Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche, where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation, dendritic cell maturation, and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naïve B cells stimulated with an agonist of Toll-like receptor 9, in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease.
Disclosure of potential conflicts of interest is found at the end of this article.
The bone marrow (BM) microenvironment contains a complex network of nonhematopoietic cells of mesodermal origin, which tightly interact with hematopoietic stem cells (HSCs) and their progenies . Within this pool of mesodermal cells, a subset of self-renewing, multipotent cells has been identified and defined as multipotent mesenchymal stromal cells (MSCs). BM MSCs provide factors required for the maintenance as well as the development of HSCs and can be induced to differentiate into various mesodermal lineages [2, –4]. They express the surface markers CD73 and CD90 in the absence of any hematopoietic antigens  and can be expanded in vitro as adherent stromal colonies without losing their self-renewal and differentiation potentials . BM MSCs have been considered as a possible tool to ameliorate HSC engraftment in an allogenic transplantation setting [7, 8]. Moreover, they have been hypothesized to possess immunomodulatory properties because they affect both the phenotype and the function of a number of cells that belong to the innate or to the adaptive immune system, namely, T and B lymphocytes, dendritic cells, and natural killer cells [9, , , , –14]. This led to the idea that these cells could be beneficial in the treatment of autoimmune diseases . However, two reports [12, 14] have shown how the interaction between MSCs and human B cells in vitro could lead to two opposite effects in terms of modulation of proliferation and differentiation capacity of B cells. Moreover, recent reports have shown how the interaction between MSCs and cells of the adaptive immune system could vary depending on the microenvironment in which the interaction is taking place [11, 16]. These conflicting results need to be carefully considered when designing potential therapeutic cellular strategies using MSCs. In this report, we investigated whether a rationale existed for considering MSCs as a possible tool for immunomodulation in systemic lupus erythematosus (SLE) . SLE is a systemic autoimmune disorder involving different components of the immune system. A hallmark of SLE is the development of somatically mutated IgG autoantibodies specific for nuclear antigens. These IgG autoantibodies are involved in pathogenic immune complexes formation and deposit in peripheral organs such as kidney, central nervous system, and skin. A number of cellular and molecular mechanisms have been invoked to explain the production of autoreactive IgG. Several lines of evidence show how breakdowns in the early and late B-cell tolerance checkpoints take place in SLE patients, supporting a causal role of B cells in the pathogenesis of the disease . We analyzed the effect of MSCs on highly purified B-cell subsets isolated from peripheral blood of normal donors and total B cells isolated from peripheral blood of pediatric SLE patients. We show that both transitional and naive B cells can efficiently respond to the toll-like receptor 9 agonist CpG 2006 in the absence of B cell receptor triggering when cocultured with MSCs. In addition, MSCs supported both proliferation and differentiation to immunoglobulin-secreting cells (ISCs) of B cells isolated from all SLE patients tested. This would imply that potentially harmful self-reactive B cells could undergo clonal expansion and exert a negative effect on the evolution of the disease.
MSCs were generated from normal donor BM samples. Briefly, mononuclear cells were isolated by Ficoll-Hypaque (Cedar Lane, Hornby, ON, Canada, http://www.cedarlanelabs.com) density gradient centrifugation. Unfractionated MNCs were plated at 20 × 106 in 13 ml of MSC medium in 75-cm2 flasks (BD Falcon, BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). The medium used was Mesencult basal medium supplemented with Mesenchymal Stem Cell Stimulatory Supplement (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). After 5 days of culture, medium containing nonadherent cells was replated. At 90% of confluence, cells were trypsinized and replaced at a density of 0.7 × 106 cells in 75-cm2 flasks. Under these conditions, cells negative for the expression of CD34, CD45, and CD14 (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) but positive for CD44 and CD73 (Becton Dickinson) were selectively expanded. MSCs were used between the 6th and 10th passage.
Peripheral blood mononuclear cells from peripheral blood of healthy donors and SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD19+ B cells were isolated by use of CD19 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer's instructions. Following stainings for lineage (CD3, CD14, and CD56; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), CD24, CD38 (Becton Dickinson), and surface IgG and IgA (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) transitional B cells were sorted by FACSAria (Becton Dickinson). Naïve, IgM memory and switch memory B cells were isolated by CD19 microbead (Miltenyi Biotec) purification followed by stainings for lineage (CD3, CD14, and CD56; Beckman Coulter), CD27 (eBioscience Inc., San Diego, http://www.ebioscience.com), and surface IgG and IgA (Jackson Immunoresearch Laboratories). The purity of the sorted B cells was >95%.
B-cell subsets were labeled with 0.5 μM 5-(and-6-)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 8 minutes at room temperature. After quenching of the labeling reaction by addition of fetal calf serum (FCS), cells were washed twice. Purified B cells (2 × 104 cells) were cocultured with 2 × 104 human BM MSCs (1:1 ratio) in a 96-well flat-bottomed culture plate with the following stimuli: 2.5 μg/ml CpG oligodeoxynucleotide 2006 (5′-tcgtcgttttgtcgttttgtcgtt-3′; TIB Molbiol, Genoa, Italy, http://www.tib-molbiol.it), 1 μg/ml soluble CD40L (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 2.5 μg/ml F(ab′)2 anti-human IgM/IgA/IgG (Jackson Immunoresearch Laboratories), and 1,000 U/ml interleukin 2 (IL-2) (Proleukin; Chiron Corp., Emeryville, CA, http://www.chiron.com). The proliferation profile of propidium iodide-negative viable CD19-positive cells was analyzed at day 4 of culture. To allow a direct quantitative comparison of B-cell proliferation in the different conditions, fluorescence-activated cell sorting (FACS) acquisitions were standardized by fixed number of calibration beads (BD Biosciences, San Diego, http://www.bdbiosciences.com).
Enzyme-Linked Immunosorbent Spot and Enzyme-Linked Immunosorbent Assay
Immunoglobulin production in culture supernatants was evaluated by enzyme-linked immunosorbent assay. Ninety-six-well flat-bottomed plates (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en) were coated with isotype-specific goat anti-human IgG, IgA, or IgM antibodies (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) diluted in 0.2 M Na2HPO4, pH 9.6, and incubated overnight at 4°C. Plates were washed and blocked with phosphate-buffered saline (PBS), 10% FCS for 2 hours at room temperature. After washing, serial dilutions of culture supernatants were added and incubated for 2 hours at room temperature. Plates were washed again, and alkaline phosphate-conjugated goat anti-human IgG, IgA, or IgM was added and incubated for 1 hour at room temperature. The reaction was developed with Sigma-Aldrich 104 substrate (Sigma-Aldrich). Plasma cells secreting IgG, IgM, or IgA were detected using an enzyme-linked immunosorbent spot assay. Briefly 96-well plates (MAIPS4510; Millipore, Billerica, MA, http://www.millipore.com) were coated with 10 μg/ml purified goat anti-human IgG, IgA, IgM (SouthernBiotech). After washing and blocking with PBS plus 1% bovine serum albumin for 30 minutes, serial dilutions of cultured B cells were added and incubated overnight at 37°C. Before plating, cultured B cells were washed five times with complete medium to eliminate the immunoglobulin present in the supernatants. Plates were washed and incubated with isotype-specific secondary antibodies, followed by streptavidin-horseradish peroxidase (Sigma-Aldrich). The assay was developed with 3-amino-9-ethylcarbazole (Sigma-Aldrich) as a chromogenic substrate.
Cytokine production was evaluated with the Bio-Plex cytokine assay according to the instruction manuals (Bio-Rad, Hercules, CA, http://www.bio-rad.com).
Human Bone Marrow-Derived Mesenchymal Stem Cells Promote Polyclonal Proliferation and Differentiation of Transitional Immature and Naïve B Cells
The human peripheral B-cell pool is composed of cells at different stages of differentiation, characterized by different requirements to differentiate into immunoglobulin-secreting cells: (a) immature transitional B cells, (b) naïve B cells, (c) IgM memory B cells, and (d) switch memory B cells. We isolated B cells from peripheral blood by positive selection with CD19 immunomagnetic beads. Further purification included sorting based on the use of monoclonal antibodies (mabs) specific for CD24 and CD38 in combination with Igs specific for cell surface IgG and IgA to isolate transitional B cells as CD19+CD24highCD38highIgG−IgA− cells (Fig. 1A). Anti-CD27 mab was used together with anti-IgG and anti-IgA for the isolation of naïve B cells as CD19+CD27−IgG−IgA− cells (Fig. 1B). To evaluate the effect of MSCs on the activation of these purified B-cell subsets, transitional and naïve B cells were activated with different stimuli either alone or in the presence of MSCs at a 1:1 ratio. BM MSCs, in the absence of stimulation, induced survival and proliferation of transitional and naïve B cells (Fig. 1C, 1D). Stimulation with CpG and IL-2, in the absence of BCR triggering and T-cell help, failed to induce significant proliferation of both transitional and naïve B cells. On the other hand, coculture with MSCs under the same conditions resulted in robust proliferation of both transitional and naïve B cells (Fig. 1C, 1D). Furthermore, the expression of CD38, a surface marker that is upregulated during plasma cell differentiation in vitro , was increased in proliferating B cells, as revealed by CFSE dilution at day 6 of culture (Fig. 2A). The Ig production was quantified in the supernatants at day 6 of culture. IgM secretion was barely detectable in cultures performed with B cells alone stimulated with CpG and IL-2, whereas upon coculture with MSCs, a significant increase of IgM concentration was detected (transitional IgM production: CpG IL-2: 0.03 ± 0.016 μg/ml, n = 5; with MSCs: 4.46 ± 2.3 μg/ml, n = 5, p = .045; naive IgM production: CpG IL-2: 0.128 ± 0.068 μg/ml, n = 5; with MSCs: 7.9 ± 3.8 μg/ml, n = 5, p = .037) (Fig. 2B). The combination of anti-Ig, CpG, soluble CD40L, and IL-2 led to proliferation of both transitional and naïve B cells. Coculture with MSCs had a strong synergistic effect with these stimuli, as revealed by the sharp increase of the percentage of cells that had undergone four or more divisions (Fig. 2C). Surface IgG and IgA were highly upregulated on transitional but not on naive B cells at day 6 after stimulation with CpG, anti-Ig, soluble CD40L, and IL-2 only when MSCs were present in the culture (Fig. 2D). Thus, MSCs could induce in vitro differentiation into antibody-secreting cells, as well as class switch recombination of immature transitional B cells stimulated with a combination of CD40L, anti-Ig, IL-2, and CpG 2006.
Polyclonal Stimulation of IgM Memory and Switch Memory B Cells Is Enhanced by BM MSCs
Human memory B cells are characterized by their ability to selectively proliferate and differentiate into plasma cells in response to polyclonal stimulation in the absence of antigen . To see whether MSCs had any influence on memory B cells, IgM memory and switch memory B cells according to the gates shown and the markers used (CD27- and IgG/A-negative and CD27- and IgG/A-positive) were isolated by cell sorting from peripheral blood of healthy donors (Fig. 3A). Purified B cells were labeled with CFSE and cocultured with MSCs at a 1:1 ratio. IgM memory and switch memory B cells responded to CpG and IL-2, as expected. When MSCs were added to the culture, an impressive increase of proliferating B cells was observed at day 4 of culture (Fig. 3B). The sustained polyclonal proliferation of memory B cells promoted by MSCs was associated with a dramatic increase in the frequency of both ISCs (Fig. 3C) (IgM memory ISC IgM CpG IL-2: 75 ± 66; with MSCs: 1,205 ± 507, n = 6, p = .0003; switch memory ISC IgG CpG IL-2: 56 ± 15.1; with MSCs: 1,306 ± 202.1, n = 6, p = .0001; ISC IgA CpG IL-2: 30 ± 6.9; with MSCs: 279 ± 82.47, n = 6, p = .0131) and Ig concentration in the culture supernatants (Tables 1, 2) at day 6 of culture. Thus, BM MSCs can efficiently sustain antigen-independent cell proliferation and differentiation into ISCs of both subsets of peripheral memory B cells.
Table Table 1.. Immunoglobulin production by memory B cells polyclonal activated
Table Table 2.. Immunoglobulin production by SLC patients
The Effect of MSCs on B-Cell Subsets Is Dependent on Cell-Cell Contact
Previous reports showed that the inhibitory effect of MSCs on T lymphocytes was mediated by soluble factors and not by cell-cell interaction . It was previously reported that BM MSCs expressed low levels of TLR9 . In addition, as shown in Figure 4B, low levels of expression of CD40 on different MSCs cell lines was detected. These data suggest that CD40L, also, may possibly promote some cytokine production by MSCs. Therefore, we further evaluated the cytokine production in culture supernatants of MSCs stimulated with CpG alone or in combination with soluble CD40L, anti-Ig, and IL-2, which were used to stimulate B lymphocytes. As shown in Figure 4A, stimulated MSCs produced cytokines known to be involved in B-cell proliferation and differentiation, including IL-6, IL-10, IL-7, IL-4, and interferon-γ. Remarkably, high levels of IL-6 (∼3 μg/ml), a cytokine that can promote proliferation and survival of plasma blasts/plasma cells , were produced by MSCs stimulated with CpG alone. The other cytokines were increased only with the combination of the stimuli mediated by CpG, CD40L, anti-Ig, and IL-2. Therefore, we evaluated the proliferative responses of polyclonally stimulated B-cell subsets in a transwell system with B cells in the lower chamber and MSCs in the upper chamber. In parallel, control B cells were kept in contact with MSCs as in the experiments described above. In spite of high IL-6 production by MSCs, when MSCs and B cells were physically separated, transitional and naïve B cells did not proliferate in response to CpG, although they did when MSCs and B cells were in contact. Moreover, IgM and switch memory B cells did not differ in the proliferative response to CpG from control cultures when MSCs were physically separated in the transwell culture (Fig. 4C; transitional CpG IL-2: 69.67 ± 15.5; with MSC: 1,364 ± 1,026, transwell: 96 ± 49, n = 3; naïve CpG IL-2: 407 ± 239, with MSCs: 1,805 ± 1,488, transwell: 515.7 ± 381, n = 3, IgM; memory CpG IL-2: 1,120 ± 333, with MSCs: 6,376 ± 866, transwell: 1,283 ± 127, n = 3; switch memory CpG IL-2: 531 ± 391, with MSCs: 3,335 ± 913, transwell: 419 ± 260, n = 3). Thus, direct cell-to-cell contact with MSCs is required to promote responses to CpG of both transitional, naïve B cells and to sustain proliferation and differentiation of memory B-cell subsets.
BM MSCs Support both Proliferation and Differentiation to Plasma Cells of Peripheral B Cells Isolated from Pediatric SLE Patients
We next evaluated the possible influence of BM MSCs on peripheral B cells isolated from 19 pediatric SLE patients, 11 with active disease and 8 in clinical remission (Table 3). Total peripheral B cells isolated by positive selection using CD19 immunomagnetic beads were labeled with CFSE and cultured in the presence of CpG and IL-2 or in the presence of a combination of anti-human Ig F(ab′)2 fragment (anti-Ig), soluble CD40L, CpG, and IL-2. MSCs enhanced B-cell proliferation scored at day 4 of culture by CFSE dilution of propidium iodide-negative cells (Fig. 5A). Analysis of IgG and IgA expression in proliferating cells revealed that proliferation of both naïve and switch memory B cells was supported by MSCs (Fig. 5B).
Table Table 3.. Clinical data SLE patients
In the patients tested, the mean ratio of proliferating cells in the presence and in the absence of MSC was 5.7 ± 1.5 and 2.5 ± 0.7 for CpG and IL-2 and anti-Ig/CD40L/CpG/IL-2 stimulation, respectively (Fig. 4D). At day 4 of culture, an increase of CD38+ cells was observed only in the presence of MSCs (Fig. 5C). Furthermore, the frequency of IgM- and IgG-secreting cells (IgA-secreting cells were undetectable) was increased (Fig. 5D, total B cells IgM ISC CpG IL-2: 484 ± 226, with MSCs: 11,260 ± 4,873, n = 3, p = .0462; IgG production CpG IL-2: 268 ± 79.9, with MSCs: 2058 ± 897.5, n = 3). Accordingly, Ig concentration in culture supernatants was augmented (Table 2). We concluded that BM MSCs can support survival, proliferation, and differentiation to antibody-secreting cells of B cells isolated from SLE patients.
SLE is a systemic autoimmune disease characterized by continuous generation of autoantibody-producing cells (i.e., autoreactive plasma cells), produced through mechanisms that are not yet fully understood. Nevertheless, it is well-established that B cells are critical in the pathogenesis of the disease through autoantibodies-dependent and -independent mechanisms. Therefore, targeting B cells to treat SLE patients is an attractive alternative to currently available pharmacological approaches . To verify whether MSCs possessed the potential to modulate autoimmune B-cell responses in SLE, we investigated the impact of their interaction in vitro with B cells isolated from healthy donors as well as from SLE patients. BM MSCs were found to enhance proliferation and differentiation into plasma cells of all peripheral B-cell subsets tested, thus raising serious concerns regarding the possible use of these cells as a therapeutic tool to downmodulate B-cell responses. The pool of circulating peripheral B cells is composed of cells at different stages of differentiation characterized by different requirements to complete their maturation into plasma cells. They include the following: (a) immature transitional B cells, (b) naïve B cells, (c) IgM memory B cells, and (d) switch memory B cells. Transitional B cells have recently been identified at a low frequency in the peripheral blood of healthy donors . They share phenotypic and functional characteristics with transitional murine B cells . They migrate from BM to the periphery and display a reduced capability of proliferating and differentiating into immunoglobulin-secreting cells as compared with naïve B cells . The antibody repertoire of these newly emigrant transitional B cells is composed mainly of self-reactive B cells that will be further selected in secondary lymphoid organs (such as the spleen), leading to a naïve B-cell pool with lower frequencies of autoreactive B cells . Both circulating human transitional B cells and naïve B cells need at least three signals to be fully activated and to undergo proliferation and differentiation into plasma cells. Namely, they require signaling via BCR, T-cell help, and appropriate cytokines (E.T., unpublished results) . The absence of response of transitional and naïve B cells to polyclonal stimulation reflects the need to avoid expansion of potentially autoreactive B cells. Our present study shows that human BM MSCs are capable of inducing immature transitional and naïve B cells to undergo proliferation and differentiation into antibody-secreting cells in response to polyclonal stimuli, in the absence of antigen. It is possible that this way leads to a possibly dangerous alteration of B-cell repertoire selection.
Notably, BM MSCs could support proliferation and differentiation to ISCs of both subsets of peripheral memory B cells. Our present data are in contrast with a previous report describing MSC-mediated inhibition of proliferation and differentiation into ISC of unfractionated human peripheral B cells . These discrepancies might reflect differences in cell purification procedures, experimental conditions, and timing of analysis. It should be stressed that our data are in line with a previous observation made by Rasmusson et al., who reported an increase in proliferation and differentiation into plasma cells of enriched B cells isolated from both peripheral blood and spleen, mediated by MSCs in vitro . Moreover, our findings match the postulated role of BM stroma in supporting not only B cells at early stages of their development but also terminally differentiated and memory B cells. In particular, human memory B cells carrying somatically mutated Ig can be found in the BM , and plasma cells migrate to the BM, in which they can be rescued in appropriate survival niches .
We tested the efficacy of i.v. MSC injection in NZB/NZW F1 mice as an animal model of SLE (F. Schena et al., manuscript in preparation). MSCs were injected intravenously three times at weeks 16, 18, and 20 of age (1.25 × 106 MSCs cells per mouse). At week 31, mice were sacrificed, and no significant differences in IgG serum autoantibody titers and proteinuria were observed in treated animals compared with the control group.
The mechanisms through which MSCs displayed their effects on B cells appear to be primarily dependent on cell-cell contact despite the fact that IL-6, a potent B-cell growth factor, was produced by MSCs after stimulation with TLR9 agonist. In all SLE patients analyzed so far, we detected a sustained proliferation and differentiation of peripheral B cells induced by MSCs in both antigen-dependent and antigen-independent stimulation. Remarkably, a perturbation of the peripheral B-cell population in children with SLE has been described in a previous report in which the expansion of memory B cells and plasma blasts was correlated with active disease . Differences in distribution of B-cell compartments detected in different patients analyzed in our study did not appear to influence the in vitro stimulatory activity of MSCs. This suggests that the functional effect exerted by MSCs on the various B-cell subsets present in SLE patients may be similar and independent on the stage of the disease. Overall, our data clearly show that BM MSCs positively influence the proliferation and differentiation into immunoglobulin-secreting cells of all peripheral B-cell subsets, raising important questions on the potential use of MSCs in autoimmune disease in which B cells play a crucial role.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
E.T. is supported by a private donation from Fausto Nicolini. This work was supported by grants awarded by the Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Sanità, and the Ministero della Salute (Ricerca Finalizzata Ministeriale 2005 “Caratterizzazione delle proprietà di immunomodulazione delle cellule mesenchimali e possibile applicazione nel trattamento delle malattie autoimmuni”). Authors' contributions were as follows: E.T. designed and performed the research, analyzed data, and wrote the paper; S.V. collected data and performed research; F.S. and F.F. participated in performing the research; and M.G., L.M., and A.M. participated in designing the research and discussion. We are thankful to E. Albanesi, Core Facilities Institute G. Gaslini, for FACS sorting.