Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro
© Nazarov et al.; licensee BioMed Central Ltd. 2013
Received: 23 April 2013
Accepted: 9 October 2013
Published: 22 October 2013
Mesenchymal stem cells (MSCs) have immunosuppressive activity. They do not induce allospecific T cell responses, making them promising tools for reducing the severity of graft versus host disease (GVHD) as well as treating various immune diseases. Currently, there is a need in the MSC field to develop a robust in vitro bioassay which can characterize the immunosuppressive function of MSCs.
Murine clonal CD4 and CD8 T cells were stimulated with cognate peptide antigen and antigen presenting cells (APCs) in the absence or presence of human MSCs, different aspects of T cell activation were monitored and analyzed using flow cytometery, real time RT-PCR and cytokine measurement.
Human MSCs (hMSCs) can alter multiple aspects of murine T cell activation induced by stimulation with specific antigen, including: reduced proliferation, inhibited or stimulated cell surface marker expression (CD25, CD69, CD44 and CD62L), inhibited mRNA expression of transcription factors (T-bet and GATA-3) and decreased cytokine expression (interferon-gamma, interleukin-10). Disappearance of activation-induced cluster formation and decreased apoptosis of CD8 T cells were also observed. Moreover, the effects are specific to MSCs; incubating the T cells with non-MSC control cell lines had no effect on T cell proliferation and activation.
Clonal murine T cells can be used to measure, characterize, and quantify the in vitro immunosuppressive activity of human MSCs, representing a promising approach to improve bioassays for immunosuppression.
Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are found in virtually all tissues and function as precursors of non-hematopoietic connective tissues with the capacity to differentiate into mesenchymal and non-mesenchymal cell lineages. They are the precursors of three main cell types of the mesodermal lineage, including osteocytes, chondrocytes and adipocytes [1–3]. These cells are commonly described as positive for CD73, CD105 and CD90 and negative for hematopoietic (CD45) and vascular (CD31) markers . Their properties have been extensively studied in recent years. Since MSCs are capable of differentiating into several cell lineages , they have been used in investigational studies to treat a variety of tissue injuries both in experimental and clinical settings [6–8].
An interesting aspect of MSCs is the finding that they exert immunoregulatory activities. MSCs from various species (human, rodents and primates) can suppress the T cell response to mitogenic and polyclonal stimuli [9, 10] and to specific peptide antigens . MSCs have a similar effect on both memory and naïve T cells , as well as both CD4+ and CD8+ subsets . The immunosuppressive effects of MSCs make them attractive candidates for a variety of cellular therapies, including treatment of immune disorders.
MSCs express low levels of MHC I and do not express MHC II or co-stimulatory molecules; they are, therefore, considered to be immune privileged cells and can be successfully transplanted across allogeneic barriers . In addition, large amounts of MSCs can potentially be generated from healthy donors. These unique properties have promoted wide application of MSCs in clinical trials to treat various immune diseases, including multiple sclerosis, Crohn’s disease, type 1 diabetes, systemic lupus erythematosus (SLE) and acute and chronic graft versus host disease (GVHD) [15, 16]. Mouse models have been used to test the efficacy for the treatment of GVHD, neurological and systemic autoimmune diseases, sepsis, and acute renal and lung injury, as well as other pathological conditions .
Due to the low frequency of MSCs in the bone marrow and the potential for allogeneic therapy, MSCs need to be extensively expanded and passaged to obtain sufficient cell numbers for cell therapies. Therefore, there is a need to understand the role of cell expansion, cell passaging, and donor differences on MSC immunosuppressive capacity. Currently, there are no robust quantitative bioassays suitable for measuring differences in immune-inhibitory activity of MSCs from different donors or at different passages, or under different conditions in large-scale tissue culture expansion. There is a related scientific need to identify the molecular mechanisms underlying MSC-mediated immunosuppression, which also requires accurate assays to measure the immunosuppressive activity of MSCs. Such methods could potentially be used to assess MSCs preparations from various donors and expansion methods or to predict MSC behavior after transplantation.
To address these issues, we developed novel immune inhibition assays using clonal murine T cell populations responding to known peptide antigens, and MSCs derived from human donors. MSCs are known to be immunosuppressive across xenogeneic barriers [18, 19], allowing us to assess the use of easily obtained clonal murine T-cells as a method to reduce variability in T-cell based in vitro immune suppression assays. Using this system we assessed the immunosuppressive activity of human bone marrow-derived MSCs (hMSCs) on antigen specific, clonal murine T cells. In our system, hMSCs clearly show dose-dependent inhibitory properties, affecting both the proliferation and the activation of antigen specific T cells. We also were able to use this system to investigate some of the molecular mechanisms that participate in cross-species immunosuppression, which may potentially shed light on allogeneic immunosuppressive activities of hMSCs.
All animal protocols and procedures were approved by the Institutional Animal Care and Use Committees at the Center for Biologics Evaluation and Research (CBER; Protocol #2011-15) and in animal facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experiments were performed according to institutional guidelines.
hMSCs were purchased (AllCells, Emeryville, CA, USA) at passage one. According to the manufacturer’s and our characterizations, MSCs were negative for hemaotopoietic lineage markers, including CD45, CD34, CD14, CD79α, CD117 and HLA-DR. MSCs were plated in T175 flasks (Greiner Bio-One, Monroe, NC, USA) at 60 cells/cm2, expanded in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 16.5% fetal bovine serum (FBS) (JMBioscience, San Diego, CA, USA), Pen Strep and L-glutamine (Invitrogen), and cultured at 37°C and 5% CO2. At 80% confluence, 0.25% Trypsin/EDTA (Invitrogen) was used to harvest MSCs. Cells were washed, then cryopreserved in freezing medium containing 5% Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, SA), 30% FBS, 1% penicillin (100 units/ml) and streptomycin (100 μg/ml) (Invitrogen) at 1 × 106 cells/ml at passage three (P3). hMSCs at P3 were thawed, cultured to 80% confluence and harvested for experiments.
The HT-1080 fibrosarcoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was plated in T175 flasks, expanded in RPMI-1640 medium supplemented with 10% FBS, Pen Strep and L-glutamine, and cultured at 37°C and 5% CO2. Cells at P3 were harvested with Trypsin/EDTA and used in experiments.
Flow cytometry analysis
hMSCs were incubated with 2.4G2 antibody at 4°C for 30 minutes to block non-specific binding. Primary antibodies used were purchased conjugated to their respective fluorochromes (phycoerythrin (PE); allophycocyanin (APC); fluorescein isothiocyanate (FITC)): anti-CD29-APC, anti-CD44-APC, anti-CD90-FITC, anti-CD73-PE (BD Biosciences, San Jose, CA, USA), anti-CD166-FITC (US Biological, Salem, MA, USA), anti-CD105-PE (Beckman Coulter, Brea, CA, USA) and anti-STRO-1-Alexa647 (Biolegend, San Diego, CA, USA). MSCs were also analyzed for negative markers, including anti-CD34-PE, anti-CD45-PE-Cy7, anti-CD14-ECD, anti-CD79α-PE-Cy5, anti-CD117-APC (Beckman Coulter) and anti-HLA-DR-FITC (BD Biosciences). hMSCs were incubated with antibody at optimized concentrations for 30 minutes at 4°C. Samples were centrifuged, washed twice with phosphate-buffered saline (PBS)/1% fetal bovine serum/0.2% sodium azide, and analyzed in single color using FACSCalibur (Becton Dickinson) or FACS Canto flow cytometers. For flow cytometry analysis of T cells, anti-CD4, anti-CD8, anti-T cell receptor (TCR) Vβ4, anti-TCR Vβ8.1/8.2, anti-CD25, anti-CD69, anti-CD62L, anti-CD44 in various fluorochrome combinations were purchased from BD Biosciences. For all surface marker analysis, TCR transgenic CD4+ T cells (BDC2.5 T cells) were first gated using CD4 and TCR Vβ4 double staining, while CD8+ TCR transgenic CD8 T cells (8.3 T cells) were gated using CD8 and TCR Vβ8.1/8.2 double staining. Annexin V/7 AAD apoptosis detection kit was purchased from BD Biosciences. CFSE (carboxyfluorescein diacetate, succinimidyl ester) was purchased from Invitrogen (Carlsbad, CA) and was used according to the manufacturer’s instructions.
NOD/ShiLtj mice, Nonobese diabetic 8.3 TCR transgenic mice (NOD 8.3) and Nonobese diabetic BDC2.5 TCR transgenic mice (NOD BDC2.5) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were maintained in specific pathogen-free conditions according to the guidelines of CBER’s Institutional Animal Care and Use Committee (IACUC). The BDC2.5 TCR transgenic CD4+ T cells specifically recognize the I-Ag7 restricted epitope derived from an islet antigen , and the 8.3 TCR transgenic CD8+ T cells specifically recognize the Kd-restricted IGRP206-214 epitope derived from the islet antigen IGRP (islet-specific glucose-6-phosphatase catalytic subunit-related protein) . These studies were approved by the IACUC of CBER.
Spleens and lymph nodes were isolated from NOD 8.3 and NOD BDC 2.5 TCR transgenic mice, then CD4 and CD8 T cells were negatively selected and purified using the mouse CD4+ T cell isolation kit and CD8+ T cell isolation kit, respectively (Miltenyi Biotec, Auburn, CA, USA). T cells were added to 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) (2 × 106 cells/well). Total splenocytes from NOD/ShiLtj mice were irradiated at 4,000 rads and added to the culture as antigen-presenting cells (APCs) (4 × 106 cells/well). In all the experiments APCs are irradiated. Islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP206-214, VYLKTNVFL) and BDC 2.5 peptides (RVRPLWVRME) were synthesized by the FDA FBR (Facility for Biotechnology Resources) core facility. Peptides were added to a concentration of 1 μg/ml per well. Next, human MSCs were trypsinized, washed and added to the wells at different T cell: MSC ratios. Ratios of 10:1 and 5:1 were found to be effective for our conditions and were used in all experiments. Cells were kept in RPMI 1640 complete medium (containing 10% FBS) in a 37°C incubator for three days after which murine T cells were harvested and analyzed.
For the immunosuppression assay using a transwell setup, hMSCs were cultured on the top level of the HTS Transwell®-24 Well plate with 0.4 μm pores (Corning, Lowell, MA USA) and the T cells together with the irradiated APCs and peptide were cultured in the bottom wells in the same ratios as described above. The cells were grown for three days at 37°C after which they were harvested and analyzed.
Supernatants were collected at Day 3 of cell culture and stored at -80°C for further analysis. Cytokine concentration was measured using the Mouse Th1/Th2 6-plex Panel kit from Invitrogen according to the manufacturer’s instructions. Samples were acquired and analyzed using a Bio Plex 200 instrument (BioRad, Hercules, CA, USA).
Real time RT-PCR
Total RNA was extracted from suspension T cells using Pure Link Micro-to Midi RNA extraction kit (Invitrogen), quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Asheville, NC, USA) and stored at -80°C for further analysis. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the RIN (RNA Integrity Number) values were all greater than 9.20. Taqman RT-PCR probes for murine transcription factors T-bet and GATA-3 were purchased (from customized probes) from Applied Biosystems (Foster City, CA). A total of 200 ng of RNA was reverse transcribed into cDNA using a High capacity cDNA Reverse Transcription kit from Applied Biosystems. cDNA was specifically amplified using the ABI 7900 instrument from Applied Biosystems. 18S rRNA was used as endogenous control in all samples. Results were analyzed using SDS 2.3 software (Applied Biosystems).
Data were analyzed using GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA), and Student’s t-test was used to compare differences between samples and groups. The differences were considered statistically significant when P-value was below 0.05.
Human mesenchymal stem cells show typical cellular and functional phenotype
The hMSC line used in this study, PCBM 1632, demonstrated tri-lineage differentiation toward adipo-, osteo- and chondro-genic lineages when using standard induction protocols (Miltenyi-Biotech) (data not shown). The MSC line expressed markers typical of hMSCs . Passage 3 hMSCs show high positive expression for MSC surface markers: CD29 (93.6%), CD44 (98.4%), CD166 (98.3%), CD90 (93%), CD73 (99.6%) and CD105 (98.6%). A small subset of hMSCs (16.9%) was positive for STRO-1, a marker thought to be associated with a clonogenic and more immunosuppressive subpopulation of MSCs .
hMSCs inhibit the proliferation of murine T cells
hMSCs affect the expression of different murine T cell activation markers
For CD62L we observed a differential effect of hMSCs on CD4+ T cells vs. CD8+ T cells. In both CD4+ T cells and CD8+ T cells with no hMSCs present, the expression of CD62L was up-regulated upon stimulation with cognate peptides. Interestingly, in CD4+ T cells the expression of CD62L is more homogeneous, whereas in CD8+ T cells we observed two peaks in the expression pattern, corresponding to a low-expressing population and a high-expressing population. The hMSCs down-regulated the expression of CD62L in CD4+ T cells (especially at the 5:1 ratio), as indicated by MFI; but in the CD8+ T cells, hMSCs co-incubation lead to an increase in the “high-expression” peak and a slight increase of MFI.
hMSCs can affect the activation of murine T cells stimulated with anti-CD3/anti-CD28 beads
Also, we have checked the impact of hMSCs on surface markers of already activated murine CD8+ T cells (8.3 T cells). As shown in Additional file 1: Figure S1, the hMSCs do not have an inhibitory effect on surface markers of activated T cells.
CD8+ T cell apoptosis is affected by hMSCs
Only a fraction of the CD4+ T cell population stained positive for Annexin V after being stimulated with a specific peptide and APCs (16%). Co-culture with hMSCs at both 10:1 and 5:1 ratios slightly increased the number of annexin V positive cells (21.7% to 25%), supporting the idea that hMSCs only marginally affect the viability of CD4+ T cells.
Conversely, stimulated CD8+ T cells showed a significant number of apoptotic cells (82.7%) after three days in culture. Incubation with hMSCs led to a decreased number of apoptotic cells (42.2%, 30%), suggesting that hMSCs seem to contribute to a better survival of these CD8+ T cells in culture. The effect seems to be dose dependent, with the ratio of 5:1 showing a more significant effect on the T cells.
hMSCs decrease the level of mRNA expression of two important transcription factors
hMSCs affect the secretion of cytokines by CD4+ and CD8+ T cells
Since Treg and Th17 cells are also important regulators in the immune system, the effects of hMSCs on these T cell subsets were checked as well. As shown in the Additional file 2: Figure S2, we did not see remarkable changes in the frequencies of Treg and Th17 cells in the presence of hMSCs.
Human fibrosarcoma cells do not have any effect on the proliferation and activation of murine T cells
To demonstrate that the inhibitory effect that hMSCs have on murine T cells is specific to the hMSCs, we also used a different adherent human cell line in our experiments, HT-1080 (human fibrosarcoma cell line). These cells have similar morphological features to hMSCs, without having any known MSC-like properties (the multi-potent ability to differentiate into osteocytes, chondrocytes and adipocytes); therefore, they are used as controls in our experiments. We used these cell lines in the same ratios as the hMSCs, and the same experimental settings.
The immuno-suppressive effect of hMSCs is cell contact-dependent
There has been widespread interest in elucidating the mechanism by which hMSC act as suppressors of the immune system. It is accepted that hMSCs act by cell-cell contact inhibition and by secreting soluble factors in vitro as well as in vivo.
We aimed at understanding the mechanism by which hMSCs affect the proliferation and activation of murine antigen specific T cells in our in vitro system. The CD4+ T cells or CD8+ T cells were cultured together with their cognate peptides and APCs in the bottom wells of a 24-well transwell system plate. In the upper wells we cultured the hMSCs in the same ratios as previously.
Due to their immunosuppressive activities, hMSCs have been used in many investigational clinical trials to investigate their potential to treat immunological disorders or inflammation-mediated pathological lesions, including Crohn’s disease, T1D and GVHD (reviewed in ). They have also been investigated in co-transfer experiments intended to improve the engraftment of allogeneic pancreatic islet transplant  and hematopoietic stem cells [32, 33]. Because of the heterogeneous nature of hMSCs, the establishment of quantitative bioassays that could detect differences between hMSCs from different donors and passages would potentially be of great value for manufacturing and MSC product assessment purposes. Currently, there is an increasing need to develop more sensitive, accurately quantitative cell-based or in vitro bioassays suitable for detecting small range differences in immune-inhibitory activity of hMSCs from different donors or at different passages in tissue culture, or under different tissue culture expansion conditions. For example, the traditionally used mixed lymphocyte reaction (MLR) is a semi-quantitative, or relatively qualitative, rather than quantitative assay; the result may be affected by many factors such as the mismatch extent of donor and recipient MHC, gender and age of donor, as well as the previous and current infectious disease status. With such inherent variability, it can be very challenging to capture minor differences in immunosuppressive activity between different lots of hMSC products using the traditional MLR method.
It has been established that the immune inhibitory activity of MSCs works across allogeneic barriers, and it has also been reported that human MSCs can home to tissues, survive and function to various extents in xenogenic models, such as in mice and rats [18, 26, 34–36]. Therefore, it is likely the immune inhibitory activity of the MSCs will work across species, at least partially. For this reason, we explored development of quantitative immune inhibition assays using clonal murine T cell populations (derived from TCR transgenic mice), known peptide antigens, and MSCs from different human donors. Compared with other existing systems, the advantages of this system include genetic and age variation between human T cell donors is eliminated; the murine donors are kept under specified pathogen free (SPF) conditions; the mouse TCRs are monoclonal with known antigen specificity; and these clonal mouse T cells are reliably available in essentially unlimited supply.
Through the work presented in the present study, we discovered that hMSCs can inhibit the activation and effector functions of mouse Ag specific T cells in response to stimulation with cognate peptide antigens as well as anti-CD3/anti-CD28. Many aspects of T cell activation are affected, such as cell surface markers CD25, CD44, CD62L, CD69, proliferation and cytokine production. The effects are intrinsic to hMSCs, since control cell lines (fibrosarcoma, hepatocellular carcinoma, fibroblasts) do not exert these activities. To our knowledge, this is the first report to demonstrate the cross-species effect of hMSCs on clonal murine T cells when they are stimulated with cognate peptide antigens.
Such an in vitro bioassay may be useful to assess the immunosuppressive activity in human MSCs from different donors, or to assess the effect of different tissue culture expansion conditions of the MSCs from the same donor on their immuosuppressive activity. It might be particularly valuable to researchers who have access to make use of the animal resources as a supplementary method when inter-donor (patient) variance is a major interference issue. Taking into consideration the fact that obtaining TCR transgenic animals and purifying mouse T cells is a relative cumbersome and probably not the most cost-effective method, this method will not be applicable to a routine cell therapy laboratory. However, if acceptable reproducibility of the assay can be achieved through optimization, potentially it might assist in informative comparison of MSC lots and different manipulation conditions.
Despite the fact that these results parallel previous findings with allogeneic MSCs, some of the results obtained in this study are not completely consistent with earlier reports. For example, it has been reported that MSCs preferentially skew the immune response toward Th2 over Th1 by inhibiting the production of TNF-α and IFN-γ by CD4+ T cells (helper T cells) and CD8+ cytotoxic T cells, while they up-regulate the expression of IL-10 and IL-4 by CD4+ and CD8+ T cells . From these results it would be expected that hMSCs inhibit the production of IFN-γ and the expression of transcription factor T-bet. However, we also observed inhibition of IL-10 and the Th2 transcription factor GATA-3. This could be due to differences between our model systems (that is, the cross-species use of hMSCs with murine T cells in this study, versus allogeneic hMSCs with human T cells; clonal T cells versus polyclonal T cells, and so on). Also, it is known that when TCR signal transduction pathways are triggered with rigorous stimuli (such as PMA and ionomycin, anti-CD3 Ab) in T lymphocytes, both Th1 and Th2 cytokines are released [37, 38], and GATA-3 expression can be turned on by TCR signals [39, 40]. Thus, it is most likely that the inhibition of Th2 cytokines as well as GATA-3 expression that we observed is merely a reflection of hMSC-mediated inhibition of TCR signaling and T cell activation.
Although the mechanisms of immune regulation have been extensively studied they are still not fully elucidated. It is well-known that allogeneic MSCs can inhibit the activation of T cells following stimulation with mitogenic or allogeneic stimulation (PHA, Con A, CD3, allogeneic PBL and so on) [9, 10, 41]. Also, peptide antigen-stimulated T cell activation can be inhibited . Several studies have suggested that these immunoregulatory effects require an initial cell-cell contact phase, but that suppressive signaling is mediated by soluble factors including transforming growth factor beta 1 (TGF-β1) , indoleamine 2,3-dioxygenase (IDO) , prostaglandin E2 (PGE2) , nitric oxide (NO) , heme oxygenase-1 (HO-1) , and insulin-like growth factor-binding proteins . In our study, the immunosuppression by hMSCs upon both CD4+ and CD8+ T cells seems to be predominantly mediated by a cell-cell contact mechanism. Perhaps the cross-species effects of hMSCs are biologically different from those in syngeneic or allogeneic systems studied by others, which need to be further clarified. We plan to test the immunosuppressive activities of mouse MSCs on murine CD4+ and CD8+ T cell clones, to investigate whether the effects we saw in this study are simply due to the difference of cytokines or soluble factors between mouse and human species.
There is controversy as to whether MSCs inhibit T cell proliferation by inducing apoptosis or not. In our experiments, we noticed a slight increase in Annexin V expression when CD4+ T cells were incubated with hMSCs, in agreement with one report that hMSCs can induce apoptosis via a mechanism involving IDO and IFN-γ . Other studies have reported that hMSCs inhibit the proliferation of T cells by non-apoptotic mechanisms, such as cell cycle arrest [13, 48]. Based on previous reports, it is surprising to see in our system that hMSCs can decrease apoptosis in activated murine CD8+ T cells. We hypothesize that hMSCs might provide certain soluble growth factors or cell-cell contact signals that favor the survival of CD8+ T cells or prevent them from activation-induced cell death. Further studies are needed to gain better understanding of the underlying mechanism.
Since experimental results obtained from in vitro systems do not always reflect the in vivo environment, they need to be confirmed by in vivo findings. Ongoing experiments are being performed in our group to further evaluate the immuno-suppressive function of hMSCs in an in vivo murine model of autoimmune type 1 diabetes. Previously, MSCs obtained from healthy mice have been shown to delay the onset of diabetes in non-obese diabetic (NOD) mice . Similarly, human MSCs lowered blood glucose levels in the STZ- (streptozotocin-) treated diabetic mice relative to untreated controls . Once our in vivo studies are finished, we may be able to correlate the in vivo inhibitory functions of hMSCs with their in vitro activities, and even identify potential biomarkers which can be used to predict the in vivo efficacy before hMSC engraftment.
In summary, we established a system where the immunosuppressive activity of hMSCs can be measured using murine clonal T cells; several biomarkers were identified which can be used to quantify the immunosuppressive activities of hMSCs, such as CD25, CD44, CD62L, CD69, proliferation, gene expression and cytokine production. Among these markers, cytokine measurement is most quantitative and easier to standardize, thus it could potentially contribute to an informative comparison of MSC lots and their potential manipulation.
Graft versus host disease
Human bone marrow-derived mesenchymal stem cells
Mean fluorescence intensity
Major histocompatibility complex
Mixed lymphocyte reaction
Mesenchymal stem cells
T cell receptor.
This work was supported by the FDA Modernizing Science grant program, the FDA medical countermeasures initiative (MCMi) as well as the Division of Cellular and Gene Therapies. Cristina Nazarov and Jessica Lo Surdo were supported through fellowship administered by the Oak Ridge Institute for Science and Education. The authors would like to thank Drs. Andrew Byrnes and Graeme Price for critically reviewing this manuscript and Jean Manirarora for assistance with the breeding and typing of the TCR transgenic mice.
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