Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SC) through the Galectin-9-Dependent Cell Contact Mechanism

Background We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SC) on different compartments of immune cells such as T cells and monocytes/macrophages in diabetes and other autoimmune diseases. However, the effects of CB-SC on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SC, we explored the modulations of CB-SC on human B cells. Methods CB-SC were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMC). Next, the activated B cells were treated in the presence or absence of coculture with CB-SC for 7 days before undergoing flow cytometry analysis of phenotypic change with different markers. RT-PCR was utilized to evaluate the levels of galectin expressions with or without treatment of activated B cells in order to find the key galectin contributing to the B-cell modulation. Results Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SC, leading to the down-regulation of immunoglobulin productions from the activated B cells. Phenotypic analysis revealed that treatment with CB-SC increased the percentage of IgD+CD27- naïve B cells, but decreased the percentage of IgD-CD27+ switched B cells. Transwell assay showed that the immune suppression of CB-SC on B cells was dependent on the manner of cell-cell contact via Gal-9 molecule, as confirmed by the blocking experiment with the anti-Gal-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were down-regulated after the treatment with CB-SC, causing the decline of mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SC. Conclusions CB-SC displayed multiple immune modulations on B cells through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advances current understanding about the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.


Introduction
Human cord blood-derived stem cells (CB-SC) display a unique phenotype with both embryonic and hematopoietic markers that distinguish them from other known types of stem cells, including hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) [1,2]. Our previous studies demonstrated that human CB-SC display multiple immune modulations on T cells and monocytes/macrophages via surface molecules and released exosomes [3,4]. Based on CB-SC's immunomodulation, we developed the Stem Cell Educator â (SCE) therapy to treat immune dysfunction-associated diseases, including type 1 diabetes (T1D), type 2 diabetes (T2D) and alopecia areata (AA) [5][6][7], through the multicenter international clinical trials in the United States, China and Spain. SCE therapy circulates a patient's peripheral blood mononuclear cells (PBMC) through a blood cell separator, cocultures their immune cells with adherent CB-SC in vitro, and then returns the "educated" immune cells back to the patient's blood circulation. Our clinical data demonstrates the safety and clinical efficacy of SCE therapy in reversing the autoimmunity, promoting the regeneration of islet β cells, and improving the metabolic control in T1D and T2D patients [5,6].
B cells have an important role in maintaining homeostasis and the adaptive immune response through antibody production, antigen presentation and the production of multiple cytokines [8,9]. Dysfunctions of B cells actively contribute to the pathogenesis of diabetes [10][11][12][13] and multiple autoimmune diseases [14,15]. For example, their roles as antibody-producing cells in systemic lupus erythematosus (SLE) [16] and antigenpresenting cells in T1D and rheumatoid arthritis (RA) have been well recognized [10,17].
Therefore, it is necessary to correct B cell-associated immune dysfunctions for the 5 treatment of autoimmune diseases. Additionally, galectins are a family of highlyconserved glycan-binding proteins expressed in different tissues, including immune and non-immune cells. In the immune system, galectins are important regulators among innate and adaptive immune responses by regulating a variety of immune cell activations, maturations and other activities. Galectins (Gal)-1，-3, and -9 have shown different effects on the functioning of T cells by modulating their development, activation and differentiation [18][19][20]. However, the actions of galectins in B cells have only recently begun to be deciphered. Gal-9(Gal-9) is a 34-39 kDa tandem-repeat type protein, which is found in immune cells, endothelial cells, and stem cells [21,22]. Increasing evidence demonstrated that Gal-9 could not only suppress T-cell activation via the Tim-3 or PD-1 receptor on T cells [23], but also could suppress B-cell activation through the B-cell receptor [24,25]. To date, our mechanistic studies have demonstrated the immune modulations of SCE therapy on the activated T cells, autoimmune memory T cells [26], regulatory T cells (Tregs) [5] and monocytes/macrophages [4,6,27]. The effects of CB-SC on B cells remained elusive. Here, we demonstrated the direct immune modulation of CB-SC on the activated B cells via Gal-9-mediated cell-cell contact mechanism, leading to the marked suppression of B-cell proliferation and phenotypic changes.

B-cell isolation and culture
Human peripheral blood mononuclear cells (PBMC) (N = 12, aged from 31 to 64 years with average at 46.83 ± 9.67 years old, male =7, female = 5) were isolated by ficollhypaque density gradient (GE Healthcare, IL, USA) from the buffy coats purchased from 6 the New York Blood Center (New York, USA). PBMC cell suspensions were pre-treated with anti-CD19-conjugated microbeads (Miltenyi Biotec, CA, USA) according to the instructions of the manufacturer. The purity of positively selected CD19 + cells was more than 95%, as assessed by flow cytometry with Korman orange-conjugated mouse antihuman CD19 monoclonal antibody (mAb) (Beckman Coulter, CA, USA). The purified CD19 + B cells were cultured in the chemical-defined and serum-free X'VIVO 15 medium (Lonza, Walkersville, MD, USA), in the absence or presence of 100 U/ml penicillin, and 100 µg/ml streptomycin.

Proliferation Assay
To examine the effects of CB-SC on B-cell proliferation, B cells were stimulated by the following combination at 37°C and 5% CO 2 conditions: the goat anti-human IgM F (ab')2 (10 µg/mL), recombinant CD40L (rCD40L 1µg/mL), IL-2 (10 ng/mL), IL-10 (20 ng/mL), Cell culture for CB-SC 7 The culture of CB-SC was performed as previously described [4,5,27]. In brief, human umbilical cord blood units were collected from healthy donors and purchased from Cryo-Cell international blood bank (Oldsmar, FL, USA). Cryo-cell has received all accreditations for cord blood collections and distributions, with hospital institutional review board (IRB) approval and signed consent forms from donors. Mononuclear cells were isolated with Ficoll-hypaque (γ = 1.077, GE Health) and red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer (Lonza, MD, USA). The remaining mononuclear cells were seeded in 150 x15 mm style non-tissue culture-treated petri dishes or non-tissue culture-treated 24-well plates at 1 x10 6 cells/mL. Cells were cultured in X'VIVO 15 chemically-defined serum-free culture medium and incubated at 37°C with 8% CO 2 for 10-14 days.

Quantitative Real Time PCR assay
The mRNA expressions of the galectin family were analyzed by quantitative real-time PCR (RT-PCR). Total RNA was extracted from CB-SC using RNeasy mini Kit (Qiagen, CA, USA). First-strand cDNA were synthesized from total RNA using an iScript gDNA  Table 1) [28]. β-actin was used as control.
Assay for antibody production 8 To detect the antibodies produced by B cells, B cells were stimulated by the following combination: goat anti-human IgM F (ab')2 (10 µg/mL), recombinant CD40L (rCD40L 1 µg/mL), IL-2 (10 ng/mL), IL-10 (20 ng/mL), and IL-21 (50 ng/mL) in the presence or absence of the treatment with CB-SC in 24-well plate, with 500 µL X'VIVO 15 chemicaldefined serum-free culture medium (Lonza, MD, USA) per well. After the treatment for 7 days, the supernatants were collected to determine the levels of antibody productions (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgM) by using LEGENDplex TM Human Immunoglobulin Isotyping Panel (Biolegend, CA, USA). The Gallios Flow Cytometer was utilized to analyze the data according to the manufacturer's recommended protocol.
To further explore the blocking effects of Gal-9 mAb, the activated B cells were characterized with cytoplasmic and mitochondrial Ca 2+ levels as well as mitochondrial membrane potential (Dym) by using flow cytometry as previously described [4]. Briefly, after the 4-hour treatment, B cells were stained with fluorescence dyes including Fluo-4 9 (ThermoFisher Scientific, MA, USA) for cytoplasmic Ca 2+ , Rhod-2 (ThermoFisher Scientific, MA, USA) for mitochondrial Ca 2+ , and tetramethylrhodamine ethyl ester (TMRE) (Abcam, MA, USA) for detection of mitochondrial membrane potential, respectively.

Western blot
Cells were prepared with radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was determined by a bicinchoninic acid (BCA) protein assay. The proteins were separated by 10% Tris-HCl gel(Bio-Rad, CA, USA) and transferred to the polyvinylidene fluoride (PVDF) membrane. The proteins were then blotted overnight with anti-human phospho-Akt and anti-human phospho-Erk1/2 mAbs (Cell Signaling, MA, USA), followed by anti-rabbit or anti-mouse horseradish peroxidase(HRP)-conjugated secondary mAb (ThermoFisher scientific) [4]. The membrane was incubated with the chemiluminescent substrate (ThermoFisher Scientifc, CA, USA) and chemiluminescent signal was detected by using ChemiDoc Imaging System (Bio-Rad, CA, USA). β-actin served as an internal control.

Flow cytometry
Phenotypic characterization of B-cell subsets was performed by flow cytometry [4,27] with specific markers including PE-conjugated mouse anti-human CD27 ( software. The normality test of samples was evaluated using the Shapiro-Wilk test. Statistical analysis of data was performed using the two-tailed paired student's t-test to determine statistical significance for parametric data between untreated and treated groups. The Mann-Whitney U test was utilized for non-parametric data. Values were given as mean ± SD (standard deviation). Statistical significance was defined as P < 0.05.

CB-SC mediate B cell suppression by cell-cell contact
Our previous studies demonstrated that multiple mechanisms contribute to the immune modulations of CB-SC on T cells, such as PD-L1/PD1-mediated cell-cell inhibition and releasing soluble factors (e.g., nitric oxide and transforming growth factor-b1) [30]. To To further prove the involvement of Gal-9 in the immune modulation of CB-SC on B cells, we performed the blocking experiment with neutralizing Gal-9 mAb. The data demonstrated that the suppression of CB-SC on the proliferation of activated B cells was markedly reversed after the treatment with the neutralizing Gal-9 mAb relative to that of 14 the Gal-9 mAb-untreated group (P = 0.025, Figure 5E). This finding indicated that Gal-9 contributed to the immune modulation of CB-SC on activated B cells.

Suppression calcium flux by CB-SC was Gal-9 dependent
The activation and proliferation of B cells are initiated by the B cell receptor (BCR), which triggers a number of signaling cascades [32]. The increase in intracellular Ca 2+ levels is one of the critical signaling pathways for tuning B-cell responses and development post the BCR activation [33]. To further explore the molecular mechanism underlying the inhibition of B-cell proliferation by the treatment with CB-SC, we examined the changes of cytosolic and mitochondrial Ca 2+ levels in CB-SC-treated B cells by flow cytometry after being stimulated with B cell-dependent activation cocktails. Using the Fluor-4 staining for cytosolic calcium, the median fluorescence intensity of Fluo-4 + activated B cells was markedly downregulated in the presence of CB-SC at the ratio of 1:5 (P < 0.05). The suppressive effect on cytosolic Ca 2+ levels was reversed after the blocking with Gal-9 mAb ( Figure 6A). The direct effect of Gal-9 on the changes of cytosolic Ca 2+ levels was further confirmed by the treatment with recombinant Gal-9 at 0.5 µg/mL (Figure 6A).
Using the Rhod-2 staining as an indicator for the mitochondrial calcium, flow cytometry demonstrated the mitochondrial Ca 2+ levels in the stimulated B cells were significantly reduced after the treatment with CB-SC at the ratio 1:5 of CB-SC to B cells ( Figure 6B).
Similar to the changes of cytosolic Ca 2+ levels, the mitochondrial Ca 2+ levels in the CB-SC-treated B cells were increased in the presence of Gal-9 mAb (Figure 6B).
Mitochondrial membrane potential (Dym) was also decreased in the stimulated B cells after the treatment with CB-SC, but upregulated after the blocking with Gal-9 mAb ( Figure  6C). The data indicated that the Gal-9-mediated Ca 2+ signaling pathway contributed to the modulation of CB-SC on the activated B cells.
Additionally, Western blotting showed that both BCR downstream molecules phospho-Akt and phospho-Erk1/2 were upregulated in the stimulated B cells without affecting their total protein levels, but markedly downregulated after the treatment with CB-SC or recombinant human Gal-9 (0.5 µg/mL). Such inhibitory effects of CB-SC on the phospho-Akt and phospho-Erk1/2 were decreased after blocking with Gal-9 mAb (Figure 6D).
These data suggest that Gal-9 expressed on CB-SC contributed to the immune modulation of CB-SC on B cells via the regulation of Ca 2+ flux and phosphorylation of Akt and Erk1/2 signaling pathways.

Discussion
Over the last 10 years, CB-SC have been utilized in multicenter international clinical trials and designated to Stem Cell Educator â (SCE) therapy for the treatment of autoimmune disease including type 1 diabetes (T1D) [5,26], alopecia areata (AA) [7], and other chronic metabolic inflammation-associated diseases (e.g., type 2 diabetes [6]). Mechanistic Researchers found that blocking B cells or impairing B cell function will significantly decrease the incidence of diabetes in NOD mice [35,36]. Additionally, the depletion of B cells with anti-human CD20 antibody (Rituximab) markedly preserved islet β-cell function and improved C-peptide levels after 1 year follow-up in recent-onset T1D patients [37].
The current study demonstrated that CB-SC markedly suppressed the proliferation of activated B cells and reduced the antibody productions in these activated B cells.
Therefore, these data suggest the clinical translational potential of Stem Cell Educator therapy to treat other B cell-mediated autoimmune diseases.
To date, the characterization of B-cell phenotype with multiparameter flow cytometry has identified several B-cell subpopulations including CD27 + IgD + non-switched memory B cells and CD27 + IgDswitched memory B cells, which may represent a biomarker for some autoimmune diseases. For instance, the percentage of switched memory B cells increased in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [38][39][40].
Our flow cytometry analysis substantiated that the percentage of switched memory B cells was markedly reduced after the treatment with CB-SC in a dose-dependent manner.
Notably, the percentage of naïve B cells (CD27 -IgD + ) was increased, highlighting the modulation of CB-SC on B-cell differentiation with the reduction of memory B cells.
To elucidate the molecular basis of SCE therapy, previous studies have identified several molecular and cellular pathways that alter autoimmune T cells and the functions of pathogenic monocytes/macrophages (Mo/Mfs) to elicit immune tolerance via: (1) the expression of autoimmune regulator (AIRE) in CB-SC, which is a master transcriptional regulator that acts to eliminate the self-antigen reactive T cells in the thymus and is controlled by the activation of the receptor activator of NF-kB (RANK) signaling pathway [41]; (2) secretion of CB-SC-derived exosomes (cbExosomes), which polarize human blood Mo/Mf into type 2 macrophages (M2) [4,27], further contributing to immune tolerance and preventing b-cell destruction; and (3) migration of platelet-derived mitochondria (pMitochondria) to islets, which are absorbed by pancreatic islets and contribute to an improved proliferation of human islet b cells [42].
Our current studies revealed the direct immune modulation of CB-SC on activated B cells through the expression of Gal-9 on CB-SC, as demonstrated by trans-well coculture and blocking experiment with anti-Gal-9 mAb. What's more, further mechanistic studies confirmed that Gal-9 expressed on CB-SC directly contributed to the regulation of Ca 2+ flux and phosphorylation of Akt and Erk1/2 signaling pathways in the stimulated B cells.
Waters and colleagues reported an increase in the oxidative phosphorylation and mitochondrial membrane potential (Dym) among the stimulated B cells [43], which was consistent with our current data showing the enhanced median fluorescence intensity of 18 TMRE staining. Notably, the Dym of stimulated B cells was substantially reduced in the Gal-9-dependent manner.
Galectins are β-galcotosid-bind lectins which can be expressed by different types of stem cells and act as regulators of immune cell function [44], especially galectin-3 and Gal-9. Galectin-3 suppresses the activation of TCR-mediated signal transduction [45], while Gal-9 binds T cell Ig mucin-3 (Tim-3) and induces negative regulate T helper 1(Th1) immunity [46]. Our current data confirmed that galectin-1, 2, 3, 4, 7, 8, and 9 were highly expressed on CB-SC, but only Gal-9 primarily contributed to the immune modulation of CB-SC on activated B cells. Gal-9 was not only located on the cellular membrane, but also acted as a soluble factor involved in the immune modulation [23]. To test this possibility, we found that CB-SC-released Gal-9 was less than 10% of total CB-SCderived Gal-9 after 3 days culture ( Figure S2). Therefore, Gal-9 expressed on CB-SC's membrane displayed more potential than the soluble form of CB-SC-secreted Gal-9 during the B-cell immune modulation. Giovannone et al reported that Gal-9 can directly bind to the poly-LacNAc-containing N-glycans on leukocyte common antigen CD45 of B cells, leading to the diminished intracellular calcium levels and ultimately inhibiting B cell activation [24]. This report was consistent with the reduction of cytosolic Ca 2+ levels in our current study. Additionally, several studies revealed the distribution of IgM on B-cell surface membranes which form the nanoscale clusters and act as BCR of primary B cells [25,47,48]. Using dual-color direct stochastic optical reconstruction microscopy (dSTORM), Cao and colleagues confirmed that Gal-9 can also directly bind to IgM-BCR of murine B cells [25], nearly resulting in a complete abolishment of the BCR activation 19 [25]. Due to the BCR-mediated Ca 2+ influx as the critical signal for B cell activation [49], the immune modulation of CB-SC on activated B cells primarily targets the regulation of intracellular Ca 2+ levels through the Gal-9-mediated pathway, leading to dampened B-cell responses and shaping their differentiation. These novel molecular mechanisms will facilitate the clinical translation of Stem Cell Educator therapy to treat T1D and other autoimmune diseases.

Conclusions
Stem Cell Educator therapy has been unutilized to treat multiple autoimmune-and inflammation-associated diseases, which pathogenesis involve in T cells, B cells, and monocytes/macrophages. The current study revealed that CB-SC displayed multiple immune modulations on B-cell proliferation and differentiation and antibody productions through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. These findings lead to a better understanding of the molecular mechanisms of Stem Cell Educator therapy to treat autoimmune diseases in clinics.

Funding
This research received no external funding.

Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate
Human cord blood units and buffy coats blood were purchased from Cryo-Cell International blood bank and the New York Blood Center (NYBC), respectively. Both Cryo-Cell and NYBC have received all accreditations for blood collections and distributions, with institutional review board (IRB) approval and signed consent forms from donors.

Consent for publication
Not applicable.

Competing Interests
Dr. Yong Zhao was inventor of Stem Cell Educator technology and has a fiducial role at Throne Biotechnologies. All other authors have no financial interests that may be relevant to the submitted work.     Western blotting showed the reduced expression of phosphorylated AKT and ERK1/2 in 29 stimulated B cells after the treatment with CB-SC or rGal-9, but upregulated after blocking with Gal-9 mAb. b-actin served as control.  at 37°C and 8% CO 2 conditions. After culture for 3 days, CB-SC cells and supernatants were collected respectively for analysis Gal-9 protein concentration. The data were given as mean ± SD of three experiments