β-Glucan induced plasma B cells differentiation to enhance antitumor immune responses by Dectin-1

Abstract

Background

B lymphocytes, essential in cellular immunity as antigen-presenting cells and in humoral immunity as major effector cells, play a crucial role in the antitumor response. Our previous work has shown β-glucan enhanced immunoglobulins (Ig) secretion. But the specific mechanisms of B-cell activation with β-glucan are poorly understood. Here, we took advantage of β-glucan to improve the antitumor immune response of B cells.

Results

In vitro experiments demonstrate that β-glucan enhance the differentiation of B220^lo CD138⁺ B cells, up-regulate co-stimulatory molecules, and increase the production of cytokines and Ig in response to various antigens. Using the Dectin-1 knockout mice, we revealed that β-glucan modulate B cell immune responses dependent on Dectin-1 receptor. In mouse models of Lewis lung cancer (LLC) tumors, combining β-glucan with programmed death-1(PD-1) blocking antibodies led to increase recruitment of CD19⁺ B cells in the tumor microenvironment (TME), higher numbers of germinal centers B cells (GC B) in the spleen and draining lymph node (DLN), elevate Ig production, and delay tumor progression.

Conclusions

These findings reveal that β-glucan can serve as a potent adjuvant to modulate B cell immune responses in a Dectin-1 dependent manner and improve immune checkpoint blockade (ICB) therapy in antitumor.

Clinical trial number

Not applicable.

Introduction

The host immune system plays a crucial role in antitumor defense, and strategies to augment clinical response have largely focused on the T cell compartment. However, multiple immune cells, including B lymphocytes, also contribute significantly to the antitumor response. B lymphocytes are key effectors of humoral immunity, responsible for producing Ig and playing essential roles as antigen-presenting cells [1]. Furthermore, B cells are an essential arm of tumor-infiltrating immune cells, observed in various solid tumors such as lung and melanoma cancers [2]. B lymphocytes not only suppress tumor progression by releasing antibodies and directly attacking cancer cells, but also contribute to the formation of tertiary lymphoid structures (TLS), which recruit immune cells to tumors and modulate antitumor immune activity [3]. B cells attack cancer cells through three methods: presenting antigens to promote T cell responses; secreting Ig to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC); and releasing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Granzyme B, and IFNγ to kill tumor cells directly [4].

Germinal centers are important in humoral immunity, located within B cell zones in TLS. TLS are transient ectopic lymphoid aggregates found in TME and tumor borders [5]. TLS contain large numbers of lymphocytes, mainly consist of T-cell-rich and B-cell-rich areas, and facilitate the differentiation of effector and memory T and B cells [6]. TLS also promote cytotoxic T lymphocyte (CTL) infiltration into tumors rapidly, enhancing the antitumor response. Patients have mature GC with tumor infiltrating B cells in TLS, which correlate with favorable outcome [7]. Amounts of single-cell RNA sequencing also demonstrated the potential functional contributions of B cells in ICB therapy [8]. As research, B cells are involved in GC formation, memory B cells generation, and long-lived plasma cells (PC) production, supported by T follicular helper (Tfh) cells and follicular dendritic cells (FDC) [9, 10]. B cells have been identified infiltrating in TME, but B cells overall functional role in cancer is incompletely understood [8].

Plasma B cells, also known as antibody-forming cells, are divided into short-lived and long-lived PC [11]. Short-lived PC, induced by exogenous mitogens, rapidly activate and proliferate, expressing unswitched or isotype-switched Ig [12]. In contrast, long-lived PC arise weeks after antigen exposure, primarily through germinal center reactions, and reside mainly in the bone marrow [13]. These long-lived PC, with half-lives exceeding six months, account for over 80% of serum Ig [14]. High-affinity antibody production requires class switching, driven by protein antigens and cytokines from T helper (Th1, Th2) cells [15].

β-Glucan, a naturally occurring polysaccharide, stimulates both innate and adaptive immune responses [16, 17]. Found in the cell walls of fungi, plants, and algae, the structure varies by source of β-glucan, leading to different immune responses [18]. It is known for its antitumor and anti-infective activities, depend on the long β-1,3-D-glucan backbone and β-1,6-glucan link side chains [19]. Previous studies have shown that whole glucan particle (WGP), purified from Saccharomyces cerevisiae cell walls, induces dendritic cell (DC) and macrophage maturation, activates T cells, and stimulates inflammatory cytokines and chemokines secretion [20, 21]. As studies demonstrated that β-glucan promote the immunological effects of antibody drugs against malignant tumor cells by activating Dectin-1 on myeloid cells and enhancing natural killer cells (NK) activity [22]. It is possible that β-glucan modulates myeloid cells and indirectly contribute into B cell responses. However, there were also reported that B-cell stimulation by β-glucan via pattern recognition receptors (PRR) resulted in activation of mitogen-activated protein kinases (MAPK) dependent pathways [23]. Stimulation of Dectin-1 on lipopolysaccharide (LPS) activated B cells resulting in selective production of IgG1 [24]. Nevertheless the specific mechanisms of B-cell activation with β-glucan are poorly understood.

Our recent research indicated that B cells surface expressed Dectin-1 and WGP-treated mice exhibited high serum Ig levels. Therefore, we hypothesized that WGP could act as a potent adjuvant, modulating B cell immune responses, enhancing Ig release, and improving humoral immunity and antitumor responses by Dectin-1. To explore the argument, we investigated the effect of WGP on B cells in vitro and evaluated its therapeutic efficacy in LLC tumor mouse models.

Materials and methods

Materials

WGP β-glucan, purified from the cell walls of Saccharomyces cerevisiae, was provided by Biothera (InvivoGen). Following alkaline and acid extraction, the cytoplasm and other polysaccharides, such as mannose, were removed to obtain intact hollow yeast cell walls composed primarily of long β-1,3 glucose polymers. The WGPs were hydrated in distilled water, sonicated to produce a single-particle suspension, and treated with 200 mM NaOH at room temperature for 20 min to eliminate any trace LPS contamination. The endotoxin level was 0.06 EU/mL, as tested by the gel-clot method (Associates of Cape Cod, East Falmouth, MA).

Mice and tumor cell lines

Female 6–8 week-old C57BL/6 wild-type (WT) mice or Dectin-1^−/− mice were purchased from Changzhou Cavens Lab Animal. CD4 ovalbumin (OVA) T-cell receptor transgenic OT-II mice were provided by Prof. Hai Qi, Tsinghua University. Mice were maintained under specific pathogen-free conditions in individually ventilated cages. Animal care and experiments were carried out in accordance with the guidelines approved by the institutional animal care and use committee (IACUC) of Nanjing Medical University. The LLC cell line and the B16-F10 melanoma cell line, derived from C57BL/6 mice, were obtained from the American Type Culture Collection. LLC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and B16-F10 cells in RPMI-1640, both supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2.

Cell preparation, isolation, and culture

Mouse splenic cells (1.5 × 10⁶/mL) were cultured in complete medium (RPMI 1640 with 10% FBS, penicillin-streptomycin, and 0.1% β-mercaptoethanol) with either 10 µg/mL LPS (Sigma-Aldrich), 100 µg/mL WGP, 100 µg/mL OVA (Sigma-Aldrich), or a combination for 3 days. LLC and B16-F10 cells (1 × 10⁶ each) were incubated for 3 days in DMEM or RPMI-1640 medium with 10% FBS. The cell-free supernatant was collected and filtered. Mouse splenic cells (1.5 × 10⁶/mL) were co-cultured with tumor supernatant for 3 days.

Splenic primary B cells were purified using negative B cell isolation kits (Invitrogen™), yielding populations of > 90%. Splenic CD4⁺ T cells were depleted using positive isolation kits (Invitrogen™), yielding populations of < 2.5%. Mouse bone marrow-derived dendritic cells (DC) were generated by culturing with 100 ng/mL of Flt-3 ligand (Invitrogen™) [25], and mouse bone marrow-derived macrophages (Mφ) were generated by culturing with 20 ng/mL of colony-stimulating factor (M-CSF) (Peprotech™) [26]. Either splenic total cells (1.5 × 10⁶/mL), purified primary B cells (1 × 10⁶/mL), depleted CD4⁺ T cells (1.2 × 10⁶/mL), or a combination of DC (2 × 10⁵/mL) and Mφ (2 × 10⁵/mL) were cultured with LPS in complete medium with or without WGP for 3 or 5 days.

Splenic cells from WT mice used in Sect. 3.2 and 3.4, splenic cells from CD4 OVA T-cell receptor transgenic OT-II mice used in Sect. 3.3, purified primary B cells from WT mice used in Sect. 3.5, purified primary B cells from WT mice or Dectin-1^−/− mice used in Sect. 3.6.

In vitro splenic cell differentiation, priming, and proliferation assay

Splenic total cells, purified B cells, depleted CD4⁺ T cells, or a combination of DC and Mφ were harvested, prepared as single-cell suspensions, and stained with fluorescein-labeled monoclonal antibodies (mAbs). For the proliferation assay, cells were labeled with CFSE (Invitrogen™) before culture. Data were acquired by flow cytometry on a FACS Canto II (BD Biosciences, San Jose, CA) and analyzed using FlowJo software Version 10.8.1 (TreeStar, Ashland, OR).

Mouse tumor models

LLC cells (3 × 10⁵) suspended in 100 µL PBS (Thermo Fisher Scientific Inc) were subcutaneously injected into the flanks of C57BL/6 mice. When tumor masses became palpable, around 6–7 days, mice were randomly assigned to control, PD1, or PD1 + WGP groups. Tumor masses were measured with calipers every other day and tumor volumes were calculated as follows: V = 1/2×the vertical length × the horizontal length². All mice were euthanized with carbon dioxide (CO₂) gas using closed chamber in accordance with the guide lines set by the IACUC when tumor length reached 15 mm, around day 21.

WGP and anti-PD-1 treatment of mice

For in vivo experiments, WT mice were treated with WGP (1 mg/100 µL PBS) for 2 weeks before examination. For the LLC tumor model, β-glucan-treated mice (PD1 + WGP) were orally administered 1 mg WGP in 100 µL PBS daily. Checkpoint blockade therapy mice (PD1, PD1 + WGP) were treated with 20 µg anti-PD-1 antibody (clone 29 F.1A12, Biolegend, San Diego, CA) in 100 µL PBS intravenously every 3 days. Independent on treatment damage, mice were treated with 100 µL PBS as control.

Tumor tissues were weighed, then minced into small pieces, and digested with a mixture of collagenase type IV, hyaluronidase, and deoxyribonuclease (Sigma-Aldrich) for 30 min at 37 °C by rotation. After filtration to remove insoluble fibers, red blood cells were lysed with red blood cell lysis buffer (Beyotime Biotechnology), and cells were washed twice with ice-cold PBS. Similarly, DLN and spleen were prepared as single-cell suspensions and stained with fluorescein-labeled mAbs.

Flow cytometry

Phenotype analysis of B cells by flow cytometry. Cells in 100 µL PBS were blocked with Fc blocking monoclonal antibody at 4 °C for 15 min. Single-cell suspensions were stained with fluorescein-labeled mouse-specific antibodies against CD3, CD4, CD44, CXCR5, PD1, CD19, B220, CD138, FAS, GL7, CD11b, Dectin-1, Major histocompatibility complex class II (MHC-II), CD86, CD80, CD40 or isotype controls (Biolegend) at 4 °C for 30 min in the dark. After two washes with PBS, cells were analyzed by flow cytometry. Immune cell subsets were characterized using specific markers, identifying plasma B cells (PC) (B220^lo CD138⁺), germinal center B cells (GC B) (CD19⁺ FAS⁺ GL7⁺), follicular helper T cells (Tfh) (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺), immunosuppressive B cells (CD11b⁺ CD19⁺), immunosuppressive plasma B cells (CD11b⁺ B220^lo CD138⁺), and regulatory plasma B cells (MHC-II^lo B220^lo CD138⁺) [27].

Cytokines and immunoglobulins assays

Cytokines in cellular supernatant and mouse serum samples were examined using the LEGENDplex™ MU Th1/Th2 Panel (8-plex) (Biolegend), a bead-based multiplex assay panel suitable for various flow cytometers. Blood samples were collected, clotted for at least 30 min, centrifuged for 20 min at 1000 g, and serum samples were stored at -20 °C. Mixed beads, detection antibodies, and SA-PE were added to each sample and standards, shaken at approximately 800 rpm at room temperature in dark. Samples were washed, and eight key targets (tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), Interleukin 2 (IL-2), IL-4, IL-5, IL-6, IL-10 and IL-13) were simultaneously quantified by flow cytometry.

Similarly, immunoglobulins in cellular supernatant and mouse serum samples were examined using the LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex) (Biolegend). Six key targets (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) were simultaneously quantified by flow cytometry, and data analysis was performed using LEGENDplex™ Data Analysis Software.

qRT-PCR

Tumor samples were treated with TRIzol reagent (Invitrogen), and total RNA was isolated and reverse transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems). Indicated mRNA levels were quantified by qRT-PCR amplification using the MyiQ single-color RT-PCR detection system (BioRad). Complementary DNA was amplified in a 20 µL reaction mixture containing 10 µL of SYBR Green PCR supermix (Invitrogen), 150 ng of complementary DNA template, and selected primers (200 nM). Data were acquired on a ViiA 7 Real-time PCR system (ABI). Primer sequences were designed with Primer Express Software Version 2.0 (Applied Biosystems) and are summarized in Supporting Information Table 1.

Statistical analysis

Data are expressed as means with standard error of the mean (SEM). Comparisons between multiple groups with independent samples were performed using ANOVA, while t-tests were used to compare two-group samples (with or without WGP treatment). Statistical significance thresholds were p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Data were analyzed using GraphPad Prism software (GraphPad, V8.0).

Results

β-Glucan up-regulates humoral immune response in vivo

To investigate the effects of β-glucan in vivo, we treated mice with WGP for 2 weeks. We found the therapy significantly increased the frequency of plasma B cells (B220^lo CD138⁺) in the spleen, whereas the frequencies of GC B cells (CD19⁺ FAS⁺ GL7⁺) and Tfh cells (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺) were not significantly altered (Fig. 1A). We also determined the cytokines and Igs release in serum. Mice treated with WGP showed up-regulated secretion of IFNγ, TNFα, IL-4, IL-5, IL-6, IgG1, IgG3 and IgM, whereas IL-2, IL-10, and IL-13 levels remained unchanged (Fig. 1B and C).

Fig. 1

β-Glucan up-regulates humoral immune response in vivo. Experimental setup (n = 5/group) with mice treated with WGP (1 mg) for 2 weeks. (A) Single cell suspensions from the spleen were stained with fluorescein-labeled mAbs. The summarized data are shown for plasma B cells (B220^lo CD138⁺), GC B cells (CD19⁺ FAS⁺ GL7⁺), and Tfh cells (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺). (B) The cytokines of mouse serum samples were collected and assayed by LEGENDplex™ MU Th1/Th2 Panel (8-plex). (C) The immunoglobulins of mouse serum samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). A representative histogram from three independent experiments with similar results is shown. (* P < 0.05, **P < 0.01, ***P < 0.001)

β-Glucan regulates B cell immune response stimulated with LPS

To assess the potential of wheat germ agglutinin (WGP) β-glucan in regulating B cell activation following stimulation by different antigens, we initially selected lipopolysaccharide (LPS) as a representative T-cell-independent antigen (TI-Ag). Subsequently, we cultured splenic cells from wild-type (WT) mice and investigated the regulatory effects of WGP β-glucan on B cells. WT splenic cells were stimulated by LPS with or without WGP in vitro for 72 h, resulting in four treated groups. Both WGP and LPS + WGP groups received 100 µg/mL WGP, then LPS and LPS + WGP groups received 10 µg/mL LPS. LPS activation led to a distinct phenotype with high expression of B cell surface markers (Fig. 2A), increased cells frequencies (Fig. 2B), and cytokines release (Fig. 2C). Cells stimulated by LPS combined with WGP showed up-regulation of surface marker MHC-II (Fig. 2A), increased frequency of B220^lo CD138⁺ B cells (Fig. 2B), improved TNFα and Igs (IgG2a, IgG2b, IgG3, IgM) release (Fig. 2C and D), but decreased CD86 expression (Fig. 2A) and reduced IFNγ and IL-10 release. IL-2, IL-5, and IL-6 levels were not significantly altered (Fig. 2C). These results suggest that WGP enhances B cell phenotypes and increases B220^lo CD138⁺ B cells frequency when stimulated with LPS.

Fig. 2

β-Glucan regulates B cell immune response stimulated with LPS. Wild type mice splenic cells were stimulated with LPS, WGP, or a combination of both in vitro for 72 h. (A) Surface marker expression of B cells was assessed by flow cytometry. (B) Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for cells (B220^lo CD138⁺), (CD19⁺ FAS⁺ GL7⁺), and (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺). (C) The cytokines of supernatant samples were collected and assayed by LEGENDplex™ MU Th1/Th2 Panel (8-plex). (D) The immunoglobulins of supernatant samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). Calculated the concentration of Ig produced by PCs (B220^lo CD138⁺) (10⁵). A representative histogram from three independent experiments with similar results is shown. (CON: black, WGP: yellow, LPS: blue, LPS + WGP: red. * P < 0.05, **P < 0.01, ***P < 0.001)

β-Glucan regulates B cell immune response stimulated with OVA

Next, we selected OVA as a T-cell-dependent antigen (TD-Ag). We then cultured splenic cells from CD4 OVA T-cell receptor transgenic OT-II mice and investigated the regulatory effects of WGP β-glucan on B cells. OT-II splenic cells were stimulated by OVA with or without WGP in vitro for 72 h, resulting in three treated groups. The OVA and OVA + WGP groups received 100 µg/mL OVA, and the OVA + WGP group received an additional 100 µg/mL WGP. B cells activated by OVA showed high expression of surface markers (MHC-II, CD86) (Fig. 3A), increased the frequencies of B220^lo CD138⁺ B cells and CXCR5⁺ PD1⁺ T cells (Fig. 3B), and up-regulated cytokines (IFNγ, TNFα, IL-6) release (Fig. 3C), but decreased CD80 expression (Fig. 3A) compared with CON group. Furthermore, we demonstrated that OVA + WGP intensified surface marker expression (CD40, CD80, CD86) (Fig. 3A), increased the frequencies of FAS⁺ GL7⁺ B cells and CXCR5⁺ PD1⁺ T cells (Fig. 3B), up-regulated TNFα and Igs release (Fig. 3C and D), but decreased B220^lo CD138⁺ B cells frequency (Fig. 3B) and down-regulated IFNγ, IL-2, and IL-6 release compared with OVA group. IL-5 and IL-13 levels were not significantly altered (Fig. 3C).

Fig. 3

β-Glucan regulates B cell immune response stimulated with OVA. OT-II mice splenic cells were stimulated with OVA, with or without WGP in vitro for 72 h. (A) Surface marker expression of B cells was assessed by flow cytometry. (B) Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for cells (B220^lo CD138⁺), (CD19⁺ FAS⁺ GL7⁺), and (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺). (C) The cytokines of supernatant samples were collected and assayed by LEGENDplex™ MU Th1/Th2 Panel (8-plex). (D) The immunoglobulins of supernatant samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). Calculated the concentration of Ig produced by PCs (B220^lo CD138⁺) (10⁵). A representative histogram from three independent experiments with similar results is shown. (CON: black, OVA: blue, OVA + WGP: prunosus. * P < 0.05, **P < 0.01, ***P < 0.001)

β-Glucan regulates B cell immune response stimulated with tumor supernatant

Furthermore, we selected tumor supernatant (LLC or B16-F10) as a TD-Ag, which also served to mimic the tumor microenvironment in vitro. Subsequently, we cultured splenic cells from WT mice and investigated the regulatory effects of β-glucan on B cells. WT splenic cells were co-cultured with supernatant from the LLC lung cancer cell line for 48 h, and treated with 100 µg/mL WGP simultaneously. LLC supernatant induced high expression of B cell surface markers (MHC-II, CD86, CD80) (Fig. 4A), increased the frequencies of B220^lo CD138⁺ B cells, FAS⁺ GL7⁺ B cells and CXCR5⁺ PD1⁺ T cells (Fig. 4B), and up-regulated cytokines (IFNγ, TNFα, IL-2, IL-6, IL-10) release (Fig. 4C) compared with CON group. Moreover, treatment with WGP resulted in an upregulation of CD40 compared to the control (CON) group (Fig. 4A). Additionally, WGP increased the frequency of B220^lo CD138⁺ B cells when compared to both the CON and LLC groups (Fig. 4B). Furthermore, WGP enhanced class switching to IgG1 compared to the LLC group, although this difference was not statistically significant (Fig. 4D). However, WGP decreased FAS⁺ GL7⁺ B cells frequency (Fig. 4B), down-regulated cytokines (IFNγ, TNFα, IL-2, IL-6, IL-10) release (Fig. 4C) compared with LLC group. The effects we observed with B16-F10 melanoma cancer cell line supernatant, WGP increased the frequencies of B220^lo CD138⁺ B cells and FAS⁺ GL7⁺ B cells, but decreased CXCR5⁺ PD1⁺ T cells frequency compared with B16 group (Fig. S1B).

Fig. 4

β-Glucan regulates B cell immune response stimulated with LLC supernatant. Wild type mice splenic cells were stimulated with supernatant from LLC, with or without WGP in vitro for 48 h. (A) Surface marker expression of B cells was assessed by flow cytometry. (B) Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for cells (B220^lo CD138⁺), (CD19⁺ FAS⁺ GL7⁺), and (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺). (C) The cytokines of supernatant samples were collected and assayed by LEGENDplex™ MU Th1/Th2 Panel (8-plex). (D) The immunoglobulins of supernatant samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). Calculated the ratio of Ig/IgA. A representative histogram from three independent experiments with similar results is shown. (CON: black, LLC: blue, LLC + WGP: red. * P < 0.05, **P < 0.01, ***P < 0.001)

β-Glucan regulates immune response of B cells co-cultured with antigen presenting cells

To further clarify the effects of WGP β-glucan on B cells, we purified B cells from WT mice splenic cells. WT cells were stimulated by LPS with or without WGP in vitro for 3 or 5 days, resulting in five groups: wild-type splenic total cells (TC), depleted primary CD4⁺ T splenic cells (-CD4⁺ T), purified primary B cells (B), B cells co-cultured with bone marrow-derived dendritic cells (B + DC), and B cells co-cultured with bone marrow-derived macrophages (B + Mφ). Combination stimulation increased B220^lo CD138⁺ B cells frequency in TC, -CD4⁺ T, and B groups (Fig. 5A and C), and decreased immunosuppressive B cells (CD11b⁺) frequency in TC and B + DC groups, but increased frequency in -CD4⁺ T and B + Mφ groups (Fig. 5B and C, S2A, S2C). Regulatory B cells (MHC-II^lo B220^lo CD138⁺) frequency decreased in TC, -CD4⁺ T, and B groups (Fig S2B, S2C). WGP up-regulated TNFα release, down-regulated IFNγ release in all groups, and affected IL-6 release differently across groups (Fig. 5D). WGP also increased Igs secretion in TC, B + DC, and B + Mφ groups, but not in depleted CD4⁺ T cells (Fig. 5E). Similar effects were observed after 5 days (Fig. S3), and CFSE dilution assays showed no significant differences in CD19⁺ B cell proliferation with or without WGP stimulation (Fig. S4).

Fig. 5

β-Glucan regulates immune response of B cells co-cultured with antigen presenting cells We separated the cells into 5 groups, including wild type mice splenic total cells (TC), depleted mouse primary CD4⁺ T splenic cells (-CD4⁺ T), purified mouse primary B cells (B), purified mouse primary B cells co-cultured with bone marrow-derived DC (B + DC), and purified mouse primary B cells co-cultured with bone marrow-derived macrophages (B + Mφ). Cells were stimulated with LPS, with or without WGP in vitro for 3 days. (A-C) Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for B cells (B220^lo CD138⁺), and immunosuppressive B cells (CD11b⁺ CD19⁺). (D) The cytokines of supernatant samples were collected and assayed by LEGENDplex™ MU Th1/Th2 Panel (8-plex). (E) The immunoglobulins of supernatant samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). Calculated the concentration of Ig produced by PCs (B220^lo CD138⁺) (10⁵). A representative histogram from three independent experiments with similar results is shown. (• LPS, ▪ LPS + WGP, * P < 0.05, **P < 0.01, ***P < 0.001)

β-Glucan regulates immune response of B cells through Dectin-1 directly

WT splenic B cells (CD3⁻CD19⁺) revealed a low but very well-defined population of Dectin-1 expressing cells (Fig. 6A). WT purified splenic primary B cells increased B220^lo CD138⁺ B cells frequency stimulated by WGP. Whereas, Dectin-1^−/− B cells were not significantly altered (Fig. 6B). Interestingly, when wild-type (WT) dendritic cells (DCs) were co-cultured with WT B cells, WGP reduced the frequencies of CD11b⁺ within the CD19⁺ B cell population (Fig. 5B and C, S3B, S3E) and of B220^lo CD138⁺ B cells (Fig. S2A, S2C, S3C, S3E). However, in groups where Dectin-1^−/− DCs were co-cultured with Dectin-1^−/− B cells, no significant alterations were observed (Fig. 6C and D). Furthermore, when Dectin-1^−/− DCs were co-cultured with WT B cells, WGP also decreased the frequencies of CD11b⁺ within the CD19⁺ B cell population (Fig. 6C) and of B220^lo CD138⁺ B cells (Fig. 6D). These findings suggest that WGP may modulate the differentiation of LPS-activated B cells through the Dectin-1 receptor.

Fig. 6

β-Glucan regulates immune response of B cells through Dectin-1 directly (A) Dectin-1 expression on wild type mice splenic B cells. Gating strategy and representative dot plots. Data shown was gated on the CD19-positive and CD3-negative B cells (red) population. (B) Wild type mice or Dectin-1^−/− mice splenic cells were purified mouse primary B cells. Cells were stimulated with LPS, with or without WGP in vitro for 72 h. Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for B cells (B220^lo CD138⁺). (C-D) Dectin-1^−/− mice bone marrow-derived DC co-cultured with B cells, which purified from wild type mice or Dectin-1^−/− mice splenic cells. Cells were stimulated with LPS, with or without WGP in vitro for 3 days. Single cell suspensions were stained with fluorescein-labeled mAbs. The summarized data are shown for immunosuppressive B cells (CD11b⁺ CD19⁺) (C), (CD11b⁺ B220^lo CD138⁺) (D). A representative histogram from three independent experiments with similar results is shown. (• LPS, ▪ LPS + WGP, * P < 0.05, **P < 0.01, ***P < 0.001)

β-glucan combined with anti-PD-1 therapy inhibits tumor growth in vivo

To investigate the in vivo antitumor effects of WGP combined with anti-PD-1 antibody, C57BL/6 mice bearing murine lung LLC were treated with oral 1 mg WGP in 100 µL PBS daily and 20 µg anti-PD-1 antibody intravenously once every 3 days. Combination therapy significantly decreased tumor burden compared to PBS therapy (Fig. 7A). Tumor-infiltrating B cells (CD19⁺) significantly increased with treatment, and GC B cell frequency increased in both spleen and DLN (Fig. 7B). Combination therapy up-regulated Igs release (IgG1, IgG2a, IgG3, IgM) (Fig. 7C), and increased mRNA levels of B-cell-related genes Fcrl5 and Ido1, but decreased Btla levels in TME (Fig. 7D). IL-5 release also significantly increased with combination therapy (Fig. S5). These data indicate that WGP enhances the effects of anti-PD-1 antibody, possibly through B cell-mediated humoral immunity, leading to delayed tumor progression.

Fig. 7

β-Glucan combined with anti-PD-1 therapy inhibits tumor growth in vivo. Experimental setup (n = 5–8/group) with mice being treated with WGP (1 mg) orally at day 7–20, or systemic anti-PD1 antibodies (20 µg) i.v. at day 7, 10, 13, 16, and 19 or combination. Mice were euthanized at day 21. (A) The tumor masses were measured with a caliper every other day once tumors were palpable, and tumor dimension volumes were calculated. Then tumor masses were photographed and weighed when the mice were euthanized. (B) Single cell suspensions from the tumor samples, DLN, or spleens, as indicated, were stained with fluorescein-labeled mAbs. The summarized data are shown. (C) The immunoglobulins of mouse serum samples were collected and assayed by LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex). (D) qRT-PCR extracted the mRNA levels. A representative histogram from three independent experiments with similar results is shown. CON: black, PD1: red, PD1 + WGP: blue. (* P < 0.05, **P < 0.01, ***P < 0.001)

Discussion

The differentiation of B cells into plasma B cells that secrete Ig is initiated by antigen activation [28]. Traditional B cell responses are categorized into two types based on the antigen stimulation: TI-Ag and TD-Ag [29]. TI-Ag, such as LPS, activate B cells via Toll-like receptor 4 (TLR4) independently of T cell assistance, leading to rapid expansion and differentiation of B cells [30]. These responses typically generate short-lived B cells producing low-affinity antibodies, although in specific cases, TI-Ag can induce the secretion of long-lived antibodies [31]. In contrast, TD-Ag are proteinaceous antigen that require T cell help for B cell activation. Upon recognition by the B cell receptors (BCR), TD-Ag are processed and presented via MHC-II to activate CD4⁺ T cells. Subsequent interactions between B cells and T cells involve CD40-CD40L co-stimulation and cytokines signaling (e.g., IL-4, IL-21), promoting B cell differentiation into memory B cells and long-lived PC within germinal centers [32]. For instance, the model antigen OVA activates CD4⁺ T cells specifically in OT-II mice. Beyond exogenous antigens, endogenous antigens like tumor antigens also activate B cells in a T cell-dependent manner. As important antigen-presenting cells, B cells can process and present tumor antigens via direct or cross-presentation pathways, forming antigenic peptide-loaded B cells [33]. Both MHC-I and MHC-II molecules are involved in presenting antigenic peptides, supporting a unified mechanism for MHC-restricted endogenous antigen presentation [34]. Additionally, B cells have been observed to activate CD4⁺ T cells even in the absence of exogenous antigen within TME [35], highlighting their role in adaptive immune responses.

In our study, we investigated the effects of various antigens (LPS, OVA, tumor supernatant) alone and in combination with the potent immune adjuvant WGP β-glucan on B cell responses in vitro. We observed that combination with WGP β-glucan stimulated, both TI-Ag and TD-Ag significantly increased the expression of B cells surface markers, particularly MHC-II and CD40. MHC-II molecules play a key role in presenting both exogenous and endogenous antigens to CD4⁺ T cells, facilitating effective immune responses. CD40-CD40L co-stimulation is essential for activating B and T cells, thereby enhancing immune responses. In addition, the effects of WGP β-glucan vary depending on the type of antigen. Notably, WGP β-glucan up-regulated the expression of B cell co-stimulatory molecules (CD40, CD80, and CD86) and MHC-II when induced by OVA, a T-cell-dependent antigen (TD-Ag). Conversely, WGP β-glucan down-regulated the expression of CD86 when stimulated by LPS, a T-cell-independent antigen (TI-Ag). Furthermore, WGP β-glucan had a limited impact on B cell surface markers when cultured with tumor supernatant from Lewis lung carcinoma (LLC) or B16-F10 melanoma cells. These findings suggest that WGP β-glucan has the potential to enhance B cell responses induced by both T-independent (TI-Ag) and T-dependent (TD-Ag) antigens, with differential effects depending on the specific antigen. We further observed that the frequencies of B220^lo CD138⁺ cells, FAS⁺ GL7⁺ cells and CXCR5⁺ PD1⁺ cells increased upon stimulation with antigens in vitro. Additionally, WGP β-glucan significantly increased the frequency of B220^lo CD138⁺ cells, particularly in responses induced by LPS and tumor supernatant. However, WGP β-glucan decreased the responses for OVA-induced germinal center B cells and follicular helper T cells. Additionally, WGP β-glucan further improved FAS⁺ GL7⁺ cells frequency compared to OVA or B16 supernatant alone. Whereas, WGP β-glucan decreased the responses for LLC supernatant. Moreover, WGP β-glucan increased CXCR5⁺ PD1⁺ cells frequency in response to OVA-induced conditions. Whereas, WGP β-glucan decreased the responses for B16 supernatant. Tfh (CXCR5⁺ PD1⁺) cells are critical for GC B (FAS⁺ GL7⁺) cells reactions, facilitating the production of high-affinity antibodies, memory B cells, and long-lived PC cells. The GC is pivotal for the humoral immune response, where somatic hypermutation occurs [36]. These data indicated that WGP β-glucan capacity for regulating cell differentiation induced by antigens, but the effects on different antigens are not completely consistent.

In our study, we also found that WGP β-glucan increased TNF-α expression induced by antigens in vitro. TNF-α, an essential inflammatory cytokine secreted during acute inflammation that regulates diverse signaling pathways, potentially influencing cell survival and apoptosis. Interestingly, TNF-α has been implicated in regulating B cell lymphopoiesis in aging and therapeutic contexts [37]. However, the down-regulation of IFN-γ implied a decreased immune capacity for antitumor. IFN-γ was also required for class-switch recombination, which switches IgM to other immunoglobulins [38]. For example, IFN-γ promotes IgG2a production in LPS-activated B cells, which exhibit potent effector functions like complement activation and antibody-dependent cellular cytotoxicity [39]. Moreover, WGP β-glucan significantly up-regulated the production of various Ig isotypes upon antigen stimulation. To exclude the effect of basal expression of Ig, which secreted by tumor cell lines (LLC or B16) in supernatant. We analyzed the production with Ig/IgA by plasma B cells.TD-Ag typically induce affinity maturation and class switching from IgM to IgG or IgA, while TI-Ag primarily induce the production of low-affinity natural antibodies such as IgM and IgG3 in mice. TD-Ag drive class switching through signals from T helper cells and cytokines, whereas TI-Ag induce class switching through signals from BCR cross-linking and various cytokines. IgA predominates at mucosal surfaces, while IgG is the major circulating antibody with stronger proinflammatory capabilities compared to IgA [40, 41]. Taken together, all of these data indicate that WGP β-glucan synergistically enhanced the differentiation of B220^lo CD138⁺ B cells, suggesting a potentiation of B cell activation and humoral immune responses.

To explore the role of different cell subsets in immune responses further, we purified primary B cells from WT mice spleens and co-cultured them with various antigen-presenting cell subsets in vitro. We found that WGP β-glucan promoted B220^lo CD138⁺ B cells differentiation, while suppressing regulatory plasma B cells (MHC-II^lo B220^lo CD138⁺) and immunosuppressive CD11b⁺ B cells in total splenic cells and purified B cells populations, particularly in co-culture with LPS-stimulated DC for 3 days. Regulatory plasma B cells are defined by their ability to produce IL-10 and IL-35, which suppress NK cells, neutrophils, and effector CD4⁺ T cell development [27]. Co-culture with DC notably suppressed CD11b⁺ B cells under WGP combined treatment. CD11b⁺ B cells are immunosuppressive cells found in Peyer’s patches during colitis, where they actively recruit Treg cells to facilitate IgA isotype switching [42]. Conversely, co-culture with macrophages inhibited PCs development induced by LPS. Macrophages can suppress direct B cells activation independently of mitogen concentration or macrophage numbers [43], although M2 macrophages can stimulate B cells survival, proliferation, and plasmablast differentiation during viral infection [44]. Additionally, WGP β-glucan increased TNF-α, IL-6, and IL-10 expression in B cells or DC/Mφ co-culture groups, essential for promoting Ig class switching. Moreover, similar effects were observed in groups treated for 5 days. Together, these findings indicate that WGP β-glucan can modulate B cells surface markers and differentiation induced by antigen, enhancing B220^lo CD138⁺ B cells frequency, regulate cytokines and Ig production.

As the result, WGP β-glucan increased the frequency of B220^lo CD138⁺ B cells with LPS-activated mouse B cell in vitro. The utilization of Dectin-1^−/− mice to further explore β-glucan modulates B cell dependent on Dectin-1 receptor. There were not significantly change for the frequency on Dectin-1^−/− B cells with WGP stimulated. Although the expression of Dectin-1 on B cell less than myeloid cells, these findings also suggested that WGP β-glucan could modulate B cell dependent on Dectin-1 receptor directly.

Notably, WGP β-glucan increased the frequency of plasma B cells, and improved the cytokines (IFNγ, TNFα, IL-4, IL-5, IL-6) secretion and Ig (IgG1, IgG3, IgM) production in vivo. Our previous studies have demonstrated that WGP β-glucan potentiated the therapeutic efficacy of anti-PD-1 antibody by reducing the differentiation of Tregs and delaying tumor progression [45]. Here, we found that WGP β-glucan also enhanced B cell infiltration into TME and increased GC B cells frequency in secondary lymphatic organs in vivo. Furthermore, studies have shown that B cell-related genes such as MZB1, JCHAIN, FCRL5, IDO1, and BTLA are significantly up-regulated in patients who respond to ICB treatment compared to non-responders [8]. In our study, WGP β-glucan treatment increased the expression of FCRL5 and IDO1, potentially enhancing responses to ICB treatment. These effects were consistent with enhanced cytokines and Ig phenotypes observed in combined treatment models. Thus, WGP β-glucan modulates B cell-mediated humoral immune responses, highlighting its potential in enhancing antitumor immunity.

In summary, our study demonstrates that WGP β-glucan enhances humoral immune responses in B cells stimulated by antigens in a Dectin-1 dependent manner. Moreover, this immune adjuvant promotes B cell infiltration into the TME, increases GC B cell differentiation in both spleen and DLN, and enhances antitumor responses. These findings elucidate the mechanisms underlying the supportive effects of WGP β-glucan in ICB therapy and highlights the pivotal role of B cell-mediated humoral immune responses in antitumor immunity.