Human Mesenchymal Stem Cell Secretome Driven T Cell Immunomodulation Is IL-10 Dependent

The Human Mesenchymal Stem Cell (hMSC) secretome has pleiotropic effects underpinning its therapeutic potential. hMSC serum-free conditioned media (SFCM) contains a variety of cytokines, with previous studies linking a changed secretome composition to physoxia. The Jurkat T cell model allowed the efficacy of SFCM vs. serum-free media (SFM) in the suppression of immunological aspects, including proliferation and polarisation, to be explored. Cell growth in SFM was higher [(21% O2 = 5.3 × 105 ± 1.8 × 104 cells/mL) and (2% O2 = 5.1 × 105 ± 3.0 × 104 cells/mL)], compared to SFCM [(21% O2 = 2.4 × 105 ± 2.5 × 104 cells/mL) and (2% O2 = 2.2 × 105 ± 5.8 × 103 cells/mL)]. SFM supported IL-2 release following activation [(21% O2 = 5305 ± 211 pg/mL) and (2% O2 = 5347 ± 327 pg/mL)] whereas SFCM suppressed IL-2 secretion [(21% O2 = 2461 ± 178 pg/mL) and (2% O2 = 1625 ± 159 pg/mL)]. Anti-inflammatory cytokines, namely IL-4, IL-10, and IL-13, which we previously confirmed as components of hMSC SFCM, were tested. IL-10 neutralisation in SFCM restored proliferation in both oxygen environments (SFM/SFCM+antiIL−10 ~1-fold increase). Conversely, IL-4/IL-13 neutralisation showed no proliferation restoration [(SFM/SFM+antiIL−4 ~2-fold decrease), and (SFM/SFCM+antiIL−13 ~2-fold decrease)]. Present findings indicate IL-10 played an immunosuppressive role by reducing IL-2 secretion. Identification of immunosuppressive components of the hMSC secretome and a mechanistic understanding of their action allow for the advancement and refinement of potential future cell-free therapies.


Introduction
Human Mesenchymal Stem Cells (hMSCs) are a promising therapeutic tool in regenerative medicine with the potential to treat a number of diseases and disorders [1,2]. However, the precise mechanisms of action remain unclear, though they are likely related to all or a combination of the following: multipotent differentiation, functional incorporation, immunomodulation, and secretion of paracrine factors [3,4]. hMSCs are of particular interest as a therapeutic tool for inflammatory diseases through their ability to suppress T cell proliferation [1,3,[5][6][7]. The suppression has been shown to be a broad spectrum and involving mitogens, peptide antigens, alloantigen-induced T cell proliferation as well as Cluster of Differentiation 3 (CD3)/CD28 antibody mediated T cell activation [8][9][10]. Proteomic profiling of serum-free conditioned media (SFCM) from human MSCs (hMSCs) revealed the presence of a range of pleiotropic biomolecules within the secretome including the vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GMCSF), and members of the interleukin (IL) family [11][12][13][14][15]. Additionally, MSCs suppress pharmacological activation of intracellular pathways of T cells, confirming that the mechanism of inhibition is a non-T-cell receptor-based pathway [16]. The suppression involves different T cell subtypes including both CD4+ and CD8+ as well as naïve T cells [4,17,18]. MSC-mediated immunosuppression was demonstrated by direct means

IL-10 Suppresses the Proliferation of Polarised Jurkat T Cells
Cell counts following activation revealed similar patterns of proliferation suppression in both SFCM and SFM +ligand (IL-4, IL-13, and IL-10) compared to SFM and SFM +Ligand+anti-Ligand (IL-4, IL-13, and IL-10) with a slight restoration (~1 fold) achieved with the neutralisation of either anti-inflammatory cytokines ( Figure 4A,C,E). Similar patterns in the reduction in MTT values were noted in both SFCM or SFM +Ligand cells compared to SFM or SFM +Ligand+anti-Ligand with no restoration achieved following neutralisation of either anti-inflammatory cytokine ( Figure 4B,D,F). To identify specific biomolecule(s) responsible for this phenotype in SFCM, cytokines with prominent anti-inflammatory properties, which we previously confirmed as being components of SFCM, were tested, including IL-4, IL-10, or IL-13 [15].  To confirm the biomolecule(s) responsible for the suppression of proliferation in polarised Jurkat T cells exposed to SFCM further, these cytokines were again individually blocked by their specific polyclonal antibodies. IL-10 neutralisation in SFCM induced restoration of proliferation in both air and physoxia environments, day 4 (SFM/SFCM +anti-IL−10 ; ~1-fold increase) ( Figure 4E). Conversely, IL-4/IL-13 neutralisation was associated with no restoration of proliferation in either normoxia or physoxia environments, day 4 [(SFM/SFCM +anti-IL−4 ; ~2-fold decrease) and (SFM/SFCM +anti-IL−13 ; ~2-fold decrease)] ( Figure  5). MTT assays showed that polarised Jurkat T cells displayed a reduced metabolism compared to non-polarised cells. Similar patterns of the suppression of metabolism were noted in both SFCM or SFM +Ligand cells compared to SFM or SFM +Ligand+antiLigand with no restoration (~1 fold) achieved following neutralisation of either anti-inflammatory cytokines. Taken together, this suggests that IL-10 in SFCM is associated with the suppression of proliferation in polarised Jurkat T cells and occurs irrespective of IL-4 and IL-13 activity. To confirm the biomolecule(s) responsible for the suppression of proliferation in polarised Jurkat T cells exposed to SFCM further, these cytokines were again individually blocked by their specific polyclonal antibodies. IL-10 neutralisation in SFCM induced restoration of proliferation in both air and physoxia environments, day 4 (SFM/SFCM +anti-IL−10 ; 1-fold increase) ( Figure 4E). Conversely, IL-4/IL-13 neutralisation was associated with no restoration of proliferation in either normoxia or physoxia environments, day 4 [(SFM/SFCM +anti-IL−4 ;~2-fold decrease) and (SFM/SFCM +anti-IL−13 ;~2-fold decrease)] ( Figure 5). MTT assays showed that polarised Jurkat T cells displayed a reduced metabolism compared to non-polarised cells. Similar patterns of the suppression of metabolism were noted in both SFCM or SFM +Ligand cells compared to SFM or SFM +Ligand+antiLigand with no restoration (~1 fold) achieved following neutralisation of either anti-inflammatory cytokines. Taken together, this suggests that IL-10 in SFCM is associated with the suppression of proliferation in polarised Jurkat T cells and occurs irrespective of IL-4 and IL-13 activity.
Further, TGFb did not inhibit the proliferation of non-polarised Jurkat T cells in SFM, indicating that activation of the T cell model is mainly linked to the presence or absence of IL-10 in SFCM, day 4 (SFM/SFM +TGFb ;~1-fold decrease) ( Figure 6).

IL-10 Suppresses IL-2 Secretion
In vivo T cell stimulation occurs following antigen presentation on the surface of antigen presenting cells and is associated with IL-2 production where subsequent proliferation leads to the launching of the immune response. In contrast, in vitro polarisation of Jurkat T cells by PMA/PHA results in an abrogation of proliferation and the production/release of IL-2. We previously demonstrated that the IL-2 release from polarised Jurkat cells was equivalent in both normoxia and physoxia ( Figure 1D) and now sought to explore if SFCM impacted its release following activation. We noted the IL-2 release following PMA/PHA treatment over 7 days which reached a maximum in SFM at day 2 [21% O 2 (5305 ± 211 pg/mL) and 2% O 2 (5347 ± 327 pg/mL)] and declined thereafter reaching a minimum at day 7 [21% O 2 (258 ± 75 pg/mL) and 2% O 2 (145 ± 32 pg/mL)] (Figure 7). SFCM suppressed IL-2 secretion in polarised Jurkat T cells in both normoxia and physoxia, day 2 [21% O 2 (2461 ± 178 pg/mL) and 2% O 2 (1625 ± 159 pg/mL)]. Following our earlier observations, we next explored whether IL-10, IL-4, and IL-13 displayed a capacity to block the IL-2 release. Cytokines added individually led to a reduction in IL-2 secretion in polarised T cells in a SFCM-comparable manner in both normoxia and physoxia environments, day 2 [(SFM/SFM +IL−4 ; >2-fold reduction) ( Figure 7A ) values for MTT assays for polarised Jurkat T cells culture SFM, SFCM, SFM +Ligand , SFM +Ligand/antiLigand AB , and SFCM +antiLigand AB in air (21% O2) and physoxia O2) environments over a 7 day period. Growth curves were obtained by haemocytometer-based counting, and the results were confirmed through MTT assays. Data expressed as mean ± SD; result represents a replicate of 3 independent experiments (n = 3). One-way ANOVA was condu with Tukey's test to determine pairwise significant difference. * p < 0.001.

IL-10 Suppresses IL-2 Secretion
In vivo T cell stimulation occurs following antigen presentation on the surfac antigen presenting cells and is associated with IL-2 production where subsequ proliferation leads to the launching of the immune response. In contrast, in v polarisation of Jurkat T cells by PMA/PHA results in an abrogation of proliferation the production/release of IL-2. We previously demonstrated that the IL-2 release f polarised Jurkat cells was equivalent in both normoxia and physoxia ( Figure 1D) and sought to explore if SFCM impacted its release following activation. We noted the release following PMA/PHA treatment over 7 days which reached a maximum in SFM day 2 [21% O2 (5305 ± 211 pg/mL) and 2% O2 (5347 ± 327 pg/mL)] and declined therea reaching a minimum at day 7 [21% O2 (258 ± 75 pg/mL) and 2% O2 (145 ± 32 pg/m and physoxia (2% O 2 ) environments over a 7 day period. Growth curves were obtained by haemocytometer-based cell counting, and the results were confirmed through MTT assays. Data expressed as mean ± SD; each result represents a replicate of 3 independent experiments (n = 3). One-way ANOVA was conducted with Tukey's test to determine pairwise significant difference. * p < 0.001. polyclonal antibodies. This again resulted in a failure to reverse the blockage of IL-2 secretion, day 2 (SFM/SFCM anti-IL−4/anti-IL−13 ; ~8 fold) ( Figure 7D).
Additionally, TGFb failed to inhibit IL-2 secretion by polarised Jurkat T cells in SFM, day 2 (SFM/SFM +TGFb <2 fold), indicating that activation of the T cell was associated with IL-10 status ( Figure 7E). These results indicate that Jurkat T-cell polarisation in SFCM is linked to IL-10 ligands highlighting the importance of IL-10 post-receptor signalling pathways.

Discussion
The application of hMSCs in clinical settings is an attractive prospect [41][42][43]. Many clinical trials to date focused on immune-mediated diseases including Crohn's disease, diabetes mellitus, graft-versus-host disease (GvHD), hepatitis, and rheumatoid arthritis [8,[44][45][46][47]. However, their first pass pulmonary engraftment potential makes the mode of action questionable [48,49]. Recent in vitro and in vivo studies confirmed that the regenerative mode of action of hMSCs is partially linked to the release of trophic factors rather than their functional incorporation [50][51][52][53]. However, the released trophic factors are a mixture of biomolecules with various functions, including pro-inflammatory, anti-inflammatory, pleiotropic cytokines, chemokines, growth factors, and angiogenic factors [15,[54][55][56]. The concentration of these cytokines varies depending on the source of hMSCs, their isolation, and the culture protocol [56][57][58]. Riberio et al. (2012) compared the composition of conditioned media collected from either adipose or umbilical-cord-derived stem cells, demonstrating an altered secretome profile and consequently differential effects on metabolic viability when applied to neuronal cell cultures [56]. The present report describes the mode of action of the hMSC secretome by testing the efficacy of SFCM itself and a Additionally, TGFb failed to inhibit IL-2 secretion by polarised Jurkat T cells in SFM, day 2 (SFM/SFM +TGFb < 2 fold), indicating that activation of the T cell was associated with IL-10 status ( Figure 7E). These results indicate that Jurkat T-cell polarisation in SFCM is linked to IL-10 ligands highlighting the importance of IL-10 post-receptor signalling pathways.

Discussion
The application of hMSCs in clinical settings is an attractive prospect [41][42][43]. Many clinical trials to date focused on immune-mediated diseases including Crohn's disease, diabetes mellitus, graft-versus-host disease (GvHD), hepatitis, and rheumatoid arthritis [8,[44][45][46][47]. However, their first pass pulmonary engraftment potential makes the mode of action questionable [48,49]. Recent in vitro and in vivo studies confirmed that the regenerative mode of action of hMSCs is partially linked to the release of trophic factors rather than their functional incorporation [50][51][52][53]. However, the released trophic factors are a mixture of biomolecules with various functions, including pro-inflammatory, anti-inflammatory, pleiotropic cytokines, chemokines, growth factors, and angiogenic factors [15,[54][55][56]. The concentration of these cytokines varies depending on the source of hMSCs, their isolation, and the culture protocol [56][57][58]. Riberio et al. (2012) compared the composition of conditioned media collected from either adipose or umbilical-cord-derived stem cells, demonstrating an altered secretome profile and consequently differential effects on metabolic viability when applied to neuronal cell cultures [56]. The present report describes the mode of action of the hMSC secretome by testing the efficacy of SFCM itself and a selection of its components: IL-4, IL-10, IL-13, and TGFb anti-inflammatory cytokines on the model Jurkat T cell line.
Oxygen concentration is an important factor for both hMSC culture and in vitro immune response models. Physoxia is a characteristic feature of hMSC niches, which are under continuous oxygen gradient exposure depending on the local tissue microenvironment, such as bone marrow (1-6% O 2 ), adipose tissue (2-8% O 2 ), and neural tissues (1-8% O 2 ) [59]. The oxygen tension is higher in most endogenous tissue compartments (4-14% O 2 ) but is still less than that of ambient 21% air oxygen [33]. Additionally, the inflammation zone is under pathological hypoxia due to activation of coagulation cascades leading to the activation of immune cells [60]. In this study, SFCM was collected in both 21% O 2 and 2% O 2 environments to simulate oxygen concentrations present in the in vivo bone marrow niches and help create a physiologically relevant in vitro immune response model. Various in vitro and in vivo studies as well as our previously published studies reported the importance of 2% O 2 collected SFCM in comparison to air oxygen SFCM [15,61,62].
Exposure of Jurkat T cells to PMA/PHA in this study resulted in their activation and indicative IL-2 secretion, abrogation of proliferation, and increases in the cell surface area. This is in line with previously published reports that T cell line exposure to mitogenic stimuli results in their differentiation and suppression of proliferation [39]. Non-stimulated Jurkat T cells displayed normal proliferation passing through the lag, log, and stationary phases whilst, upon activation, the cells reached a plateau after the lag phase showing no log or stationary phases.
It was reported that the immunosuppressive activity of MSCs is mainly mediated through soluble factors [63]. However, there is lack of clarity surrounding the specific signalling biomolecules responsible for immunosuppression, making the exact mechanism of action questionable [64,65]. The secretome profile is a mixture of complex proteinbased bioactive factors, including the stem cell factor (SCF), IL-6, IL-8, IL-10, IL-12, IFNγ, prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), hepatocyte growth factor (HGF), and transforming growth factor -b1 (TGFb1) [11,55,58,66]. Di Nicola et al. confirmed that the in vitro immunosuppressive activity of MSCs could be reverted through blocking the effects of both TGFb and HGF [4]. However, neutralisation of either TGFb or HGF individually resulted in minimal restoration of the immune response, while their addition in combination achieved a comparable immunosuppression to MSCs [67]. Failure to achieve immunosuppression with TGFb alone was reported by different in vitro studies [68][69][70]. Mori et al. concluded that TGFb mediates its action synergistically with HGF through JNK-dependent Smad2/3 phosphorylation at their promotor regions [71]. In the present study, TGFb failed to induce immunosuppression, confirming that TGFb has no role in SFCM-mediated immunosuppression. Jurkat T cells were proliferative in SFM +TGFb and produced IL-2 following PMA/PHA activation, confirming that TGFb alone has no immunosuppressive properties. Interestingly, cyclosporine A, a potent immunosuppressive agent, was previously associated with elevated levels of intracellular TGFb and its receptor, suggesting that its mechanism of action is mainly linked to TGFb [72]. These studies indicate that there is a relationship between the mode of action of TGFb and HGF and could explain the failure to respond to SFM +TGFb in the present study.
Naïve T cell proliferation is regulated by IL-2 and CD25 (IL-2 receptor alpha subunit (IL-2Ra)), whilst their maturation is under the control of different cytokines, including IL-2, IL-12, and IFNγ which promote Th1 differentiation and IL-4, IL-5, IL-9, IL-10, and IL-13 which promote Th2 differentiation [73]. Hence, IL-4 and IL-13 are part of the differentiation mechanism of T cells, sharing 25% structural similarity, characterised by receptor overlapping phenomena via sharing the receptor subunit (IL-4Rα) for their signal transduction; therefore, IL-13 can induce many functional properties of IL-4 [73,74]. In the present study, the immunosuppression achieved by either IL-4 or IL-13, when individually tested on Jurkat T cells, was confirmed by the suppression of proliferation and reduction in IL-2 production. However, neutralisation of IL-4/IL-13 individually, or in combination, induced no restoration in the immunosuppressive activity of SFCM, suggesting that alternative pathways could be responsible for the immunomodulation achieved by SFCM. Dupilumab, a human monoclonal antibody for the treatment of asthmatic patients, targets both IL-4 and IL-13; the mechanism of this antibody is based on the inhibition of IL-4/IL-13 engage-ment with the α subunit of the IL-4 receptor [75]. Despite successful immunosuppression achieved by dupilumab and the failure to achieve immune response restoration following IL-4/IL-13 neutralisation in SFCM, their participation in immunosuppression is not negligible, indicated by the suppression of proliferation and revoked IL-2 secretion by Jurkat T cells. Cytokine cross-reactivity and receptor affinity are likely responsible, noting that SFCM is a mixture of different cytokines, and most cytokines which are present in SFCM, such as, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, show IL-4 receptor sharing [73].
IL-4/IL-13 share post-receptor latent proteins and the translation pathway (STAT6), and the translocation of this second messenger protein to the nucleus results in the transcription of anti-inflammatory genes [73,74]; moreover, pharmacological targeting of either cytokine alone achieved limited therapeutic activity in comparison to combined therapy [73]. However, IL-10 mediated its immunosuppressive activity through a distinct (STAT3) post-receptor translation pathway [23]. Various reports suggest that IL-10 has a unique capacity to block the synthesis of proinflammatory cytokines, including TNFa, IFNγ, IL-1B, and IL-6 [23,24]. Moreover, IL-10 post-receptor translation pathways involve the induction of more than one post-receptor translation pathway including STAT1, STAT3, and STAT5. STAT1 and STAT5 are not involved directly in IL-10 receptor stimulation; however, their knockout is associated with the modulation of the cellular response to IL-10 [23]. These results are conflicting, and clarification is required to dissect the immunosuppression activity of SFCM through IL-10 receptor blocking or JAK1 knockout rather than simple polyclonal antibody neutralisation.
The critical role of the potent anti-inflammatory cytokine IL-10 during host infection involves the modulation of both innate and adaptive immunity through the suppression of T cells, NK, and macrophages, helping to protect from the effects of excessive and prolonged inflammation [23,[76][77][78][79]. Conversely, persistent viral infection results in the upregulation of IL-10, leading to impairment of the T cell response and a lack of efficient clearance of pathogens from the host [80]. Regulation of IL-10 and the effects on T cell homeostasis are therefore critical to the proper functioning of the immune response. Previous studies show that rheumatoid arthritis patients display lower serum levels of IL-10 compared to healthy patients [81]. In addition, IL-10 secreting MSCs demonstrated enhanced cell survival and therapeutic benefits in models for Duchenne muscular dystrophy (DMD) [82]. The ability of MSCs to migrate, accumulate, and exhibit both immunosuppressant and antiapoptotic effects through the secretion of IL-10 at sites of injury makes them an attractive prospect in both cell and cell-free therapies for diseases with chronic inflammatory pathologies [82,83].

Materials and Methods
4.1. Cell Line Culture 4.1.1. hMSCs hMSCs were isolated and expanded from human bone marrow aspirate (BMA) using an adherence-based methodology [35]. Three donor human BMAs (two male and one female, ages 20-36) were purchased from Lonza, USA and seeded at a density of 1 × 10 5 mononuclear cells/cm 2 on fibronectin pre-coated culture flasks in Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 5% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine, 1% (v/v) non-essential amino acids (NEAA), and 1% (v/v) Penicillin-Streptomycin-Amphotericin B (PSA) (Lonza, Slough, UK). Seeded flasks were incubated in humidified incubators with distinct oxygen tensions of 21% O 2 or 2% O 2 . After 7 days, half of the media volume was removed and replaced with fresh antibiotic-free growth medium followed by a complete media change after a further 7 days. Media was then changed every 3 days until confluent. Once confluent, hMSC were enzymatically passaged with 1% Trypsin/EDTA (Lonza, Slough, UK) at 1:2 split ratios. Passage 1 cells and their CM were used for all experiments.
SFCM was produced by washing 70% confluent hMSC seeded T75 flasks with phosphatebuffered saline (PBS) followed by the addition of 15 mL serum-free media (SFM) consisting of DMEM supplemented with 1% L-glutamine (v/v) and 1% NEAA (v/v). For conditioning, 12 of 17 20 mL of SFM were added to hMSC cultures and incubated for 24 h. Conditioned media was then collected, centrifuged for 10 min at 300× g, and stored at −80 • C as SFCM. Prior to use, SFCM was thawed and filtered (0.2 µm). All SFCM was produced from hMSC cultures at passage 1. Time taken for hMSC isolation from BMA plating, expansion, passaging, and confluency at passage 1 was 28-29 days (28 days for donors 1 and 2, 29 days for donor 3) and was consistent between the oxygen concentrations.
Positive expression for CD73, CD90, and CD105 and negligible expression for CD14, CD19, CD34, CD45, and HLA-DR surface molecules were confirmed, as well as trilineage differentiation potential according to ISCT guidelines, and were published previously [15].

Jurkat T Cells
The Jurkat cell line (ATCC clone E6-1) was cultured in suspension in tissue culture flasks. Cells were passaged by centrifuging at 180× g for 3 min; the supernatant was removed and the cell pellet re-suspended in fresh growth media (GM) consisting of Roswell Park Memorial Institute (RPMI)-1640 (Lonza, Slough UK) supplemented with 10% (v/v) FBS, 1% L-glutamine, and 1% NEAA. Cells were passaged and seeded at a density of 1 × 10 5 cells/mL with media changes performed twice per week. Seeded flasks were incubated in humidified incubators with distinct oxygen tensions of either 21% O 2 or 2% O 2 .

Jurkat Cell Activation
Jurkat cells were seeded at 5 × 10 5 cells/mL for activation through the addition of 50 ng of Phorbol Myristate Acetate (PMA) (Sigma-Aldrich, Gillingham, UK) and 1 µg of Phytohaemaglutinin (PHA) (Sigma-Aldrich, Gillingham, UK) to the cell culture media. The activation was performed for a period of 24 h in a humidified incubator at 37 • C at either 21% O 2 or 2% O 2 .

Cell Counting
Haemocytometer cell counts were performed on time-point samples over 7 days to determine the rate of cell growth from an initial seeding density of 2 × 10 5 cells/mL. Cell counts were performed on the cells in different conditions, including, activated and inactivated cells in their GM, SFM, and SFCM, and under different oxygen tensions, 2% O 2 and 21% O 2 . Counts were performed on triplicate flasks.

MTT Assay
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay (Sigma-Aldrich, Gillingham, UK) was used to determine metabolic activity. Jurkat cells were seeded in triplicate at a concentration of 2 × 10 5 cells/mL before incubation in MTT for 4 h at 37 • C as per manufacturer's instructions. A total of 50 µL of DMSO were added to each well to dissolve formazan crystals, and the plate was incubated again for 45 min. The optical density (O.D.) of each sample was determined by reading at 570 nm on a Synergy2 plate reader (BioTek, Cheadle, UK).

Cytospin
The Cytospin technique was used to assess Jurkat cell morphology. To prepare Cytospin slides for imaging, 5 × 10 4 cells were centrifuged and the supernatant removed, and pellets were washed twice in cold PBS and resuspended in 1 mL PBS. The cells were then pipetted into the Cytofunnel (Fisher Scientific, Loughborough, UK) and centrifuged by Cytospin-centrifuge (Fisher Scientific, Loughborough, UK) at 300× g for 2 min. Slides were then removed and air dried for 15 min and fixed with 95% ethanol for 15 min. Following air drying, the slides were stained with May-Grünwald solution (Sigma-Aldrich, Gillingham, UK) for 5 min, washed, and further staining with Giemsa solution (Sigma-Aldrich, Gillingham, UK) for 15 min. A xylene mounting agent was placed over the slides to fix the cover slides. Images were captured, and the cell surface area was calculated for 100 cells using ImageJ software (NIH, Maryland, MD, USA).

ELISA Assay
ELISA assays were conducted for the detection of IL-2 (PeproTech, London, UK #900-K12), IL-4 (PeproTech, London, UK #900-M14), and IL-10 (PeproTech, London, UK #900-M21). Standard serial dilutions and culture media samples were loaded in triplicate into an overnight pre-coated surface with cytokine specific capture antibodies. Blocking was carried out for 1 h using 1% BSA-blocking buffer, followed by a 2 h incubation with a diluted detection antibody mixture and 30 min with a diluted avidin-HRP conjugate. Each step was accompanied by forcibly discarding the contents and four washing steps with diluted detergent buffer. Finally, an enzymatic reaction initiated by the addition of an ABTS-substrate (2,2 -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), leading to a bluish-green colour development within 5-15 min during which a visible signal was detected at 405 nm via a plate reader. The concentrations of unknown samples were determined by the interpolation of the standard calibration curves for each component.

Statistical Analysis
Statistical analysis was conducted between SFCM and SFM using a two-sample t-test for most of the measured parameters. For comparison of more than 2 groups, a one-way ANOVA with Tukey's multiple comparisons test was performed to determine pair-wise statistical significance; p ≤ 0.05 was considered significant. The analysis was performed using GraphPad Prism 6 (San Diego, CA, USA). Unless otherwise stated, all values quoted in the results are mean ± standard deviation (SD).

Conclusions
Collectively, the present findings support the suggestion that IL-10 in hMSC SFCM plays an immunosuppressive role in Jurkat T cell proliferation and activation. The specificity of IL-10 in this role was confirmed through the inability of IL-4 and IL-13 neutralisation in SFCM to restore Jurkat cell proliferation. Further, it was demonstrated that TGFb has no role in SFCM-mediated immunosuppression. The identification of specific immunosuppressive components of the hMSC secretome and the development of a mechanistic understanding of their action pave the way for the development of more controlled cell-free therapies in the future, expanding the treatment options available for patients suffering from diseases with an inflammatory component. Funding: Financial support was provided by the Ministry of Higher Education and Scientific Research, Iraq (S1443) and the Guy Hilton Asthma Trust.