INVOLVEMENT AND REGULATION OF INDOLEAMINE 2,3-DIOXYGENASE IN THE IMMUNO-MODULATORY ACTIVITIES OF UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELLS
Ting Lo1, Shu-Ching Hsu3, Chiao-Hui Chuang 2, Nai-Chih Hsu3, *Oscar K. Lee 2,4
1. Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
2. Department of Orthopaedics and Traumatology, Veterans General Hospital- Taipei and School of Medicine, National Yang-Ming University, Taiwan
3. Vaccine Research and Development Center, National Health Research Institutes, Miaoli, Taiwan
4. Stem Cell Research Center, National Health Research Institutes, Miaoli, Taiwan
* Authors for correspondence ()
Abstract
We have previously reported the existence of mesenchymal stem cells (MSCs) in human umbilical cord blood (UCB) and it was recently found that MSCs from other sources possess immuno-regulatory capabilities. The purpose of this study is to explore the possible roles and mechanisms of UCB-MSCs in the regulation of immune system. It was found that after co-culture, uMSC could inhibit MLR response in a dose-dependent manner. Furthermore, IL-10R was induced in uMSCs while co-cultured with T lymphocyte. The addition of IL-10 could enhance the expression of IDO of uMSCs in the co-culture system. Neutralizing antibody against IL-10 ameliorated the inhibitory effects of UCB-MSCs. Taken together, UCB-MSCs modulated the immune system by secreting IDO and through the activation of its downstream signaling pathways.
Introduction
Mesenchymal Mesenchymal stem cells (MSCs) have unique self-renewal and multi-lineage differentiation capabilities and they are able to replenish a variety of specific cell types. Moreover, it has been found that MSCs possessed low immunogenicity and, when MSCs were co-cultured with allogeneic T-lymphocytes in vitro, they inhibited proliferation of allogeneic T cells, suggesting the regulatory activities of MSCs in the immune system. Indoleamine 2,3-dioxygenase (IDO) is an enzyme which catabolizes the essential amino acid L-tryptophan. The degradation of tryptophan, which is required for cell proliferation, was the mechanism of IDO-induced T cell suppression. It has been reported that expression of IDO was exploited by the mammalian fetal allograft to suppress T cell activity and prevent rejection1. Besides, it was further shown that expression of IDO in antigen-presenting cells may control autoreactive immune responses2. The purpose of this study is to elucidate the roles of IDO in the immuno-modulatory activities of umbilical cord blood-derived MSCs (uMSCs).
Materials and Methods
Culture of Human UCB-MSCs
MSCs were obtained from human umbilical cord blood (UCB) using the methods previously reported6. Briefly, mononuclear cells were first harvested from UCB by density gradient with Ficoll-Paque (Amersham-Pharmacia, Piscataway, NJ, USA; 1.077g/cm3), then negative immuno-selection was performed using an immuno-depletion kit (RosetteSep®, StemCell Technologies, Vancouver, BC, Canada), as per manufacturer’s instructions. The cells were plated in non-coated tissue culture flasks (Becton Dickinson) in the expansion medium which consists of Iscove’s modified Dulbecco’s medium (IMDM, Gibco BRL, Grand Island, NY, USA) and 20% Fetal Bovine Serum (FBS, Hyclone, Logan, UT, USA), supplemented with 10ng/ml bFGF, 100 U penicillin, 1000 U streptomycin, and 2mM L-glutamine (Gibco BRL). Cells were allowed to adhere overnight and non-adherent cells were washed out with medium changes. Medium changes were carried out twice weekly thereafter. To obtain single cell-derived, clonally-expanded MSCs, limiting dilution was performed. Cells were serially diluted and plated on to 96-well plates (Becton Dickinson) in expansion medium at the final density of 30 cells per 96-well plate (0.3 cell/well). Cell pellets from each well were re-suspended and re-plated at the initial density of 3,000 cells/cm2. When 50-60% of confluence was reached, cells were trypsinized, counted and sub-cultivated. Immuno-phenotyping was performed to confirm the surface phenotype of UCB-MSCs; they were positive for CD73, CD105, CD166, CD44, CD29, SH-2, SH-3, SH-4, but were negative for CD14, CD34, and CD45 (data not shown). Differentiation capabilities of UCB-MSCs into bone, fat, and cartilage cells were confirmed prior to further experiments using the protocols previously described6.
Isolation of human T lymphocytes
Human T lymphocytes were prepared from peripheral blood of healthy donors (n=3). Mononuclear cells were fractionated by density gradient centrifugation with Ficoll-Paque (1.077g/cm3), and the mononuclear cells were passed through a nylon wool fiber column and non-adherent cells were collected. T lymphocytes were then obtained from non-adherent cells with a human CD3+ isolation kit (Dynal Biotech, Norway) according to the manufacturer’s instructions. In each of the assays described in this study, the purity of CD3+ T lymphocytes was 99.63 % ± 0.16 and CD14+ cell purity was less than 0.73% ± 0.02%. The viability of purified T lymphocytes or monocytes was determined by ethidium bromider/acridine orange vital staining and showed > 99 % cell viability at the time of harvest (data not shown).
Immunoblotting
T cells were harvested after co-cultured with UCB-MSCs or the addition of IL-10. Cells were washed twice with PBS buffer and then lysed in lysing buffer (1% P-40, 100mM NaCl, 20mM Tris-HCL, 10mM NaF, 1mM Sodium orthovanadate, and 30nM sodium glycerophosphate) (Roche, Germany) and incubated on ice for 20 min. The lysates were centrifuged at 10,000 rpm for 5 min, and total protein was obtained from the supernatants. Protein concentration was measured with the Lowry protein assay. For Western Blot, 10μg of protein sample was mixed with equal volume loading buffer and incubated in boiling water for 10 minutes. The sample proteins were analyzed by 10% SDS-polyacrylamide gel, and the gels were run at 150 Volts for 60 to 90 minutes. The proteins were then transferred to nitrocellulose transfer membranes (BIO-RAD, USA). The membranes were blocked with 2% non-fat milk powder overnight at 4oC, then incubated with primary antibody（Anti- IDO, or b-actin antibody, Upstate, USA）diluted 1000-5000-fold in diluted buffer at room temperature for 1 hour. The membranes were then washed three times with 0.05% Tween-20/ PBS, incubated for 30 min. with horseradish peroxidase (HRP)-conjugated antibody diluted 2000-fold (upstate, USA) in 0.05% Tween-20/ PBS containing 1% BSA. After washing three times with 0.05% Tween-20/ PBS, the proteins were visualized with ECL reagent and by exposure to BioMax MR Films (BIO-RAD, USA). A quantitative analysis was performed using Image Master ID Elite software system (Pharmacia Biotech, USA).
Mixed leukocyte reactions (MLRs)
In MLR culture, T lymphocytes were prepared as responders as described above, and the cells were washed three times with complete culture media (CCM), consisting of RPMI-1640 supplemented with L-glutamine (2-mM), penicillin (100 unit/ml), streptomycin (100 μg/ml), Hepes (10 mM) (Gibco, Life technologies, USA) and 10% fetal bovine serum (HyClone, USA). UCB-MSCs (0.25, 0.5, 1, or 10 x 104) were used as stimulators and they were treated with 0.5 mg/ml mitomycin C before being cultured with the T cells. All the assays were done in triplicates. Phytohemagglutinin (PHA; 5 mg/ml, Sigma) was added into culture to induce T cell-proliferation and activation. Proliferation was evaluated with the reduction ratio of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenase of viable cells to blue formzan products which can be measured spectrophotometrically. At the end of co-culture, 5 mg/ml MTT solution was added and then crystal formzan was dissolved in acid-isopropanol. Optical density was measured in a microplate spectrophotometer (SPECTRAmax 340PC384, Molecular Devices, Sunnyvale, CA, USA) at 570 nm.
Flow cytometric analysis
In order to discover whether regulatory T cells are involved in the immuno-modulation of UCB-MSCs mediated by IL-10, IL-10 was added to T cells for 5 days. For surface antigen phenotyping, T cells were detached and stained with fluorescein- or phycoerythrin-coupled antibodies and analyzed with Epics XL (Beckman Coulter, USA) as standard protocol. The FITC-, PE-, PE- Cy5.5-, and Biotin- conjugated Rat or mouse monoclonal antibodies against human antigens such as CD3, CD4, CD8, and CD25 were purchased from Becton Dickinson and e-Bioscience (USA). FITC- and Biotin- conjugated hamster, rat or mouse IgG1, IgG1a, IgG1k, IgG2a k; IgG2b isotype standard antibodies were used as control antibodies.
Statistical analysis
For the present experiment, an ANOVA analysis was used to evaluate the effect of MSCs on proliferation of activated lymphocytes. The Student t test for paired data was used to test the probability of significant differences among experimental and control groups.
Preliminary Results
Figure 1
Figure 1. uMSC could inhibit MLR response in a dose-dependent manner. uMSC could inhibit MLR response in a dose-dependent manner. Human allogenic T cells or different dosage of uMSCs treated mitomycin C were co-cultured with responder T cells for 5 days.
Figure 2
Figure 2. T cell proliferation could be inhibited by either uMSCs secreted soluble factors or direct cell-cell contact with uMSCs. T cell proliferation could be inhibited by either uMSCs secreted soluble factors or direct cell-cell contact with uMSCs.
Figure 3
Figure 3. Program death ligand 1 (PD-L1) and it’s receptor, PD-1 were expressed on uMSC and T cells in the co-cultured system. Program death ligand 1 (PD-L1) and it’s receptor, PD-1 were expressed on uMSC and T cells in the co-cultured system.
Figure 4
Figure 4. IL-10R was induced in uMSCs while co-cultured with T lymphocyte. IL-10R was induced in uMSCs when co-cultured with T lymphocyte. Adding IL-10 neutralized Abs in to the co-cultured system could reverse the inducible expression of CD210.
Figure 5
Figure 5. The expression of IDO was induced in uMSCs when co-cultured with T cells. The expression of IDO was induced in uMSCs when co-cultured with T cells.
Figure 6
Figure 6. IL-10 could enhance the expression of IDO of uMSCs in the co-culture system. IL-10 could enhance the expression of IDO in the uMSC-T cell co-cultured system. However, IL-10 treatment only could not induce IDO in MSCs without culturing with T cells. Lane 1 : co-cultured MSC + IL-10 treatment. Lane 2 : IL-10R-positive co-cultured MSC + IL-10 treatment. Lane 3 : MSC only + IL-10 treatment.
Figure 7
Figure 7. IL-10 neutralization antibodies reduced IDO expression of MSCs in the co-culture system. IL-10 neutralization Ab will reduce IDO expression in the MSC-T cell co-cultured system.
Figure 8
Figure 8. Either IL-10 or PD-1 neutralized antibodies could reverse the inhibition effect on T cells. T cell response was suppressed when co-cultured with MSC. Either IL-10 or PD-1 neutralized Abs could reverse the inhibition effect on T cells. T :T-cell only. A :Allogenic T-cells. M :MSC. I :IL-10 neutralization Ab in culture medium at day 5. P : PD-1 neutralization Ab in cultured medium at day 5. The percentage means the decrease compared to positive control (T+A).
Figure 9
Figure 9. Functional activity assay of IDO expression was performed to confirm the increase of kynurenine, product of tryptophan degradation, in MSCs. Functional activity assay of IDO expression in MSCs was performed by measuring the production of kynurenine after tryptophan was metabolized. HeLa cell was a positive control of IDO expression.
Discussion and future work
The absence of MHC class II confers MSCs with the potentials to escape recognition by alloreactive CD4+ T cells18, 34-35. However, how MSCs evade allorejection and why they possessed the hypoimmunogenic properties underlying this action is still not completely clear. The critical observation here is the involvement of IDO in the immuno-regulation of UBC-MSCs.
Secretion of the soluble cytokines from MSCs plays an important role in the suppression of the immune system. It is evident that MSCs do not constitutively express IL-2, IL-3, IL-4 and IL-536-37. On the other hand, it has been shown that MSC do constitutively express mRNA for cytokines such as interleukin (IL)-6, -7, -8, -11, -12, -14, -15, -27, leukemia inhibitory factor, macrophage colony stimulating factor, and stem cell factor38. IL-10 has a well-documented role in T cell regulation and in the promotion of the regulatory or suppressive phenotype. It has been shown in the literature that IDO was exploited by the mammalian fetal allograft to suppress T cell activity and prevent rejection 11. Besides, it was further shown that expression of IDO in antigen-presenting cells may control autoreactive immune responses.
Immune responses can be monitored by status of cell activation in T lymphocytes, therefore, TCR signaling, co-stimulatory molecules and presence of cytokines are the most common checking events to have the evaluation. The immune-suppressive mechanisms of MSCs were involved in cell contact dependent and independent interaction had been reported, but the detail molecular regulation of cell-cell contact in suppressing immune responses was still unclear.
Here we have shown the induction of CD28 in T lymphocytes after PHA stimulation was dramatic inhibited by MSCs. In our study, IDO was indeed involved in the immuno-regulatory activities of uMSCs in an inducible and regulated fashion. Most of our assays suggest the events happened in UCB-MSCs -cultured T cells have the similar profiles to addition of IL-10 into allogeneic T lymphocytes in the absence of UCB-MSCs. Our finding provided the evidences to have the perspective on the significance of the role of IDO in modulating immune activity of UCB-MSCs. However, further efforts are required for the detailed regulatory machinery of IDO expression in uMSCs.
References:
1. Caplan AI. Mesenchymal stem cells. Orthop Res. 1991; 9:641-650.
2. Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem. 1994; 56:283-294.
3. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284:143-147.
4. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone. 1992; 13:81-88.
5. Hayneswoth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone. 1992; 13:69-80.
6. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004; 103:1669-1675.
7. Kogler G, Sensken S, Airey JA, Trapp T, Muschen M, Feldhahn N, Liedtke S, Sorg RV, Fischer J, Rosenbaum C, Greschat S, Knipper A, Bender J, Degistirici O, Gao J, Caplan AI, Colletti EJ, Almeida-Porada G, Muller HW, Zanjani E, Wernet P. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med. 2004; 200:123-135