloer M, Jonuleit H, Schild H, Schmitt E, and Bopp T. 2015. Protein kinase CK2 enables regulatory T cells to suppress excessive T2 responses in vivo. Nat Immunol 16:267–275. [DOI] [PubMed] [Google Scholar]
  • 29.de Bourayne M, Gallais Y, El Ali Z, Rousseau P, Damiens MH, Cochet C, Filhol O, Chollet-Martin S, Pallardy M, and Kerdine-Romer S. 2016. Protein kinase CK2 controls T-cell polarization through dendritic cell activation in response to contact sensitizers. J Leukoc Biol 101:703–715. [DOI] [PubMed] [Google Scholar]
  • 30.Hashimoto A, Gao C, Mastio J, Kossenkov A, Abrams SI, Purandare AV, Desilva H, Wee S, Hunt JT, Jure-Kunkel M, and Gabrilovich DI. 2018. Inhibition of casein kinase 2 disrupts differentiation of myeloid cells in cancer and enhances the efficacy of immunotherapy in mice. Cancer Res 78:5644–5655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Larson SR, Bortell N, Illies A, Crisler WJ, Matsuda JL, and Lenz LL. 2020. Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection. Front Immunol 11:59026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Rickert RC, Roes J, and Rajewsky K. 1997. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res 25: 1317–1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rebholz H, Zhou M, Nairn AC, Greengard P, and Flajolet M. 2013. Selective knockout of the casein kinase 2 in d1 medium spiny neurons controls dopaminergic function. Biol Psych 74: 113–121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Schmitt TM, and Zuniga-Pflucker JC. 2002. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17: 749–756. [DOI] [PubMed] [Google Scholar]
  • 35.Yoon SO, Zhang X, Berner P, Blom B, and Choi YS. 2009. Notch ligands expressed by follicular dendritic cells protect germinal center B cells from apoptosis. J Immunol 183: 352–358. [DOI] [PubMed] [Google Scholar]
  • 36.Hammad H, Vanderkerken M, Pouliot P, Deswarte K, Toussaint W, Vergote K, Vandersarren L, Janssens S, Ramou I, Savvides SN, Haigh JJ, Hendriks R, Kopf M, Craessaerts K, de Strooper B, Kearney JF, Conrad DH, and Lambrecht BN. 2017. Transitional B cells commit to marginal zone B cell fate by Taok3-mediated surface expression of ADAM10. Nat Immunol 18: 313–320. [DOI] [PubMed] [Google Scholar]
  • 37.Hampel F, Ehrenberg S, Hojer C, Draeseke A, Marschall-Schroter G, Kuhn R, Mack B, Gires O, Vahl CJ, Schmidt-Supprian M, Strobl LJ, and Zimber-Strobl U. 2011. CD19-independent instruction of murine marginal zone B-cell development by constitutive Notch2 signaling. Blood 118: 6321–6331. [DOI] [PubMed] [Google Scholar]
  • 38.Piazza F, Manni S, Arjomand A, Visentin A, Trentin L, and Semenzato G. 2020. New responsibilities for aged kinases in B-lymphomas. Hematol Oncol 38: 3–11. [DOI] [PubMed] [Google Scholar]
  • 39.Gomes AM, Soares MV, Ribeiro P, Caldas J, Povoa V, Martins LR, Melao A, Serra-Caetano A, de Sousa AB, Lacerda JF, and Barata JT. 2014. Adult B-cell acute lymphoblastic leukemia cells display decreased PTEN activity and constitutive hyperactivation of PI3K/Akt pathway despite high PTEN protein levels. Haematologica 99: 1062–1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Song C, Ge Z, Ding Y, Tan BH, Desai D, Gowda K, Amin S, Gowda R, Robertson GP, Yue F, Huang S, Spiegelman V, Payne JL, Reeves ME, Gurel Z, Iyer S, Dhanyamraju PK, Xiang M, Kawasawa YI, Cury NM, Yunes JA, McGrath M, Schramm J, Su R, Yang Y, Zhao Z, Lyu X, Muschen M, Payne KJ, Gowda C, and Dovat S. 2020. IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia. Blood 136: 1520–1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Piazza F, Manni S, Ruzzene M, Pinna LA, Gurrieri C, and Semenzato G. 2012. Protein kinase CK2 in hematologic malignancies: reliance on a pivotal cell survival regulator by oncogenic signaling pathways. Leukemia 26: 1174–1179. [DOI] [PubMed] [Google Scholar]
  • 42.Manni S, Brancalion A, Mandato E, Tubi LQ, Colpo A, Pizzi M, Cappellesso R, Zaffino F, Di Maggio SA, Cabrelle A, Marino F, Zambello R, Trentin L, Adami F, Gurrieri C, Semenzato G, and Piazza F. 2013. Protein kinase CK2 inhibition down modulates the NF-kappaB and STAT3 survival pathways, enhances the cellular proteotoxic stress and synergistically boosts the cytotoxic effect of bortezomib on multiple myeloma and mantle cell lymphoma cells. PLoS One 8: 1–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Attanavanich K, and Kearney JF. 2004. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J Immunol 172: 803–811. [DOI] [PubMed] [Google Scholar]
  • 44.Wen L, Brill-Dashoff J, Shinton SA, Asano M, Hardy RR, and Hayakawa K. 2005. Evidence of marginal-zone B cell-positive selection in spleen. Immunity 23: 297–308. [DOI] [PubMed] [Google Scholar]
  • 45.Pillai S, Cariappa A, and Moran ST. 2005. Marginal zone B cells. Annu Rev Immunol 23: 161–196. [DOI] [PubMed] [Google Scholar]
  • 46.Cariappa A, Tang M, Parng C, Nebelitskiy E, Carroll M, Georgopoulos K, and Pillai S. 2001. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14: 603–615. [DOI] [PubMed] [Google Scholar]
  • 47.Pierre F, Chua PC, O’Brien SE, Siddiqui-Jain A, Bourbon P, Haddach M, Michaux J, Nagasawa J, Schwaebe MK, Stefan E, Vialettes A, Whitten JP, Chen TK, Darjania L, Stansfield R, Bliesath J, Drygin D, Ho C, Omori M, Proffitt C, Streiner N, Rice WG, Ryckman DM, and Anderes K. 2011. Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer. Mol Cell Biochem 356: 37–43. [DOI] [PubMed] [Google Scholar]
  • 48.Mandato E, Nunes SC, Zaffino F, Casellato A, Macaccaro P, Tubi LQ, Visentin A, Trentin L, Semenzato G, and Piazza F. 2018. CX-4945, a selective inhibitor of casein kinase 2, synergizes with B cell receptor signaling inhibitors in inducing diffuse large B cell lymphoma cell death. Curr Cancer Drug Targets 18: 608–616. [DOI] [PubMed] [Google Scholar]
  • 49.Ranganathan P, Vasquez-Del Carpio R, Kaplan FM, Wang H, Gupta A, VanWye JD, and Capobianco AJ. 2011. Hierarchical phosphorylation within the ankyrin repeat domain defines a phosphoregulatory loop that regulates Notch transcriptional activity. J Biol Chem 286: 28844–28857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Cheng P, Kumar V, Liu H, Youn JI, Fishman M, Sherman S, and Gabrilovich D. 2013. Effects of Notch signaling on regulation of myeloid cell differentiation in cancer. Cancer Res 74: 141–152. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1710007-supplement-1.pdf (1.4MB, pdf)

RESOURCES