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作者：Chun-Jung Changa, Men-Luh Yenb, Yao-Chang Chenc, Chih-Cheng Chiend,e, Hsing-I. Huangd,f, Chyi-Huey Baig, B. Linju Yena作者单位：aStem Cell Research Center, National Health Research Institutes, Zhunan, Taiwan;bDepartment of Primary Care Medicine and Department of Obstetrics/Gynecology, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan;cDepartments of Laboratory Medicine an
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【摘要】
2 I; P$ V- g' P6 D Several types of nonhematopoietic stem cells, including bone marrow mesenchymal stem cells (BMMSCs) and embryonic stem cells, have been shown to have immunosuppressive properties. We show that human placenta-derived multipotent cells (PDMCs), which are isolated from a source without ethical concern and harbor multilineage differentiation potential, have strong immunosuppressive properties. PDMCs suppress both mitogen-induced and allogeneic lymphocyte proliferation in both CD4 and CD8 populations. The immunosuppression seen with PDMCs was significantly stronger than that with BMMSCs. Both PDMCs and BMMSCs express indoleamine 2,3-dioxygenase, but only PDMCs are positive for intracellular human leukocyte antigen-G (HLA). Mechanistically, suppression of lymphocyte reactivity by PDMCs is not due to cell death but to decreased cell proliferation and increased numbers of regulatory T cells. Addition of neutralizing antibodies to interleukin-10 and transforming growth factor (TGF)-ß partially restored lymphocyte proliferation. Unlike BMMSCs, PDMCs treated with interferon- for 3 days only very minimally upregulated HLA-DR. On the contrary, PD-L1, a cell surface marker that plays an inhibitory role in T-cell activation, was upregulated and TGF-ß expression was seen. The immunosuppressive properties of PDMCs, along with their multilineage differentiation potential, ease of accessibility, and abundant cell numbers, may render these cells as good potential sources for future therapeutic applications. 8 m6 C- M/ e6 ?4 ?- r) l7 u2 J
【关键词】 Mesenchymal stem cell Placenta Multilineage differentiation Immunosuppression Mixed lymphocyte culture Interferon- Human leukocyte antigen class I G Indoleamine -dioxygenase
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8 g+ F' c% ^# q: \( m/ l9 L' YStem cells are specialized cells capable of self-renewal as well as multilineage differentiation. Both embryonic stem cells (ESCs) and adult stem cells, including mesenchymal stem cells (MSCs), have been isolated from human sources, and ex vivo propagation for either population can be achieved with relative ease .! J8 k9 V4 w# ^1 A4 z, }
* M+ i- H* k) Z& ?, kRecent research, however, is revealing that MSCs can suppress immune reactivity of allogeneic lymphocytes, both in vitro and in vivo. Allogeneic BMMSCs from humans, baboons, and mice have been shown to decrease the immune response of lymphocytes in vitro ., U+ N0 D5 b7 W* @( w. ^. w* \ x
7 w: |% r- x" Q' b- r2 {! T$ x( BAlthough the recent data regarding immunosuppressive effects of BMMSCs are promising, the fact remains that these cells are rare and numbers decrease with increasing donor age . We therefore studied the immunological characteristics of PDMCs and found that these cells have potent inhibitory effects. Comparisons with BMMSCs were performed, and the mechanisms behind the immunosuppression by PDMCs were explored.
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MATERIALS AND METHODS, Q$ T; b0 @5 R% V6 x! @! \
4 q) U" a- P$ F% f! jCell Culture5 l5 K* e! Z6 W" t! [0 I
2 N2 J# a" N2 O& O* QPlacenta-Derived Multipotent Cells. Term (38¨C40 weeks gestation) placentas from healthy donor mothers were obtained with informed consent approved according to the procedures of the institutional review board. The cells were isolated as previously reported . For use as third-party cells in mixed lymphocyte cultures (MLCs), PDMCs and BMMSCs were inactivated with mitomycin C (Sigma-Aldrich).0 v8 {6 }1 u4 f* ^6 o
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Differentiation studies were carried out as previously described . All reagents were from Sigma-Aldrich. Briefly, for adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 µM isobutyl-methylxanthine, 1 µM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com), 10 µM insulin, and 60 µM indomethacin. Osteogenic differentiation was achieved by culturing cells in complete medium along with 0.1 µM dexamethasone, 10 mM ß-glycerol phosphate, and 50 µm ascorbate. Neurogenic differentiation was induced by culturing cells in serum-free medium with the addition of 10¨C6 M retinoic acid. Cells were stained for oil red and alizarin red for visualization of adipogenic and osteogenic differentiations, respectively.- Q1 E: J, x+ F, Z& d/ e
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Generation of Human Mature Dendritic Cells. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (1.077 g/ml; Invitrogen) density gradient centrifugation. Monocytes were purified from PBMCs by using the MACS (magnetic cell sorting) CD14 Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com/nn.404,company.html). Briefly, PBMCs were incubated with anti-CD14 magnetic beads in 4¡ãC for 20 minutes. After incubation, the PBMCs and magnetic beads complex were washed with phosphate-buffered saline (PBS) containing 1% FBS and isolated to CD14 and CD14¨C fractions by AutoMACS (Miltenyi Biotec). The purity was determined to be more than 85% CD14 by flow cytometric analysis. Cells (1 x 106 per milliliter) were cultured subsequently for 7 days in RPMI-1640 (Invitrogen) and 10% FBS (HyClone) with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1,000 U/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and interleukin-4 (IL-4) (500 U/ml; R&D Systems Inc.). Cytokines were replenished every other day. After 7 days, cells were washed by PBS, and the medium was changed. To induce maturation of monocyte-derived cells to mature dendritic cells (mDCs), lipopolysaccharide (1 µg/ml; Sigma-Aldrich) was added for another 48 hours of culture with GM-CSF and IL-4.
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& O3 S9 I1 Z* HIsolation of Human CD4 and CD8 Cells. CD4 and CD8 cells were isolated from PBMCs by using the MACS CD4 and CD8 cell isolation kits (Miltenyi Biotec) in the same methodology as reported above. Briefly, PBMCs were incubated with the appropriate magnetic beads in 4¡ãC for 20 minutes. After the incubation, the PBMCs and magnetic beads complex were washed with PBS containing 1% FBS and isolated to positive and negative fractions by AutoMACS (Miltenyi Biotec). The positive fraction was collected, and the purities of the CD4 and CD8 T lymphocytes were demonstrated to be greater than 98% by flow cytometric analysis. T cells were cultured in RPMI-1640 (Invitrogen) and 10% FBS (HyClone).
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Immunophenotyping of Cells
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Cells were detached with trypsin/EDTA, washed, and resuspended in PBS with 1% FBS. The cells (1 x 105 per milliliter) were then stained for 30 minutes on ice with saturating amounts of fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated monoclonal antibodies. Antibodies recognizing the following human antigens were used: CD4, CD8, CD14, CD25, CD34, CD40L, CD80, CD86, CD122, CD90/Thy-1, CD117/c-kit, CD166/ALCAM, PD-1, PD-L1, HLA-ABC, and HLA-DR (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com); CD13, CD29, and CD44 (Dako Denmark A/S, Glostrup, Denmark, http://www.dako.dk); CD133 (Miltenyi Biotec); Foxp3 (Serotec Ltd., Oxford, U.K., http://www.serotec.com); and CD105/endoglin/SH-2 and stage-specific embryonic antigen-4 (SSEA-4) (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww). Antibodies against the human antigen SH-3 were purified; the SH-3 hybridoma cell line was acquired from American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org). For analysis of T regulatory cells (Tregs), responder PBMCs were harvested before and after 3 days of mixed lymphocyte culture (MLCs) and were double-stained in the following combinations: CD4/CD25, CD4/CD122, CD4/Foxp3, CD8/CD25, and CD8/CD122. Each analysis included the appropriate FITC- and PE-conjugated isotype controls. All analyses were done on a Becton Dickinson FACScan laser flow cytometric system (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) equipped with a Macintosh PowerMac G3 personal computer (Apple Computer, Inc., Cupertino, CA, http://www.apple.com) using CellQuest software (BD Biosciences).
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9 s5 p) `, {4 l% o( _4 J) yImmunofluorescence
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2 j" ^- v; D pCultured cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature and permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) for 10 minutes. Primary antibodies against indoleamine 2,3-dioxygenase (IDO) and HLA-G were purchased from Serotec Ltd. Samples were first incubated with the primary antibodies at 4¡ãC overnight, rinsed three times with PBS, and incubated for 60 minutes at room temperature with FITC- or PE-conjugated secondary antibodies. All samples were stained with 4',6-diamidino-2-phenylindole (Molecular Probes Inc., Eugene, OR, http://www.invitrogen.com) for 5 minutes. Staining was visualized under a fluorescence microscope (Nikon Corporation, Tokyo, http://www.nikon.com).
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MLCs and Cell Proliferation Assay8 {. a7 \! s2 H7 K; r6 X8 Y3 }: ^, v
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PDMCs were plated into 96-well plates (5 x 104 cells per milliliter) containing responder cells (CD4, CD8 T cells, or PBMCs) and stimulator cells (PBMCs, mDCs, or 4 µg/ml phytohemagglutinin ; Sigma-Aldrich). Stimulator cells were inactivated with mitomycin C (Sigma-Aldrich). Inactivated third-party cells (PDMCs or BMMSCs) were added directly to plates. For Transwell experiments, MLCs were carried out in 24-well plates with third-party cells plated on the transwell. After 72 hours of coculture, 100 µl of cells from each well was transferred to new 96-well plates with 10 µl of Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan, http://www.dojindo.co.jp) added to each well. After incubation for 1¨C4 hours at 37¡ãC, the absorbance was measured at 450 µm with a microplate reader (Molecular Devices Corporation, Sunnyvale, CA, http://www.moleculardevices.com) using a reference wavelength of 600 nm.
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7 _/ E0 w2 T4 f0 i; k7 }' @+ _9 k2 aCarboxyfluorescein Diacetate, Succinimidyl Ester Labeling and Analysis
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/ B( k& L: |7 S* Y0 WPBMCs and CD4 and CD8 T cells were labeled with 2.5 µmol/l of carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) for 10 minutes at 20¡ãC in PBS with 0.1% bovine serum albumin (BSA). After washing twice with 1% BSA in PBS, the cells were resuspended in RPMI-1640 (Sigma-Aldrich) 10% FBS (HyClone) and incubated at 20¡ãC for another 10 minutes. MLCs were performed in the following manner: PDMCs were plated into 24-well plates (5 x 104 cells per milliliter) containing CFSE-labeled PBMCs, CD4, or CD8 T cells (5 x 105 cells per milliliter) with mDCs (5 x 104 cells per milliliter) or PHA (4 µg/ml) with or without IL-2 (50 U/ml; R&D Systems Inc.) in RPMI-1640 (Invitrogen) and 10% FBS (HyClone). After 72 and 96 hours, the T cells were harvested and washed twice with PBS containing 1% FBS. Analysis of cell division was performed by flow cytometry. To assess the effects of interferon- (IF-), PDMCs were pretreated with 100 ng/ml of IF- (R&D Systems Inc.) for 48 hours prior to MLCs. In experiments using neutralizing antibodies to IL-10 and transforming growth factor-ß (TGF-ß; R&D Systems Inc.), the antibodies were added to MLCs for 3 days of incubation after which T-cell proliferation was assessed by flow cytometry.+ B2 N6 u) W" ?& Y8 \
: Z2 S- o0 m) g5 q2 ^8 t6 |9 O* }: GApoptosis
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# ]* o+ _+ G/ P4 \. q3 i! ~3 ^+ cThe apoptosis assay for T cells was performed with the annexin V/propidium iodide (PI) staining kit (Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche.de/diagnostics/index.htm). Briefly, cells were harvested by centrifugation (1,200 rpm, 5 minutes), and the medium was discarded. The lymphocytes were then recovered, stained with FITC-conjugated annexin V and PI, and analyzed by flow cytometry.& Z4 O/ I# f& W! r2 t2 D, R
: c2 ]- t* [, g3 b/ \5 w$ UNatural Killer Cell Cytotoxicity Assay2 @4 u# X2 ~% o, X1 d2 Q% y5 E
' B9 B- e& |3 d" N# v5 UPurified natural killer cells (NKCs) were obtained from PBMCs by negative selection with the MACS NKC Isolation Kit (Miltenyi Biotec). The degree of purity was measured with fluorescence-activated cell sorting analysis with labeled antibodies against CD56 (Becton, Dickinson and Company). K562 (ATCC), a well-characterized chronic myeloic lymphoma cell line known for its NKC sensitivity caused by a lack of major histocompatibility complex (MHC) class I molecules, was used as a positive target cell, and MCF-7 (ATCC), a breast cancer cell line known to be a poor NKC target was added, cells were further incubated for 1 hour, and fluorescence was measured in a fluorescence reader (Molecular Devices Corporation).
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7 v2 {: c: R# r! A- L; uReverse Transcription-Polymerase Chain Reaction
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Total mRNA was extracted using Trizol (Invitrogen) and reverse-transcribed using the SuperScript III First-Strand (Invitrogen). Primers were as follows: IL-4, forward primer 5'-CAACTTTGTCCACGGACAC-3', reverse primer 5'-TCCAACGTACTCTGGTTGG-3'; IL-10, forward primer 5'-ATGCCCCAAGCTGAGAACCAAGACCCA-3', reverse primer 5'-AAGTCTCAAGGGGCTGGGTCAGCTATCCCA-3'; TGF-ß, forward primer 5'-CTATCCACCTGCAAGACTATCGAC-3', reverse primer 5'-GGAGCTGAAGCAATAGTTGGTGTC-3'; and ß-actin positive control, forward primer 5'-TGGCACCACCTTCTACAATGAGC-3', reverse primer 5'-GCACAGCTTCTCCTTAATGTCACGC-3'. Polymerase chain reaction (PCR) was carried out with 25-µl reaction volumes of Platinum PCR SuperMix (Invitrogen) and 60 to approximately 80 ng of cDNA template. The annealing temperature was 60¡ãC, and amplification was set for 30 cycles. The PCR was performed in a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com).
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4 O. L0 w4 a' SStatistical Analysis
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Statistical analysis was performed with the statistical SPSS 12.0 software (SPSS Inc., Chicago, http://www.spss.com). The t test (t distribution) was used to test the probability of significant differences between samples. Statistical significance was defined as p
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RESULTS
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Immunophenotyping and Differentiation of PDMCs
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PDMCs isolated from human term placenta showed fibroblastic morphology and exhibited many cell surface markers common to BMMSCs , was found intracellularly in PDMCs but not BMMSCs (Fig. 1D).
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Figure 1. Immunophenotyping and differentiation of PDMCs. (A): Cell surface markers of undifferentiated PDMCs assessed by flow cytometry. (B): Phase-contrast, oil red stain, and alizarin red stain for PDMCs differentiated into (i) neural- (magnification x400), (ii) adipocytic-, and (iii) osteoblastic-lineage cells, respectively (magnification x100). (C): Immune-related cell surface markers of PDMCs and BMMSCs assessed by flow cytometry. (D): Immunofluorescent staining for IDO (FITC) and HLA-G (PE) in PDMCs versus BMMSCs (magnification x50). Scale bars: 200 µm. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; HLA-G, human leukocyte antigen, class I, G; IDO, indoleamine 2,3-dioxygenase; PDMC, placenta-derived multipotent cell; PE, phycoerythrin.
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PDMCs Suppress Lymphocyte Reactivity After Mitogen or Alloantigen Stimulation
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It has been demonstrated that BMMSCs suppress the immune response of PBMCs when stimulated with alloantigens . To determine whether PDMCs can also suppress immune response, we performed MLCs stimulated with either PHA or allogeneic PBMCs and cocultured with or without PDMCs. Third-party PDMCs from four different donors were used, and BMMSCs were also used for comparison. In all experiments, lymphocyte proliferation was suppressed to a significant extent when PDMCs or BMMSCs were added to the MLCs (Fig. 2A). Compared with BMMSCs, PDMCs were able to suppress lymphocyte proliferation to a significantly greater extent.
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Figure 2. Effect of human PDMCs and BMMSCs on PBMC proliferation. (A): PBMCs (5 x 104 cells per milliliter) were incubated with third-party cells (5 x 104 cells per milliliter BMMSCs or PDMCs from four donors A, B, C, and D) and stimulated with mitogen (PHA, black column) or allogenic PBMCs (gray column; white column, medium-only negative control). Cell proliferation was assessed by CCK-8 (see Materials and Methods) after 3 days of MLCs. Comparisons were made between medium control versus BMMSCs (*), and BMMSCs versus the average value of the four PDMC samples (**) for corresponding stimulators (i.e., mitogen-stimulated BMMSC MLCs were compared with mitogen-stimulated PDMC MLCs). Statistical significance and standard deviation were calculated from three experiments. (B): NKC cytotoxicity assay: Isolated NKCs were incubated with K562, MCF-7, or PDMCs, and cell lysis (%) was assessed. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; MLC, mixed lymphocyte culture; NKC, natural killer cell; O.D., optical density; PBMC, peripheral blood mononuclear cell; PDMC, placenta-derived multipotent cell; PHA, phytohaemagglutinin.1 D7 c; _, W/ K3 s5 m
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PDMCs Are Not Lysed by NKCs
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+ C6 M ~% ^) }8 i" T4 p5 A* OAlong with T lymphocytes, NKCs play an important role in transplant rejection. We therefore tested whether NKCs were able to lyse PDMCs. Using K562, which is a well-established NKC target, and MCF-7, reported to be a poor NKC target , we observed that NKC-specific lysis of PDMCs was similar to that of MCF-7; lysis of MCF-7 was approximately threefold less than lysis of K562 (Fig. 2B).5 t; \+ }5 U+ P+ z0 G' a v
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Suppression of Lymphocyte Reactivity by PDMCs Is Not Due to Apoptosis but to Decreased Cell Proliferation and Increased Numbers of Tregs6 B! Z! V( `4 c& {
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The mechanism behind PDMC suppression of lymphocyte reactivity was further investigated. To rule out the possibility that the suppressive effect was due to lymphocyte cell death, cell apoptosis was assayed by flow cytometric analysis of PI and annexin V staining. No significant difference was seen between control MLCs lymphocytes and PDMC-cocultured lymphocytes (Fig. 3A). Next, we investigated whether cell division was affected. To assess this, PBMCs were stained with the cell surface dye CFSE prior to antigen stimulation with PHA and IL-2. Lymphocyte cell division was assessed after 3 days of MLCs. In MLCs cocultured with either PDMCs or BMMSCs, decreased lymphocyte cell division could be seen (Fig. 3B).
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Figure 3. Mechanisms of PDMC suppression of lymphocyte reactivity (flow cytometric analysis). (A): Lymphocyte apoptosis. PBMCs were stimulated with PHA and cocultured with PDMCs for 3 days, then stained with annexin V/PI. (B): Lymphocyte proliferation. PBMCs were stained with CFSE then subjected to stimulation with PHA and/or IL-2, and cocultured with or without third-party PDMCs or BMMSCs. (C): Induction of CD4 CD25high cells. Responder PBMCs were double-stained for CD4 and CD25 in mixed lymphocyte culture cocultured with or without PHA and/or PDMCs for 3 days. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; CFSE, carboxyfluorescein diacetate, succinimidyl ester; IL-2, interleukin-2; PBMC, peripheral blood mononuclear cell; PDMC, placenta-derived multipotent cell; PHA, phytohaemagglutinin; PI, propidium iodide.
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We also investigated whether the inhibitory effects of PDMCs involved Treg cells, which are CD4 CD25high T cells capable of modulating tolerance in the immune response , we also found a threefold increase in CD4 /Foxp3 lymphocytes when third-party PDMCs were added to MLCs (supplemental online Fig. 2).. T3 { k: q6 b; s+ c4 N! A' s4 `
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PDMCs Have Similar Inhibitory Effects on CD4 and CD8 T Cells, and the Inhibitory Effects Are Seen with Either Nonspecific Mitogen or Alloantigen Stimulation) H& M3 F2 ^: d4 n. w9 E7 w* @7 c
# R$ a) t3 Z2 z% ~To further clarify the inhibitory effects of PDMCs, we investigated whether the immune suppression was directed at a particular T-cell population. We used AutoMACS magnetic beads to separate T cells into CD4 and CD8 populations. These separated populations of CD4 (Fig. 4A) and CD8 (Fig. 4B) T cells were then stimulated by both a nonspecific mitogen, PHA, or allogeneic mDCs. Analyzing cell proliferation by flow cytometry, we found that PDMCs can reduce proliferation in either T-cell population. Moreover, proliferation was inhibited whether mitogens or allogeneic mDCs, which are professional antigen-presenting cells (APCs), were used. When stimulated with mDCs, PDMCs appear to have a stronger inhibitory effect on CD4 than CD8 cells.
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Figure 4. Immunosuppressive activity of PDMCs on CD4 and CD8 T lymphocytes. CD4 (A) and CD8 (B) T cells were isolated from PBMCs and stained with CFSE. Lymphocytes were stimulated with PHA or allogeneic mature DCs and cocultured with or without PDMCs for 3 days before flow cytometry analysis. Abbreviations: CFSE, carboxyfluorescein diacetate, succinimidyl ester; DC, dendritic cell; PBMC, peripheral blood mononuclear cell; PDMC, placenta-derived multipotent cell; PHA, phytohaemagglutinin.
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$ _" C. a6 ?6 w- z& IImmunosuppression by PDMCs on Both CD4 and CD8 Lymphocytes Is Partially Restored with Addition of Neutralizing Antibodies to IL-10 and TGF-ß
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- L$ r) Q+ N. NMLCs in which third-party PDMCs were seeded on to Transwell did not show any change in the inhibitory effects (supplemental online Fig. 3), implicating a role for secreted factors in immunosuppression. Thus, to examine which soluble factors are involved in PDMC immunosuppression, we performed MLCs with the addition of neutralizing antibodies to two anti-inflammatory cytokines, TGF-ß and IL-10. Antibodies to either cytokine were able to partially reverse the inhibitory effects of PDMCs on both CD4 and CD8 lymphocyte proliferation (Fig. 5).# ^" G: @2 I& V( h
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Figure 5. Effects of TGF-ß and IL-10 neutralizing antibodies on PDMC immunosuppression. Magnetic bead-separated populations of CD4 and CD8 T cells were stained with CFSE, stimulated with PHA, and cocultured with PDMCs for 3 days. Neutralizing antibodies to TGF-ß and IL-10 were added to the MLC, and flow cytometry was performed. Abbreviations: CFSE, carboxyfluorescein diacetate, succinimidyl ester; IL-10, interleukin-10; MLC, mixed lymphocyte culture; PDMC, placenta-derived multipotent cell; PHA, phytohaemagglutinin; TGF-ß, transforming growth factor-ß.- _) T% W" _( g( D2 _8 e$ p
5 C" i0 y8 s& [Pretreatment with IF- Enhances the Inhibitory Effects of PDMCs0 |: Q& h) P6 f
/ B; s( }4 e u4 b# Q9 Y9 XTransplantation of the allograft will elicit a cascade of host responses in vivo, including secretion of IF-, one of the most potent inflammatory cytokines known . This was seen with PDMCs but not BMMSCs (Fig. 6A).
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Figure 6. Effect of IF- on inhibitory effects of PDMCs. PDMCs and BMMSCs were treated with IF- for 3 days, followed by (A) flow cytometric analysis of HLA-DR and PD-L1 expression and (B) reverse transcription-polymerase chain reaction for TGF-ß expression. (C): Magnetic bead-separated populations of CD4 and CD8 T cells were stained with CFSE, stimulated with PHA, and cocultured with PDMCs with (red lines) or without (black lines) 3 days of IF- pretreatment. Neutralizing antibodies to TGF-ß and IL-10 were added to mixed lymphocyte cultures, and flow cytometric analysis was performed. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; CFSE, carboxyfluorescein diacetate, succinimidyl ester; HLA, human leukocyte antigen; IF-, interferon-; IL-10, interleukin-10; PDMC, placenta-derived multipotent cell; PHA, phytohaemagglutinin; TGF-ß, transforming growth factor-ß.( W7 b, O( j+ z2 Y
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We also investigated whether IF- treatment of PDMCs resulted in any changes in expression of the anti-inflammatory cytokines TGF-ß, IL-4, and IL-10. Reverse transcription-PCR showed a strong increase in the expression of TGF-ß in PDMCs after 3 days of IF- treatment (Fig. 6B). In contrast, for BMMSCs, there appears to be constitutive expression of TGF-ß, levels of which did not change after IF- treatment. No expression of IL-4 or IL-10 was detected in PDMCs or BMMSCs, either with or without IF- treatment (data not shown).
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: p3 e; m" v9 WFinally, we examined whether these phenotypic changes induced by IF- would enhance the immunosuppressive effects of PDMCs. Using CFSE-labeled CD4 and CD8 T cells, MLCs were performed with third-party PDMCs treated either with or without IF-, and with the addition of IL-10- and TGF-ß-neutralizing antibodies. IF--treated PDMCs are able to resuppress the lymphocyte proliferation restored by the neutralizing antibodies, whereas untreated PDMCs were not able to do so (Fig. 6C).- R* w' ^8 [% G4 D- g5 v
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DISCUSSION
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0 K3 e. a( U7 m$ I yAlthough immunosuppressive effects are best established for BMMSCs , was found intracellularly in PDMCs but not BMMSCs (Fig. 1D); this may be a contributing factor in the stronger immunosuppression seen with PDMCs, as well as in making these cells a poor target for NKC lysis (Fig. 2B). Although based on in vitro data, our results would suggest that PDMCs (being of fetal origin) may harbor an "immunologic advantage," which would be of significant benefit regarding future clinical use of these cells. Further research, including in vivo studies, is necessary for the corroboration of these effects before therapeutic use can be considered.: M) u! y; { H- y7 i
, R- h2 h% L; p5 c' }/ P }( N* wHost response to foreign antigen stimulation includes the production of proinflammatory cytokines and chemokines, recruitment of inflammatory cells to the site of infection, and activation of cytotoxic T lymphocytes and NKCs. Although these responses help to slow or eradicate the spread of the pathogen, such responses when uncontrolled can result in severe inflammation and host tissue damage. It is now clear that, concurrently, an immune-tolerant response is also induced that helps to maintain homeostasis of the immune system. One of the key players mediating tolerance is Tregs, a subpopulation of T cells that are CD4 CD25high and have immune-tolerant properties . Although we did not find such a population of CD8 CD122 cells (data not shown), this may be due to interspecies differences. More specific research is needed at this point to clarify these issues and show a mechanistic connection regarding Tregs and third-party stem/progenitor cells., L, G: C- [) p+ s% Z2 v
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The immunosuppressive effects of PDMCs appear to be effective against mDCs, which are the most potent APCs and are crucially involved in allograft rejection. APCs are specialized immune cells capable of recognizing and processing antigens .
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: H! r5 o4 w6 d2 G% ] [The majority of studies to date show that cell-cell contact is not necessary for the inhibitory effects of BMMSCs . Indeed, the addition of neutralizing antibodies to IL-10 as well as TGF-ß partially reversed the inhibitory effects of PDMCs. The fact that only a partial reversal was seen may be indirect evidence that multiple mechanisms are responsible for the immunosuppression of PDMCs, some of which we have shown in this paper.
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1 ~, ?& W4 f8 o4 f. uAlthough IF- is an important mediator in the host inflammatory response to allograft transplantation, we found that pretreatment with this cytokine actually enhanced the immunosuppressive effects of PDMCs. PDMCs increase the secretion of TFG-ß and upregulate the inhibitory cell surface marker PD-L1 after IF- treatment. PD-L1 is a ligand of the recently discovered PD-1 pathway, which when activated leads to inhibition of T-cell receptor-mediated lymphocyte proliferation and cytokine secretion .
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5 M: _7 M( ?8 D/ QCONCLUSION
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PDMCs show wide-ranging in vitro immunosuppressive effects against various types of antigens and across both CD4 and CD8 cells. IF- stimulation of PDMCs actually enhanced the inhibitory effects rather than reduced it. PDMCs are an easily accessible and abundant supply of multipotent cells procured from a source without ethical controversy. The immunosuppressive properties now identified add further support for the consideration of these cells as a clinically viable source for cell therapy.
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DISCLOSURES
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Y-C.C., C-C.C., H-I.H., and B.L.Y. have jointly filed for U.S. patent application no. 11/032,153 and Taiwan patient application no. 94,100,920.
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' g" w9 c5 D) h2 L, H' }ACKNOWLEDGMENTS, e7 e6 f6 q# B" b7 l/ z* _
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This work was supported in part by an intramural grant from the National Health Research Institutes (94A1-SCPP05-002). C-J.C. and M-L.Y. contributed to this work equally.) H5 }$ [7 F, T. \6 l, G
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发表于 2015-8-27 14:01
