Research ArticleExperimental Studies

Anti-tumor Effect of Activated Canine B Cells With Interleukin-21 and Anti-B Cell Receptor

SO-YOUNG SUR, GA-HYUN LIM, SU-MIN PARK, KYOUNG-WON SEO and HWA-YOUNG YOUN
Anticancer Research September 2023, 43 (9) 4007-4014; DOI: https://doi.org/10.21873/anticanres.16588

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Abstract

Background/Aim: Recently, novel studies on the pivotal role of B cells in the tumor-microenvironment and anti-tumor immunity have been conducted. Additionally, Interleukin-21 (IL-21) and anti-B cell receptor (BCR) have been reported to stimulate B cells to secrete granzyme B, which exhibits cytotoxic effects on tumor cells. However, the direct anti-tumor effect of B cells is not yet fully understood in the veterinary field. This study is the first attempt in veterinary medicine to identify the immediate effect of B cells on tumor suppression and the underlying mechanisms involved. Materials and Methods: Canine B cells were isolated from peripheral blood and activated with IL-21 and anti-B cell receptor (BCR). The canine leukemia cell line GL-1 was co-cultured with B cells, and the anti-tumor effect was confirmed by assessing the changes in cell viability and apoptotic ratio. Results: When B cells were activated with IL-21 and anti-BCR, the secretion of granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increased. Simultaneously, the viability of GL-1 cells decreased, and the apoptotic ratio increased, particularly when co-cultured with activated B cells. Conclusion: The results demonstrated the direct anti-tumor effect of granzyme B-and TRAIL and its enhanced potential of B cells to inhibit tumor cell growth after activation with IL-21 and anti-BCR. This study is the first study dealing with immunomodulation in the canine tumor micro-environment.

Key Words:

The immune system and modification of the tumor microenvironment play crucial roles in cancer development and growth (1). Cancer arises from the accumulation of genetic and epigenetic mutations that sustain proliferative signaling, inhibit of growth suppressors, and compromise immune responses (2). The role of the immune system in cancer development has led to a breakthrough in immunotherapeutic strategies and has been validated for several types of cancer treatments, including cytokine therapies and anti-cancer vaccines (3).

Conventionally, only cytotoxic T cells and natural killer (NK) cells produce cytokines, upregulate inhibitory molecules, and directly kill tumor cells (3). However, recently, the direct and indirect roles of B cells in anti-tumor responses have been appreciated (4-6). B cells have diverse functions ranging from producing antibodies to presenting antigens (7). In a tumor-specific microenvironment, B cells exhibit pro-tumoral effects by expressing cytokines such as interleukin 10 (IL-10), interleukin 35 (IL-35), and transforming growth factor-β (TGF-β) or inhibitory molecules such as programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) (8-10). Conversely, B cells also inhibit tumor progression by producing tumor-specific antibodies and effector cytokines, presenting antigens to effector cells, and directly expressing cytotoxic molecules (11). Tumor-infiltrating B cells are known to secrete antibodies that are against tumor-specific antigens and mediate tumor lysis by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (12). Furthermore, B cells also promote T cell responses by presenting antigens to helper T cells or directly activating cytotoxic T cells. Moreover, B cells directly kill tumor cells via granzyme B, perforin, and TRAIL (13).

Granzymes, serine proteases stored in the cytotoxic granules of immune cells, are released to eliminate infected transformed target cells. Granzymes are of numerous types, with granzymes A and B being the most abundant (14). Granzyme B, which inserts into the target cell’s plasma membrane and cleaves after an aspartate residue, activates the apoptotic pathway by proteolyzing multiple caspases such as caspase 3 pathway or cleaving some key caspase substrates (15). It is also transported to the nucleus and cleaves multiple nuclear substrates via an unknown pathway (16). Several studies suggest that granzyme-secreting B cells function in early cell-mediated immune responses associated with inflammation and cancer (17, 18). Granzyme-positive B cells infiltrate various solid tumors (19-21). While the tumor-infiltrated B cells are associated with tumor progression, granzyme B-secreting B cells are known to contribute to the anti-tumor immune response (17).

TRAIL is a member of the tumor necrosis factor (TNF) family that induces apoptosis by binding to TRAIL receptors (22). TRAIL can be transmembrane or soluble protein in various tissues and cells, but is mainly expressed on the surface of immune cells, where it acts as an inducer of apoptosis of target cells (23). TRAIL binds to the death receptor 4/5 (DR4/5) to initiate the extrinsic pathway of cell death, which mainly involves the caspase pathway. The intrinsic pathway, which starts in the mitochondria, activates caspase proteins and the p53 pathway to initiate cell death (24). TRAIL induces apoptosis in various cancer cells without affecting normal cells (25-27). Cytotoxicity against tumor cells increases when B cells express high levels of TRAIL (28). Thus, TRAIL has been considered a target for anti-tumor therapy (29).

IL-21 and anti-BCR have been reported to play pivotal roles in determining B-cell activity. IL-21 induces proliferation of B cells and promotes immunoglobulin production (30). IL-21 also induces the differentiation of B cells into long-lived and antibody-secreting plasma cells and stimulates B cells to express the active form of granzyme B (17). A study showed that B cells activated properly with both IL-21 and anti-BCR increased granzyme B production and subsequently exerted a significant cytotoxic effect on tumor cells (31).

This study aimed to evaluate the anti-tumor effects of canine B cells activated with IL-21 and anti-BCR. Only a few studies have been performed on the anti-cancer immunity of B cells. To our knowledge, this is the first study on canine B cells. We examined the interaction between the canine leukemia cell line (GL-1) and canine B cells isolated from peripheral blood that were activated by IL-21 and anti-BCR. The ability of B cells to express granzyme B and TRAIL was evaluated, and their anti-tumor effect on GL-1 cells was assessed using cell viability and apoptosis assays.

Materials and Methods

Canine leukemia B cell line and cell culture. The well-characterized canine leukemia B cell line GL-1 was obtained from Prof. YukoGoto-Koshino (Tokyo University, Japan). The GL-1 cell line was maintained in Roswell Park Memorial Institute (RPMI)-1640 (Welgene, Gyeong-San, Republic of Korea) medium supplemented with 10% fetal bovine serum (FBS; Gibco®, Paisley, Scotland), 1% solution of 10,000 units/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 μg/ml streptomycin (Sigma-Aldrich) (PS). The cell lines were cultured at 37°C and 5% CO2. The media were refreshed every other day, and cells were sub-cultured when their confluency reached 80%.

Isolation of canine B cells. Canine cluster of differentiation 21 positive (CD21+) B cells were isolated from canine peripheral blood mononuclear cells (cPBMCs). With the owners’ consent, blood samples were obtained from healthy dogs visiting the Seoul National University Veterinary Medical Teaching Hospital and participating in a blood donation program. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (protocol no. SNU-221102-1). Using the remaining white blood cell layer, Ficoll-Paque PREMIUM (Cytiva Life Sciences™, Marlborough, MA, USA) was used to isolate cPBMCs, following the manufacturer’s instructions. The white blood cell layer was mixed with an equal amount of Dulbecco’s phosphate-buffered saline (DPBS) solution (Welgene) and placed onto Ficoll-Paque PREMIUM in accordance with the manufacturer’s guidelines. After centrifugation at 480 × g for 30 min at room temperature (RT) with a low brake, the isolated mononuclear cell layer was aspirated and treated with red blood cell lysis buffer (Sigma-Aldrich) at RT for 5 min. Cells were then washed with DPBS and centrifuged at 780 × g for 10 min. CD21 antibody (dilution, 1:100; Invitrogen, Carlsbad, CA, USA) and anti-mouse immunoglobulin G microbeads (Miltenyi Biotec, Auburn, CA, USA) with MACS (Miltenyi Biotec) were used to isolate canine B cells according to the manufacturer’s instructions. MACS separation buffer (Miltenyi Biotec) was used. The canine CD21 antibody was added to cPBMCs at 4°C for 1 h. CD21+ B cells were collected by centrifugation at 780 × g for 10 min. For magnetic labelling, 20 μl of anti-mouse IgG microbeads and 80 μl of MACS separation buffer were added to the CD21+ B cells, which were incubated for 15 min. Magnetically labelled CD21+ B cells were collected after centrifugation at 780 × g for 10 min. After washing the LS column by flowing 3 ml of MACS separation buffer, CD21+ B cells were added to the LS column for isolation. After another wash with 3 ml of MACS separation buffer, CD21+ B cells were collected in 6 ml of MACS separation buffer.

B cell activation with anti-BCR and IL-21. The obtained CD21+ B cells were stimulated with anti-BCR (Jackson ImmunoResearch Inc., West Grove, PA, USA) and IL-21 (Biotechne, Minneapolis, MN, USA). Cells were seeded at a density of 2×105 cells/ml in 6 well plates (SPL Life Science, Po-Cheon, Republic of Korea). Anti-BCR and IL-21 were added to the activation group at a working concentration of 10 μg/ml and 100 ng/ml, respectively. The cells were incubated at 37°C in 5% CO2 for 16 h for the activation.

B cell phenotyping analyses. After IL-21 and anti-BCR stimulation, B cells were fixed with ice-cold 70% alcohol overnight at 4°C. After fixation, B cells were washed thrice with ice-cold DPBS. They were then incubated for 30 min at 4°C with antibodies against FITC-conjugated CD86 (1:100; BioLegend, San Diego, CA, USA) and phycoerythrin-conjugated CD80 (1:100; BioLegend). After three washes with ice-cold DPBS, flow cytometry was performed within 1 h using FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using FlowJo v10.8.1 software (BD Biosciences).

Co-culture system. Approximately 1×106 GL-1 cells were seeded in each well of 6 well plates (SPL). GL-1 cells were divided into three groups: negative control group without B cells, another group co-cultured with naïve B cells, and one group co-cultured with activated B cells. The 0.4-μm pore size inserts were placed in each well, and the insert was preconditioned with the media for one hour. Then, naïve and activated B cells were seeded onto the insert at a density of 5×105 cells/well, at a ratio of 2:1 (GL-1:B cell). All groups of cells were incubated at 37°C in 5% CO2 for 24 h. The media of each insert was harvested for enzyme-linked immunosorbent assay (ELISA) of Granzyme B and TRAIL, and GL-1 cells were evaluated for cell viability and harvested for further apoptosis assays.

Enzyme linked immunosorbent assay. The media obtained from naïve and activated B cells were centrifuged at 300 × g for 5 min. The supernatant layer was separated and stored at −20°C. The Canine Granzyme B ELISA kit (Mybiosource.com, San Diego, CA, USA) and Canine Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) ELISA Kit (Reddot Biotech, Houston, TX, USA) were used. All ELISA procedures were performed according to manufacturer’s instructions.

Cell viability assay. The Cell Counting Kit-8 (CCK-8) (Dong-in Biotech, Seoul, Republic of Korea) assay was used to confirm the anti-tumor effect of B cells. GL-1 cells were seeded at a density of 1×106 cells/well in 6 well plates, and B cells were added at a ratio of 2:1 (GL-1:B cell). After incubation for 24 h, the CCK assay was performed according to the manufacturer’s instructions. Absorbance at 450 nm wavelength was determined using a spectrophotometer (Epoch Microplate Spectrophotometer; BioTek Instruments, Winooski, VT, USA).

Apoptosis analysis. Three groups of GL-1 cells, with or without B cell activation, were harvested and washed three times with cold DPBS. An Annexin V-FITC apoptosis detection kit (Enzo Life Science, Farmingdale, NY, USA) was used to detect apoptotic cells according to the manufacturer’s protocol. The cells were resuspended in 1× binding buffer and stained with Annexin V-FITC and PI (dilution 1:20) for 15 min at RT in the dark. Flow cytometry was performed within 1 h using FACS Aria II (BD Biosciences), and the results were analyzed using FlowJo v10.8.1 software (BD Biosciences).

Statistical analysis. GraphPad Prism (version 9.3.1) software (GraphPad Software, San Diego, CA, USA) was used for the statistical analyses. Data were analyzed using the Student’s t-test and one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. The data are presented as mean value±standard deviation. Statistical significance was set at p-value <0.05.

Results

B cell phenotyping. In this study, cPBMCs were obtained using Ficoll solution, and CD21+ B cells were isolated using the MACS positive selection kit (Miltenyi Biotec). To confirm whether the isolated cells were genuine B cells, a flow cytometric assessment of CD80 and CD86 was performed (32, 33). The results showed the expression of CD80 and CD86 on the surface of isolated cPBMC by flow cytometry; thus, the isolation of B cells from cPBMC was confirmed (Figure 1A).

Figure 1.
Figure 1.

Expression of CD80 and CD86 assessed using flow cytometry. (A) Surface expression of CD80 and CD86 markers on canine B cells. Histograms represent the expression of CD80 and CD86 in naïve CD21+ B cells. (B) Surface expression of CD80 and CD86 markers on canine IL-21 and anti-BCR stimulated B cells. Histograms represent the expression of both CD80 and CD86 in the IL-21 and anti-BCR stimulated B cells. BCR: B cell receptor; CD: cluster of differentiation; IL: interleukin.

B cell activation with IL-21 and anti-BCR. In accordance with previous studies, isolated human B cells stimulated with IL-21 and anti-BCR show increased anti-tumor effects in breast cancer cells (31). After the activation of B cells with IL-21 and anti-BCR (Figure 2A), B cells were assessed using flow cytometry to determine whether the isolated and activated cells were genuine B cells. Both CD80 and CD86 were expressed in the stimulated cells, confirming that they were B cells (Figure 1B).

Figure 2.
Figure 2.

B cell activation with IL21 and anti-BCR. (A) The anti-tumor effect of B cells in the tumor micro-environment. B cells suppress tumor progression by secreting granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). (B) Schematic representation of the two-dimensional co-culture of B cells and GL-1 cells using the Transwell assay. GL-1 cells were co-cultured with naïve B cells and activated B cells.

After activation, B cells and GL-1 cells were co-cultured using a transwell system (Figure 2B). The supernatant was acquired and stored to verify alterations in the levels of granzyme B and TRAIL secretion. ELISA analyzed the levels of secreted granzyme B and TRAIL. The results showed that granzyme B levels in naïve B cells were 0.3360±0.04088 pg/ml, whereas those in activated B cells were 0.4510±0.09614 pg/ml. Therefore, we confirmed a statistically significant increase in granzyme B secretion in activated B cells (Figure 3A, p<0.01. The level of TRAIL in naïve B cells was 231.2±1.261 pg/ml, and that in activated B cells was 708.3±60.15 pg/ml. Notably, TRAIL secretion by activated B cells was higher than that of naïve B cells (Figure 3B, p<0.0001).

Figure 3.
Figure 3.

Determination of the levels of granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) using ELISA. (A) Compared to the naïve B cell group, the activated B cell group displayed an increase in granzyme B levels. (B) TRAIL levels increased in the activated B cell group. Values are expressed as mean±standard deviation. Statistical significance was determined using the Student’s t-test: **p<0.01, ****p<0.0001.

Cytotoxic effect of activated B cells on a canine leukemia B cell line. The cytotoxicity of activated B cells was confirmed by assessing the viability of the GL-1 cells. The viability of naïve GL-1 cells without any activation, GL-1 cells co-cultured with naïve B cells, and GL-1 cells co-cultured with B cells activated with IL-21 and anti-BCR were assessed using the CCK-8 assay. The results showed that the viability of GL-1 cells was significantly reduced when co-cultured with both naïve B cells and activated B cells (Figure 4). The viability of GL-1 cells co-cultured with naïve B cells and activated B cells was 86.04±5.683% and 75.82±7.967%, respectively. Thus, a significant reduction in the viability of GL-1 cells was observed when co-cultured with B cells that were activated with IL-21 and anti-BCR (control vs. naïve B cell, p<0.05; naïve vs. activated B cell, p<0.01; control vs. activated B cell <0.0001).

Figure 4.
Figure 4.

GL-1 cell viability assay. Cell viability of GL-1 cells that were co-cultured with B cells was assessed using the CCK-8 assay. The viability of GL-1 cells decreased when co-cultured with B cells. GL-1 cells co-cultured with activated B cells showed more reduction in viability compared with the group co-cultured with naïve B cells. Statistical significance was determined using the one-way analysis of variance (ANOVA) test: *p<0.05, **p<0.01, ***p<0.001.

Apoptosis effect of activated B cells in a canine leukemia B cell line. Annexin V/PI staining and flow cytometry were performed to confirm the apoptotic effect of activated B cells on GL-1 cells. The results showed that apoptosis increased in GL-1 cells after co-culture with activated B cells (Figure 5A and B). The apoptotic ratios for the negative control and GL-1 cells co-cultured with naïve B cells were 2.793±0.8211% and 2.490±0.3030%, respectively. Compared with them, GL-1 cells co-cultured with B cells that were activated with IL-21 and anti-BCR showed an apoptotic ratio of 5.830±1.059%, which indicated a statistically significant increase in apoptotic ratio (naïve vs. activated B cells, p<0.01; Control vs. activated B cells, p<0.001). When these results and the anti-tumor mechanisms of granzyme B and TRAIL described previously are combined, the increase in the apoptotic ratio may be promoted by the cytotoxicity of granzyme B and TRAIL secreted by B cells.

Figure 5.
Figure 5.

Determination of the apoptotic ratio using flow cytometry. (A) Apoptosis of GL-1 cells increased when co-cultured with B cells activated with IL-21 and anti- B cell receptor (BCR). (B) Quantification of data shown in A. Statistical significance was determined using the one-way analysis of variance (ANOVA) test: **p<0.01, ***p<0.001; ns: Not significant.

Discussion

Tumor-infiltrating B cells contribute to anti-tumor immunity through multiple mechanisms. The major roles of B cells in the tumor microenvironment are to present tumor-specific antigens to effector cells and secrete numerous cytokines to activate helper and cytotoxic T cells. Their novel roles in triggering the complement cascade, mediating phagocytosis of tumor cells, and inducing NK cells without antibody intervention have also been elucidated (32, 34). In recent studies, the novel anti-tumor roles of B cells have been studied. These studies found that B cells directly contribute to tumor killing by secreting granzyme B and TRAIL (17, 31, 35). Additionally, IL-21 and anti-BCR have also been reported to exert anti-tumor effects in leukemia and breast cancer cell lines by inducing the secretion of granzyme B (6, 31, 36). With the independence of the classical T-cell pathway, IL-21 stimulates B cells to express and secrete the active form of granzyme B (35). Therefore, we hypothesized that stimulation of canine B cells with IL-21 and anti-BCR can result in enhanced granzyme B and TRAIL expression and suppression of tumor cell viability. Canine B cells were separated and stimulated with IL-21 and anti-BCR, and the direct contact-mediated anti-tumor effect of B cells was examined.

In this study, we isolated B cells from the peripheral blood of healthy dogs, stimulated them with IL-21 and anti-BCR, and subsequently confirmed their anti-tumor effects. Prior to this, the isolated cells were confirmed to be B cells. CD80 and CD86 are proteins mainly expressed in antigen-presenting cells (37). Multiple studies have shown that tumor-infiltrating B cells express CD80 and CD86, with the primary function of antigen presentation (32, 38). Based on this information, the expression of CD80 and CD86 was confirmed by flow cytometric analysis of separated cells. Thus, we confirmed that the cells used in the next experiments were genuine B cells. Flow cytometry was conducted after stimulation with IL-21 and anti-BCR. CD80 and CD86 were also expressed in stimulated cells, confirming the cells used for the next experiments were B cells.

In this study, we confirmed that granzyme B and TRAIL secretion increased in cells activated with IL-21 and anti-BCR. IL-21 and anti-BCR have previously been shown to stimulate granzyme B synthesis in human B cells. Via the STAT3 pathway, IL-21 and anti-BCR are known to induce the differentiation of B cells to granzyme B-expressing B cells (39, 40). Anti-BCR has been reported to sensitize B cells to apoptosis induced by TRAIL (41), but its effect on TRAIL secretion has not been fully clarified. For the first time, this study confirmed an increase in soluble TRAIL secretion from B cells stimulated with IL-21 and anti-BCR. Based on these results and previous studies, IL-21 and anti-BCR are considered to increase the secretion of granzyme B and TRAIL by canine B cells, and further research on the pathway involved should be conducted.

Furthermore, compared with the control GL-1 group, both groups GL-1 cells co-cultured with B cells showed decreased cell viability. Moreover, compared with the group co-cultured with naïve B cells, viability was significantly reduced in the group co-cultured with stimulated B cells. To confirm that the cytotoxicity of GL-1 cells was induced by granzyme B and TRAIL, an apoptotic assay using flow cytometry was performed. GL-1 cells co-cultured with B cells activated with IL-21 and anti-BCR showed a significant increase in apoptosis. This may provide supporting evidence that granzyme B and TRAIL produced by B cells contribute to the cytotoxicity of GL-1 cells via the apoptotic pathway.

Granzyme B and TRAIL have been reported to induce tumor cell apoptosis via various mechanisms. In cancer cells, Granzyme B induces direct proteolytic processing and activation of executioner procaspase-3 and -7, permeabilizes the mitochondrial outer membrane, proteolyzes multiple housekeeping proteins, including some pro-survival membrane receptors, and translocates into the nucleus to cleave multiple intranuclear proteins (42). Previous studies reported that the soluble form of TRAIL induces apoptosis in tumor cells via the extrinsic and intrinsic apoptotic pathways. The extrinsic pathway is initiated by binding to the death receptor (DR) and induces apoptosis by activating effector caspases, whereas the intrinsic pathway is initiated by mitochondria and is mediated by the formation of an apoptosome and activation of the effector caspase pathway (43). Binding of TRAIL to its receptor recruits FAS-associated proteins with death domains and caspase 8 to form a functional death-inducing signaling complex; thus, induces apoptosis by releasing multiple mitochondrial proteins and forming a functional apoptosome. With the sequential activation of caspase 9 and caspase 3, cleavage of a large number of intracellular targets results in apoptosis of the target cancer cells (15, 44). In this study, stimulation with IL-21 and anti-BCR improved the anti-tumor effects of B cells. Combined with previous studies, we concluded that the anti-tumor effect is probably due to granzyme B and TRAIL secreted by activated B cells.

This study has several limitations. First, activated B cells have only been applied to one type of canine cancer cell line, and a variety of malignant cell lines may be used in follow-up studies. Additionally, the anti-tumor effect was demonstrated by confirming an increase in apoptosis, whereas the exact pathway or molecules affected has not yet been determined. Therefore, further studies are required.

Conclusion

We activated canine B cells with IL-21 and anti-BCR and verified the alterations in their anti-tumor effects. The expression and secretion of granzyme B and TRAIL of B cells increased with activation, and when these activated B cells were co-cultured with GL-1 cells, cell viability of GL-1 cells decreased by inducing apoptosis.

To the best of our knowledge, this is a novel study that elucidated the anti-tumor effects of canine B cells isolated from peripheral blood. Contrary to conventional studies that mainly focused on cell therapy, we conducted research based on the immunomodulators, IL-21 and anti-BCR. In this study, we explored the potential of this novel immunotherapy for cancer treatment in veterinary medicine. Additional in vivo experiments and early clinical trials should be conducted to establish their clinical significance.

Acknowledgements

The Authors are grateful to the Research Institute for Veterinary Science, Seoul National University and the BK21 PLUS Program for Creative Veterinary Science Research.

Footnotes

  • Authors’ Contributions

    GH Lim and SM park participated in the conception and design of the experiments. SY Sur, GH Lim and SM park participated in acquisition, analysis, and interpretation of data. SY Sur and GH Lim wrote the main manuscript text. HY Youn supervised the project. All Authors reviewed the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received May 30, 2023.
  • Revision received June 30, 2023.
  • Accepted July 3, 2023.

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Anti-tumor Effect of Activated Canine B Cells With Interleukin-21 and Anti-B Cell Receptor
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