Immunomodulatory Effects of Mesenchymal Stem Cells on T- and B-Cells in a Quiescent State in a Chronic Experimental Model of Autoimmune Encephalomyelitis

Rafael Coveñas, MSc, PhD

Universidad de Salamanca, Instituto de Neurociencias de Castilla y León (INCYL), Laboratorio 14, c/ Pintor Fernando Gallego, 1, 37007 Salamanca, Spain; Tel. 34-923-294400; ext 5315; Fax: 34-923-294549; E-mail: covenas@usal.es ; mangasam@usal.es

INTRODUCTION

Although, there are still many problems to be solved before cell therapy can be used reliably to repair mature central nervous system (CNS) lesions, one strategy that could be used in multiple sclerosis (MS) is the transplantation of stem cells. Several types of stem cells have been studied and characterized: embryonic stem cells, neural stem cells, haematopoietic stem cells, non-haematopoietic stem cells and bone marrow stromal cells.^{1,2,3,4,5,6,7} In this regard, neural precursor cells were the first candidates for cell-based therapy in neuroinflammation.^8,9 However, the invasiveness associated with harvesting neural precursor cells, together with the reduced number of cells that can be obtained, may limit their clinical application. A potential alternative to neural precursor cells is mesenchymal stem cells (MSC). Recently, the actions exerted by adipose-derived mesenchymal stromal cells,^{10,11,12,13,14} placenta-derived mesenchymal stem cells,^15,16 Wharton’s jelly mesenchymal stem cells,¹⁷ human decidua-derived mesenchymal stem cells¹⁸ and human umbilical cord blood mesenchymal stromal cells¹⁹ have been reported in experimental autoimmune encephalomyelitis (EAE) animal models.

MSC have been characterized by their multipotentiality and their capacity for immunomodulation, thus raising great hopes in regenerative medicine. These cells could be ideal candidates for application in human diseases, since they offer significant practical advantages: they are obtained from different adult tissues and can readily be cultured and expanded; they are multipotent and self-renewing. They have the capacity to differentiate into mesodermal-derived²⁰ or into neural-like and glial-like cells²¹; they have been shown to have immunoregulatory properties^{10,12,13,14,17,18,22,23}; they secrete factors that may stimulate endogenous neural stem cells in the CNS^24,25; they can be safely injected autologously without the need for immunosuppression²⁶; they decrease the number of infiltrating inflammatory cells, preserving axons and ameliorating demyelination,^15,19,27,28 ameliorate neuroinflammation^11,29 and exert a neurotrophic action.¹⁶ All these features make MSC suitable for therapy in autoimmune diseases. In fact, the beneficial effects of MSC have been reported in EAE models.^{25,30,31,32,33,34} Most of these studies were carried out in mice and in Lewis rats, and this means that to date the immunomodulatory effects of MSC in EAE Lewis 1A rats have not been studied. However, EAE Lewis 1A rats have been widely used to study the effects of new drug candidates for MS. Lewis 1A rats belong to the Major Histocompatibility Complex (MHC) congenic Lewis rat strain. The effects of MSC on T- and antigen-presenting cells have been studied in depth. Nevertheless, and contrary to their well-known effect on T-cells and antigen-presenting cells, the effect of MSC on B-cells remains unclear. B-cells are critical for myelin oligodendrocyte glycoprotein (MOG)-induced EAE, but are redundant in MOG (35-55)-induced EAE.^35,36 This is important, because B-cells play a critical role in human MS.³⁷ Moreover, MSC have been implicated in B-cell development in bone marrow, spleen and lymphoid follicles,³⁸ exerting a negative control on B-cell lymphopoiesis. In addition, it has been reported that B-cell proliferation is inhibited by MSC,³⁸ although this has not been confirmed by other authors.³⁹ The distribution of MSC in bone marrow and secondary lymphoid organs allows an intimate interaction between both cell subsets, which contributes to normal lymph node development⁴⁰ as well as to the support of tumor B-cells in follicular lymphomas.⁴¹ Therefore, the study of the effect of MSC on B-lymphocytes is necessary to increase our insight into autoimmune processes such as MS. In addition to the demonstration of the immunomodulatory effects of MSC on B- and T-lymphocytes in the EAE model, the effects of such cells on the abolition or not of EAE episodes must be studied. Another important point to make clear is the expression of α4 integrins. In this sense, murine bone marrow (BM)-MSC do not express α4 integrins,⁴² and one study on BM-MSC focusing on EAE lesions has provided conflicting results regarding the capacity of BM-MSC to migrate into inflammed CNS.^{25,30,31,32,43} In a mouse model of EAE, it has been reported the synergic effect (e.g., the expression of the brain-derived neurotrophic factor was increased) of BM-MSC when these cells were combined with fasudil⁴⁴ (this combination reduced the severity of EAE in comparison with fasudil or BM-MSC alone); that rapamycin increased the immunomodulatory properties of BM-MSC,⁴⁵ playing the latter combination an important role in neuroprotection, and that the combination of resveratrol and BM-MSC increased the immunomodulatory effects (pro-inflammatory cytokines were suppressed and anti-inflammatory ones were increased).⁴⁶ Moreover, in a rat model of EAE, it has been demonstrated that the combination therapy of BM-MSC and EGb761 (a Ginkgo biloba extract) increased the neuroprotective effects, inhibited the secretion of pro-inflammatory cytokines and decreased the disease severity and the inflitrated cells.⁴⁷ Finally, in a mouse model of EAE, it has been reported that MSC controlled the induction of T-cells with a regulatory phenotype and the inhibition of pro-inflammatory T-cells.⁴⁸

In the light of the foregoing data, we tested the therapeutic potential of intravenously injected BM-MSC from healthy donors in a chronic EAE model (Lewis 1A rats). We studied in vitro the immunomodulatory effects of MSC on the B- and T-lymphocytes of both control and EAE animals and the effects of intravenously administered MSC on EAE episodes.

MATERIALS AND METHODS

Animals and Induction of EAE

Lewis 1A female rats (n=23) aged 10-11 weeks (weight around 190 g) obtained from CERJ Janvier (France) were used. The animals were kept under standardized lighting and temperature conditions and had free access to food and water. They remained for at least ten days in their cages before the experiments. The aminals were weighed and scored according to the scale described below in “Animal groups and clinical evaluation of EAE” 6 days/week throughout the experiments. The experimental design, protocols, and procedures were performed under the guidelines of the ethical and legal recommendations of Spanish and European law. The study was also approved by the experimental research commission of the University of Salamanca (Spain).

EAE rats were immunized with a solution containing 50 µg of MOG and complete Freund’s adjuvant (ACF), to which heat-inactivated Mycobacterium tuberculosis H37RA had been added. The rats were anaesthetized with isofluorane and the solution was injected intradermally at the base of the tail.

Animal Groups and Clinical Evaluation of EAE

Animals were classified in different groups: 1) Control animals (group 1; n=3); 2) EAE animals (group 2; n=5); 3) EAE animals to which MSC were administered before the induction of EAE (group 3; n=5); 4) EAE animals to which MSC were administered after the induction of EAE (group 4; n=5); and 5) EAE animals to which MSC were administered before and after the induction of the EAE (group 5; n=5).

The animals were weighed and scored according to a previously described scale⁴⁹ 6 days/week throughout the experiments. The neurological signs of EAE were assessed and scored using this scale: 0, no signs; 1, tail weakness or tail paralysis; 2, hind leg paraparesis or hemiparesis; 3, hind leg paralysis or hemiparalysis; 4, complete paralysis (tetraplegy). Any animal reaching a moribund state was immediately perfused (see below) in order to avoid suffering. At the end of the experiment, all the animals were deeply anaesthetized with urethane (1 g/kg, intraperitoneal) and perfused via the ascending aorta with 50-100 ml of cold physiologic saline (0.9% NaCl) and then with 500 ml of cold 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.2).

Cell Isolation

MSC were isolated from the femora and tibias of wild-type female Lewis 1A rats (aged 10-11). MSC were harvested and placed in culture in DMEM containing 10% fetal bovine serum and 1% penicillin streptomycin. Cells were cultured at 37º C in a humidified atmosphere in the presence of 5% CO₂. Cultures were incubated at a final concentration of 3 x 10⁶ nucleated cells per ml at 37° C in a 5% CO₂ humidified incubator for 72 h. Non-adherent cells were aspirated on day 3 and the adherent population was cultured to achieve the maximum number of fibroblast colony-forming units prior to initial passage. Twice a week, adherent cells were fed by complete replacement of the medium. When the layer was confluent, cells were detached by treatment with trypsin and then subcultured at a concentration of 10,000 cells/cm² until confluence. This process was repeated at least 3 times in order to obtain a sufficient number of cells for analysis. All cultures were used between the third and fifth passages.

Adipogenic, osteogenic and condrogenic differentiation was induced as previously described⁵⁰ and recommended by the International Society of Cell Therapy (ISCT) consensus.⁵¹ Phenotypic characterization was performed using the following monoclonal antibody combinations: Fluorescein (FITC)/Phycoerythrin (PE)/Peridin chlorophil protein-Cyanine-5 (PerCP-Cy5)/Allo-phycocyanin (APC): CD45/CD106/CD90.1/MHC II. Data acquisition was performed with a FACScalibur^TM flow cytometer and data analysis with the Paint-A-Gate program.

B- and T-lymphocytes were obtained from splenocytes from healthy donor rats using cell sorting. Cells were stained with CD45RA and CD3, following the manufacturer’s instructions, for B- or T-cells, respectively. For isolation, a FACSAria Cell Sorting flow cytometer was used. Positively selected cells contained >95% B- or T-cells, as assessed by flow cytometry.

Administration of MSC

Animals were deeply anaesthetized with isofluorane. MSC were administered intravenously in the tail vein. Depending on the animal group (3, 4 or 5), the rats were infused at the base of the tail with a solution of sterile phosphate-buffered saline (PBS) containing MSC. MSC were infused two weeks before the induction of EAE (groups 3 and 5); eighteen days after induction (groups 4 and 5); and five weeks after the induction of EAE (groups 4 and 5).

Cell Viability Assays

10⁵ MSC were seeded in the plates and after 12 hours, 10⁶ positively selected B- or T-lymphocytes were seeded in the 96-well culture plate. Cells were cultured for 3 days in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. The effect of MSC on B-cell or T-cell growth was assessed by measuring the 3-(4.5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) dye absorbance of the cells. For this, 10⁵ B-cells or T-cells/100 mL were plated in triplicate onto 96-well tissue culture dishes in culture medium with or without 10⁴ MSC. MTT absorbance was assessed on the third day. Three wells were analyzed for each condition, and the results were presented as means±SD of triplicates.

For the detection of apoptosis, an AnnexinV-PE/7 amino-actinomycin (7-AAD) apoptosis detection kit was used. A minimum of 10⁶ lymphocytes were washed and resuspended in binding buffer (1:10 diluted in PBS), maintaining a cell concentration of 1×10⁶ /mL. Annexin V-PE and 7-AAD, 5 mL each, were added for 15 minutes. In order to identify B- and T-lymphocytes, anti-CD45RA-FITC and anti-CD3-APC were also added. For each condition, 50,000 events were collected and analyzed. The samples were acquired using Trucount^TM Tubes, which contain a calibrated number of fluorescent microbeads. The absolute count of annexin V-PE plus 7-AAD-negative cells was calculated using the following equation: (number of events in region containing annexin V-PE plus 7-AAD negative cells/number of events in the absolute count bead region) x (number of beads per-test/test volume). The Win MDI software was used for analysis.

Proliferation Assays

Studies were also performed on B-lymphocytes and T-lymphocytes cell cycles and DNA contents. For this, 5×10⁵ lymphocytes were cultured for 2 days. The cells were stained with 500 ml of solution B containing 0.5 g/L of RNAse; this solution was added for 10 minutes in the dark. Finally, 500 ml of solution C, containing 0.42 g/L of propidium iodide, was added to each tube and the cells were incubated in the dark for 15 minutes. After this period, measurements of DNA cell contents were performed on a FACScalibur^TM flow cytometer. A minimum of 20,000 events were acquired. For the analysis of the distribution of the cells along the cell cycle phases, the model included in the ModFit LT^TM software program was used after excluding cell debris and cell doublets in a FSC/FL2 area and a FL2 width/FL2 area dot plot, respectively.

Immunophenotipic Characterization

5×10⁴ MSC were seeded in the chamber of a 48-transwell plate and after 12 hours, 5×10⁵ positively selected B-lymphocytes were seeded in the chamber. The cells were cultured for 3 days in 1 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin.

T-cell activation was analyzed using the following monoclonal antibody combinations: FITC/PE/PerC-Cy5/APC: -/IL-4/-CD3, -/IFN-γ/-CD3. Standard intracellular cytokine staining was performed using a leukocyte activation cocktail—a ready-to-use polyclonal cell activation mixture with phorbol ester PMA (Phorbol 12-Myristate 13-Acetate), a calcium ionophore (ionomycin), and the protein transport inhibitor GolgiPlug™ (brefeldin A)—following the manufacturer’s instructions. After two washes with staining buffer, samples were first stained extracellularly with anti-CD3 before they were fixed and permeabilized for intracellular staining with phycoerythrin (PE)-conjugated anti-IL-14 or PE-conjugated anti-IFN-γ. Isotype-matched PE- and APC-conjugated monoclonal antibodies (mAbs) of irrelevant specificity were tested as negative controls. B-cell maturation was analyzed using the following monoclonal antibody combinations: FITC/PE/PerC-Cy5/APC: CD45RA/-CD86/-CD27. After staining for the antigens, cells were washed and resuspended in 0.5 ml of PBS until their acquisition in the flow cytometer. Data acquisition was performed on a FACSCalibur^TM flow cytometer using the CellQuest^TM software program, gated on the live population, and was analyzed using the Win MDI software. Analysis of both intracellular cytokines and the percentage of cells positive for surface antigens was performed on the gated population using the CellQuest^TM software.

RESULTS

General Considerations

After analysing their morphology and phenotype, rat MSC can be said to fulfill the requirements established by the International Society of Cellular Therapy (ISCT).⁵¹ Moreover, rat MSC expressed the vascular cell adhesion molecule CD106 (VCAM) to a slight extent; this could explain the migratory effect of MSC. MSC promoted T- and B-lymphocyte survival in all cases studied and did not modify the T- or B-lymphocyte cell cycles in a quiescent state. The results also confirmed that MSC modulated EAE through the production of soluble cytokines. That is, the presence of MSC increased IFN-γ production and decreased IL-4 production in a different way. Thus, in vitro MSC decreased EAE by immunomodulating the T-helper 1 (Th1)/Th2 response. The presence of MSC modulated CD27/CD86 expression; they increased CD27/CD86 expression on the T-cells of healthy animals to a certain extent, and they significantly decreased CD27/CD86 expression in EAE animals.

Regarding the clinical evaluation of the EAE, it was observed that either MSC decreased the EAE clinical score or the animals did not develop EAE when those cells were infused prior to the induction of EAE. Finally, it is important to note that in order to confirm the results found in this study more animals must be used and a detailed statistical analysis must also be conducted. This procedure could confirm some of the data shown in the results section of this preliminary study.

Isolation, Differentiation and Characterization of MSC

The isolation of BM-derived MSC was accomplished by culturing BM cells obtained from femora and tibias. MSC formed a heterogeneous population of cells that proliferated in vitro as plastic-adherent cells, had a fibroblast-like morphology, and formed colonies in vitro. After 2-3 passages, cells with a fibroblastic appearance reached confluence (Figure 1A, 1B). An enrichment in MSC was documented 1 month later by positive staining of cultured cells with anti-CD90.1 (Thy 1) and a slight expression of CD106 (V-CAM). However, they were negatively stained for CD45 (hematopoietic lineage marker) and MHC class II molecules (Figure 1C, 1D).

Figure 1. MSC Morphology at the Time of the First Passage (A), and at the Time of the Second Passage (B). MSC from Controls Proliferated and Acquired a
Spindle-Shaped Morphology (arrow). C: Immunophenotypic Assays of MSC from Healthy Donors. R1 Represents the Selection of the Percentages of Viable MSC. D: Differential Expression of the Markers Analyzed between Controls (blue line) and the MSC Phenotype (red line).

Immunomodulatory Effects of MSC on T- and B-Cells

To investigate the immunomodulatory effects of purified MSCs, myelin-sensitized lymphocytes (obtained from EAE rats and healthy donor rats) were cultured with MSC at 1:10 concentration. For this purpose, purified T- or B-lymphocytes were cultured for 48 or 72 hours with or without MSC in 96- or 48-well plates. MSC increased the viability of T- and B-cells in all cases studied. As shown in Figures 2 and 3, respectively, the presence of MSC increased T-cell (Figure 2) and B cell viability (Figure 3). Similar results were obtained in the rest of the experimental groups analysed after 3 days of culture. These results are shown in Table 1. Analyses with MTT confirmed those findings (data not shown) (Figures 4 and 5).

Figure 2. T-Cell Viability Assessed by Annexin/7AAD Staining in the Absence (A to C) or Presence (D to F) of MSC in some Cases Analysed. CD45RA cells were Excluded and CD3 Cells Positively Selected. The First Two Dot Blots are from the Healthy Donor Animal Group, the Second two from the EAE Animal Group, and the Third Two are from EAE Animals to which MSC were Administered before and after the Induction of EAE. Percentage of
Annexin/7ADD-Positive Cells for Samples Cultured with (violet column) or without (maroon column) MSC. (1) Control animals (group 1); (2) EAE
Animals (group 2); (3) EAE Animals to which MSC were Administered before the Induction of EAE (group 3); (4) EAE Animals to which MSC were
Administered after the Induction of EAE (group 4); and (5) EAE Animals to which MSC were Administered before and after the Induction of EAE (Group 5) (G).

Figure 3. B-Cell Viability Assesed by Annexin/7AAD Staining after 4 Days of Culture
Increased Significantly in the Presence of MSC. CD45RA Cells were Selected. B-Cell Viability Assessed by Annexin/7AAD Staining in the Absence (A to C) or Presence (D to F) of MSC in some Cases Analysed. The First Two Dot Blots are from the Healthy Donor Animal Group, the Second Two Dot Blots are from the EAE Animal Group, and the Third
Two Dot Blots are from the EAE Animals to which MSC were Administered before and after the Induction of EAE. G: Annexin/7ADD-Positive Cells for Samples Cultured with Violet Column) or without (Maroon Column) MSC: (1) Group 1; (2) Group 2; (3) Group 3; (4) Group 4; and (5) Group 5 of Experimental Animals.

Figure 4: Cell Cycle of T-Cells Cultured in the Absence of MSC (A-B) or in the Presence of MSC (C-D). The First Two Dot Blots are from Group 1 of the Experimental Animals, and the Next Dot Blots
are from Group 5 of the Experimental Animals. No Changes in the T-Cell Cycle were Observed in any Case.

Figure 5: The same as in Figure 4 but Referring to the B-Cell Cycle.

In order to analyse the production of soluble cytokines, the production of T-cell cytokines with or without MSC was studied. In the cases studied, MSC modulated the production of soluble cytokines in a different way. In control animals, MSC maintained or decreased the production of soluble IFN-γ, whereas in the EAE animals they increased its production (Figure 6). The opposite occurred with the production of IL-4 (Figure 7). Healthy T-lymphocytes secreted more IL-4 with MSC in the culture than EAE T-lymphocytes (Figure 8).

Figure 6: Percentage of IFN-g (Blue E)-Positive T-Cells after 3 Days of Culture without (A-C) MSC or with (D-F) MSC. Group 1 of the Experimental Animals (A, D), Group 3 of the Experimental Animals (B, E), and Group 5 of the Experimental Animals (C, F).

Figure 7: Percentage of IL-4 (Blue Events)-Positive T-Cells after 3 Days of Culture without (A-C) MSC or with (D-F) MSC. Purified CD3-Positive Cells
were Analysed. The First Two Blots are from Group 1 of the Experimental Animals, the Second Two Blots are from Group 3 of the Experimental Animals, and the Third Two Blots are from Group 5 of the Experimental Animals.

Figure 8: Increase in Positive Cells After the Culture of T-lymphocytes in the Absence of MSC (Violet Column) or in Presence of MSC for IFN-γ (Maroon Column) or IL-4 (Yellow Column), for the Different Cases Analysed. (1) Group 1; (3) Group 3; (4) Group 4; and (5) Group 5 of Experimental Animals.

MSC modulated CD27/CD86 expression in B-cells. MSC increased the percentage of CD27-positive cells in healthy donors, whereas MSC significantly decreased CD27 expression in animals in which the clinical score was decreased due to MSC infusion (Figure 9). The same occurred with CD86 expression (Table 2).

Figure 9: Analysis of CD27/CD86-Positive B-Cells in the Absence of MSC (A-C) or in the Presence
of MSC (D-F) for 3 Days of Culture. The First Two Dot Blots are from Healthy Donor; the Next Ones
are from EAE Experimental Animals and the Last Ones are from EAE Animal to which MSC were
Administered before and after the Induction of EAE. G: Increase in Positive Cells after Culture of B Lymphocytes in the Absence of MSC (Violet Column) or in the Presence of MSC (Maroon Column) for CD27/CD86 Expression: (1) Group 1; (2) Group 2; (3) Group 3; (4) Group 4; and (5) Group 5 of
the Experimental Animals.

Clinical Evaluation of EAE

Non-EAE immunized animals (group 1) showed a clinical score of 0. However, EAE-induced animals (groups 2-5) developed a mean clinical score of 2.2, with a standard deviation of 0.7 (the animals that did not develop the disease were not considered in the statistical data: one animal from group 3 and another animal from group 5). It is noteworthy that the animals infused with MSC prior to EAE immunization either failed to develop EAE or their EAE clinical scores were reduced. In contrast, the animals that received MSC after the induction of EAE developed a normal course of EAE.

In sum, rat MSC expressed the vascular cell adhesion molecule CD106 to a slight extent. MSC promoted T- or B-lymphocyte survival but did not modify the T- or B-lymphocyte cell cycles in the quiescent state. The results also confirmed that MSC modulated EAE though the production of soluble cytokines. In vitro, MSC decreased the EAE by immunomodulating the Th1/Th2 response. Moreover, MSC controlled CD27/CD86 expression in different ways. Animals infused with MSC prior to the EAE immunization did not develop EAE, or their EAE clinical scores were decreased, whereas animals that received MSC after the induction of EAE developed a normal EAE course.

DISCUSSION

MSC belong to a more recent field in the range of experimental therapies currently being developed to treat MS. While interest in the use of MSC was originally due to their potential capacity to differentiate into different cell lineages, recent work demonstrating their interesting immunological properties has led to a revised concept, envisaging their use for immunomodulatory purposes. These properties have already been exploited in the clinical setting for the treatment of severe autoimmune diseases. It has been reported that MSC inhibit monocyte-derived dendritic cell maturation as well as T-lymphocyte proliferation.^52,53,54,55 MSC have been reported to inhibit the proliferation of T-cells through an MHC-independent mechanism,⁵⁶ leading to the arrest of cell division.⁵³ Although, the mechanisms involved in this inhibition of T-cell proliferation are still poorly understood, a veto-like activity has been reported,⁵⁷ and a role for soluble molecules—including prostaglandin E2,⁵⁴ transforming growth factor,⁵⁸ and indoleamine 2,3-dioxygenase⁵⁹—has been proposed. It has been reported that MSC increase the survival of unstimulated T-cells and inhibit the proliferation of activated T-cells.⁶⁰ This is in accordance with previous studies indicating that MSC arrest T-cells in the G1 phase of the cell cycle⁵³ and that this effect is mediated by the inhibition of cyclin D2 and the upregulation of p27kip1.

It is known that MSC inhibit B-cell proliferation and differentiation⁶¹; that MSC may induce both the expansion and differentiation of B-cells stimulated with an agonist of the Toll-like receptor (TLR) 9 in the absence of B cell receptor triggering⁶²; and that MSC increase antibody secretion by human B-cells stimulated with lipopolysaccharide, cytomegalovirus, or varicellazoster virus.⁶³ More recently, it has been described that MSC promote the survival and inhibit the proliferation and maturation of B-cells.⁶⁴ These effects are mediated through the activation of MAPK pathways such as pErk 1/2 and p38. Here, it has been shown that MSC play a critical role in the immune regulation in EAE. Thus, we provide evidence that MSC given to Lewis 1A rats can, in vitro, support cell survival when T- or B-cells are in a quiescent state, leading to cell apoptosis. First, we observed that T- or B-cells cultured under non-stimulated conditions, a cell population physiologically prone to spontaneous cell death, are significantly rescued from apoptosis by the presence of MSC. Other authors have suggested that the protective effect of MSC mainly targets the “death receptor” pathway of apoptosis, through the downregulation of the Fas receptor and the Fas ligand on arrested T-cells.⁶⁰ In this sense, and in the same manner, the increased viability induced by MSC in resting B-cells could explain the higher production of IgG upon weak stimulation, as shown in previous studies.^63,64 However, we analysed the effect of MSC on T- or B-cell cycles and, contrary to the results showing that MSC can hamper T-cell proliferation through the inhibition of cell division and subsequent accumulation of cells in the G0 phase of the cell cycle,⁶⁵ we did not find any difference between T- or B-cells cultured with or without MSC. This is also in part due to the absence of stimulating conditions and the low T- or B-cell proliferation capacity in the quiescent state.

However, there is a marked increase in the expression of TLR in MS brain lesions and cerebrospinal fluid mononuclear cells as well as in EAE brain lesions.^66,67 TLR3 signals cause Th1 polarization with increased IFN-γ secretion concomitant with increased CD4 T-cell death.⁶⁸ TLR signals are therefore potent modulators of microglial activation programs. The MSC-induced improvement was accompanied by changes in neural cell responses, with increased oligodendrocytes and decreased astrocytes in lesioned areas as well as changes in spleen cell responses.³² In active EAE, the predominant response is mediated through Th1 pro-inflammatory cells and the expression of their associated cytokines. In animals that received human BM-MSC, there was a significant reduction in pro-inflammatory cytokines, including IL-17, IFN-γ, IL-2, IL-12p70, and TNF-α, and a significant increase in anti-inflammatory cytokines, including IL-4 and IL-5. Moreover, it has been reported that MSC can suppress the T-cell proliferative response against TCR-dependent and -independent polyclonal stimuli.⁴³ Such an effect was paralleled by a significant suppression of IFN-γ and TNF-α production by activated T-cells, supporting the notion of a profound inhibition of the Th1 response by MSC. Compatible with this, we observed that MSC regulated the balance of T lymphocytes between Th1/Th2 and modified the cytokines released during EAE. Thus, the presence of MSC increased the production of IFN-γ by T-cells in the EAE animals. However, this increase was not seen in the healthy animals or in the EAE animals belonging to experimental group 5 (animals to which MSC were administered before and after the induction of the EAE). Moreover, the production of IL-4 increased in all cases analysed. The most important aspect was the increase in the production of soluble IL-4 by T-cells in the EAE animals of group 5. This means that MS is a T-cell-mediated autoimmune disease, involving inflammatory demyelination of the CNS by CD4+ T-cells specific for myelin oligodendrocyte glycoprotein and other CNS autoantigens.⁶⁹

In recent years, B-cells have emerged as a novel therapeutic target for treating MS, and clinical data with rituximab, as a B cell-depleting monoclonal antibody-based therapy, provide reciprocal conceptual support for a prominent role of B-cells in the pathogenesis of MS.⁷⁰ Memory B-cells are significantly different from naïve B-cells, and the production of different effector cytokine profiles is a fundamental characteristic distinguishing naïve (CD27−) from memory (CD27+) human B-cells.⁷¹ Memory and naïve B-cells are considered to play different roles in immune regulation. However, the roles of memory and naïve B-cell subsets in MS have not yet been elucidated. Moreover, CD80 and CD86 are major costimulatory signals for T-cell activation, and variations in the expression of these proteins are likely to influence immune regulation in MS.⁷² Here, we examined the expression of CD86 and CD27 in B-cells by flow cytometry. In this regard, a high expression of CD27/CD86 was observed in the EAE animals of experimental group 5 versus healthy animals. In culture, this increment decreased in the presence of MSC. In the present study, we observed that subsets of memory and naïve B-cells differ between EAE animals and healthy control animals, and these differences could be exploited in the search for targets in MS therapies. Taken together, these results suggest that naïve and memory B-cell subsets play different active roles in the regulation of normal immune responses and indicate that abnormalities in these functions may contribute to MS. Moreover, the effect of MSC on B-cells antigen expression suggested that MSC could modulate the disease, which partly depended on the day on which the MSC were injected.

Finally, the clinical course of EAE was significantly ameliorated in the animals treated with MSC (intravenous administration). In this sense, it seems that the beneficial effects of MSC only appear if these cells are infused prior to the induction of EAE.

ACKNOWLEDGEMENTS

This work has been supported by Gemacbio (Saint Jean d’Illac, France), the Institut pour le Developpement de la Recherche en Pathologie Humaine et Therapeutique (IDRPHT, Talence, France), Red de Terapia Celular de Castilla y León (Spain), Consejería de Educación (Junta de Castilla y León, Spain) (SA099A08) and the INCYL-Federación de Cajas de Ahorro de Castilla y León (Spain). Arturo Mangas was supported by the Red de Terapia Celular de Castilla y León (Spain). The authors wish to thank to Professor Javier Yajeya (University of Salamanca, Spain) for his help in the studies carried out on the sciatic nerve motor conduction velocity. The authors also wish to thank Nicholas Skinner (University of Salamanca, Spain) for supervising the English text.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

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