The Effect of Novel Selenopolysaccharide Isolated from Lentinula edodes Mycelium on Human T Lymphocytes Activation, Proliferation, and Cytokines Synthesis

Polysaccharides isolated from Lentinula edodes are bioactive compounds with immunomodulatory properties. In our previous studies from L. edodes mycelium, we have isolated a selenium(Se)-enriched fraction (named Se-Le-30), a mixture of linear 1,4-α-glucan and linear 1,3-β- and 1,6-β-glucans. In this study, we analyzed the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs) and on the production of cytokines by peripheral blood mononuclear cells (PBMCs). Se-Le-30 had effects on T cell proliferation induced by Abs against CD3 and CD28. It significantly inhibited the proliferation of CD3-stimulated CD4+ and CD8+ T cells and enhanced the proliferation of CD4+ T cells stimulated with anti-CD3/CD28 Ab. Moreover, Se-Le-30 downregulated the number of CD3-stimulated CD4+CD69+ cells, CD4+CD25+ cells, as well as CD8+CD25+ cells, and upregulated the expression of CD25 marker on CD4+ and CD8+ T cells activated with anti-CD3/CD28 Abs. Furthermore, Se-Le-30 enhanced the synthesis of IFN-γ by the unstimulated and anti-CD3/CD28-stimulated PBMCs, inhibited synthesis of IL-2 and IL-4 by CD3-stimulated cells, and augmented the synthesis of IL-6 and IL-10 by unstimulated, CD3-stimulated, and CD3/CD28-stimulated PBMCs. Together, we demonstrated that Se-Le-30 exerts immunomodulatory effects on human T lymphocytes. These observations are of importance for the prospective use of Se-Le-30 in research or as a therapeutic compound.

Lentinan is one of the widely studied polysaccharides from L. edodes. It is a highly purified β-(1→6) branched β-(1→3)-glucan with molecular weight (Mw) of 1.153 × 10 3 g/mol, which is clinically used as an adjuvant in cancer therapies in some Asian countries [2].
It has been documented that the biological activity of fungal polysaccharides depends on their source (mycelium or fruiting bodies), methods of extraction, composition, Mw, branching degrees, and helical conformation [5][6][7]. Polysaccharides with higher Mw and triple helical conformation show stronger biological activity, which may be explained by

Proliferation Assay
Isolated PBMCs were counted. Up to 1 × 10 7 cells were washed, resuspended in 1 mL of RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 5% heatinactivated human serum (Sigma, Saint Louis, MO, USA) and transferred to a 14 mL Falcon tube. Next, 110 µL of Phosphate-Buffered Saline (PBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was added to 5 µL of CFSE stock solution. CFSE was added to the cells suspension, vortexed, and incubated at 25 • C, for 7 min in the dark. After incubation, cells were washed twice in PBS with 10% human serum. PBMCs (1 × 10 5 cells/well) were cultured in 96-well flat-bottom plates (Greiner Bio-One, Kremsmünster, Austria) for 5 days in the following variants: (1) cells stimulated with Dynabeads coated with anti-CD3/CD28 Abs (ratio 2:5, Gibco, MA, USA); (2) cells stimulated with anti-CD3 Ab (plates were pre-coated with 0.75 µg/mL of Ab, BD Pharmingen, San Diego, CA, USA); and (3) unstimulated cells (control). PBMCs were incubated in the presence of Se-Le-30 (100 µg/mL) or without polysaccharide adding an equivalent amount of water for injection instead (control cultures). After 5 days, cells were collected and transferred to 4 mL polypropylene Falcon tubes, washed in PBS, and resuspended in 100 µL of 1:400 Zombie Violet™ stock solution (BioLegend, San Diego, CA, USA). Cells were incubated for 20 min at room temperature in the dark and washed with 2 mL BD Pharmingen Stain Buffer (BSA, BD Biosciences, San Jose, CA, USA). Next, they were resuspended in 100 µL of Stain Buffer, and labelled with anti-CD3, anti-CD4, and anti-CD8 Abs (15 min at room temperature in the dark). After incubation, cells were washed with 2 mL of Stain Buffer, resuspended in 100 µL of PBS with 0.01% sodium azide (Sigma, Saint Louis, MO, USA), and acquired with a DxFlex flow cytometer (Beckman Coulter, Brea, CA, USA) using CytExpert software (Beckman Coulter). Flow cytometry data were analyzed using FlowJo software (v. 10.8.1; BD, Ashland, OR, USA). The division index was calculated as the total number of divisions divided by the number of cells at the start of the culture. The proliferation index was calculated as the total number of divisions divided by the number of cells that went into division. The expansion index was calculated as the total number of cells divided by the number of cells at the start of the culture. The replication index was calculated as the total number of divided cells divided by the number of cells that went into division.

Multiplex Cytokine Profiling
Cytokines detection in cell culture supernatants was performed by Luminex ® Multiplex Assay. 2 × 10 5 of PBMCs were cultured for 24 h in variants described above. Initially, dynabeads coated with CD3/CD28 Abs have been removed by a magnet. Cell cultures were transferred to 4 mL polypropylene tubes and centrifuged. Cell-free supernatants were recovered and stored at −80 • C for future analysis.
Before analysis, Luminex calibration and verification were performed using MAGPIX Calibration Kit and MAGPIX Performance Verification Kit (Merck Millipore, Darmstadt, Germany). The concentration of IL-2, IL-4, IL-6, IL-10, and interferon (IFN)-γ was measured on MAGPIX (Merck Millipore, Darmstadt, Germany) with Luminex-based bead array MILLIPLEX ® Human Cyto Panel A (Merck Millipore, Darmstadt, Germany) according to manufacturer's instructions. Standards and quality controls were run on the same plate as analyzed supernatants. Data were analyzed using xPONENT software (Luminex Corp., Austin, TX, USA).

Cell Viability Assay
On 96-well flat-bottom plates, 2 × 10 5 of PBMCs were seeded (Greiner Bio-One, Kremsmünster, Austria) and cultured for 24 h at 37 • C in a humidified atmosphere with 5% CO 2 , in the presence of Se-Le-30 (100 µg/mL) and with an equivalent amount of medium and water for injection as controls. After incubation, cells were harvested, washed in 2 mL of PBS, resuspended in 100 µL of Zombie Violet™ (BioLegend, San Diego, CA, USA) stock solution at a ratio of 1:400, incubated for 20 min at room temperature in the dark, then washed with 2 mL of Stain Buffer and labeled with mouse anti-human CD3 Ab (CD3-PerCP, Clone SK7, BD Biosciences, San Jose, CA, USA) in 100 µL of Stain Buffer for 15 min. Next, cells were washed in 2 mL of Stain Buffer, resuspended in 100 µL of PBS wit 0.01% sodium azide, and acquired with DxFlex flow cytometer. For each variant, the percentage of CD3 + T lymphocytes positive for Zombie Violet dye was recorded and compared to control cultures.

Statistical Analysis
Statistical analysis of acquired data and their visualization were performed using GraphPad Prism 9.4.0 (GraphPad Software). The normality of the data sets distribution was tested using Kolmogorov-Smirnov, Shapiro-Wilk, Anderson-Darlin, D'Agostino, and Pearson tests. The data set was considered to have a normal distribution when each of the applied tests had a prediction value higher than 0.05. Determination for outliers was performed using both ROUT (Q = 1%) and Grubbs' (α = 0.05) methods. The Student t-test was performed when the distribution of differences was normal, and the Wilcoxon test was used when the distribution of differences was not normal. A p-value of < 0.05 (*) was considered statistically significant, and p < 0.01 (**), or p < 0.001 (***) as highly significant. Graphs are presented as mean ± SEM (standard error of the mean).

Effects of Se-Le-30 on Human CD4 + and CD8 + T Cells Proliferation
The effects of Se-Le-30 on the proliferation of human T cells are shown in Figure 1. Polysaccharide significantly inhibited the proliferation of CD4 + T cells stimulated with anti-CD3 Ab (division index: p = 0.0003; proliferation index: p = 0.0003; expansion index: p = 0.0141; replication index: p = 0.0095; percentage of cells divided: p < 0.0001) and enhanced proliferation when cells were stimulated with anti-CD3/CD28 Abs (division index: p = 0.0216; percentage of cells divided: p = 0.0033). Similarly, Se-Le-30 inhibited the proliferation of CD8 + T cells stimulated with anti-CD3 Ab (division index: p = 0.0012; proliferation index: p = 0.0123; expansion index: p = 0.0058; replication index: p = 0.0146; percentage of cells divided: p < 0.0001) but had no effect on double stimulated CD8 + T cells proliferation (p > 0.05). It must be noted that the mode of activation and possibly anti-CD3 Abs concentration was different for the stimulation with Abs against CD3 only and double stimulation with Abs against CD3 and CD28. proliferation of CD8 + T cells stimulated with anti-CD3 Ab (division index: p = 0.0012; proliferation index: p = 0.0123; expansion index: p = 0.0058; replication index: p = 0.0146; percentage of cells divided: p < 0.0001) but had no effect on double stimulated CD8 + T cells proliferation (p > 0.05). It must be noted that the mode of activation and possibly anti-CD3 Abs concentration was different for the stimulation with Abs against CD3 only and double stimulation with Abs against CD3 and CD28. Effects of Se-Le-30 on the proliferation of human CD4 + and CD8 + T cells stimulated with anti-CD3 Ab (top row) and anti-CD3/CD28 Abs (bottom row). PBMCs from 13 healthy donors were stimulated and treated with Se-Le-30 (100 μg/mL) for 5 days. In each experiment, the division index, proliferation index, expansion index, replication index, and percentage of divided cells were calculated by using FlowJo software. Statistical differences were considered when p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Points on bar charts represent experiments conducted with individual donors.

Effects of Se-Le-30 on Human CD4 + and CD8 + T Cells Activation
Upon T cell activation, several of their cell surface markers are upregulated. CD69 is a very early marker, which can be detected on the surface of T cells 2-3 h after activation. CD25 (interleukin-2 receptor, IL-2R) is an early activation marker, with increased cell surface expression 12-24 h after activation [27].
The effect of Se-Le-30 on the presence of CD69 marker on T cells is presented in Figure 2. When cells were stimulated with anti-CD3 Ab, the polysaccharide significantly decreased the percentage of CD4 + CD25 + T cells after 12, 24, and 48 h (p = 0.059, p = 0.0253, and p = 0.0219, respectively). When cells were stimulated with CD3/CD28 Abs, Se-Le-30 upregulated the percentage of CD4 + CD69 + T cells after 24 and 48 h of culture (p = 0.0080, and p = 0.0015, respectively). The number of anti-CD3-stimulated CD8 + cells expressing the CD69 marker did not change after 12, 24, and 48 h of culture (p > 0.05), however, polysaccharide upregulated the percentage of anti-CD3/CD28-stimulated CD8 + CD69 + T cells after 24 and 48 h of culture (p = 0.0065, and p = 0.0090, respectively).

Figure 1.
Effects of Se-Le-30 on the proliferation of human CD4 + and CD8 + T cells stimulated with anti-CD3 Ab (top row) and anti-CD3/CD28 Abs (bottom row). PBMCs from 13 healthy donors were stimulated and treated with Se-Le-30 (100 µg/mL) for 5 days. In each experiment, the division index, proliferation index, expansion index, replication index, and percentage of divided cells were calculated by using FlowJo software. Statistical differences were considered when p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Points on bar charts represent experiments conducted with individual donors.

Effects of Se-Le-30 on Human CD4 + and CD8 + T Cells Activation
Upon T cell activation, several of their cell surface markers are upregulated. CD69 is a very early marker, which can be detected on the surface of T cells 2-3 h after activation. CD25 (interleukin-2 receptor, IL-2R) is an early activation marker, with increased cell surface expression 12-24 h after activation [27].
The effect of Se-Le-30 on the presence of CD69 marker on T cells is presented in Figure 2. When cells were stimulated with anti-CD3 Ab, the polysaccharide significantly decreased the percentage of CD4 + CD25 + T cells after 12, 24, and 48 h (p = 0.059, p = 0.0253, and p = 0.0219, respectively). When cells were stimulated with CD3/CD28 Abs, Se-Le-30 upregulated the percentage of CD4 + CD69 + T cells after 24 and 48 h of culture (p = 0.0080, and p = 0.0015, respectively). The number of anti-CD3-stimulated CD8 + cells expressing the CD69 marker did not change after 12, 24, and 48 h of culture (p > 0.05), however, polysaccharide upregulated the percentage of anti-CD3/CD28-stimulated CD8 + CD69 + T cells after 24 and 48 h of culture (p = 0.0065, and p = 0.0090, respectively).
The effect of Se-Le-30 on the presence of CD25 marker on T cells is presented in Figure 3. When cells were stimulated with anti-CD3 Ab, the polysaccharide significantly decreased the percentage of CD4 + CD25 + T cells after 12, 24, and 48 h (p = 0.059, p = 0.0253, and p = 0.0219, respectively). When cells were stimulated with CD3/CD28, Abs Se-Le-30 increased the percentage of CD4 + CD25 + T cells after 24 h and 48 h (p = 0.0032, and p = 0.0090, respectively). The number of anti-CD3-stimulated CD8 + cells expressing the CD25 marker did not change after 12 and 48 h of culture (p > 0.05) but slightly increased after 24 h of stimulation (p = 0.0501). Conversely, when cells were stimulated with anti-CD3/CD28 Abs, Se-Le-30 increased the percentage of CD8 + CD25 + T cells (after 24 h: p = 0.0039, and after 48 h: p = 0.0095). The effect of Se-Le-30 on the presence of CD25 marker on T cells is presented in Figure 3. When cells were stimulated with anti-CD3 Ab, the polysaccharide significantly decreased the percentage of CD4 + CD25 + T cells after 12, 24, and 48 h (p = 0.059, p = 0.0253, and p = 0.0219, respectively). When cells were stimulated with CD3/CD28, Abs Se-Le-30 increased the percentage of CD4 + CD25 + T cells after 24 h and 48 h (p = 0.0032, and p = 0.0090, respectively). The number of anti-CD3-stimulated CD8 + cells expressing the CD25 marker did not change after 12 and 48 h of culture (p > 0.05) but slightly increased after 24 h of stimulation (p = 0.0501). Conversely, when cells were stimulated with anti-CD3/CD28 Abs, Se-Le-30 increased the percentage of CD8 + CD25 + T cells (after 24 h: p = 0.0039, and after 48 h: p = 0.0095).

Effects of Se-Le-30 on Cytokines Production by PBMCs
The effects of Se-Le-30 on cytokines secretion by PBMCs are shown in Figure 4. Polysaccharide had no influence on IFN-γ secretion by anti-CD3-stimulated PBMCs (p > 0.05), however, significantly upregulated its secretion in anti-CD3/CD28-stimulated cells

Effect of Se-Le-30 on Human CD3 + T Cells Viability
The effects of Se-Le-30 on T cells viability are shown in Figure 5. Polysaccharide did not reduce CD3 + T cells viability after 24 h of culture. The percentage of dead cells did not differ statistically between control cultures and Se-Le-30 cultures.
bar charts represent experiments conducted with individual donors.

Effect of Se-Le-30 on Human CD3 + T Cells Viability
The effects of Se-Le-30 on T cells viability are shown in Figure 5. Polysaccharide did not reduce CD3 + T cells viability after 24 h of culture. The percentage of dead cells did not differ statistically between control cultures and Se-Le-30 cultures.

Discussion
Over the past few years, multiple studies have confirmed that various L. edodes polysaccharides can modulate the immune system through the activation of numerous signaling pathways. We have previously demonstrated that Se-Le-30, a selenium-enriched lentinan analog, significantly inhibited the proliferation of human T cells stimulated with anti-CD3 Ab [20,26,28]. In the present study, we have further compared the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by CD3 or CD3/CD28 Abs, as well as on their secretion of cytokines. The results of this study not only confirmed our previous observations regarding anti-CD3 Ab stimulation, but also demonstrated that when lymphocytes were stimulated with a dual signal (i.e., anti-CD3/CD28 Abs), Se-Le-30 enhanced their activation and proliferation: it increased the percentage of divided CD4 + T cells and their division index (please see Figure 1). These findings are in line with the results described by Wang et al. [29], who reported that lentinan, a β-1,3-branched β-1,6-D-glucan, increased the number of CD3 + CD4 + and CD3 + CD8 + T cells in patients with non-small cell lung cancer treated with chemotherapy. Moreover, this glucan inhibited the synthesis of IL-10, and enhanced IFN-γ. Conversely, our study indicated that Se-Le-30 increased the secretion of both IL-10 and IFN-γ in CD3/CD28-stimulated cells. In another randomized study, L. edodes was administered orally to healthy young adults and it was shown that fungus ingestion resulted in an increase in the proliferative potential of T lymphocytes, upregulated the expression of CD69 activation marker on T cells, and enhanced the production of IL-10 [30]. Similarly, it has been found that L. edodes extract consumption increased the plasma levels of IL-10 in healthy men exposed to exercise-induced skeletal muscle damage, but had no effect on the levels of IL-6 [31]. In the present study, we have found that Se-Le-30 increased IFN-γ secretion by PBMCs,

Discussion
Over the past few years, multiple studies have confirmed that various L. edodes polysaccharides can modulate the immune system through the activation of numerous signaling pathways. We have previously demonstrated that Se-Le-30, a selenium-enriched lentinan analog, significantly inhibited the proliferation of human T cells stimulated with anti-CD3 Ab [20,26,28]. In the present study, we have further compared the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by CD3 or CD3/CD28 Abs, as well as on their secretion of cytokines. The results of this study not only confirmed our previous observations regarding anti-CD3 Ab stimulation, but also demonstrated that when lymphocytes were stimulated with a dual signal (i.e., anti-CD3/CD28 Abs), Se-Le-30 enhanced their activation and proliferation: it increased the percentage of divided CD4 + T cells and their division index (please see Figure 1). These findings are in line with the results described by Wang et al. [29], who reported that lentinan, a β-1,3-branched β-1,6-D-glucan, increased the number of CD3 + CD4 + and CD3 + CD8 + T cells in patients with non-small cell lung cancer treated with chemotherapy. Moreover, this glucan inhibited the synthesis of IL-10, and enhanced IFN-γ. Conversely, our study indicated that Se-Le-30 increased the secretion of both IL-10 and IFN-γ in CD3/CD28-stimulated cells. In another randomized study, L. edodes was administered orally to healthy young adults and it was shown that fungus ingestion resulted in an increase in the proliferative potential of T lymphocytes, upregulated the expression of CD69 activation marker on T cells, and enhanced the production of IL-10 [30]. Similarly, it has been found that L. edodes extract consumption increased the plasma levels of IL-10 in healthy men exposed to exercise-induced skeletal muscle damage, but had no effect on the levels of IL-6 [31]. In the present study, we have found that Se-Le-30 increased IFN-γ secretion by PBMCs, which is consistent with data obtained in a study in patients receiving L. edodes mycelia extract combined with cancer immunotherapy [32]. Similarly, elevated serum IFN-γ levels were reported in a group of healthy adults administered rice bran fermented with L. edodes [32]. However, in contrast to our findings, there was no evidence of an effect of L. edodes on the regulation of IL-2, IL-4, and IL-10 secretion [33]. Another well-studied polysaccharide obtained from the mycelium of L. edodes is AHCC ® , an active hexose-correlated compound which contains 20% of α-1,4-glucans [34]. Similar to Se-Le-30, AHCC ® was found to increase the secretion of IFN-γ and TNF-α by CD4 + and CD8 + T cells of healthy adults [35]. Moreover, this glucan increased the proliferation of CD8 + T cells in adults receiving the influenza vaccine [36].
On the other hand, some studies did not confirm the effect of L. edodes β-glucans on cytokines secretion in humans [37,38]. However, it should be noted that in these studies, the source of β-glucans was orally administrated substances, which might suggest that this type of administration might be ineffective to induce the effect of regulating the secretory activity of PBMCs.
Data on the immunomodulatory effects of glucans isolated from L. edodes in humans are limited, however, numerous studies have been conducted to evaluate its properties in animal models. Chen S. et al. demonstrated that three polysaccharide fractions with different molecular weights isolated from L. edodes reverted immune suppression in mice and upregulated splenic T lymphocytes proliferation in response to Concanavalin A and LPS [5]. The polysaccharide with the lowest molecular weight of 14-35 kDa was found to be the most effective [5]. In another study, the immunomodulatory properties of the synthetic analogue of lentinan basic unit-glucohexose have been analyzed. It was demonstrated that it upregulated CD69 expression on CD4 + and CD8 + T lymphocytes and increased the number of IFN-γ producing CD8 + T lymphocytes in mice [39]. These results are consistent with our study in humans. Most studies on the immunomodulatory properties of lentinan and its analogues have shown its immune-enhancing properties, however, there are some reports suggesting the immunosuppressive activity of L. edodes polysaccharides as well.
McCormack and colleagues demonstrated that lentinan reduced serum levels of IL-4, IL-6, and IL-10 in rats [40]. Moreover, it was found that this drug enhanced the expansion of CD8 + T lymphocytes, which is similar to our findings in humans [41].
The binding of β-glucans to dectin-1 affects both CD8 + T lymphocytes and CD4 + T lymphocytes activation, resulting in enhanced granzyme production in CD8 + lymphocytes and differentiation of CD4 + into Th1 and Th17 phenotype [56,57]. Binding of β-glucans to CR3 results in the activation of phagocytes and NK cells, which promotes phagocytosis and cytotoxic degranulation. This may help to overcome tumor resistance to these forms of effector mechanism and lead to higher secretion of IL-6 and IFN-γ [12]. It has been suggested that 1,4-α-d-glucans do not affect macrophages via CR3 binding [58], but activation of T lymphocytes up-regulates expression of CR3 [44,45]. Interestingly, it was found that blocking the CD11b subunit of CR3 contributed to T lymphocyte proliferation inhibition after stimulation with anti-CD3 Abs [44]. Evidently, this finding allows us to hypothesize that Se-Le-30 might block CR3 and thus inhibit the proliferation of T lymphocytes when stimulated with anti-CD3 Ab only. Especially considering that CR3 expression on T lym-phocytes is enhanced after activation. Dual CD3/CD28 stimulation would also be affected, but less significantly. This might be reflected in a higher division index and percentage of divided cells, while having no impact on other proliferation indexes.
As mentioned above, dectin-1, CR3, and TLRs are implicated in the recognition of β-glucans by immune cells. Interestingly, recent research has shown that β-glucans may stimulate an immune response by binding to CD28 on the surface of T cells and that this stimulation is potentiated by CD3 activation. Cormer and colleagues explored interactions of β-1,3 glucans with the CD28 receptor and found that glucan molecules insert themselves into a channel on the surface of CD28 and moreover diffuse around the receptor, coming into contact with different regions of the protein [62]. This is another potential mechanism underlying the observed immunomodulatory effects of Se-Le-30.

Conclusions and Future Perspectives
Se-Le-30, a fraction of polysaccharides isolated from mycelium of L. edodes is a mixture of linear 1,4-α-glucan and linear 1,3-and 1,6-β-glucans. In vitro models demonstrated that Se-Le-30 exerts immunomodulatory effects on human T lymphocytes, however, the direction of its biological activity depends on the type of cell stimulation.
Moreover, it has been revealed that Se-Le-30 upregulated the production of IL-6 and IL-10 in PBMCs, regardless of the type of stimulation. Human PBMCs include lymphocytes, NK cells, monocytes, and dendritic cells, therefore, it is possible that other than T cell population is responsible for the observed activity. Future studies focusing on the mechanism of action of Se-Le-30, including identification of intracellular signaling pathways, as well as in vitro analyses with purified CD3 + T cells and other PBMC cell types are warranted.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author without any restrictions.

Conflicts of Interest:
The authors declare no conflict of interest.