ORIGINAL RESEARCH published: 06 June 2019 doi: 10.3389/fimmu.2019.01288 Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted Extracellular Vesicles Laura Carreras-Planella 1,2 , Marta Monguió-Tortajada 1,3 , Francesc Enric Borràs 1,2,4 and Marcella Franquesa 1,4* 1 REMAR-IVECAT Group, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain, 2 Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona, Barcelona, Spain, 3 ICREC Research Program, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain, 4 Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell Edited by: communication and typically reflect the phenotype of the cell of origin. MSC-EVs could Martin Johannes Hoogduijn, Erasmus University resemble MSC functions, and are being contemplated as an improved alternative to Rotterdam, Netherlands the MSC-based immunomodulatory therapy. In the present work, we focused on the Reviewed by: factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the Federica Casiraghi, immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were Istituto Di Ricerche Farmacologiche Mario Negri, Italy isolated from MSC-conditioned medium (CM) using size-exclusion chromatography Evren Alici, (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell Karolinska Institute (KI), Sweden Myung-Shin Jeon, proliferation was compared to that of the whole MSCs. Co-culture with MSC or Inha University, South Korea unfractionated CM induced naïve and CD24hi CD38hi , IL-10 producing (Breg) phenotypes *Correspondence: on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, Marcella Franquesa inducing a naïve phenotype, and even though they did not induce the shift toward a mfranquesa@igtp.cat CD24hi CD38hi population, MSC-PF fostered IL-10 production by B cells. Conversely, Specialty section: MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs This article was submitted to induced CD24hi CD38hi B cells to a similar extent of that of MSC, but not bona fide Bregs Alloimmunity and Transplantation, a section of the journal since they did not produce IL-10. Our results show that B cell modulation by MSC is Frontiers in Immunology partially mediated by soluble factors other than EVs. Received: 22 January 2019 Keywords: mesenchymal stromal cells, exosome, regulatory B cell, immunosuppression, memory B cell, Ev Accepted: 21 May 2019 isolation Published: 06 June 2019 Citation: Carreras-Planella L, INTRODUCTION Monguió-Tortajada M, Borràs FE and Franquesa M (2019) Mesenchymal stem or stromal (MSC) are immunomodulatory toward numerous immune cell types Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted in vitro as well as in vivo (1–3). We recently showed their ability to induce regulatory (Breg) and Extracellular Vesicles. naïve B cells while reducing activated and memory B cells (4). While the exact mechanism of action Front. Immunol. 10:1288. remains unclear (5), both cell-contact and secreted factors are needed for MSC modulation of B doi: 10.3389/fimmu.2019.01288 cells (6, 7). Some cytokines and growth factors have been identified as key mediators amid secreted Frontiers in Immunology | www.frontiersin.org 1 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells factors, but more recently the focus has been put on Bead-Based Flow Cytometry extracellular vesicles (EVs). EVs are membrane nanovesicles The EV-enriched SEC fractions were determined by beads- that carry molecules reflecting the phenotype and functions based flow cytometry according to the presence of CD9 and of the cells of origin (8). MSC-derived EVs have been shown CD90 following the previously described procedure (11, 15) to emulate their effect on B cells and other immune cells (Figure 1B). Briefly, EVs were coupled to 4 µm aldehyde/sulfate (9–11). However, parameters related to the EV isolation method latex microspheres and were then labeled with the fluorochrome- -including purity- are key to downstream analyses. Widely used conjugated antibodies anti-CD90-PE-Cy7 or indirectly labeled techniques such as ultracentrifugation (UC) or precipitating with the primary antibodies anti-CD9 (Clone VJ1/20) and or agents-based methods cause the co-precipitation of EVs with the IgG isotype control and secondary antibody FITC-conjugated other potentially confusing soluble molecules (12), whilst size- Goat F(ab’)2 Anti-Mouse IgG. The MSC-EV fractions with the exclusion chromatography (SEC) is being considered the method highest CD9 and CD90 MFI were pooled to obtain highly of choice to highly enrich functional EVs (13). enriched MSC-EV preparations. The purpose of the present study is to use SEC to dissect the role of MSC-EV from secreted soluble factors in order to deepen Cryo-Transmission Electron Microscopy (Cryo-TEM) in the mechanisms of B cell immunomodulation by MSC. The presence of EVs in EV-enriched fractions was confirmed by cryo-transmission electron microscopy (cryo-TEM) as previously described (16). MATERIALS AND METHODS Protein Quantification Mesenchymal Stem or Stromal Cell Protein elution was checked by reading the absorbance (Abs.) at 280 nm of each fraction using Nanodrop R ND-1000 Isolation and Cell Culture (Thermo Scientific) to pool fractions with the highest protein Subcutaneous adipose tissue was obtained from patients concentration and obtain MSC-PF preparations. undergoing heart surgery in University Hospital Germans Trias i Pujol (HUGTiP). Informed consent was obtained from all B Cells subjects, and the study protocol conformed to the principles Isolation From Tonsils outlined in the Declaration of Helsinki. Mesenchymal stem or Tonsils were obtained from children undergoing routine stromal cells (MSC) were isolated from fat tissue as previously tonsillectomy after the informed consent of their legal tutors described (4, 14). MSC, which were used in passages between 3 (HUGTiP). The study protocol followed the principles of and 10, were cultured in αMEM (Sigma Aldrich) supplemented the Declaration of Helsinki. To obtain B cells, tonsils were with 10% FBS (Lonza), penicillin (100 IU/ml, Cepa S.L., Madrid, mechanically disaggregated with a scalpel, washed with Spain), streptomycin (100 mg/ml, Normon Laboratories S.A., PBS, mononuclear cells were isolated by ficoll differential Madrid, Spain) and 2 mM L-Glutamine (Sigma Aldrich). centrifugation (GE Healthcare) and frozen (liquid N2 ). For the experiments, tonsil cells were thawed and negatively sorted (MACS, Miltenyi Biotech) to obtain the CD43− population Preparation of Conditioned Medium (mature inactivated B cells). Two million MSC were seeded in cell culture flasks with 15 ml of complete medium depleted from fetal bovine serum (FBS)- Activation and Culture derived EVs (11). To deplete medium from FBS-EVs, 20% One hundred-thousand B cells were seeded in flat bottom 96 well FBS complete medium (αMEM +1% P/S +2 mM L-Glutamine) plates, T-cell-like-stimulated with IL-2/anti-IgM/anti-CD40 and was ultracentrifuged at 100,000 × g for 16 h in polypropylene co-cultured with 10,000 MSC for 7 days in a 10:1 B:MSC ratio as ultracentrifugation tubes (Beckman coulter, Brea, CA). The previously described (4, 7). supernatant was collected and filtered through a 0.22 µm filter B cells were also cultured with MSC-CM, MSC-EV, or MSC- (Sarstedt, Germany) to sterilize the medium, which was finally PF. Of each condition, 100 µl of MSC-EV or MSC-PF were added diluted with αMEM medium to the final concentration of 10% to the culture. This volume corresponded to EVs and PF secreted FBS for cell culture. After 48 h, the medium was collected and by 50,000 MSC which is 5 times the amount of MSC added in centrifuged at 400 × g and 2,000 × g to eliminate cells and cell the B cell-MSC co-culture. In the dose-dependency experiments, debris, respectively, to obtain MSC-conditioned medium (CM). half (50 µl) or one tenth (10 µl) of the initial volume of CM or PF conditions were used. Extracellular Vesicles and Soluble Protein B Cell Subsets and Proliferation Assessment by Separation and Analysis Flow Cytometry Size-Exclusion Chromatography After 7 days, B cells were collected and processed for flow MSC-CM was concentrated using a 100 kDa ultrafiltration unit cytometric analysis to analyze B cell populations (FACS (Amicon Ultra, Millipore, Millerica MA) and fractioned by Canto II, BD Biosciences) as previously described (4, 7). SEC using columns of 1 ml sepharose CL-2B (Sigma Aldrich). Viability was assessed by 7AAD staining (BD Biosciences) Figure 1A schematically depicts the followed protocol, which can and quantified as CD19+ 7AAD− cells among the lymphocyte be read in detail in Monguió-Tortajada et al. (15). population. In separate experiments, B cells were labeled with Frontiers in Immunology | www.frontiersin.org 2 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells FIGURE 1 | (A) Workflow of the methodology used to isolate MSC-EV and MSC-PF by SEC from MSC-CM. (1) Supernatant was collected after 48 h of MSC culture and (2) sequentially centrifuged at 400 × g for 5 min and at 2,000 × g for 10 min to exclude cells and cell debris, respectively. The obtained MSC-CM was partially kept for experimental use in B cell culture and the rest was then (3) concentrated by 100 kDa ultrafiltration. (4) Concentrated MSC-CM was loaded into a 1 ml SEC (Continued) Frontiers in Immunology | www.frontiersin.org 3 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells FIGURE 1 | sepharose column and (5) 100 µl fractions (up to 20) were collected immediately after loading. (B) Representative plot showing the SEC elution profile according to protein concentration and median fluorescence intensity (MFI) of EV markers of each SEC fraction. Fractions with the highest CD9 and CD90 MFI or were pooled together, similarly to fractions with the highest protein concentration, to obtain the MSC-EV or MSC-PF preparations, respectively. (C) Cryo-TEM was used to analyze MSC-EV size and morphology. Black arrows point EV in the preparations over the TEM grid. Scale bars = 0.5 µm (left) and 0.2 µm (right). (D) Gating strategy followed to define B cell subsets by flow cytometry. Doublets and debris were excluded by FSC-A/FSC-H and FSC-A/SSC-A, respectively, by gating on singlets and lymphocyte populations. Alive B cells were further gated as CD19+ 7AAD− , from which each subset was defined: naïve (CD27− IgD+ ), non-switched memory (CD27+ IgD+ ), switched memory (CD27+ IgD− ), activated/memory (CD24hi CD38int/lo ), and Breg (CD24hi CD38hi ). Bottom right panel shows a representative proliferation plot where cell generations are indicated with dashed lines. For analysis, the percentages of total proliferated cells (G1 onwards) and G4+ proliferated cells (G4 onwards) were calculated. FIGURE 2 | Flow cytometry was used to measure (A) percentages of alive B cell (CD19+ 7AAD− ); (B) percentages of B cell subsets: naïve (CD27− IgD+ ), non-switched memory (CD27+ IgD+ ), switched memory (CD27+ IgD− ); (C) the percentage of Breg (CD24hi CD38hi ) and the ratio between Breg (CD24hi CD38hi ) and activated/memory (CD24hi CD38int/lo ) for each condition, and (D) in a dose-dependency experiment for PF and CM conditions. Each dot represents a different combination of MSC and B cells donors. Statistical significance (p < 0.05) was determined by Kruskall-Wallis with Dunn’s multi-comparison test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). MSC, mesenchymal stem cells; CM, conditioned medium; EV, extracellular vesicles; PF, protein fraction. Violet CellTrace (Invitrogen) for 20 min at 37◦ C to analyze RESULTS proliferation by dye dilution. Figure 1D shows the gating strategy followed. EV Isolation and Analysis MSC-CM obtained after 48 h of culture was collected concentrated and fractionated by SEC. Fractions eluted IL-10 and TNFα Quantification by ELISA from SEC were evaluated for CD9 and CD90, which are known After co-culture, supernatants were harvested and stored at EV (8) and MSC-EV markers (17), respectively (Figure 1A). −80◦ C for later IL-10 and TNFα quantification using dedicated As previously described by our group (11), we consistently ELISA kits (U-CyTech). observed that SEC EV-enriched fractions were free from the bulk of soluble proteins, that eluted in later fractions. MSC-EV were Data Analysis further characterized by Cryo-TEM, confirming the presence of All the experiments were performed with an allogenic co-culture nanosized EV (Figure 1C). of MSC and B cells from eleven and five different donors, respectively. Technical duplicates were used. Flow cytometry data B Cell Survival Relies on was analyzed by FlowJo v10 software (FlowJo LLC, Ashland, Cell-To-Cell Contact OR). Statistical analysis was performed with Prism v6 software We have previously shown that MSC increase B cell survival (GraphPad, La Jolla, CA). Kruskall-Wallis with Dunn’s multi- compared to non-stimulated B cells but also compared to T comparison test was used to determine statistical differences cell-like activated B cells (IL-2/anti-IgM/anti-CD40) (4). In the between groups (p < 0.05 was considered significant). current setting, the number of living B cells (CD19+ 7AAD− ) Frontiers in Immunology | www.frontiersin.org 4 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells was significantly increased in the co-cultures with MSC but not in the other conditions, compared to stimulated B cells alone (Figure 2A). B Cell Plasticity Mediated by MSC Is Independent of MSC-EVs B cell population analysis by flow cytometry revealed that MSC- CM and MSC-PF retained B cells in a naïve state similarly to MSC contact, and reduced non-switched and switched memory B cells. MSC-EV did not change B cell populations of stimulated B cells (Figure 2B). The MSC-PF but Not MSC-EV Is Partially Responsible of Breg Induction We next assessed the induction of Breg, phenotypically defined here as CD24hi CD38hi CD19+ B cells and by secretion of IL-10 and low secretion of TNFα. The CD24hi CD38hi phenotype was only significantly induced by MSC co-culture. Since activated and memory B cells represent the CD24hi CD38int/lo subset, we defined the ratio CD24hi CD38hi /CD24hi CD38int/lo as an index between transitional immunosuppressive (CD24hi CD38hi ) and inflammatory activated/memory (CD24hi CD38int/lo ) B cell populations. In this case, MSC significantly generated a higher immunoregulatory ratio than any of the other conditions (Figure 2C). To further confirm that the paracrine effect of MSC on Breg polarization is mediated by the PF of the CM, a dose-dependency experiment was performed. We observed that the percentage of CD24hi CD38hi cells and the ratio CD24hi CD38hi /CD24hi CD38int/lo were higher when the highest dose of PF and CM was added to cells, showing that this effect is related to the CM and its soluble protein-enriched fractions FIGURE 3 | Concentration of IL-10 and TNFα in the culture medium after 7 (Figure 2D). Moreover, the PF as well as the CM could increase days of B cell culturing with the different conditions measured by ELISA. Each the percentage of non-switched and switched memory subsets in dot represents a different combination of MSC and B cells donors. Statistical a dose-dependent manner (data not shown). significance (p < 0.05) was determined by Kruskall-Wallis with Dunn’s In line, the IL-10 concentration of MSC co-culture condition multi-comparison test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). MSC, mesenchymal stem cells; CM, conditioned medium; EV, extracellular vesicles; was also significantly higher than in any other condition, PF, protein fraction. except for MSC-PF. MSC-PF significantly induced more IL- 10 secretion than MSC-EV and B cells alone. Noticeably, TNFα was secreted at very low concentrations in all conditions (<20 pg/ml) (Figure 3). DISCUSSION Assessment of B Cell Subsets Proliferation In the present work we studied the role of the MSC secreted We also compared the effect of whole MSC with their secreted factors on B cell immunomodulation. We isolated EVs by factors in terms of B cell subsets proliferation. In our setting, SEC, which allows to obtain purer EV preparation to properly after 7 days >80% of B cells proliferated in all conditions with discriminate it from the non-EV soluble fraction. few changes between groups (data not shown), so we focused Ultracentrifugation (UC) without any extra purification step on the study of 4th generation onwards proliferation (G4+). has been the solely and most widely used method for EV MSC co-culture allowed B cell proliferation G4 onwards whilst isolation, as reflected in the majority of published articles. MSC-CM and its derived fractions did not reach the same However, UC is not an appropriate method to isolate EV percentage, even though the differences did not reach statistical since it affects the biophysical properties and co-precipitate significance. We further observed a trend of MSC-EV compared EV with other particles (proteins, membrane fractions. . . ) that MSC-PF to reduce proliferation of naïve, non-switched are interfering with the subsequent molecular or functional memory B cells and Bregs while increasing switched memory analyses (18–20). Recently, more refined techniques for EV proliferation (Figure 4). purification have made their way into the field which are slowly Frontiers in Immunology | www.frontiersin.org 5 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells FIGURE 4 | Percentage of proliferation from the 4th generation onwards (G4+ proliferation) of the different B cell subsets measured by dilution of Violet CellTrace dye by flow cytometry. The different subsets were gated from alive B cells (CD19+ 7AAD− ). Naïve, CD19+ CD27− IgD+ ; non-switched memory, CD27+ IgD+ ; switched memory, CD27+ IgD− ; Breg, CD24hi CD38hi . No statistically significant differences were found between conditions by Kruskall-Wallis with Dunn’s multi-comparison test. changing the paradigm about the properties and functional time frame, since factors are secreted continuously throughout characteristics of EV. Within these methods, SEC stands out 7 days in the co-culture. To counterbalance this effect, and for its better preservation, higher yield and purity, and ease because a well-defined quantification of EVs is missing in the for EV isolation (12, 21). Several groups have assessed the field, the amount of MSC-EV and MSC-PF used was coming differences between UC and SEC for downstream analyses, from a proportionally higher number of cells than those used concluding that UC co-precipitate EVs and soluble proteins in the co-culture setting. This approach avoids the potential hampering the discrimination of their individual effect in underestimation of the effect of EV or soluble factors due to an functional studies (11, 13, 22, 23). insufficient amount. Conditioned medium (MSC-CM) was fractionated by SEC to The lack of unique markers for Breg complicates their obtain EV-enriched and non-EV soluble protein fractions, and analysis. While the transitional phenotype CD24hi CD38hi their distinct effect on B cell subsets and proliferation after T- appears as one of the most accepted, IL-10 production cell like activation was analyzed. MSC-CM and MSC-PF had is still the key feature to define Breg. In our hands, the a comparable immunomodulatory effect to that of MSC on transitional phenotype did not fully correlate with IL-10 B cells—increasing the proportion of naïve B cells and IL-10 secretion, since the percentage of CD24hi CD38hi was only production and reducing memory B cell phenotype proportions. increased in the MSC co-culture setting compared to B In contrast, MSC-EV did not exert any effect on activated B cells. cells alone, while IL-10 concentration was significantly Our results contrast with those obtained before that postulate increased in both the co-culture as well as with MSC-PF. MSC-EV as important mediators for B cell immunomodulation However, we experimentally observed that the ratio transitional (9, 24). One of the key points of this difference is the method used (CD24hi CD38hi )/activated-memory B cells (CD24hi CD38int/lo ) to isolate and purify the EVs. correlated better with IL-10 production. This argues for the The effect of MSC-PF is similar but to a lower level than hypothesis that a favorable balance between immunomodulatory MSC co-culture. This can be due to the fact that the modulation and pro-inflammatory phenotypes is needed for of B cells by MSC is partially cell-contact dependent, but it immunomodulation, rather than fostering immunomodulatory also could be explained by differences in B cells’ stimulation phenotypes alone. Frontiers in Immunology | www.frontiersin.org 6 June 2019 | Volume 10 | Article 1288 Carreras-Planella et al. MSC-EV Do Not Immunomodulate B Cells Proliferation experiments rendered a similar percentage la investigació clínica de l’Hospital Universitari Germans of proliferating cells in B cells alone and MSC conditions. Trias i Pujol. However, as the viability of B cells alone was significantly lower, the total number of proliferated B cells was higher AUTHOR CONTRIBUTIONS in the MSC co-culture. Results were not significantly different between MSC-CM or its derived fractions, but LC-P contributed to collection of data analysis and the mean proliferation percentage was lower than in the interpretation and manuscript writing. MM-T and FB co-culture, suggesting a cell-contact based mechanism to contributed to data analysis and interpretation, and final fulfill an optimal MSC conditioning. Still, we observed a approval of manuscript. MF conception and design, trend of MSC-CM and MSC-PF to better reflect the effect of collection of data, data analysis and interpretation, and MSC co-culture compared to MSC-EV supporting the idea manuscript writing. that MSC-PF better reflects the immunomodulatory effect of MSC. ACKNOWLEDGMENTS Using SEC as one of the most refined current techniques to separate CM’s EV and soluble proteins, we can conclude that the This work was supported in part by Fundació La Marató de partial effect of MSC soluble factors on B cell immunomodulation TV3 (201516-10, 201502-30), SGR programme of Generalitat de is preferentially induced not by EV but rather by the protein- Catalunya (2017-SGR-301 REMAR Group), ISCIII-REDinREN enriched fractions. The actual factors responsible of the effect are (RD16/0009 Feder Funds) and Instituto Carlos III project still under investigation. PI17/00335, integrated in the National R+ D+ I and funded by the ISCIII and the European Regional Development Fund. LC-P by the Spanish Government FPU grant (Formación de ETHICS STATEMENT Personal Universitario, FPU17/01444); MM-T is sponsored by the PERIS (SLT002/16/00234) from the Generalitat de Catalunya; This study was carried out in accordance with the FB is a researcher from Fundació Institut de Recerca en Ciències recommendations of Guideline for Good Clinical Practice de la Salut Germans Trias i Pujol, supported by the Health from the Comitè d’Ètica de la investigació clínica de l’Hospital Department of the Catalan Government (Direcció General de Universitari Germans Trias i Pujol with written informed Recerca i Innovació, Department Salut, Generalitat de Catalunya) consent from all subjects. All subjects gave written informed and MF is funded by the Catalan Health Department (Generalitat consent in accordance with the Declaration of Helsinki. de Catalunya) contract PERIS (SLT002/16/00069); the authors The protocol was approved by the Comitè d’Ètica de also want to thank Miriam Morón-Font for the graphical art help. REFERENCES functions. J Extracell Vesicles. (2015) 4:27066. doi: 10.3402/jev.v4. 27066 1. Hoogduijn MJ, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, 9. Budoni M, Fierabracci A, Luciano R, Petrini S, DiCiommo V, Muraca M. The et al. The immunomodulatory properties of mesenchymal stem cells and immunosuppressive effect of mesenchymal stromal cells on b lymphocytes their use for immunotherapy. Int Immunopharmacol. (2010) 10:1496–500. is mediated by membrane vesicles. Cell Transplant. (2012) 22:369–79. doi: 10.1016/j.intimp.2010.06.019 doi: 10.3727/096368912X653309 2. Le Blanc K, Mougiakakos D. Multipotent mesenchymal stromal cells 10. 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