SEARCH

SEARCH BY CITATION

Keywords:

  • Immunogenicity;
  • Immunosuppression;
  • Adult stem cells;
  • Cell adhesion molecules;
  • Immunotherapy;
  • Interleukin;
  • Progenitor cells;
  • Stem cell-microenvironment interactions

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Human skin-derived precursors (hSKPs) are multipotent somatic stem cells that persist within the dermis throughout adulthood and harbor potential clinical applicability. In this study, we investigated their immunogenicity and immunosuppressive features, both in vitro and in vivo. As such, this study provides a solid basis for developing their future clinical applications. We found that hSKPs express HLA-ABC molecules, but not HLA-DR, rendering them poorly immunogenic. Using a coculture set-up, we could further demonstrate that hSKPs inhibit the proliferation of allogeneic activated T cells and alter their cytokine secretion profile, in a dose-dependent manner. Cotransplantation of hSKP and human peripheral blood leukocytes (PBL) into severe combined immune-deficient mice also showed a significant impairment of the graft-versus-host response 1 week post-transplantation and a drastic increase in survival time of 60%. From a mechanistic point of view, we found that hSKPs require cell contact as well as secretion of soluble inhibitory factors in order to modulate the immune response. The expression/secretion levels of these factors further increases upon inflammation or in the presence of activated T cells. As such, we believe that these features could be beneficial in a later allogeneic clinical setting, because rejection of engrafted allogeneic hSKP might be delayed or even avoided due to their own promotion of a tolerogenic microenvironment. Stem Cells 2014;32:2215–2228


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Human skin-derived precursors (hSKPs) are multipotent dermal stem cells that share many properties with embryonic neural crest stem cells [1, 2]. In particular, hSKPs are able to generate peripheral neuronal cells such as Schwann cells [3, 4] and catecholaminergic neurons [5] and mesenchymal cell types such as adipocytes, chondrocytes, and osteocytes [1, 6, 7]. In addition, hSKPs persist throughout adulthood in the human dermis [1, 8]. They can be isolated and expanded in large quantities from small skin biopsies of abdomen [8], breast [8], arm [9], foreskin [10], face [11], and scalp [11, 12] tissue. Importantly, preceding research showed that the gene expression profiles of hSKP populations derived from different skin sources are not only virtually identical, but also very distinctive from mesenchymal stromal cells (MSC) [10, 13]. As such, these cells represent key candidates for cell-based therapy and their clinical potential is currently being investigated in preclinical animal models of spinal cord injury [3, 14] and bone repair [6]. However, in any transplantation scenario, immunocompatibility needs to be considered. Yet, until now, no study has investigated the immunogenicity of hSKP. Furthermore, immune regulation by nonlymphoid cells, including different types of stem cells as well as fibroblast-like cells, is described as being an important component to maintain immune system tolerance [15-18]. These immunomodulatory cells possess immunosuppressive properties [15-18] through the regulation of different immune cells via activation of disparate mechanisms [15-18]. These mechanisms include the production of immunoregulatory factors including prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), interleukin (IL)-10, leukemia inhibitory factor (LIF), and HLA-G [15, 19, 20]. However, whether an allogeneic graft is accepted or rejected by the host's immune system mainly depends on its expression of major histocompatibility complex (MHC) antigens. These MHC antigens act as the primary targets of the immune system and are part of a two-signal-mediated activation of lymphocytes, including costimulatory molecules in order to trigger a full immune response leading to allograft rejection [21]. As such, an immune rejection response against allogeneic grafts derived from hSKP could present a major problem in a clinical setting unless their rejection can be delayed or avoided due to the promotion of a tolerogenic microenvironment. In this study, we define for the first time the immunophenotype of hSKP and investigate whether hSKPs are able to suppress the immune response, both in vitro and in vivo, of allogeneic activated peripheral blood mononuclear cells (PBMCs). Hereto, the interferon (IFN)-γ, IL-10, and immunoglobulin (Ig) G levels as well as the expression of costimulatory surface molecules were evaluated. Furthermore, we investigated the proliferation of allogeneic-activated PBMC and determined the survival rate of mice undergoing a graft-versus-host reaction, both in the presence and absence of hSKP. We also determined the impact of an inflammatory environment on the immunological properties of hSKP since (necro-)inflammation is often present in situations where cellular replacement therapy is required as for example in degenerative neurological disorders [22-24]. These inflammatory conditions, and more specifically the presence of the cytokine IFN-γ, might result in an increase of MHC class II antigens in hSKP-derived grafts and facilitate their rejection [25].

In order to understand why hSKPs are able to impair the immune response, we further determined the expression and secretion levels of several immune inhibitory factors (HGF, LIF, and PGE2) in hSKP under different experimental conditions, including inflammation and third party mixed leukocyte reactions (MLR). Finally, we investigated whether hSKPs require cell contact in order to perform their immune modulatory function.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Isolation and Cultivation of hSKP

hSKPs are isolated and subcultivated as previously described [10]. As all samples are derived from young children, informed consent of both parents is obtained. All experiments are approved by the Ethical Commission of the UZ-Brussels. The mean age of the donors is 3 ± 2 years (male, range 1–7 years old) and samples of a total of 12 donors were used throughout the experiments. Approximately 2 weeks after isolation, hSKP spheroids are broken down to single cells using 0.2 mg/ml Liberase DH (Roche, Vilvoorde, Belgium, http://www.roche-applied-science.com) for 12 minutes at 37°C. They are subsequently cultivated in hSKP growth medium supplemented with 5% (vol/vol) fetal bovine serum (FBS; Hyclone, Thermo Scientific, Erembodegem, Belgium, http://www.thermoscientific.com) to promote adherence of the cells to the plastic. The next day, the attached hSKPs are washed and further cultivated in hSKP growth medium without FBS until further use. A cell density of 2 × 104 cells per square centimeter is applied. hSKPs between passages 2 and 4 are used for further experiments.

qRT-PCR

The extraction, quantification, and reverse transcription of total RNA, the purification of cDNA purification as well as the qPCR reaction is performed as previously described [10]. The used gene expression assays are listed in Supporting Information Table S1. The estimation of the qPCR efficiency as well as the selection of the most stable reference gene(s) is conducted as previously described [10]. According to geNorm (Biogazelle NV, Zwijnaarde, Belgium, http://www.biogazelle.com), all six reference genes are sufficiently stable (M < 1.5). Therefore, the minimal optimal number of reference targets to be used in this experiment is 1 (V < 0.15). As such, GAPDH is further used to normalize the qPCR data. The expression of the target genes in hSKP is calculated as the percentage relative to the normalized hydroxy-methylbilane synthase (HMBS) expression.

Microarray Data Analysis

Total RNA is extracted using the TriPure Isolation Reagent (Roche, Vilvoorde, Belgium, http://www.roche-applied-science.com) and quantified at 260 nm using a Nanodrop spectrophotometer (Thermo Scientific, Erembodegem, Belgium, http://www.thermoscientific.com). The microarrays are performed as previously described using Affymetrix (Santa Clara, CA, http://www.affymetrix.com) Human Genome U133 plus 2.0 arrays [10]. Background correction, summarization (median polish), and normalization (quantile) are done with robust multiarray analysis [26]. Genes with a fold change >2 and p-value < 0.05 are selected for further analysis. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE48757. Gene ontology enrichment analyses are performed using the PARTEK (Saint Louis, MO, http://www.partek.com) Genomics Suite (version 6.6). Processes with an enrichment score >3 are considered to be significantly enriched (p < 0.05). Determination of upstream regulators (UR) and modulated canonical pathways is performed by ingenuity pathways analysis (version SEP 2011; Ingenuity Systems, Qiagen, Redwood City, CA, http://www.ingenuity.com) using Benjamini-Hochberg (B-H) multiple testing corrected p-values (fold change >2; B-H p-value < .05). An activation z-score is calculated as a measure of functional and translational activation in upstream regulators analysis. An absolute z-score of below (inhibited) or above (activated) 2 was considered as significant. The p-value of overlap is indicative for the percentage of modulated genes consistent with the activation status of the upstream regulator and is considered significant below 0.05.

Immunocytochemistry

Immunostainings are performed as previously described [27]. The respective primary and secondary antibodies are listed in Supporting Information Table S2.

Flow Cytometry

hSKPs are phenotypically characterized by flow cytometry as previously described [28] on a MacsQuant analyzer (Miltenyi Biotec, Leiden, The Netherlands, http://www.miltenyibiotec.com) using conjugated monoclonal antibodies against (a) hSKP cell surface markers, (b) endothelial and stromal surface markers, (c) human leukocyte antigens, (d) costimulatory molecules, (e) cell-cell interaction-associated molecules, and (f) other relevant immune regulatory molecules. Furthermore, the T-cell expression profile of the major costimulatory molecules involved in T-cell activation is assessed during a coculture with hSKP. As such, the percentage of positive T lymphocytes is determined for each marker. Intracellular staining is accomplished by treating the cells first with TrypleSelect solution (Lonza, Basel, Switzerland, http://www.lonza.com) followed by washing in MACS buffer (Miltenyi Biotec, Leiden, The Netherlands, http://www.miltenyibiotec.com). Thereafter, hSKPs are fixed and permeabilized using the FIX & PERM reagents according to the manufacturer's instructions (Life Technologies, Gent, Belgium, http://www.lifetechnologies.com). After one wash step in MACS buffer, intracellular staining of hSKP is done by incubation with the corresponding monoclonal antibody for 30 minutes at room temperature. Next, hSKPs are washed in MACS buffer and analyzed by flow cytometry. The conjugated monoclonal antibodies are listed in Supporting Information Table S2.

Proinflammatory Stimulation

The impact of an inflammatory environment on hSKP is evaluated as previously described [29]. Briefly, hSKPs are stimulated for 18 hours using a cocktail of proinflammatory cytokines: 25 ng/ml IL-1β (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), 1 × 103 U/ml IFN-γ, 50 ng/ml tumor necrosis factor alpha (TNFα), and 3 × 103 U/ml IFN-α (all from Prospec, Inc., Ness-Ziona, Israel, http://www.prospecbio.com) and consequently characterized by flow cytometry.

Acquisition of Immune Cells for In Vitro Assays

Peripheral blood (PB) samples are collected from healthy donors after informed consent was obtained. PBMCs are isolated by Ficoll-Hypaque (Sigma-Aldrich, Diegem, Belgium, http://www.sigmaaldrich.com) gradient centrifugation of PB. Total CD3+ T lymphocytes are purified by positive selection using the MACS system (Miltenyi Biotec, Leiden, The Netherlands, http://www.miltenyibiotec.com) according to the manufacturer's instructions. The purity of the selected cells is always above 95% as determined by flow cytometry.

Immunogenicity Assay

To assess their immunogenicity, allogeneic hSKPs (1 × 105; irradiated 25 Gy) are cultured for 5 days in the presence of PBMC as responder cells (1:1 cell ratio). Then, the proliferation of PBMC is assessed by 5-bromo-2-deoxy-uridine (BrdU) incorporation (Roche, Vilvoorde, Belgium, http://www.roche-applied-science.com).

Immunosuppressive Assays

To assess their immunosuppressive potential, MLRs are conducted in the presence (1:1 cell ratio) and absence of hSKP. During MLR, irradiated (25 Gy) allogeneic PBMC are used to stimulate CD3+ T-lymphocytes (1 × 105). Upon initiation, the MLR is added to plated (1-day-ahead cocultures) irradiated hSKP and incubated for 5 days.

To evaluate the dose-dependent immunosuppressive effect of hSKP, T cells are first activated with 5 µg/ml of phytohemagglutinin (PHA; Thermo Scientific, Erembodegem, Belgium, http://www.thermoscientific.com) and 20 U/ml of interleukin-2 (IL-2; Biotest AG, Dreieich, Germany, http://www.biotest.de). Subsequently, different T-cell/hSKP ratios (5:1; 10:1; 20:1 cell ratios) are investigated.

To determine the importance of direct cell contact in the suppressive effect observed for hSKP, a Transwell system (Transwell Permeable Supports Corning, Tewksbury, MA, http://www.corning.com) is used to separate T-lymphocytes and hSKP (5:1 cell ratio). The system consists of a polycarbonate membrane with a 0.4 µm pore size avoiding any direct contact between hSKP and T cells, but allowing soluble factors to reach the cells. The lower chamber of each well contains adherent hSKP and stimulated T cells are plated in the upper chamber. As HGF is the major immune-regulatory molecule that is expressed and positively modulated by hSKP, 20 µg/ml of HGF neutralizing antibodies (R&D Systems, Abingdon, UK, http://www.rndsystems.com) is added to the cocultures to investigate whether HGF is a key player in the immune suppressive effect of hSKP.

BrdU-Based Proliferation Assay

Lymphocyte proliferation is assessed by BrdU incorporation using a colorimetric assay according to the manufacturer's instructions (Roche, Vilvoorde, Belgium, http://www.roche-applied-science.com). Hereto, 50 mM BrdU is added to the cocultures at day 4. T-cell proliferation is expressed by the proliferation index, which is defined as the ratio between the optical density of activated T-cell proliferation and the optical density of nonactivated T cells, after eliminating the background.

Carboxyfluorescein Succinimidyl Ester-Based Proliferation Assay

T-cell proliferation is assessed by CFDA-SE (carboxyfluorescein diacetate N-succinimidyl ester) labeling using the CellTrace CFSE Cell proliferation kit (Life Technologies, Gent, Belgium, http://www.lifetechnologies.com) and following the protocol described by the manufacturer. Briefly, 107 CD3+ lymphocytes are stained with 10 µM of CFDA-SE prior to their coculture with hSKP. After 5 days of coculture, the carboxyfluorescein succinimidyl ester (CFSE)-based fluorescence is analyzed by flow cytometry. T cells are activated with 5 µg/ml of PHA (Thermo Scientific, Erembodegem, Belgium, http://www.thermoscientific.com) and 20 U/ml of IL-2 (Biotest AG, Dreieich, Germany, http://www.biotest.de).

Cotransplantation of PBMC and hSKP in Severe Combined Immune-Deficient Mice

Human PBMC (with or without CFSE labeling) are isolated from a buffy coat (Blood Transfusion Centre—Ghent) by isopycnic density gradient centrifugation. Hu-PBL-severe combined immune-deficient (SCID) mice are produced essentially as described before [30]. Briefly, 1 day before transplantation, nonobese diabetic (NOD)-SCID mice (NOD/LtSz-Prkdcscid/Prkdcscid) receive total body irradiation (3 Gy) and are injected intraperitoneally with 1 mg of TM-β1; a rat monoclonal antibody that targets the β-chain of the murine IL-2 receptor. It was previously shown that TM-β1 pretreatment efficiently depletes mouse natural killer cells in vivo [31]. In the spleen of the NOD-SCID mice either 5 × 106 PBMC, 1 × 106 hSKP, or a mixture of 5 × 106 PBMC and 1 × 106 hSKP (5:1 cell ratio) is injected. One week after transplantation, mouse EDTA plasma is collected and the concentration of human IL-10 and IFN-γ is determined using the human IL-10 and human IFN-γ Enhanced Sensitivity Flex Set CBA test (BD Biosciences, Erembodegem, Belgium, http://www.bdbiosciences.com), respectively. Human IgG is measured with a human IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, http://www.bethyl.com) according to the protocol provided by the manufacturer. The percentage CFSEneg and CD45+ cells in the spleen and blood is determined by flow cytometry.

Cytokine Secretion

The production of relevant immunoregulatory factors by hSKP during their coculture with allogeneic activated T cells (MLR) and/or after their inflammatory stimulation is assessed by ELISA quantification. HGF (RayBiotech, Norcross, GA, http://www.raybiotech.com), IL-10, IFN-γ, LIF, and PGE2 (R&D Systems, Abingdon, UK, http://www.rndsystems.com) levels are determined in culture medium according to the manufacturer's instructions.

Statistical Analysis

For the in vitro experiments, the results are expressed as the mean ± SEM. Statistical comparisons are performed using the Wilcoxon test for paired samples. A p-value less than 0.05 was considered statistically significant. To assess whether the in vivo observed difference in human IFN-γ, IL-10, and IgG levels between the different transplantation groups is statistically significant, the data are analyzed using the unpaired nonparametric two-tailed Mann-Whitney U test (Prism v5.0d, Graph-Pad Software, La Jolla, CA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Immunophenotype of hSKPs

To characterize the investigated cell population, the expression of hSKP markers is assessed. Immunocytochemistry shows that the cells used in these experiments indeed express the typical hSKP markers, being the neural crest-related transcription factors paired box 3 (Pax3), snail homolog 1 (Snail), muscle segment homeobox 1 (Msx1), and the intermediate filament protein nestin (Fig. 1A) [1]. This distinguishes them from bone marrow MSC (Supporting Information Fig. S1). In addition, flow cytometry shows that the investigated cell population is homogeneous as indicated by the high percentage of cells staining positive for nestin (95.33% ± 1.45%), vimentin (99.00% ± 0.00%), and fibronectin (96.33% ± 0.14%) (Fig. 1B). To determine the immunophenotype of hSKP, flow cytometry was performed for a number of typical cell surface markers. Immunostainings for endothelial and stromal surface markers show that hSKPs do not express CD34 (0.60% ± 0.09%), but that CD90 and CD105 are expressed by 79.20% ± 6.97% and 48.63% ± 9.70% of the cell population, respectively (Fig. 2A). Furthermore, expression of the human leukocyte antigens HLA-ABC (96.11% ± 1.46%) and intracellular (i) HLA-G (77.28% ± 4.56%) is observed for most hSKP. In contrast, nearly no expression could be detected for HLA-DR (1.16% ± 0.06%), membrane-bound (m) HLA-G (3.00% ± 0.50%), and the leukocyte common antigen CD45 (1.84% ± 0.27%) (Fig. 2B). The only costimulatory molecule found to be expressed, although only in a limited fraction of hSKP, is CD40 (16.30% ± 4.66%). No significant expression is observed for CD80, CD86, CD134, and CD252 (Fig. 2C). As interactions between immune cells and immunomodulating cells are of key importance, we further investigated the expression by hSKP of cell-cell interaction-associated molecules, commonly referred to as cell adhesion molecules (CAM). The results show that several CAM are constitutively and highly expressed in hSKP including CD29 (96.30% ± 0.95%), CD44 (97.57% ± 0.35%), CD49e (99.00% ± 0.00%), CD54 (57.57% ± 5.43%), and CD166 (67.16% ± 4.6%). CD58 (38.25% ± 2.82%), CD62E (16.83% ± 1.06%), and CD146 (11.63% ± 3.32%), however, are only found in a smaller percentage of the cells. In contrast, no constitutive expression of CD31, CD102, and CD106 could be observed (Fig. 2D, 2E). With respect to relevant immunoregulatory molecules, flow cytometry reveals that the majority of hSKP expresses high levels of CD73 (99.00% ± 0.00%) and HO-1 (79.00% ± 7.82%), but not of CD39, CD200, CD200R, CD271, CD274, and CXCR4 (Fig. 2F).

image

Figure 1. Characterization of hSKP. hSKPs are characterized (A) by the expression of the neural crest-related transcription factors Pax3, Msx1, and Snail and the neural progenitor marker nestin using immunocytochemistry (scale bar = 50 µm) and (B) by the stable expression of the cell surface markers fibronectin, vimentin, and nestin using flow cytometry both in normal and in proinflammatory conditions. The values are expressed as mean ± SEM of six different hSKP donors. Abbreviations: hSKPs, human skin-derived precursors; Msx1, muscle segment homeobox homolog 1; Pax3, paired box 3; Snail, snail homolog 1.

Download figure to PowerPoint

image

Figure 2. Immunophenotype of hSKP in normal and inflammatory conditions. The immunological profile of hSKP is determined in normal and inflammatory conditions by flow cytometry for (A) endothelial and stromal surface markers, (B) human leukocyte antigens, (C) costimulatory molecules, (D, E) cell-cell interaction-associated molecules, and (F) immune regulatory molecules. The values are expressed as mean ± SEM and originate from six different hSKP donors. * Significantly increased percentage of hSKP expressing the protein of interest versus controls (p-value < .05). ** Significantly increased percentage of hSKP expressing the protein of interest versus controls (p-value < .01). Abbreviations: CD, cluster of differentiation; CXCR4, stromal cell-derived factor 1 receptor; HLA, human leukocyte antigen; HO, heme oxygenase; hSKPs, human skin-derived precursors; I, intracellular; m, membrane-bound.

Download figure to PowerPoint

hSKPs Adjust Their Immunophenotype in an Inflammatory Environment

To assess the impact of an inflammatory environment on the immunological phenotype of hSKP, cells are stimulated with a cocktail of proinflammatory cytokines. At the mRNA level, analysis of microarray data using ingenuity pathway analysis software confirms the activation state of the proinflammatory cytokines IFN-γ, TNF, IFN-α2, and IL-1β in proinflammatory stimulated hSKP (hSKP-INFL) versus the control condition (z-score > 2). At least 70% of the genes known to be affected by these cytokines are modulated consistent with their activation in hSKP-INFL (p-value of overlap < .05). Furthermore, gene ontology analysis shows, within the class of biological processes, a strong enrichment of the group of immune system processes. Within this group, several gene systems related to antigen presentation and immune response activation are strongly enriched (enrichment score > 3; Supporting Information Fig. S2). Several canonical pathways related to immunology are also found to be significantly modulated in hSKP by inflammation (B-H p-value < .05) of which the antigen presentation pathway is the most affected (Table 1). As a result, 1,871 and 2,779 probesets are found to be significantly upregulated and downregulated in hSKP-INFL, respectively (p-value < .05 and fold change >2; Supporting Information Tables S3, S4). At the protein level, no loss of cell surface markers, expressed by default in hSKP, could be observed in hSKP-INFL. More specifically, the hSKP markers nestin, vimentin, and fibronectin remain unchanged (Fig. 1B). In addition, no significant changes are observed for the expression of the stromal surface markers CD90 and CD105 (Fig. 2A), the human leukocyte antigens HLA-ABC and iHLA-G (Fig. 2B), the cell adhesion molecules CD29, CD44, CD49e, and CD166 (Fig. 2D, 2E) and the immune regulatory molecules CD73 and HO-1 (Fig. 2F). Compared to hSKP under normal conditions, under inflammatory stimulation, hSKPs significantly increase their expression of mHLA-G (22.00% ± 2.40%) (Fig. 2B), the costimulatory molecule CD40 (57.14% ± 3.01%) (Fig. 2C), the cell adhesion molecules CD54 (98.80% ± 0.20%), CD58 (92.50% ± 1.50%), CD62E (79.66% ± 3.05%), and CD106 (74.00% ± 4.02%) (Fig. 2D, 2E), and the immune regulatory molecule CD274 (77.86% ± 3.54%) (Fig. 2F). In addition, a nonsignificant, albeit twofold increased expression of CD146 (39.33% ± 7.70%), is observed (Fig. 2E). Even after inflammatory stimulation, hSKPs remain negative for CD34 (Fig. 2A), HLA-DR and CD45 (Fig. 2B), the costimulatory molecules CD80, CD86, CD134, and CD252 (Fig. 2C), the cell adhesion molecules CD31 and CD102 (Fig. 2D, 2E), and the immune regulatory molecules CD39, CD200, CD200R, CD271, and CXCR4 (Fig. 2F).

Table 1. Proinflammatory stimulation of hSKP modulates several of their immunology-related pathways
URPredicted activation stateActivation z-scorep-Value of overlapNo. of modulated genes consistent with activation of URTotal no. of genes modulated by URPercentage of modulated genes consistent with activation of URFold change of UR
IFNGActivated9.4732.63E-1819326572.831.1
TNFActivated9.0561.36E-1322932770.034.9
IFNA2Activated8.1281.21E-24799880.611.1
IL1BActivated7.6451.93E-0811817069.414.2
Modulated canonical pathways in hSKP by inflammationB-H p-valueNo. of modulated genes in pathwayTotal genes in pathwayPercentage of modulated genes in pathway
  1. hSKPs are exposed for 18 hours to a cocktail of proinflammatory cytokines (IL-1β, IFN-γ, TNFα, and IFN-α). Microarray analyses confirm the activated status of these proinflammatory cytokines (z-score > 2); ≥70% of the genes known to be affected are modulated consistent with their activation (p-value of overlap <.05). In hSKP, several canonical pathways related to immunology are significantly modulated by inflammation (B-H p-value <.05). The data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE48757. The results represent three different hSKP donors.

  2. Abbreviations: B-H, Benjamini-Hochberg; hSKPs, human skin-derived precursors; UR, upstream regulator.

Inflammation versus control
Antigen presentation pathway4.35E-06214052.50
Interferon signaling7.40E-045520027.50
IL-9 signaling5.83E-03164040.00
CD40 signaling5.83E-03247034.29
GM-CSF signaling5.83E-03246835.29
IL-3 signaling1.20E-02257433.78
fMLP signaling in neutrophils1.64E-023312925.58
CD27 signaling in lymphocytes1.72E-02195733.33
Role of NFAT in regulation of the immune response1.72E-024519822.73
IL-6 signaling1.72E-023512428.23
IL-17 signaling2.19E-02247432.43
Macropinocytosis signaling2.19E-02237630.26
NF-kB activation by viruses2.40E-02248229.27
IL-15 signaling2.40E-02216731.34
OX40 signaling pathway2.40E-02179418.09
TREM1 signaling2.74E-02197126.76
NF-kB signaling2.84E-024317524.57
Role of PKR in interferon induction and antiviral response2.90E-02154632.61
Activation of IRF by cytosolic pattern recognition receptors3.07E-02207227.78
Cytotoxic T lymphocytes-mediated apoptosis of target cells3.48E-02188521.18
IL-2 signaling3.51E-02185831.03
IL-15 production3.94E-02113135.48
PI3K signaling in B lymphocytes4.16E-023414024.29
Regulation of IL-2 expression in activated and anergic T lymphocytes4.50E-02238925.84
Clathrin-mediated endocytosis signaling4.79E-024619623.47

hSKPs Impair the Activation and Proliferation of PBMC

hSKPs do not initiate an allogeneic lymphocyte proliferative response as no significant proliferation of PBMC could be observed during their coculture (data not shown). Interestingly, hSKPs, used as a third-party cell, modulate the allogeneic lymphocyte response during a MLR in vitro. Moreover, hSKPs induce the suppression of lymphocytes as indicated by the absence of expanded T-cell colonies (Fig. 3A, 3B). As a consequence, a significant 50% inhibition of T-cell proliferation (Fig. 3C; from 8.71 ± 0.80 to 4.54 ± 0.48) is achieved. hSKPs are also able to alter the cytokine secretion profile of stimulated T-lymphocytes as well as their expression of different lymphocyte costimulatory molecules. More specifically, the in vitro secretion of the proinflammatory cytokine IFN-γ by allogeneic activated T cells is significantly decreased whereas the in vitro anti-inflammatory cytokine IL-10 levels are significantly increased in the presence of hSKP (Fig. 3D, 3E). Upon stimulation, a high percentage of T cells expresses the costimulatory molecules CD27 (72% ± 15%), CD134 (75% ± 8%), and CD154 (40% ± 5%). However, in the presence of hSKP, the percentage of T cells expressing CD27 (29% ± 5%), CD134 (28% ± 5%), and CD154 (17% ± 3%) is significantly decreased by more than twofold (Fig. 3F–3H).

image

Figure 3. hSKPs suppress allogeneic activated T-cell proliferation and activation in vitro. Allogeneic MLR are performed in the presence and absence of hSKP for 5 days. The mean proliferation index is determined using BrdU incorporation. Production of cytokines is assessed on cell supernatants. (A): Phase-contrast photomicrograph of proliferating allogeneic activated T cells. (B): Phase-contrast photomicrograph of allogeneic activated T-cell proliferation in the presence of hSKP. (C): Mean proliferation index of T-lymphocytes in the absence and presence of hSKP. In the presence of hSKP, allogeneic activated T-cell proliferation is suppressed. (D): Presence of hSKP decreases the secretion of the proinflammatory cytokine IFN-γ by T cells. (E): Presence of hSKP increases the secretion of the anti-inflammatory cytokine IL-10 by T cells. * Significantly decreased activated T-lymphocyte proliferation versus controls (p-value < .05). ** Significantly increased or decreased secretion level of cytokine versus controls (p-value < .01). The values are expressed as mean ± SEM; six different hSKP donors were involved. (F–H): Flow cytometric histogram plots of different lymphocyte costimulatory molecules expressed on the surface of not-stimulated (NST) and stimulated T cells in the absence (ST) or presence of hSKP (ST+hSKP). The results shown are representative for three different hSKP donors. Abbreviations: hSKPs, human skin-derived precursors; IFN, interferon; IL, interleukin; MLR, mixed leukocyte reaction; NST, not-stimulated T cells; ST, stimulated T cells; ST+hSKP, stimulated T cells in the presence of hSKP.

Download figure to PowerPoint

Next, cotransplantation of human PBMC and hSKP (cell ratio 5:1) into severe combined immunodeficiency mice shows a significant impairment of the activation of both T and B cells 1 week post-transplantation. Activated T cells, in the absence of hSKP, secrete into the plasma significant amounts of IFN-γ (2.63 ± 0.51 ng/ml) and IL-10 (13.36 ± 3.60 pg/ml) whereas activated B cells produce significant levels of IgG (192.96 ± 27.70 µg/ml). However, in the presence of hSKP, significantly lower plasma levels of IFN-γ (0.31 ± 0.06 ng/ml), IL-10 (0.88 ± 0.04 pg/ml), and IgG (37.80 ± 8.53 µg/ml) are detected (p-value < .001). Importantly, hSKPs do not significantly produce IFN-γ (<0.82 pg/ml), IL-10 (<0.82 pg/ml), and IgG (<20 µg/ml) as all plasma levels remain below the limit of quantification in the absence of PBMC (Fig. 4A–4C). The proliferation of CFSE-labeled PBMC seems to be impaired in vivo in the presence of hSKP as a significantly approximately twofold lower percentage of CFSEneg cells is found to circulate in the blood of cotransplanted animals 1 week post-transplantation (Fig. 4D). Furthermore, a significant impairment of the expansion of the CD45+ leukocyte compartment of PBMC is observed in the presence of hSKP. Indeed, a significant reduction of both the percentage and absolute number of resident CD45+ cells is noted in the spleen of cotransplanted mice. More specifically, 6.97 ± 2.13 × 106 CD45+ cells can be detected in the spleen of mice that are transplanted with PBMC, whereas only 1.17 ± 0.50 × 106 CD45+ cells are found after cotransplantation with hSKP (Fig. 4E, 4F). These immunosuppressive effects of hSKP eventually lead to a significant increase in the survival of mice suffering from a graft-versus-host reaction, inflicted by the transplanted xenogenic human PBMC. In the absence of hSKP, the median survival time is estimated at 17.5 days after intrasplenic injection of 5 × 106 PBMC whereas upon cotransplantation with 1 × 106 hSKP the animals survive for at least 28 days (Fig. 4G).

image

Figure 4. hSKPs impair PBMC proliferation, activation, and maturation in vivo. PBMC, hSKP, or their mixture (5:1 cell ratio) is injected in the spleen of irradiated NOD-SCID mice. One week post-transplantation, production of human cytokines and human IgG is measured in collected plasma samples. The presence of hSKP decreases the plasma levels of (A) the proinflammatory cytokine IFN-γ, (B) the anti-inflammatory and B cell inducing cytokine IL-10 and (C) human IgG produced by activated B cells. In addition, the presence of hSKP decreases (D) the proliferation of circulating PBMC, represented by CFSEneg cells in the blood, and (E, F) the maturation of PBMC in the spleen, represented by CD45+ cells. (G): Cotransplantation of PBMC with hSKP (5:1 cell ratio) significantly prolongs the survival of animals suffering from a graft-versus-host reaction in contrast to the control condition (PBMC only). This is shown by a Kaplan-Meier plot. The results represent three independent experiments. * Significantly decreased in the presence of hSKP versus control (p-value < .05). ** Significantly decreased in blood versus spleen (p-value < .05). Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; hSKPs, human skin-derived precursors; LOQ, limit of quantification; PBMC, peripheral blood mononuclear cell.

Download figure to PowerPoint

hSKPs Exhibit the Machinery Required for Immunomodulation and Immunosuppression

In order to understand why hSKPs are able to impair the activation of T and/or B cells, mechanistic insight is needed. Therefore, qPCR is performed to first determine the presence of key factors involved in immunoregulation. It is found that hSKP constitutively express transcripts that are required to modulate an immune response, namely hSKPs express PTGS1, PTGS2, and HGF at similar, or even higher levels, compared to the well-known housekeeping gene HMBS (Fig. 5A). In contrast, constitutive HO-1 and LIF expressions are approximately 10-fold lower compared to HMBS (Fig. 5A). Interestingly, hSKPs also significantly differ from bone marrow-derived MSC in their expression of these immune regulatory molecules. More specifically, hSKPs express significantly higher levels of HGF, PTGS1, and HLA-G compared to bone marrow-derived MSC whereas the gene expression of LIF is significantly lower (Supporting Information Fig. S1B). In order to attribute to hSKP immunomodulating and/or -suppressing properties, it must be shown that hSKPs express and secrete a panel of immunoregulatory factors. Consequently, the secretion of HGF, PGE2, and LIF by hSKP has been assessed by ELISA. It is found that the production of these factors, which either is constitutive or inductive, is modulated by hSKP and depends on the surrounding microenvironment (Fig. 5B–5D). As such, HGF is constitutively secreted by hSKP (1,104 ± 31 pg/ml) and in response to a proinflammatory signal or during MLR coculture, HGF secretion is further significantly increased by more than 5.9-fold (6,547 ± 398 pg/ml) and 2.5-fold (3,049 ± 144 pg/ml), respectively (Fig. 5B). The constitutive secretion of HGF is also more than threefold higher compared to bone marrow-derived MSC (Supporting Information Fig. S1C). In contrast, PGE2 and LIF are not constitutively secreted by hSKP, but their secretion is highly induced in the presence of MLR (Fig. 5C, 5D). Indeed, during MLR, PGE2 and LIF secretion levels reach values of 11,018 ± 726 pg/ml and 726 ± 116 pg/ml, respectively (Fig. 5C, 5D). Proinflammatory stimulation does not have a significant impact on secretion of LIF by hSKP (Fig. 5D), but significantly induces PGE2 secretion levels (9,037 ± 533 pg/ml) (Fig. 5C). Important to mention is the fact that no secretion of HGF, LIF, or PGE2 could be detected in the absence of hSKP (Fig. 5B–5D).

image

Figure 5. hSKPs exhibit the machinery required for immunomodulation. The relative normalized expression of genes involved in immunoregulation and immunomodulation is determined in hSKP. The expression of the target genes in hSKP is calculated as the percentage relative to the normalized HMBS expression. Cytokine production is measured in cell supernatants of hSKP cultured alone, in the presence of a MLR or stimulated by an inflammatory environment (ELISA). (A): hSKPs express several genes involved in immunomodulation. (B): hSKPs constitutively secrete HGF and that secretion is significantly increased during inflammation and MLR. (C): PGE2 secretion by hSKP is induced during inflammation and MLR. (D): hSKPs secrete LIF during MLR. * Significantly increased secretion level of cytokine versus control values (p-value < .05). ** Significantly increased or decreased secretion level of cytokine versus control values (p-value < .01). The results are expressed as mean ± SEM. Samples of six different hSKP donors were used. Abbreviations: HGF, hepatocyte growth factor; HMBS, hydroxy-methylbilane synthase; HO, heme oxygenase; hSKPs, human skin-derived precursors; LIF, leukemia inhibitory factor; MLR, mixed leukocyte reaction; PG, prostaglandin; PTGS, prostaglandin-endoperoxide synthase.

Download figure to PowerPoint

In order to unambiguously ascribe immunosuppressive effects to hSKP, it is further investigated whether these effects (a) are dependent on hSKP cell numbers, (b) require cell contact, and (c) are a result of secretion of soluble inhibitory molecules. As such, stimulated CD3+ T cells are labeled with CFSE and cultured under different conditions in the presence and absence of hSKP (Fig. 6A–6J). In the presence of hSKP, not only the proliferation of activated T cells is significantly lower but also the inhibition of T-cell proliferation is dependent on the T-cell/hSKP cell ratio. More specifically, a ratio of 5:1 results in a T-cell inhibition of 55% ± 3%, whereas ratio's of 10:1 (36% ± 2%) and 20:1 (18% ± 1%) both result in a significantly lower inhibition of the proliferation of activated T cells (Fig. 6A–6D, 6I). Furthermore, it is noted that hSKPs require direct cell contact to optimally perform their immunosuppressive effects since a significantly lower T-cell inhibition could be observed when a transwell system is being used (24% ± 2%) in which direct cell contact between both cell populations is avoided (Fig. 6A, 6B, 6H, 6J). Finally, addition of HGF-neutralizing antibodies to the cocultures shows that when HGF—secreted by hSKP—is neutralized, a significantly lower T-cell inhibition (27% ± 5%) could be observed (Fig. 6A, 6B, 6E, 6F, 6J).

image

Figure 6. The importance of cell contact and ratio, and the role of HGF in the immune suppressive effect of hSKP. CFSE-labeled T cells are cocultured with hSKP at different T-cell/hSKP ratios (5:1; 10:1; 20:1). The importance of cell contact and the role of HGF in the suppressive effect are evaluated at 5:1 T-cell/hSKP ratio. CFSE fluorescence was analyzed by flow cytometry. Representative CFSE histogram plot for (A) stimulated T cells in the absence of hSKP, (B–D) stimulated T cells in the presence of hSKP at T-cell/hSKP cell ratios of 5:1, 10:1, and 20:1, respectively, (E, F) stimulated T cells treated with anti-HGF neutralizing antibodies in the absence and presence of hSKP, (G) not-stimulated T cells, and (H) stimulated T cells in the presence of hSKP without cell contact. The presence of hSKP significantly decreased the proliferation of stimulated T cells and depended on (I) cell ratio, (J) cell contact, and secretion of HGF. The data are presented as the mean percentage of T-cell proliferation ± SEM and samples came from three different hSKP donors. * Significantly decreased percentage T-cell proliferation in the presence of hSKP versus stimulated T cells (p-value < .05). ** Significantly increased percentage T-cell proliferation versus stimulated T cells in the presence of hSKP at 5:1 cell ratio (p-value < .05). Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; HGF, hepatocyte growth factor; hSKPs, human skin-derived precursors.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

hSKPs are a multipotent stem cell population that resides within the dermis throughout adulthood and shares several properties with embryonic neural crest stem cells [1, 3-6, 32]. In addition, they express several transcription factors also found in neural crest stem cells [33] including Pax3, Snail, Msx1, the intermediate filament proteins nestin and vimentin, and the glycoprotein fibronectin [1]. Most importantly, they are characterized by a high expression of Pax3 [1], a transcription factor involved in the neural crest-driven development of craniofacial structures [34] and melanocytes [35], and nestin [36], a marker for progenitor cells of the nervous system [37] that clearly distinguishes them from bone marrow-derived MSC. As hSKP can be easily isolated, virtually from any patient and expanded in large quantities for long-term use [1, 38], they have the potential for clinical cell therapy [4, 6]. However, until now, no data exist concerning their immunological features. Yet before hSKP can be used in a clinical setting, it is essential to first establish their immunophenotype, identify potential immunomodulating properties, and define the impact of a proinflammatory environment on their immunological features. In this study, we could show that hSKPs exhibit an endothelial/stromal cell profile similar to that of MSC. Indeed, independent of inflammation, hSKP uniformly did not express the endothelial marker CD34 and were constitutively positive for the stromal cell-associated markers CD90 and CD105, which are important to ensure and maintain an efficient immunomodulating cell population [39, 40].

Immunogenicity of hSKP

Clinical use of hSKP implies that these cells should be poorly to nonimmunogenic and thus their immunological profile needs to be defined. We show here for the first time, that during coculture with PBMC, hSKP failed to induce an allogeneic T-lymphocyte response. In addition, hSKP constitutively expressed high levels of HLA class I (HLA-ABC), but lacked the expression of HLA class II (HLA-DR) and CD45 molecules which are required for T-lymphocyte activation. Importantly, although proinflammatory stimulation significantly modulated the antigen presentation pathway in hSKP at the mRNA level, no impact on their HLA-ABC and HLA-DR expression was observed at the protein level. Due to the absence of HLA-DR, hSKP did not possess antigen-presenting functions and could therefore not activate lymphocytes. Interestingly, a small percentage of hSKP constitutively expressed the costimulatory molecule CD40, a transmembrane receptor of the TNF superfamily that is present on a variety of antigen-presenting cells [41, 42]. CD40 binds to its ligand CD40L, which is transiently expressed on activated T cells under inflammatory conditions, to facilitate the immune response [43]. Under proinflammatory conditions, a significant inducible expression of CD40 was observed for hSKP. This increase can be ascribed to the presence of IFN-γ in the culture medium, which is a well-known inducer of CD40 transcription [44]. However, increase of CD40 expression is not sufficient to solely induce a full T-lymphocyte activation since a number of other costimulatory molecules are additionally needed as this activation pathway is tightly regulated by a subset of inhibitory molecules [45, 46]. In contrast, even upon proinflammatory stimulation of hSKP, we could not observe any expression of the panel of costimulatory molecules critical for immune response activation, including CD80, CD86, CD134, and CD252. Therefore, even in the presence of inflammation, hSKP could be considered as poorly immunogenic because they are not able to initiate a full immune response due to the lack of several crucial signals.

Immunosuppression

In addition to be poorly immunogenic, hSKPs possess valuable immunosuppressive features, both in vitro and in vivo. Indeed, they downmodulated the expression of the lymphocyte costimulatory surface molecules CD27, CD134, and CD154 on stimulated T cells. Also, in the presence of hSKP, (a) the intensity of an allogeneic MLR was substantially reduced in vitro, (b) the in vivo activation of T cells was significantly suppressed as evidenced by reduced IFN-γ plasma levels, and (c) the in vivo secretion of human IgG by activated B cells was drastically reduced, confirming the potential of hSKP to suppress an immune response. Moreover, besides a significantly lower percentage of human CFSEneg PBMC in the blood circulation, we also found a significantly lower number of CD45+ leukocytes in the spleen upon cotransplantation with hSKP. These results show a clear immunosuppressive effect of the graft-versus-host reaction by hSKP that led to a 60% increase in survival time.

Mechanisms of Immunosuppression

Having demonstrated that hSKPs are able to inhibit allogeneic lymphocyte responses, we further explored the possible mechanisms underlying these effects. Therefore, we screened hSKP for a panel of surface and soluble molecules known to regulate an immune response.

Cellular Interactions

It was found that hSKPs require direct cell contact with T cells in order to optimally perform their immunosuppressive effect on T-cell proliferation. Direct cell interactions between hSKP and T cells are possible due to the expression of a large number of surface molecules that are known to be responsible for cellular interactions via binding to receptors on T cells. More specifically, CD44, a receptor for hyaluronic acid, as well as the integrin very late activation protein (VLA)-β (CD29) and VLA-α5 (CD49e) was constitutively expressed by all hSKP, whereas the intercellular adhesion molecule 1 (CD54), lymphocyte function-associated antigen 3 (CD58), leukocyte endothelial cell adhesion molecule 2 (CD62e), melanoma cell adhesion molecule (CD146), and activated leukocyte cell adhesion molecule (CD166) were only expressed in a smaller number of hSKP. Interestingly, we showed that hSKPs do not express other cell surface molecules such as the platelet endothelial cell adhesion molecule 1 (CD31), the intercellular adhesion molecule 2 (CD102), and the vascular cell adhesion molecule 1 (CD106). Upon exposure of hSKP to a proinflammatory environment, a selective increased expression of CD54, CD58, and CD62E was observed. In particular, we demonstrated the notable induction of CD106 expression. This is in agreement with previous reports stating that the expression of adhesion molecules is dependent on culture conditions [47] and can be modulated under certain circumstances including inflammation [48]. In addition, interactions between immune effector cells and target cells are mainly regulated by CAM. As such, hSKPs are able to actively participate in inflammatory and immune reactions by adapting their functional pattern of CAM in response to the microenvironment [49-52]. In general, these observations argue that hSKPs express a large and modulated panel of CAM allowing them to sense different environmental signals, in particular to interact with activated immune cells and to respond to any inflammatory stimulus.

Modulation of Helper T-Cell Subset Balance

The identification of helper T (Th)-cell subsets has greatly improved our understanding of the regulation of immune effector functions. The Th subset differentiation is known to be regulated by the cytokine environment [53]. In particular, we identified HGF as the most prominent immune-regulatory molecule that is expressed and positively modulated by hSKP. It is known to be a potent immunomodulatory factor that inhibits dendritic cell function along with differentiation of IL-10-producing T cells, a decrease in IL-17-producing T cells, and downregulation of surface markers of T-cell activation [54].

In our in vitro coculture model, hSKP seemed to influence the Th subset balance by altering the cytokine profile of T-lymphocytes. By decreasing the T-lymphocyte secretion of IFN-γ, hSKP might have attenuated the differentiation of naive CD4+ T cells into Th1 effectors. In parallel, hSKP stimulated the T-lymphocyte production of IL-10 during an in vitro MLR. This correlates with the observation that, when HGF was neutralized in culture, a significant reduction of T-cell inhibition was observed. These in vitro observations suggest that hSKP, by modulating the T-lymphocyte cytokine balance, attenuate the differentiation toward Th1 effectors, but rather promote the polarization of the immune reaction toward an IL-10 producing Th subset response in the presence of allogeneic activated T cells. In vivo cotransplantation of hSKP and PBMC in irradiated SCID mice resulted in a similar decrease in IFN-γ plasma levels as observed in vitro. However, IL-10 levels were also found to be decreased in the presence of hSKP, which is in contrast to our in vitro observations, but strongly correlates with the significantly reduced secretion of human IgG [55] and thus impairment of B-cell activation. As such, these data suggest that, depending on the cellular composition of the microenvironment, hSKPs differently modulate the immune system in order to induce a tolerogenic environment. As such, modulation of the Th subset balance by hSKP seems to represent one of their key immunomodulation mechanisms.

Surface Immunoregulatory Molecules

Cell-cell contact through the expression of surface immunoregulatory molecules is another type of immunosuppressive mechanism. In order to modulate an immune cell response, hSKP might generate adenosine which is considered to be an important immunosuppressive mediator by inducing the expression of the ectonucleotidases CD39 and CD73 [56]. Yet hSKP did not express CD39, whereas CD73 was uniformly expressed. None of these ectonucleotidases were responsive to inflammatory signals. Furthermore, hSKP, even in the presence of a proinflammatory environment, did not induce the expression of the CD200/CD200R molecule pattern reported to display immunosuppressive and anti-inflammatory effects [57].

HO-1 is the rate-limiting enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and carbon monoxide. Along with its cytoprotective properties, HO-1 has also immunoregulatory features [58]. Moreover, it is known that HO-1 acts as an inducible defence mechanism against oxidative stress during inflammation by decreasing TNFα plasma levels coupled to a significant increase in IL-10 levels [59]. Altogether, by highly expressing HO-1, hSKPs contribute to the inhibition of a lymphocyte reaction.

HLA-G is a nonclassic major histocompatibility complex Class Ib molecule that was initially shown to play a major role in feto-maternal tolerance. HLA-G is now considered as a tolerogenic molecule that actively participates in the control of the immune response [60]. Both membrane and intracellular HLA-G are expressed by hSKP and are probably contributing to their immunosuppressive effects on T-lymphocytes. Functionally, HLA-G exhibits its immunoregulatory properties by inhibiting the activity and proliferation of CD4+/CD8+ T cells, promoting a shift of the Th1/Th2 balance toward Th2 polarization and inducing regulatory T cells [61]. Expression of HLA-G is regulated in response to microenvironmental factors such as stress and cytokines (IL-10 and LIF). In our system, both HLA-G and IL-10 were present and modulated. It has been postulated that HLA-G and IL-10 generate a tolerogenic loop as they upregulate the expression of each other and consequently consolidate the inhibitory effect [62].

hSKP also showed a highly increased expression of CD274, also known as programmed death ligand 1 (PD-L1), under proinflammatory conditions. Lymphocyte activation is tightly regulated by a subset of inhibitory molecules. Among these molecules, CD274 is described to negatively interfere with an immune response [63]. The receptor of this ligand, PD-1, is an immunoinhibitory receptor expressed by activated T cells. Engagement of PD-1 by PD-L1 leads to the inhibition of T-cell receptor-mediated lymphocyte proliferation and cytokine secretion [64]. The upregulation of CD274 in hSKP could be involved in the inhibition of lymphocyte proliferation and may be also associated with the decrease of IFN-γ in parallel to the increase of IL-10 levels as observed for T cells. This is in accordance with previous reports showing that increased (over)expression of PD-L1 results in a significantly reduced proliferation of activated T cells, a lower production of IFN-γ and a higher concentration of IL-10 [65]. In addition, upregulation of CD274 expression may represent a mechanism by which hSKP counterbalance the upregulation of CD40 as observed in an inflammatory context and finally lead to lymphocyte inhibition. Altogether, PD-L1 (CD274) expression could be one of the modes of action of hSKP to modulate an immune response.

Release of Soluble Immunoregulatory Factors

The expression of immunoregulatory factors was observed at both gene and protein levels. hSKP constitutively expressed the genetic machinery required to produce these factors. During the last years, protective properties have been ascribed to HGF and seem to be associated with an anti-inflammatory effect [66]. Here, we found that, although hSKP already secreted high amounts of HGF, its secretion still increased upon inflammation or in the presence of allogeneic activated T cells. Importantly, some anti-inflammatory and immunosuppressive effects have been reported for HGF. The underlying mechanism of the latter includes suppression of Th1 differentiation and promotion of regulatory T-cell generation [66, 67].

Prostaglandins (PG) and in particular PGE2 are molecules derived from fatty acids playing important roles in regulating and modulating inflammatory immune responses. The biosynthesis of PGE2 is regulated by two cyclooxygenases PTGS1 (COX1) and PTGS2 (COX2) and is sensitive to proinflammatory cytokines [68]. hSKP also highly expressed both enzymes and might represent a source of PGE2. Under normal conditions, hSKP did not produce PGE2. Yet upon inflammation and in the presence of allogeneic activated T cells, hSKP started to secrete high amounts of PGE2. These data suggest that hSKP might use PGE2 to modulate the immune response. PGE2 are known to favour Th2-like cytokine secretion profiles by inhibiting both Th1-associated proliferation and production of IFN-γ, and in the same time, by enhancing the production of the Th2-associated cytokines such as IL-10 [69].

LIF is a major anti-inflammatory molecule and has also a direct role in the regulation of adaptive immune tolerance. Previous observations report that LIF is involved in feto-maternal as well as transplantation tolerance [70, 71]. hSKP did not secrete LIF under normal circumstances, but showed elevated levels in the presence of allogeneic activated T cells, indicating that LIF may be released to modulate the lymphocyte response.

Overall, our data show that the immunomodulating properties of hSKP rely on a combined action of mediators such as cell contact and soluble immunosuppressive factors (e.g., HGF). As such, a local suppressive microenvironment is created altering the immune response that ultimately leads to a significant improvement in survival time of mice suffering from a graft-versus-host reaction inflicted by human PBMC. Indeed, by altering the lymphocyte cytokine balance (IL-10 vs. IFN-γ), expressing membrane regulatory molecules (CD73, HO-1, HLA-G, and CD274) and by secreting soluble immunosuppressive factors (HGF, PGE2, and LIF), hSKPs are able to modulate both, inflammatory and immune reactions.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

hSKP may be considered immunologically privileged as they do not express the complete pattern of molecules required to fully activate T-lymphocytes. Specifically, they lack expression of molecules related to antigen presentation such as HLA-DR, CD80, CD86, CD134, and CD252. By expressing a variety of adhesion molecules, hSKP can easily interact and respond to their cellular environment. Moreover, hSKPs are able to suppress the allogeneic activation of T-lymphocytes and as a consequence thereof also the production of IgG, resulting in an improved health status of animals suffering from a graft-versus-host reaction. They are also sensitive to an inflammatory environment, and in particular to T-cell-derived proinflammatory cytokines. As a consequence, hSKPs adjust their phenotype and their expression of immunoregulatory mediators to optimally respond to the presented challenges. We believe that due to these features hSKP may constitute a valuable and easily accessible cell source for cell therapeutic strategies requiring immunosuppression and/or as a poorly immunogenic multipotent stem cell population for cellular replacement therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

We thank Lieven Verhoye, Peter Vander Linden, Margit Henry, and Susan Rohani for their excellent technical assistance. This work was supported by Grants of the Fund for Scientific Research in Flanders (FWO-Vlaanderen), the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), Le Fonds National de la Recherche Scientifique (FNRS), the Research Council (OZR) of the Vrije Universiteit Brussel, the Ghent University (GOA 01G01712), Wetenschappelijk Fonds Willy Gepts from the UZ Brussel and from BRUSTEM, an impulse programme of the Institute for the encouragement of Scientific Research and Innovation of Brussels (ISRIB), and HEPRO-2, an Interuniversity Attraction Pole programme of the Belgian Science Policy Office (BELSPO).

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

J.D.K.: concept and design of in vitro/in vivo studies, provision of study material, collection of data, data analysis and interpretation, and manuscript writing; P.M.: concept and design of in vivo studies, collection of data, data analysis and interpretation, and manuscript writing; G.R.: concept and design of in vitro, collection of data, and data analysis and interpretation; R.M.R. and S.B.: provision of study material, collection of data, and data analysis and interpretation. K.M.: concept and design of transcriptomics, collection of data, and data analysis and interpretation; V.D.B.: provision of study material, collection of data, and manuscript writing; A.S.: administrative support of transcriptomics, financial support of transcriptomics, and manuscript writing; G.L.-R.: design of in vitro/in vivo studies, administrative support of in vivo studies, financial support of in vivo studies, and manuscript writing; T.V.: administrative support of in vitro studies, financial support of in vitro studies, manuscript writing; L.L.: administrative support of in vitro studies, financial support, and manuscript writing; V.R.: concept and design of in vitro/in vivo studies, administrative support, financial support, data interpretation, manuscript writing, and final approval of manuscript; M.N.: concept and design of in vitro/in vivo studies, provision of study material, collection of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. V.R. and M.N. are equally contributing senior authors.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1692-sup-0001-Suppfig1.tif12939KSupporting Information Figure 1
stem1692-sup-0002-Suppfig2.tif5475KSupporting Information Figure 2
stem1692-sup-0003-Supptbl1.doc37KSupporting Table 1
stem1692-sup-0004-Supptbl2.doc72KSupporting Table 2
stem1692-sup-0005-Supptbl3.xls308KSupporting Table 3
stem1692-sup-0006-Supptbl4.xls421KSupporting Table 4

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.