2022 Volume 45 Issue 7 Pages 926-933
B cells are types of lymphocytes that are involved in the production of antibodies against pathogens. They also deliver and present antigens for the priming of T cells. Recently, we developed an in vivo splenic marginal zone (MZ) B cell-targeting liposomes decorated with polyethylene glycol (PEG) containing a hydroxyl-terminus group (HO-PEG-Lip). In an expansion of a previous study, we used HO-PEG-Lip as an in vitro antigen delivery tool to splenic B cells to test the ability of this formulation to overcome the limitations of the poor antigen uptake ability of B cells for implantation. To achieve our purpose, various factors were optimized. These factors include cell number, liposome concentration, pre-opsonization of liposomes, fresh serum concentration, and incubation time, all of which affect the extent of interaction between liposomes and B cells. As a result, we confirmed that the HO-PEG-Lip required incubation at 37 °C for at least 20 min with 50% mouse fresh serum followed by a subsequent incubation at 37 °C for at least another 30 min with splenic B cells. By using such a loading system, fluorescein isothiocyanate (FITC)-labeled ovalbumin (OVA), a model antigen, encapsulated in HO-PEG-Lip could be efficiently loaded into splenic B cells. In addition, HO-PEG-Lip and FITC-labeled OVA encapsulated in HO-PEG-Lip were efficiently associated with MZ-B cells with high levels of complement receptors (CRs) rather than follicular B cells with low levels of CRs. These results propose a novel and useful system to efficiently load antigens into B cells in vitro by taking advantage of complement systems.
Advances of biomedical science have led to a paradigm shift in drug design from simple small molecules to more complicated systems. One of the recent promising approaches is therapy that utilizes living cells.1) Thanks to advancements in recent research that have revealed the mechanisms of cell differentiation and the functions of each cell, various functional cells have become applicable as drugs for the treatment of certain diseases in clinical settings. For example, cell-based vaccine is a promising strategy that induces immune responses against infections and cancers by injecting antigen-loaded antigen presenting cells (APCs) such as dendritic cells (DCs).2,3) Another example of cell-based therapy is chimeric antigen receptor T cells,4) which are engineered T cells that express artificial T cell receptors. Following implantation, these cells recognize specific antigens and kill target tumor cells without the involvement of the major histocompatibility complex. These therapies usually strengthen the native physiological functions of cells via physical and gene manipulations, such as the loading of antigens or transfecting artificial receptors, and, thus, they impart potent therapeutic effects after implantation. The selection of cells most suited for therapy and efficient methods that can be used to manipulate these cells have been dominant factors in developing these efficient cell-based therapies.
In addition to DCs, B cells are now thought to have potential utility for cell-based therapies in the treatment of infectious diseases, cancers, and many other life-threatening maladies. B cells are a type of lymphocyte that functions as APCs.5,6) Antigen-specific B cells stimulate CD4+ T cells for humoral immune responses and also CD8+ T cells via cross-presentation for cellular immune response.7) Also, large numbers of B cells are easy to obtain via non-invasive methods that involve separation from the blood of donors and animals. Furthermore, an efficient in vitro cultivation strategy to generate ready-to-use B cells has already been developed via activation through CD40/CD40L pathways.8) Regardless of these benefits, the use of B cells for cell-based therapy has continued to encounter obstacles. In general, B cells have a low ability to uptake substances such as antigens.9) Therefore, efficient methods must be developed to accomplish the loading of a large number of substances into B cells.
To achieve the efficient loading of antigen into B cells in vitro, we proposed the use of liposomes modified by polyethylene glycol (PEG) with a hydroxyl group on its terminal (HO-PEG-Lip) as an antigen carrier. We have reported that HO-PEG-Lip activates the complement system via an alternative pathway, resulting in opsonization with complement component 3 (C3).10) B cells are known to express complement receptors (CRs) CR1 and CR2 on their surface.11) These developments led us to assume that HO-PEG-Lip opsonized with C3 could be efficiently associated with B cells via CR-mediated endocytosis. As an expansion of our previous work, in the present study we attempted to develop a splenic B cell-targeted system for loading antigens in vitro via the use of HO-PEG-Lip. We investigated the association of HO-PEG-Lip with murine splenic B cells under certain in vitro conditions to reach a maximized association of HO-PEG-Lip with B cells. Our in vitro delivery system was intended to deliver the antigens to B cells in vitro, which would be a useful tool in the development of B cell-based therapies.
Hydrogenated egg phosphatidylcholine (HEPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (CH3O-PEG2000-DSPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[hydroxyl (polyethylene glycol)-2000] (HO-PEG2000-DSPE) were purchased from Nippon Fine Chemical Co., Ltd. (Hyogo, Japan). Cholesterol (Chol), and Hoechst 33342 were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Lipophilic carbocyanine dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Thermo Fisher Scientific (MA, U.S.A.). Fluorescein isothiocyanate isomer I, and ovalbumin (OVA) were purchased from Merck (Darmstadt, Germany). All other reagents were of analytical grade.
AnimalsMale C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan) and were 5–8 weeks old at the beginning of each experiment. All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of Tokushima University (Approved No. T2019-47).
Preparation of DiI-Labeled PEGylated LiposomesDiI-labeled hydroxy PEG-modified liposomes (DiI-HO-PEG-Lip) and DiI-labeled methoxy PEG-modified liposomes (DiI-CH3O-PEG-Lip) were prepared via microfluidic mixing. Briefly, HEPC and Chol with either HO-PEG2000-DSPE or CH3O-PEG2000-DSPE were dissolved in ethanol (organic phase) at a molar ratio of 1.94 : 1 : 0.06. Lipophilic dye, DiI, (1% total phospholipid) was also added into the organic phase to label the liposomes. The organic phase was rapidly mixed with the aqueous phase (N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffered saline (HBS)) using a Nanoassemblr® (Precision NanoSystem, Vancouver, Canada) at a flow rate ratio of 1 : 3 with a total flow rate of 9 mL/min. To remove ethanol in the liposome solutions, the resultant mixture was dialyzed with Biotech CE Tubing (molecular weight cut off 300 kDa) (Repligen, MA, U.S.A.) for 12 h against saline at room temperature. The mean diameter and the zeta potential of the resultant liposomes were determined using a ZETASIZER NANO equipped with 633 nm He-Ne laser (Malvern, Worcestershire, U.K.). For the dynamic light scattering (DLS) measurements, liposomes were diluted with phosphate buffered saline (PBS) to a final concentration of 0.1 mM phospholipids in a disposable cuvette. All samples were measured at a scattering angle of 173° (backscatter) using the “General purpose” analysis model and the default size analysis parameters as well as a refractive index of 1.59 for the polystyrene particle matrix as sample parameter. For the measurement of zeta potential, samples were diluted with PBS to a final concentration of 0.5 mM phospholipids in a clear disposable zeta cell. The values for the mean diameter and zeta potential of the DiI-HO-PEG-Lip were 119.8 nm (PdI 0.135) and −6.05 mV, respectively. The values for the mean diameter and zeta potential for the DiI-CH3O-PEG-Lip were 113.9 nm (PdI 0.063) and −6.62 mV, respectively. Phospholipid concentrations in the liposome preparations were determined via colorimetric assay.12)
Preparation of OVA-Containing PEGylated LiposomesTo assess the uptake of antigen, fluorescein isothiocyanate (FITC)-labeled OVA, a model antigen, was encapsulated in HO-PEG-Lip. Briefly, non-labeled OVA was dissolved in 0.1 M sodium carbonate buffer (pH 9) to prepare a 4 mg/mL solution. Fluorescein isothiocyanate isomer I was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg/mL solution. The fluorescein isothiocyanate isomer I solution (50 µL) was added to the OVA solution (1 mL). The mixture was incubated at 4 °C for 8 h. In order to stop the reaction, NH4Cl was added to a final concentration of 50 mM with incubation at 4 °C for a further 2 h. The reaction mixture was dialyzed with Seamless Cellulose Tubing, size 18 (FUJIFILM Wako Pure Chemical Corporation), against saline at room temperature for 24 h to remove unbound FITC.
Liposomes were prepared via microfluidic mixing, as described above. An organic phase (ethanol) that included lipids (HEPC:Chol:HO-PEG2000-DSPE or HEPC:Chol:CH3O-PEG2000-DSPE = 1.94 : 1 : 0.06 as molar ratio) was mixed using a Nanoassemblr® system with an aqueous phase (1.5 mg/mL FITC-OVA in HBS) at a flow rate ratio of 1 : 3 with a total flow rate of 9 mL/min. To remove ethanol from the liposome solutions, the resultant mixture was dialyzed with Biotech CE Tubing (molecular weight cut off 300 kDa) for 12 h against saline at room temperature. Unencapsulated FITC-OVA was removed by size exclusion chromatography (Sepharose CL-4B, GE Healthcare, Uppsala, Sweden). The mean diameter and the zeta potential of the resultant liposomes were determined using a ZETASIZER NANO. The values for mean diameter and zeta potential of the OVA-containing HO-PEG-Lip were 159.5 nm (PdI 0.096) and −7.84 mV, respectively. The values for mean diameter and zeta potential of the OVA-containing CH3O-PEG-Lip were 150.3 nm (PdI 0.086) and −9.68 mV, respectively. Phospholipid concentrations in the liposome preparations were determined via colorimetric assay.12) Encapsulated FITC-OVA in the liposomes was determined by Lowry protein assay (DC protein assay kit, Bio-Rad Laboratories, CA, U.S.A.). The loading amount of OVA in OVA-containing HO-PEG-Lip was 28.6 µg OVA/µmol phospholipids and the loading amount of OVA in OVA-containing CH3O-PEG-Lip was 52.7 µg OVA/µmol phospholipids.
In Vitro Interaction of Liposomes with Murine Splenic B CellsTo obtain a murine spleen cell suspension, spleen was collected from Male C57BL/6N mice. Spleen fragments were prepared and then pressed through a cell strainer (100 µm pore size, Greiner Bio-One, Kremsmünster, Austria) into an RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cosmo BIO Co., Ltd., Tokyo, Japan), 100 units/mL of penicillin, 100 µg/mL of streptomycin (FUJIFILM Wako Pure Chemical Corporation), and 50 µmol/L 2-mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation). The dispersed spleen cells were centrifuged at 300 × g for 7 min. In order to lyse red blood cells, 2 mL of ammonium chloride lysis buffer (0.83% NH4Cl) was added to the cell pellet, which was let stand for 3 min. After the lysis of red blood cells, the cell suspension was washed by centrifugation and resuspended with RPMI medium. The number of cells was counted using a CellDrop BF (DeNovix, Wilmington, DE, U.S.A.).
The resultant spleen cells were incubated at 37 °C with a mixture of either DiI-HO-PEG-Lip or DiI-CH3O-PEG-Lip in the presence of either fresh mouse serum or heat-inactivated serum (56 °C, 30 min). Various factors were changed such as cell numbers, liposome concentration, fresh serum concentration for pre-opsonization of liposomes, and incubation time, which could have affected the extent of interaction between liposomes and B cells. After the incubation, the spleen cells were washed with cold PBS (−) and stained with a combination of FITC-labeled anti-mouse CD21/35 (eBioscience, CA, U.S.A.) and Alexa Fluor 647-labeled anti-mouse CD23 (BioLegend, San Diego, CA, U.S.A.). To identify which subset of splenic B cells took up the liposomes, the cells were analyzed by flow cytometry (Gallios, Beckman Coulter, CA, U.S.A.). The data were further analyzed using Kaluza software (Beckman Coulter). Marginal zone B cells (MZ-B) were gated on CD21high and CD23− populations, and follicular B cells (FO-B) were gated on CD21low and CD23+ populations.
In Vitro Interaction of FITC Labeled OVA with Murine Splenic B CellsMurine spleen cell suspension was prepared as described above. Naïve B cells were positively isolated from the cell suspension by using B220 MicroBeads on an auto MACS pro Separator (Milteny Biotech, Teterow, Germany). Either FITC-OVA containing HO-PEG-Lip or FITC-OVA containing CH3O-PEG-Lip (100 µL, 10 µg OVA, 1.7–3.1 mM phospholipids) were pre-incubated with 50% fresh mouse serum (400 µL) for 20 min at 37 °C. Then, the B cells (500 µL, 1 × 106 cells), isolated from spleen cells, were added into the incubation mixture and incubated for another 30 min at 37 °C. Cells were washed twice with PBS (−) and stained with a combination of APC-labeled anti-mouse CD21/35 (BioLegend) and PE-labeled anti-mouse CD23 (eBioscience). To identify which subset of splenic B cells took up the liposomes, the cells were analyzed by flow cytometry (Gallios). The data were further analyzed using Kaluza software. Marginal zone B cells (MZ-B) were gated on CD21high and CD23− populations, and follicular B cells (FO-B) were gated on CD21low and CD23+ populations.
Confocal Microscopic Observation of B Cells Loaded with DiI-HO-PEG-LipMurine spleen cell suspension was prepared as described above. Naïve B cells were positively isolated from the cell suspension by using B220 MicroBeads on an auto MACS pro Separator (Milteny Biotech). DiI-HO-PEG-Lip and DiI-CH3O-PEG-Lip (100 µL, 1 mM) were separately pre-incubated with 50% fresh mouse serum (400 µL) for 20 min at 37 °C. Then, the B cells (500 µL, 1 × 106 cells), isolated from spleen cells, were added and incubated for another 30 min at 37 °C. Cells were washed twice with PBS (−) and fixed with 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 10 min at room temperature. After washing twice with PBS (−), Hoechst 33342 staining solution (20 µM) was added to the cells, which were then incubated for 10 min at room temperature. The cells were washed twice with distilled water and observed under a confocal laser-scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany).
Statistical AnalysisAll values are expressed as the mean ± standard deviation (S.D.) Statistical analysis was performed using a two-tailed unpaired t test and one-way ANOVA followed by a Tukey post hoc test using GraphPad InStat software (GraphPad Software, CA, U.S.A.). The levels of significance were set at * p < 0.05, ** p < 0.01, and *** p < 0.001.
B cells in the spleen are mainly divided into subsets of either FO-B cells expressing low CR1/2 or MZ-B cells expressing high CR1/2.13) The association of HO-PEG-Lip with splenic FO-B cells and MZ-B cells was analyzed by flow cytometry following 1 h of incubation with spleen cells in the presence of fresh naïve mouse serum (Fig. 1). In the FO-B cells, the fluorescence intensities of DiI associated with HO-PEG-Lip were entirely low even if the cell numbers were decreased (Fig. 1A). Compared with the FO-B cells, strong fluorescence intensities of DiI were detected in the MZ-B cells. Similar to the result of FO-B cells, the intensities were decreased with increases in the number of cells in the range of cell numbers we tested. According to the results shown in Fig. 1A, the numbers of spleen cells were fixed at 1 × 106 cells/mL to study the effect of lipid concentration on the association of HO-PEG-Lips with splenic FO-B cells and MZ-B cells. In the FO-B cells, the fluorescence intensities of DiI were entirely low even if the lipid concentrations were increased to the range of the lipid concentrations in HO-PEG-Lips we tested (Fig. 1B). The DiI intensities gradually increased with increases in the lipid concentration of up to 1 mM and then gradually decreased to 10 mM. In the MZ-B cells, relative to the FO-B cells, stronger fluorescence intensities of DiI were detected. The intensities gradually increased with increases in lipid concentration of up to 1 mM and then gradually decreased to 10 mM. Therefore, for subsequent experiments, the lipid concentration of HO-PEG-Lip was set at 1 mM.
(A) Effect of cell numbers on the interaction between HO-PEG-Lips and either splenic follicular B cells (FO-B) or marginal zone B (MZ-B) cells. DiI-HO-PEG-Lip (100 µL, 1 mM) were incubated for 1 h at 37 °C with different numbers of spleen cells (900 µL, 1 × 106, 5 × 106 and 1 × 107 cells) in the presence of 5% fresh mouse serum. Mean fluorescence intensity (MFI) of DiI associated with either FO-B cells (CD21low and CD23+ population) or MZ-B cells (CD21high and CD23− population) was analyzed by flow cytometry. (B) Effect of the phospholipid concentration in HO-PEG-Lip on the interaction between HO-PEG-Lip and either splenic FO-B or MZ-B cells. DiI-HO-PEG-Lip with different concentrations (100 µL, 0.1, 0.5, 1, 5, 10 mM) were incubated for 1 h at 37 °C with spleen cells (900 µL, 1 × 106 cells) in the presence of 5% fresh mouse serum. Fluorescence intensity of DiI of HO-PEG-Lip associated with either FO-B cells or MZ-B cells was analyzed by flow cytometry. The data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01.
The pre-opsonization effect of HO-PEG-Lip on the interaction between HO-PEG-Lip and B cells was studied. Incubating DiI-labeled HO-PEG-Lip with B cells in the presence of 5% fresh mouse serum without pre-opsonization resulted in a slight association of DiI with FO-B cells and a more significant association of DiI with the MZ-B cells (Fig. 2A). When HO-PEG-Lip was pre-opsonized via pre-incubation with 10% fresh mouse serum and subsequently incubated with splenic B cells, the fluorescence intensities of DiI were entirely increased approximately 3-fold in both FO-B cells and MZ-B cells. Interestingly, when DiI-labeled HO-PEG-Lip was pre-incubated with mouse heat-inactivated serum, which killed the complement activity, before incubation with B cells, the associations of DiI with FO-B cells and MZ-B cells disappeared (data not shown). This indicates that activation of the complement system by HO-PEG-Lip plays an important role in the interaction between HO-PEG-Lip and B cells. Then, the effect of fresh serum concentration for pre-incubation on the interaction between HO-PEG-Lip and B cells was studied further. In FO-B cells, the fluorescence intensities of DiI were gradually increased with increases in the serum concentration of up to 50% in the range of serum concentration we tested (Fig. 2B). Compared with FO-B cells, higher fluorescence intensities of DiI were detected in MZ-B cells. The fluorescence intensities were gradually increased with increases in the serum concentration of up to 50% in the range of serum concentration we tested. Finally, for subsequent experiments, the concentration of the fresh mouse serum was set at 50%.
(A) DiI-HO-PEG-Lip (100 µL, 1 mM) was incubated with spleen cells (900 µL, 1 × 106 cells) for 1 h at 37 °C in the presence of fresh mouse serum. Alternatively, DiI-HO-PEG-Lip (100 µL, 1 mM) was pre-incubated in the media containing 10% fresh mouse serum (400 µL) for 30 min at 37 °C. Then, spleen cells (500 µL, 1 × 106 cells) were added to the reaction mixture and further incubated for another 30 min at 37 °C. (B) DiI-HO-PEG-Lip (100 µL, 1 mM) was incubated for 30 min at 37 °C with the medium (400 µL) in the presence of different concentrations of mouse serum (10, 20, 30, 40, and 50%), respectively. Then, spleen cells (500 µL, 1 × 106 cells) were added to the reaction mixture and incubated for another 30 min at 37 °C. The association of DiI-HO-PEG-Lip with FO-B and MZ-B cells was analyzed by flow cytometry. The data represents the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
The effect of the pre-incubation time of HO-PEG-Lip with fresh mouse serum on the interaction between HO-PEG-Lip and B cells was studied (Fig. 3A). In the FO-B cells, fluorescence intensities of DiI associated with HO-PEG-Lip were slightly increased with increases in time (up to 20 min) for pre-incubation with fresh mouse serum (Fig. 3A). In the MZ-B cells, relative to FO-B cells, the fluorescence intensities of DiI were entirely high and slightly increased in a pre-incubation time-dependent manner (up to 20 min). When time was increased for incubation with the pre-incubated HO-PEG-Lip and spleen cells (Fig. 3B), in both FO-B cells and MZ-B cells, fluorescence intensities of DiI were gradually increased. The fluorescence intensities were higher in MZ-B cells than in FO-B cells. According to the results shown in Fig. 3, the times for the pre-incubations of HO-PEG-Lip with fresh mouse serum and with spleen cells were set at 20 and 30 min, respectively.
(A) DiI-HO-PEG-Lip (100 µL, 1 mM) was pre-incubated for 10, 20 or 30 min at 37 °C with 50% fresh mouse serum (400 µL). Then, spleen cells (500 µL, 1 × 106 cells) were added and incubated for another 30 min at 37 °C. (B) DiI-HO-PEG-Lip (100 µL, 1 mM) was pre-incubated with 50% fresh mouse serum (400 µL) for 20 min at 37 °C. Then, spleen cells (500 µL, 1 × 106 cells) were added and incubated for another 10, 20, 30, or 60 min at 37 °C. The association of DiI-HO-PEG-Lip with either FO-B cells or MZ-B cells was analyzed by flow cytometry. The data represents the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Following several studies described above, the optimal conditions needed to gain efficient loading of HO-PEG-Lip to splenic B cells included HO-PEG-Lip incubation with 50% fresh mouse serum for 20 min at 37 °C, which was followed by incubation of spleen cells with the pre-incubated HO-PEG-Lip for another 30 min at 37 °C. The Fig. 4A insertion pictures from confocal microscopy reveal that under the optimal conditions several red spots appeared on the surface of the cells. These red spots indicate the association of DiI-labeled HO-PEG-Lip with B cells, and no such spots of were observed when using DiI-labeled CH3O-PEG-Lip. Similarly, in DiI-labeled HO-PEG-Lip, larger fluorescence intensities were detected by flow cytometry in total B cells (Fig. 4A, left panel). This association was not detected, however, in DiI-labeled CH3O-PEG-Lip (Fig. 4A, left panel). In DiI-labeled HO-PEG-Lip, the fluorescence intensities were higher in MZ-B cells than in FO-B cells (Fig. 4A, right panel), which is consistent with the results shown in Figs. 1–3. To further confirm the issue of whether our developed and optimized method with HO-PEG-Lip can load encapsulated antigen to splenic B cells, FITC-labeled OVA, a model antigen, encapsulated into PEGylated liposomes (HO-PEG-Lip or CH3O-PEG-Lip) and pre-incubated with 50% fresh mouse serum, was incubated with spleen B cells. In HO-PEG-Lip, larger fluorescence intensities of FITC were detected in FO-B cells and MZ-B cells, but not in CH3O-PEG-Lip (Fig. 4B). In HO-PEG-Lip, the fluorescence intensities were larger in MZ-B cells than in FO-B cells. These data clearly indicate that we could dial in the optimal conditions to achieve antigen loading to B cells in vitro by means of complement-activating PEGylated liposomes (HO-PEG-Lip).
(A) DiI-HO-PEG-Lip and DiI-CH3O-PEG-Lip (negative control) (100 µL, 1 mM) was separately pre-incubated with 50% fresh mouse serum (400 µL) for 20 min at 37 °C. Then, B cells (500 µL, 1 × 106 cells) were isolated from spleen cells and added with incubation for another 30 min at 37 °C. Association of DiI-labeled PEG-Lip with total B cells was observed under confocal microscopy. The typical images from 3 independent experiments appear as inserts. Scale bars in the images represent 5 µm. Association of DiI-labeled PEG-Lip with total B cells, FO-B cells, or MZ-B cells was analyzed by flow cytometry. (B) Either FITC-labeled OVA-containing HO-PEG-Lip or FITC-labeled OVA-containing CH3O-PEG-Lip (100 µL, 10 µg OVA, 1.7–3.1 mM phospholipids) was pre-incubated with 50% fresh mouse serum (400 µL) for 20 min at 37 °C. B cells (500 µL, 1 × 106 cells), isolated from mouse spleen, were then added and incubated for a further 30 min at 37 °C. Associations of FITC-OVA with either FO-B cells or MZ-B cells were analyzed via flow cytometry. The data represent the mean ± S.D. (n = 3). ** p < 0.01 *** p < 0.001.
The aim of this study was to develop a useful method to achieve loading antigen to splenic B cells in vitro. In this study, HO-PEG-Lip, pre-incubated with fresh mouse serum, was efficiently associated with splenic B cells—particularly with MZ-B cells (Figs. 2–4). Actually, FITC-OVA, a model antigen, encapsulated in HO-PEG-Lip was also associated with splenic B cells following pre-incubation with fresh mouse serum (Fig. 4B). The optimized incubation conditions are as follows. The first step involved the opsonization of nanocarriers. To accomplish this, HO-PEG-Lip (100 µL, 1 mM) was incubated at 37 °C with 50% mouse naïve serum (400 µL) for 20 min. The second step involved the association of opsonized nanocarriers with splenic B cells, and an incubation mixture containing opsonized nanocarriers was mixed and incubated at 37 °C with the B cells (500 µL, 1 × 106 cells/mL) for another 30 min. We employed liposomes modified with PEG containing a hydroxyl group on the terminal (HO-PEG-Lip), which has been confirmed to activate the complement system via an alternative pathway that accomplishes opsonization with C3.10) Because B cells are known to express CR1 and CR2 on their surface,11) it is assumed that the opsonized HO-PEG-Lip will efficiently associate with B cells. Our in vitro delivery system delivers antigens to B cells in vitro, which should be a useful tool for the development of B cell-based therapies.
Pre-incubation of HO-PEG-Lip with fresh mouse naive serum at 37 °C was indispensable for the association of HO-PEG Lip with splenic B cells (Figs. 2–4). We previously reported that HO-PEG-Lip activates the complement system, while CH3O-PEG-Lip does not.10) C3 is central to the complement activation. In general, C3b, an activated C3 fragment, is capable of reacting with, and covalently coupling to, hydroxyl groups on the target surface.14) Therefore, the incubation of HO-PEG-Lip with fresh naive serum would result in tagging the surface of HO-PEG-Lip with C3b, which is referred to as opsonization. There are mainly 2 subsets of B cells in the spleen; FO-B cells that express low levels of CR1 and CR2 and MZ-B cells that express high levels of CR1 and CR2.13) CR1 and CR2 are receptors for C3-derived activation fragments (C3b, iC3b, C3d, C3dg).15) In addition, MZ-B cells possess higher phagocytic ability since they are the cells responsible for the trapping of blood-borne pathogens in the spleen.16) Accordingly, the opsonization of HO-PEG-Lip via complement activation and the high expression of CR1/2 along with the higher phagocytic activity of MZ-B cells could be a major cause for the specific association of HO-PEG-Lip with MZ-B cells throughout this study (Figs. 2–4). This concern was confirmed by the observation that the association of HO-PEG-Lip to splenic B cells was diminished by the heat-inactivation of mouse serum at 56 °C for 30 min, which also inactivates activity in the complement system.
It appears that short-term periods of pre-incubation of HO-PEG-Lip with mouse naïve serum (20 min) (Fig. 3A) and for incubation of the opsonized HO-PEG-Lip with splenic B cells (Fig. 3B) is sufficient to load the antigens in HO-PEG-Lip to splenic B cells in vitro (Fig. 4B). Andersson et al.17) have reported that complement activation via the alternative pathway and the binding of C3 (opsonization) against a model biomaterial surface (polystyrene) occurs within 20 min. In addition, Hess et al. have observed that after the capturing of opsonized substances via CRs, B cells start to internalize the substances within 10 min.18) Actually, images of confocal microscopy indicate that B cells internalize the opsonized HO-PEG-Lip (Fig. 4A) presumably via CRs on their surface. These results indicate that the short-term incubation periods we set in this study are sufficient to achieve complement activation by HO-PEG-Lip and recognition of the opsonized HO-PEG-Lip by B cells.
With the recent development of cell manipulation technology, cell-based therapies are expected to become more popular. B cells have shown promise for use in cell-based therapies due to usability and many functions. Attempts to load therapeutic agents into B cells in vitro have been conducted in several previous studies. However, many of those attempts were achieved using expensive targeting elements such as antibodies and virus vectors. For example, Zhang et al. reported that B cells loaded with antigen encoding viral vector tends to induce antigen-specific immunity in mouse models after implantation.19) However, such attempts sometimes result in low loading efficacy and are time consuming. The method proposed in the present study has shown results superior to those previous methods due to a process that by comparison is simpler, shorter (within 50 min), and less expensive. In addition to such advantages, in general, liposomes can encapsulate various therapeutic agents such as small molecules, peptides, proteins, and nucleic acids.20) Therefore, our method of using nanocarriers such as HO-PEG-Lip would be an excellent platform for B cell-based therapies.
Manipulating the native physiological functions of cells enhances the efficacy of cell-based therapies. The main function of B cells is to produce high-affinity antibodies and also to present antigens to T cells.5) For example, manipulating a B cell’s ability to produce antibodies via the previously studied practice of transducing B cells with whole sequences of a new antibody21) makes those cells useful for the prevention and treatment of infectious diseases and cancers. Also, loading antigen with B cells allows those cells to act as cell-based vaccines.22) Toward the development of cell-based vaccines, DCs have been utilized as capable APCs.2) However, B cells have superior characteristics to DCs in population size,23) which makes it easy to isolate them from the body. Also, B cells have superior ability to migrate to lymphoid tissues after implantation compared with DCs.19) Therefore, B cell-based vaccines could be an alternative to DC-based vaccines. Therefore, in future studies we intend to apply this HO-PEG-Lip loading system to those strategies and confirm its therapeutic efficacy.
In conclusion, we were able to determine the optimal cultivation conditions for the loading of B cell-targeting carriers, which involved the loading of HO-PEG-Lip into splenic MZ-B cells. Our proposed system delivers antigens to B cells in vitro by taking advantage of the innate humoral immune system, which is referred to as the complement system, and would be a useful tool for the development of B cell-based therapies.
This study was in part supported by a Grant-in-Aid for Young Scientists (15K18921), and a Grant-in-Aid for Transformative Research Areas (A) (Publicly Offered Research) (21H05526) from the Takeda Science Foundation; by the Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan; by a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University; and, by JST SPRING Grant No. JPMJSP2113, Japan.
The authors declare no conflict of interest.
