Quiescent B Cells Acquire Sensitivity to Cell Cycle Arresting Agents by B Cell Receptor Stimulation

Abstract

For the treatment of autoimmune diseases, depletion of B cells specific for auto-antigens is important because they will be a source of plasmablasts/plasma cells to produce autoantibodies. However, because some types of B cells like naïve B cells and memory B cells are at quiescent phase, they are insensitive to anticancer drugs which exert cytotoxicity by arresting the cell cycle. Here we show that B cell receptor (BCR) stimulation increases the sensitivity of anticancer drugs by promoting the proliferation of quiescent B cells. The BCR stimulation to primary naïve B cells enhanced sensitivity to several anticancer drugs which arrest the cell cycle through different mechanisms. The present results indicated that combination of the BCR stimulation and anticancer drugs is a promising strategy for the antigen-specific depletion of pathogenic quiescent B cells.

INTRODUCTION

Hallmark of autoimmune diseases are generation of antibodies against endogenous antigens. The autoantibodies are involved in the progression of symptoms.¹⁾ Thus, self-reactive B cells that can differentiate into autoantibody-producing plasmablasts/plasma cells have been recognized as therapeutic targets for the treatment of autoimmune disorders.²⁾ At present, antibodies against CD19, CD20, and CD22 that specifically target B cells including naïve B cells (NBC) and memory B cells (MBC) are widely used clinically and have been shown to be very effective in treating a variety of autoimmune diseases.³⁾

Anticancer drugs which arrest the cell cycle such as cyclophosphamide have also been used for the treatment of autoimmune diseases.⁴⁾ The drugs target T cells and B cells, which are sensitive to the anticancer drugs due to their rapid proliferation.⁵⁾ However, the drugs will not be effective for NBC and MBC which are basically in a quiescent state in the cell cycle.

Recently, strategy to reenter cancer stem cell (CSC) into the cell cycle has been gathering attention because it provides sensitivity to anticancer drugs to CSC.^6,7) It has been found that several molecules such as granulocyte macrophage colony-stimulating factor⁸⁾ and agonists for peroxisome proliferator-activated receptor γ⁹⁾ stimulate CSC to reenter into the cell cycle. Thus, combination of these stimulants with the cell cycle-arresting anticancer drugs has been used for clinical trials for the treatment of myeloid leukemia.⁶⁾

It is known that quiescent NBC and MBC initiate the cell cycle via the intracellular signaling through B cell receptor (BCR) triggered by the recognition of antigens.¹⁰⁾ Thus, the BCR signaling in NBC and MBC has potential to sensitize toward anticancer drugs. In this study, this hypothesis was examined for the first time using B cells in a proliferative state prepared from quiescent B cells by BCR-stimulation.

MATERIALS AND METHODS

Materials

Mertansine (DM-1) and SN-38 were purchased from Cayman CHEMICAL (MI, U.S.A.). Monomethyl auristatin E (MMAE) was purchased from Chem Scene (NJ, U.S.A.). Paclitaxel was purchased from AdipoGen LIFE SCIENCES (CA, U.S.A.). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (MO, U.S.A.).

Mice, B Cell Isolation, Cell Culture

C57BL/6J mice were purchased from CLEA Japan (Japan). Mice were bred and maintained under specific pathogen–free conditions and used at 8–12 weeks of age. Animal care and experiments were approved by the animal committee of Kyushu University, and all experiments were conducted according to the approved guidelines. For B-cell isolation, resting B cells were purified from spleen by negative selection with anti-CD43 magnetic beads (Miltenyi Biotech). The purified B-cell population was >95% positive for B220. B cells were cultured in RPMI-1640 (Wako, Japan) supplemented with 10% fetal bovine serum (FBS) (CAPRI CORN), 2-mercapto ethanol (50 µM, Sigma-Aldrich), penicillin (100 U/mL, Nacalai Tesque, Japan), streptomycin (100 µg/mL, Nacalai Tesque), L-N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES) (1 mM, Nacalai Tesque), MEM nonessential amino acid (Nacalai Tesque), and L-sodium pyruvate (100 mM, Nacalai Tesque) (complete medium). All cells were cultured in a humidified atmosphere containing 5% CO₂ in air at 37 °C.

Flow Cytometry Analysis

B cells were washed with phosphate buffered saline (PBS) containing 2% FBS and stained with propidium iodide (PI) (final concentration 1 µg/mL, Nacalai Tesque). Cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter). The acquired data were processed using FlowJo software.

B Cell Viability after BCR Stimulation

For B cell stimulation, purified B cells (1 × 10⁶ cells/mL) were cultured in complete medium and stimulated with 20 µg/mL of anti-immunoglobulin M (IgM) F(ab)′₂ (Jackson Immunoresearch). Viability of B cells after incubation with or without anti-IgM F(ab)′₂ was determined as PI negative cells (gating strategy is shown in Supplementary Fig. S1).

Fig. 1. Hypothetical Working Mechanism of Cell Cycle Arresting Anticancer Drug on B Cell Receptor (BCR) Stimulated B Cells

Cytotoxicity Assessment

Cytotoxicity of anti-cancer drugs to B cells was assessed by using flow cytometry analysis. For B cell stimulation, purified B cells (1 × 10⁶ cells/ml) were cultured in complete medium and stimulated with 20 µg/mL of anti-IgM F(ab)′₂ and with or without 20 ng/mL of BAFF (R&D Systems). Simultaneously, anticancer drug (paclitaxel, MMAE, DM-1 or SN-38) was added to culture medium containing B cells and anti-IgM F(ab)′₂. After 72 h of incubation, cells were wash with PBS containing 2% FBS and stained with PI and subjected to flow cytometry.

Statistical Analysis

Statistical analyses were performed by one-way ANOVA. Differences with p values < 0.05 were considered significant.

RESULTS

First, we established the in vitro system to evaluate the effect of the BCR signaling on the susceptibility increase of the primary NBC toward anticancer drugs. NBC was purified by negative selection method from mice splenocyte. Because NBC thus obtained does not survive in vitro without suitable cell signalings, we examined the effect of the following two kinds of cell signalings for the maintenance of the cell viability. The BCR signaling is known to induce proliferation as well as survival of B cells, while the signaling through BAFF receptor by BAFF solely induces survival on B cells.¹¹⁾ Figure 2 A shows the viability of NBC after 72 h in the presence of either BCR signaling or BAFF receptor signaling. BCR signaling was induced by F(ab)′₂ of anti-IgM to crosslink BCR. The both signalings increased the cell viability about 50% from the cells with neither signal (approx. 10%). The 50%-viability of NBC attained by BAFF is simply due to the survival. In contrast, the 50%-viability by anti-IgM should be derived from the summation of the proliferation and survival signals via BCR. To check the effect of proliferation signal by anti-IgM, we evaluated the viability of NBC cultured with or without anti-IgM at different time points. The increase of the cell viability of NBC was clearly observed from 48 h (Fig. 2B). The 50%-viability is enough to afford the evaluation of cytotoxicity of anticancer drugs toward NBC.

Fig. 2. (A) Cell Viability of Naïve B Cells Cultured in Medium for 72 h (Control) or in the Medium Containing Either Anti-IgM F(ab)′₂ or BAFF for 72 h

Results are expressed as mean ± standard deviation (S.D.) (n = 3). (B) Time course of cell viability of naïve B cells incubated with or without anti-IgM F(ab)′₂. Results are expressed as mean ± S.D. (n = 3). Data are representative of two independent experiments.

We examined the effect of anticancer drugs on the viability of NBC in the presence of anti-IgM at 72 h. Here we used four kinds of anticancer drugs which arrest the cell cycle by different mechanisms. As shown in Fig. 3, all the anticancer drugs reduced the cell viability in a dose-dependent manner. This reduction should be due to the competition of the cell cycle-arresting by the anticancer drugs with the progression of the cell cycle by anti-IgM. The extent of cytotoxicity of each drug toward NBC reflected its reported IC₅₀ value toward cancer cells. IC₅₀ value of each drug has been reported as follows: 0.2–5 nM (MMAE),^12,13) 0.2–2 nM (DM-1),¹⁴⁾ 2.5–7.5 nM (paclitaxel),¹⁵⁾ 13–720 nM (SN-38).¹⁶⁾

Fig. 3. BCR-Stimulated B Cells Are Sensitive to Anti-cancer Drugs; B Cells Were Treated with Four Different Drugs (MMAE, DM-1, Paclitaxel, SN-38) in the Presence of Anti-IgM F(ab)′₂

Cell viability values are given in percentage with the untreated control as 100%. The results are expressed as mean ± S.D. (n = 3); * p < 0.05; N.S., not significant. (One-way ANOVA). Data are representative of three independent experiments.

To examine whether the cytotoxic effect of the anticancer drugs depends on the cell cycle, the NBC maintained by BAFF-induced survival signal was used. As shown in Fig. 4, the effects of the two drugs are negligible or weak for the BAFF-stimulated NBC. In contrast, the NBC with the additional BCR signal increased the sensitivity toward the drugs. These results indicated that NBC was relatively resistant to the anticancer drugs in the resting state but they became susceptible to the drugs in the proliferation state.

Fig. 4. Switching of Sensitivity of B Cells toward MMAE and SN-38 from Resting State Maintained by BAFF to Proliferation State Induced by BAFF + anti-IgM

Cell viability values are given in percentage with the untreated control as 100%. Data are expressed as mean ± standard error of the mean (S.E.M.) (n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001; N.S., not significant. (One-way ANOVA). Data are pooled from two independent experiments.

DISCUSSION

Here we examined the hypothesis that the BCR-stimulation to NBC raises the sensitivity to anticancer drugs. We used four kinds of anticancer drugs with different working mechanisms to arrest the cell cycle, i.e., inhibition of microtubule organization (MMAE, DM-1, paclitaxel) and inhibition of TOPO isomerase (SN-38). The anticancer drugs showed negligible or weak effect on NBC without the BCR signaling (Fig. 4). However, the BCR signaling increased the sensitivity of NBC toward the anticancer drugs irrespective of their working mechanisms (Figs. 3, 4). These results clearly verified our hypothesis. Based on our finding, it will be able to propose a new strategy to treat autoimmune diseases, i.e., the BCR-stimulation-induced sensitization of the quiescent B cells toward anticancer drugs.

At present, anti-CD20 antibodies are used for the treatment of the autoimmune diseases by depleting B cells that potentially produce autoantibodies. However, because the anti-CD20 antibodies non-specifically kill all B cells which express CD20, they cause immunocompromise for infection.³⁾ In contrast, BCR-mediated “wake up” strategy proposed here has potential to achieve antigen-specific B cell killing. In the current study, BCR stimulating molecule (anti-IgM) and anticancer drugs were added separately, which may show risk of incomplete killing of B cells. To avoid such risk, BCR stimulating molecule and anticancer drug should be conjugated to completely kill the BCR-stimulated B cells. In this case, antigen should be used as BCR-stimulating molecule for the B cell clone-specific targeting. Such work will be conducted in our group. Recently, Ichikawa et al. reported that an antigen-modified liposome loaded with anticancer drug suppressed the antibody production in allergy model mice.¹⁷⁾ They utilized the antigen for the specific delivery of the anticancer drug to the antigen-specific B cells. The working mechanism of their system to kill B cells may include our finding.

CONCLUSION

Here we examined for the first time the effect of the BCR signaling in the quiescent NBC on the susceptibility to several anticancer drugs which arrest the cell cycle. The BCR signaling was induced by crosslinking BCR via anti-IgM. We found that the BCR signaling raised the sensitivity toward anticancer drugs irrespective of their working mechanisms. Because the BCR signaling can be induced by using antigens, antigen-specific activation of the quiescent B cells may be possible to target causative B cells that secrete autoantibodies. Thus, combination of the antigen-specific B cell activation by antigens and cell cycle arresting anticancer drugs may be an effective strategy to treat these diseases.

Acknowledgments

This work was supported by Japan Agency for Medical Research and Development (AMED) (JP19ek0410044 and JP19gm6110004) to Y.B., open innovation platform for industry-academia co-creation to Y.K., and Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JP19K08883, JP21H00429, JP21H05530, JPMJFR210S, JP22H03112 to S.T., JP20H05876 to T.M., and JP18H02626 to Y.B.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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