Reasearch Awards nomination

Email updates

Keep up to date with the latest news and content from Stem Cell Research & Therapy and BioMed Central.

Open Access Research

CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death

Eun Mi Lee12, Sunghoon Hurh1, Bumrae Cho1, Kook-Hwan Oh3, Seung U Kim4, Charles D Surh5, Jonathan Sprent6, Jaeseok Yang7, Jae Young Kim8 and Curie Ahn137*

Author Affiliations

1 Transplantation Research Institute, Seoul National University Medical Research Center, College of Medicine Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea

2 Department of Medicine, Program of Immunology, The Graduate School, College of Medicine Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea

3 Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea

4 Medical Research Institute, Chungang University School of Medicine, 102 Heukseok-ro, Dongjak-gu, Seoul 156-755, Korea

5 Department of Immunology & Microbial Science, The Scripps Research Institute, La Jolla, CA, USA

6 Immunology and Inflammation Research Programme, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

7 Transplantation Center, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea

8 Department of Biological Science, Gachon University of Medicine and Science, 191 Hambangmoe-ro, Yeonsu-gu, Incheon 406-799, Korea

For all author emails, please log on.

Stem Cell Research & Therapy 2013, 4:56  doi:10.1186/scrt206


The electronic version of this article is the complete one and can be found online at: http://stemcellres.com/content/4/3/56


Received:19 September 2012
Accepted:9 May 2013
Published:21 May 2013

© 2013 Lee et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

Neural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host.

Methods

To investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro.

Results

Significantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells.

Conclusions

We present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.

Keywords:
Neural stem cells; Co-stimulatory molecules; Immune escape mechanism

Introduction

Neural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. Derivation methods to produce neuronal cells with specific functions from NSCs, such as dopaminergic neurons, have also been established [1]. Therefore, it is highly likely that NSCs will become the first choice for stem cell treatment.

Most cellular and organ transplantation between two unrelated individuals results in graft rejection. However, it has been often found that stem cell transplantation could escape the graft rejection [2-4], presumably by inducing apoptosis of T cells via Fas–Fas ligand (FasL) interaction [5].

Transplantation of rodent embryonic stem cells (ESCs) into allogeneic recipients indicated that they may have immune-privileged properties. The injection of undifferentiated rat ESC-like cells into fully MHC-mismatched rats led to the induction of donor-specific immunological tolerance [6]. Moreover, undifferentiated allogeneic murine ESCs led to engraftment of these cells in the thymus, spleen and liver, with establishment of mixed hematopoietic chimerism [7]. This also proved that ESCs represent a good research tool for possible therapeutic applications in solid organ transplantation because of their capability, when given alone, to induce tolerance to semi-allogeneic solid organ allograft [8].

NSC transplantation could be applied to the brain because this organ is subject to less surveillance by the immune system than other parts of the body, and like brain cells NSCs may display characteristics of cells with immune privilege. Consistent with this idea, T cells that infiltrate around transplanted NSCs in a stroke model exhibited little alloimmune response [9], suggesting NSCs have a strong potential to protect cells of the central nervous system against harmful T-cell infiltration [10]. In a related study, the systemic injection of neuronal stem/precursor cells provided a remarkable amelioration of the pathological features of experimentally induced autoimmune encephalomyelitis and other preclinical models of neurological disorders [11,12].

In this paper, we have investigated the mechanisms responsible for NSC-induced immune tolerance. Our studies indicate a two-stage model whereby human NSCs induce apoptosis of allogeneic CD4+ T cells. First, CD70 expressed constitutively on NSCs engage CD27 on CD4+ T cells and induce upregulation of FasL expression on the CD4+ T cells. Second, T cells thereafter undergo apoptosis from fratricide; that is, through Fas–FasL interaction in the CD4+ T cells themselves.

Materials and methods

Cell culture

The immortalized human embryonic neural stem cell line, HB1.F3 was generated in a previous study by transfection of primary cells from fetal human telencephalon tissue of 14 weeks gestation with amphotropic, replication-incompetent retroviral vector containing v-myc[13]. Primary NSCs were provided by Professor Kim (Korea University, Seoul, Korea) [14,15]. HB1.F3 cells and primary NSCs were cultured in DMEM with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), and 1% antibiotic–antimycotic solution (Gibco, Carlsbad, CA, USA). Human umbilical cord blood mesenchymal stem cells purchased from MEDIPOST Co., Ltd (Seoul, Korea) were used. Human umbilical cord blood-derived mesenchymal stem cells were isolated and expanded as described previously [16,17]. Cultures were maintained in a humidified incubator at 37°C containing 5% CO2.

CD4+ and CD8+ T-cell preparation

Whole blood was collected from healthy volunteer donors (n = 55, age: 24.85 ± 3.52, sex: male/female = 36/19). The institutional review board of the Seoul National University Hospital approved this study, and all volunteers provided written informed consent. Peripheral blood mononuclear cells were isolated by Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ) density gradient centrifugation of peripheral venous blood [18]. The peripheral blood mononuclear cells were separated into CD4+ and CD8+ T-cell populations by magnetic-activated cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany).

Co-culturing T cells with neural stem cells

To determine NSC-dependent apoptosis, CD4+ or CD8+ T cells were cultured with HB1.F3 at a density of 5 × 104:5 × 103, 5 × 104:1 × 104, 5 × 104:2.5 × 104, 5 × 104:5 × 104, and 5 × 104:1 × 105 cells/well on 96-well plates in well media. Adherent NSCs and nonadherent T cells were separately harvested at the indicated times and washed in 1× PBS.

Analysis of T-cell apoptosis

Apoptotic cell death was assessed using Annexin V staining coupled with propidium iodide (BD PharMingen, San Diego, CA, USA). T cells were cultured with HB1.F3 at a various densities or times. T cells were washed twice with 1× PBS. Then 2 × 105 to 5 × 105 T cells were added to a 12 mm test tube and washed twice in 1 ml of 1× binding buffer. Cells were mixed gently with 5 μl Annexin V–fluorescein isothiocyanate and incubated at 4°C in the dark for 5 minutes. After washing with binding buffer, 10 μl of the 20 μg/ml propidium iodide solution (end concentration 1 μg/ml) and 190 μl binding buffer were added to the cells. Cells were immediately analyzed by fluorescence-activated cell sorting (FACS).

Flow cytometry

Adherent NSCs were trypsinized and resuspended in staining buffer (0.1% BSA in PBS) to reach a final concentration of 5 × 105 to 1 × 106 cells/ml. The cells were incubated for 20 minutes on ice with the following antibodies: anti-CD27 (O323), anti-CD70 (Ki-24), anti-Fas (APO-1), anti-FasL (NOK-1), anti-PD-1 (J105), anti-PD-L1 (MIH1), anti-TR-1 (DJR1), anti-TR-2 (DJR2-4), and anti-TRAIL (RIK-2). After incubation, the cells were washed twice with staining buffer and resuspended. The stained cells were analyzed by FACS.

Semiquantitative RT-PCR

Total RNA was prepared from CD4+ T cells using the RNA isolation kit (QIAGEN Inc, Valencia, CA, USA). Two micrograms of RNA were reverse-transcribed. The reaction mixture consisted of 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 U of RNaseOUT™, 1 mM dNTP, 2.5 μM of oligo(dT)20, 200 U of SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and the remaining volume was diethylpyrocarbonate-treated water for a total volume of 20 μl. The mixture was incubated at 50°C for 1 hour, and at 85°C for 5 minutes. For semiquantitative PCR, the reaction mixture consisted of 10 mM Tris–HCl, 50 mM KCl, 0.1% Triton X-100, 2 mM MgCl2, 0.4 mM dNTP, 0.2 pmol sense and antisense primer, 2.5 U Taq DNA polymerase and 3 μg cDNA. Primer sequences are provided in Additional file 1.

Additional file 1. a table presenting primer sequences for the RT-PCR.

Format: DOCX Size: 19KB Download fileOpen Data

HLA-A, HLA-B and HLA-DR allele on HB1.F3 cells

We examined HLA-A, HLA-B, and HLA-DR alleles on HB1.F3 cells using the PCR sequence-specific oligonucleotide method (Dynal RELI™ HLA typing kit, Dynal Biotech, Wirral, U.K.) [19]. The results are provided in Additional file 2.

Additional file 2. a table presenting phenotype and gene frequencies of HLA-A, HLA-B, HLA-DR loci on HB1.F3 cells defined by DNA typing method.

Format: DOCX Size: 13KB Download fileOpen Data

Treatment of anti-human Fas ligand mAbs

CD4+ T cells were incubated with HB1.F3 for 24 hours with a series of concentrations of neutralizing Fas ligand mAb (NOK-2): 0, 0.01, 0.1, 0.5, and 1 μg/ml.

Treatment of actinomycin D and cycloheximide

We treated a transcription inhibitor, actinomycin D (1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA), on CD4+ T cells for 1 hour before co-culture with HB1.F3. The cells were co-cultured for 0 to 18 hours, and death ligand on CD4+ T cells was assessed at the transcriptional level by RT-PCR. We treated a translation inhibitor, CHX (10 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) on T cells for 12 hours before co-culture with HB1.F3. The cells co-cultured for 12 hours, and intracellular Fas ligand expression on CD4+ or CD8+ T cells and HB1.F3 was assessed by FACS.

Treatment of anti-human CD27 or CD70 mAbs

CD4+ T cells were incubated with HB1.F3 for 12 hours with a series of concentrations of anti-CD27 (LG.7F9) or anti-CD70 mAb (BU69): 0, 0.01, 0.1, 0.5, and 1 μg/ml.

Combinatory treatment of anti-CD27, anti-PD-L1, or anti-CD40 mAb

CD4+ T cells were incubated with HB1.F3 for 24 hours with 0.5 μg/ml of anti-CD27 (LG.7F9), anti-PD-L1 (MIH-1), or anti-CD40 mAb (5C3).

Statistical analysis

All results are expressed as the mean ± standard deviation. The statistical significance of differences between group means was determined using Student’s t test.

Results

Human neural stem cells induce CD4+ T-cell apoptosis

To assess the extent of allogeneic response against NSCs, the in vitro response of human T cells was measured towards the fetal NSC line HB1.F3 [20]. Surprisingly, the majority of human T cells displayed morphology of apoptotic cells within 24 hours upon incubation with HB1.F3 (Figure 1A). Apoptosis of T cells commenced within 6 to 12 hours and reached the maximum at 24 hours after co-culturing with HB1.F3 (Figure 1B). The induction of cell death was prominent for CD4+ T cells, affecting ~30 to 40% above the background, but was negligible for CD8+ T cells (Figure 1B). The extent of CD4+ T-cell death increased with a higher ratio of HB1.F3 to T cells, while the extent of CD8+ T-cell apoptosis did not rise with elevated HB1.F3 ratio (Figure 1C). In addition to HB1.F3, primary NSCs induced CD4+ T-cell apoptosis. NSCs appear unique in their ability to induce apoptosis of CD4+ T cells, because other types of cells, including fibroblasts, epithelial cells, and even stem cells of another lineage (mesenchymal stem cells), did not induce apoptosis of CD4+ T cells (Figure 1D).

thumbnailFigure 1. Human neural stem cells (HB1.F3) induce T-cell apoptosis. (A) The morphology of CD4+ T cells after the co-culture with HB1.F3 was characteristic of apoptotic cells: blebbing and shrinkage of cytoplasm (scale bar: 20 μm). (B) CD4+ T cells showed maximal apoptosis at 24 hrs (■, AV+/PI- and AV+/PI+ cells), however the total dead cells of T cells increased by time dependent manner (, total of AV+/PI-, AV+/PI+, and AV-/PI+ cells). (C) The degree of CD4+ T-cell apoptosis occurred in an HB1.F3 density-dependent manner. (D) Contrary to CD4+ T-cell apoptosis by pNSCs or HB1.F3, the apoptosis levels of CD4+ T cells by HEK-293, Detroit 551, and human umbilical cord blood-derived mesenchymal stem cells did not significantly differ from each other. MSC, mesenchymal stem cell; NSC, neural stem cell.

Fas–Fas ligand interaction is involved in neural stem cell-induced T-cell apoptosis

To determine the mechanism of T-cell apoptosis mediated by NSCs, we analyzed for expression of death-inducing molecules Fas, FasL, PD-1, PD-L1, TRAIL receptor-1, TRAIL receptor-2, and TRAIL on HB1.F3, as these molecules were previously reported to be present on stem cells [21-24]. HB1.F3 cells expressed high levels of Fas and TRAIL receptor-2 on cell surface, but not FasL, TRAIL, and PD-1 (Figure 2A). Since human PBL do not express FasL [25], T cells presumably upregulated FasL in order to be susceptible to Fas-mediated cell death by NSCs. To confirm this notion, FasL expression on T cells was analyzed after co-culture with HB1.F3 cells. FasL expression on the cell surface was slightly upregulated on the majority of CD4+ T cells and a small fraction (~7.3%) of cells expressed high levels of FasL (109.96 ± 11.52) (Figure 2B,C). The peak of FasL upregulation was at 12 hours post incubation with HB1.F3. In contrast to CD4+ T cells, FasL upregulation was minimal on CD8+ T cells (Figure 2C). After blocking of Fas-FasL interaction with an anti-FasL mAb (NOK-2) inhibited NSCs-induced CD4+ T cell apoptosis in a dose-dependent manner (Figure 2D).

thumbnailFigure 2. Effect of Fas ligand expression on HB1.F3-induced T-cell apoptosis. (A) Fas, PD-L1, and TR-2 (TRAIL receptor-2) were expressed on HB1.F3 cells. (B) CD4+ T cells, CD8+ T cells, and HB1.F3 cells constitutively did not express Fas ligand (FasL). FasL expression on CD4+ T cells co-cultured with HB1.F3 was induced at 12 hours, but FasL expression on CD8+ T cells was much less dramatic. The FasL level of CD4+ T cells was higher than CD8+ T cells in a small population. (C) FasL (median fluorescence intensity) on CD8+ T cells did not significantly change (dark shading) during the co-culture period, whereas marked FasL upregulation on CD4+ T cells was detected (no shading). (D) The inhibition of CD4+ T cells apoptosis showed anti-FasL mAbs dose-dependent manner.

Neural stem cells induce Fas ligand upregulation on T cells

Induction of FasL was evident also at the mRNA level in CD4+ T cells within 12 hours after interaction with HB1.F3 cells (Figure 3A). FasL mRNA upregulation was blocked by actinomycin D, indicating new synthesis of FasL (Figure 3A). Figure 3B and C shows the intracellular FasL expression levels on co-cultured CD4+ T cells and CD8+ T cells, and HB1.F3 without cycloheximide and with cycloheximide. Intracellular FasL upregulation was higher on CD4+ T cells than CD8+ T cells. Upregulation of FasL on CD4+ T cells was blocked by cycloheximide, but not CD8+ T cells. Intracellular FasL expression on CD4+ T cells and co-cultured HB1.F3 were reduced about 50% (46.91 vs. 22.43, 13.14 vs. 6.66; Figure 3B). On the other hand, intracellular FasL expression on CD8+ T cells and co-cultured HB1.F3 was increased slightly (3.04 vs. 17.82, 1.38 vs. 5.16; Figure 3C). Cycloheximide is assumed to affect the Fas-induced apoptosis on CD8+ T cells and HB1.F3.

thumbnailFigure 3. The effect of Actinomycin D and cycloheximide on death ligand expression levels on CD4+ T cells and HB1.F3. (A) Actinomycin D (Act D) abolished induction of Fas ligand (FasL) transcription on CD4+ T cells at 12 hours, but TRAIL has no differences in the co-culture period. (B), (C) Intracellular FasL expression levels on co-cultured CD4+ T cells and CD8+ T cells, and HB1.F3 without cycloheximide (CHX−) and with cycloheximide (CHX+). Intracellular FasL expression on CD4+ T cells and co-cultured HB1.F3 were reduced about 50% (46.91 vs. 22.43, 13.14 vs. 6.66 (B)). On the other hand, intracellular FasL of CD8+ T cells and co-cultured HB1.F3 were increased slightly (3.04 vs. 17.82, 1.38 vs. 5.16 (C)).

CD70–CD27 interaction is involved in neural stem cell-induced CD4+ T-cell death

To determine the molecular interactions responsible for induction of FasL on CD4+ T cells, we assessed expression of several co-stimulatory molecules on HB1.F3 and T cells by RT-PCR. HB1.F3 constitutively expressed CD70 mRNA, but not other co-stimulatory molecules, including 4-1BBL, CD80, CD86, ICOSL, and OX40L (Figure 4A). The expression pattern of these co-stimulatory molecules on NSCs was confirmed by FACS analysis, which showed that the majority of both HB1.F3 and primary NSCs expressed CD70, but not other molecules (Figure 4B and data not shown). As reported previously [26], freshly purified human CD4+ and CD8+ T cells were found to express mRNA for CD27, the receptor for CD70, and other co-stimulatory molecules, including CD28, HVEM and ICOS (Figure 4A). In addition, as reported previously [27], CD27 expression was found on nearly on all human CD4+ T cells, but only ≈ 50% on CD8+ T cells (Figure 4B). The presence of CD70 on NSCs and CD27 on T cells led us to consider the possibility that CD70–CD27 interaction could play an important role in NSC-induced apoptosis of the T cells. To this end, the effect of anti-CD27 or anti-CD70 blocking antibodies was evaluated. The presence of either blocking antibody inhibited NSC-mediated death of CD4+ T cells in a dose-dependent manner, but only at ~50% of the level, with anti-CD70 blocking antibodies mediating a slightly stronger inhibition (Figure 4C). A synergistic effect of the two blocking antibodies was observed as the presence of both blocking antibodies did lead to an enhanced, but incomplete, inhibition of apoptosis (Figure 4C). Otherwise, we found the expression of PD-L1 on HB1.F3 (as in Figure 2A). In a related study, PD-L1-induced effector T-cell apoptosis was known as a mechanism of immune privilege of corneal allografts [28]. The CD40 system also regulates apoptosis, positively or negatively, depending on the target cells. We therefore confirmed whether PD-L1 and CD40 were involved in T-cell apoptosis by HB1.F3. Figure 4D shows the inhibitory effects by the addition of anti-CD40 or anti-PD-L1 mAb with anti-CD27 mAb. Also, after treatment with anti-CD27 mAbs, the number of FasL-positive CD4+ T cells was markedly reduced to the baseline level (Figure 4E).

thumbnailFigure 4. CD70-CD27 interaction was involved in HB1.F3-induced T cell apoptosis. (A) While constitutive expression of CD70 mRNA was detected on HB1.F3, other co-signaling molecule mRNA was not detected. T cells constitutively expressed CD27 as well as several the other co-signaling molecules such as CD28, CTLA4, HVEM, and ICOS. (B) CD70 expression on HB1.F3 and CD27 on T cells was also confirmed by fluorescence-activated cell sorting (FACS). CD27 expression level on CD4+ T cells were above 90%, but CD8+ T cells were about 50%. (C) Addition of anti-CD27 and CD70 mAbs resulted in a dose-dependent inhibition of the CD4+ T-cell apoptosis but did not inhibit the apoptosis completely (■, CD4+ T cells only; ○, CD4+ T cells co-cultured w/ HB1.F3 + isotype control mAbs); ●, CD4+ T cells co-cultured w/ HB1.F3 + anti- CD27 or anti-CD70 mAbs). (D) To assess the impact of anti-CD40 and PD-L1 mAbs on the CD4+ T-cell apoptosis, we measured the CD4+ T-cell apoptosis ratio after anti-CD27, anti-CD40 or anti-PD-L1 mAbs treatment. This showed an inhibitory effect of the CD4+ T-cell apoptosis compared with the media control group (□, CD4+ T cells only; ■, CD4+ T cells w/ HB1.F3). (E) FasL expression level on CD4+ T cells reduced about 50% by addition of anti-CD27 mAb.

Discussion

Immune escape induced by stem cells is partly due to reduced expression of several cell surface molecules of immune activation on stem cell surfaces. For example, reduced alloreactivity caused by lack of MHC II and reduced MHC I expression on ESCs has been reported [29]. Others have reported that stem cells induced T-cell apoptosis by Fas–FasL interaction, which had been observed to be one of the immune escape mechanisms on T cells [5,30].

CD4+ T-cell apoptosis seemed to continuously occur even 24 hours after the co-culture, because the number of propidium iodide-positive cells continuously increased even after the number of Annexin V-positive cells reached its peak at 24 hours. However, it has not been clearly demonstrated whether this reaction was caused by direct interaction between the FasL expressed on the surface of NSCs and Fas on CD4+ T cells. Similar to other researchers' observations of human ESCs in various stages of differentiation [2], we could not detect either FasL mRNA or surface protein by FACS in NSCs. FasL expression on HB1.F3 was examined using FACS analysis and immunocytochemistry. Both results showed an absence of FasL on HB1.F3, which was similar to the previous results of a study performed with ESCs [29]. Direct Fas–FasL interactions between NSC and CD4+ T cells are therefore less likely to occur (see Additional file 3). NSCs must therefore have provoked Fas–FasL-mediated CD4+ T-cell apoptosis through an indirect pathway.

Additional file 3. a figure showing FasL expression on CD4+ T cells (left) and apoptosis of CD4+ T cells (right) increased by anti-CD3 (OKT-3, 1 μg/ml; eBioscience, San Diego, CA, USA) and anti-CD28 (CD28.2, 1 μg/ml; BD Pharmingen, San Diego, CA, USA) agonistic antibodies, and recombinant CD27L (1 μg/ml; R&D Systems) treatment (no shading, CD4+ T + anti-CD3; solid shading, CD4+ T + msCD27L; vertical shading, CD4+ T + anti-CD28; diagonal shading, CD4+ T + anti-CD3 + msCD27L; diagonal crosshatch, CD4+ T + anti-CD3 + msCD27L + anti-CD28). The experiment shown is representative of three independent experiments with three different blood donors.

Format: TIFF Size: 1.3MB Download fileOpen Data

In addition, transplant rejection occurs through recognition of foreign antigens presented by MHC molecules expressed on antigen-presenting cells, a process that can be mimicked by triggering T cells with CD3-specific mAb [31]. We used anti-CD3 (OKT3) and anti-CD28 (CD28.2) mAbs, and recombinant mouse CD27L (Minneapolis, MN, USA) to stimulate T cells in a manner that partially mimics stimulation by antigen-presenting cells. We then checked FasL expression and apoptosis of CD4+ T cells. Our previous data showed an increase of FasL-positive cells and apoptotic CD4+ T cells by antigen presentation by T-cell receptor and co-stimulatory signals. However, the median fluorescence intensity of FasL and the apoptosis level varied by blood donor, incubation time, and the combinations of antibodies and recombinant protein (see Additional file 4).

Additional file 4. a figure showing expression of FasL on co-cultured NSCs with allogeneic T cells was examined using FACS analysis (NOK-1) and immunocytochemistry (G247-4). (A) NSCs constitutively did not express FasL. We were able to check the expressions of FasL after IL-1β treatment on NSCs (positive control). (B) FasL expression on co-cultured NSCs with allogeneic T cells was not detected.

Format: TIFF Size: 1.7MB Download fileOpen Data

In this paper, we demonstrated that CD70–CD27 interaction was involved in NSCs-induced CD4+ T cell apoptosis. Originally, CD27 is a cell surface glycoprotein belonging to the TNF receptor superfamily, which can provide stimulatory signals for both cell growth and apoptosis. CD70–CD27 interactions between most immune cells produce T cell expansion and the development of effector cytotoxic or memory T cells [32]. On the other hand, it has been reported that CD27 can bind apoptosis-inducing factor (Siva 1), an intracellular mediator of apoptosis [33,34], but the role of this interaction for the function of CD27 is yet to be resolved, because ligation of CD27 generally does not limit but rather contributes to the expansion of activated lymphocytes [35].

CD70 expressed on malignant cells showed novel function in immune escape [36]. Human brain tumor cells, such as malignant glioma like or glioblastoma multiforme, express CD70 [37], and this CD70 – but not TNFα or FasL – initiated T-cell death through the receptor-dependent pathway [38]. However, the exact mechanism of this process is not yet fully understood.

Levels of inhibition by both antibodies were otherwise incomplete, implicating the existence of other causes for apoptosis in addition to CD70–CD27 interaction. In CD70-mediated T-cell apoptosis, the role of Siva, a pro-apoptotic molecule, was identified, as well as soluble mediators such as transforming growth factor beta [32,39]. We evaluated the expression of Siva protein using western blotting. Siva was increased at 48 hours in CD4+ T cells and the NSC co-culture system. We therefore think that, at least in our system, apoptosis was mainly induced by the FasL upregulation by CD27–CD70 ligation between NSCs and CD4+ T cells in the early phase (see Additional file 5).

Additional file 5. a figure showing expression of Siva on co-cultured T cells with NSCs. Co-cultured CD4+ T-cell lysate was tested with anti-Siva antibodies (clone C-20; Santa Cruz, CA, USA) by western blotting. α-tubulin was used as a loading control.

Format: TIFF Size: 549KB Download fileOpen Data

We then examined cytokine profiles from the supernatant at various co-culture times (1, 3, 6, 12, 18, and 24 hours). Several cytokines (such as IL-1β, TNFα, IL-4, IL-10, IFNγ, IL-5, and IL-13) were shown to be negative in the co-culture periods. Moreover, IL-6 increased in a time-dependent manner. However, the IL-6 level is similar in the NSC-only culture group. In addition, the supernatant of the CD4+ T-cell-only culture group was shown to be negative for IL-6. Therefore, IL-6 might require NSC self-renewal and progenitor cell division and differentiation [40-42]. As in ESCs, lack of co-stimulation [9,43] or participation of soluble mediators could play a role in T-cell immune escape. However, activation-induced cell death was less likely to play a role [44] since CD4+ T-cell apoptosis in our experiments reached its peak in 24 hours.

Finally, we have demonstrated that FasL expression on CD4+ T cells was significantly increased as a consequence of CD70–CD27 ligation. Antibody blocking experiments also confirmed that FasL expression on CD4+ T cells was CD70–CD27 dependent. NSCs therefore probably induce CD4+ T-cell apoptosis in two stages: CD70–CD27 ligation between NSCs and CD4+ T cells, which induces FasL expression on some CD4+ T cells, followed by Fas–FasL-mediated CD4+ T-cell apoptosis.

Conclusion

We demonstrated CD70–CD27 ligation to be an initiating step for NSC-induced CD4+ T-cell apoptosis. NSCs probably induce CD4+ T-cell apoptosis in two stages: CD70–CD27 ligation between NSCs and CD4+ T cells, which induces FasL expression on some CD4+ T cells, followed by Fas–FasL-mediated CD4+ T-cell apoptosis. Therefore, if it is possible to maintain persistent expression of CD70 using a gene delivery technique, this will be an exciting option to maintain immune escape in NSC transplantation.

Abbreviations

BSA: Bovine serum albumin; DMEM: Dulbecco’s modified eagle’s medium; ESC: Embryonic stem cell; FACS: Fluorescence-activated cell sorting; FasL: Fas ligand; IFN: Interferon; IL: Interleukin; mAb: Monoclonal antibody; MHC: Major histocompatibility complex; NSC: Neural stem cell; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; RT: Reverse transcriptase; TNF: Tumor necrosis factor.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

EML was responsible for conception and design, in vitro cell work, collection and assembly of data, manuscript writing, and final approval of the manuscript. SH was responsible for RT-PCR analysis and interpretation of data. BC was responsible for western blot analysis and interpretation of the data. K-HO was responsible for review design and interpretation of the data. SUK was responsible for material support (HB1.F3). CDS was responsible for manuscript writing and review, interpretation of the data, and redesign research. JS was responsible for review of the manuscript, interpretation of the data, and redesign research. JY was responsible for review design, interpretation of the data, and redesign research. JYK was responsible for conception and design, interpretation of the data, and manuscript writing, CA was responsible for conception and design, interpretation of the data, manuscript writing, and final approval of manuscript. EML and CA were responsible for the final manuscript and all authors read and approved the final manuscript.

Acknowledgements

The authors gratefully acknowledge Sun Ha Paek (Department of Neurosurgery, College of Medicine, Seoul National University, Korea) and Jong-Hoon Kim (Division of Biotechnology, Korea University, Korea) for supply of the NSCs. This work was supported by the Ministry of the Knowledge Economy (grants 2009-67-10033838) and a grant from Hanwha Chemical Corporation (Project No. 0411–20070011).

References

  1. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD: Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells.

    Nat Biotechnol 2000, 18:675-679. PubMed Abstract | Publisher Full Text OpenURL

  2. Li L, Baroja ML, Majumdar A, Chadwick K, Rouleau A, Gallacher L, Ferber I, Lebkowski J, Martin T, Madrenas J, Bhatia M: Human embryonic stem cells possess immune-privileged properties.

    Stem Cells (Dayton, Ohio) 2004, 22:448-456. Publisher Full Text OpenURL

  3. Robertson NJ, Brook FA, Gardner RL, Cobbold SP, Waldmann H, Fairchild PJ: Embryonic stem cell-derived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance.

    Proc Natl Acad Sci U S A 2007, 104:20920-20925. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  4. Koch CA, Geraldes P, Platt JL: Immunosuppression by embryonic stem cells.

    Stem Cells (Dayton, Ohio) 2008, 26:89-98. Publisher Full Text OpenURL

  5. Singh RK, Varney ML, Buyukberber S, Ino K, Ageitos AG, Reed E, Tarantolo S, Talmadge JE: Fas-FasL-mediated CD4+ T-cell apoptosis following stem cell transplantation.

    Cancer Res 1999, 59:3107-3111. PubMed Abstract | Publisher Full Text OpenURL

  6. Fandrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS, Parwaresch R, Zavazava N, Binas B: Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning.

    Nat Med 2002, 8:171-178. PubMed Abstract | Publisher Full Text OpenURL

  7. Bonde S, Zavazava N: Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients.

    Stem Cells (Dayton, Ohio) 2006, 24:2192-2201. Publisher Full Text OpenURL

  8. Imberti B, Casiraghi F, Cugini D, Azzollini N, Cassis P, Todeschini M, Solini S, Sebastiano V, Zuccotti M, Garagna S, Redi CA, Noris M, Morigi M, Remuzzi G: Embryonic stem cells, derived either after in vitro fertilization or nuclear transfer, prolong survival of semiallogeneic heart transplants.

    J Immunol 2011, 186:4164-4174. PubMed Abstract | Publisher Full Text OpenURL

  9. Lee HJ, Kim KS, Kim EJ, Choi HB, Lee KH, Park IH, Ko Y, Jeong SW, Kim SU: Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.

    Stem Cells (Dayton, Ohio) 2007, 25:1204-1212. Publisher Full Text OpenURL

  10. Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L, Salani G, Martinello M, Cattalini A, Bergami A, Furlan R, Comi G, Constantin G, Martino G: Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism.

    Nature 2005, 436:266-271. Publisher Full Text OpenURL

  11. Pluchino S, Zanotti L, Brambilla E, Rovere-Querini P, Capobianco A, Alfaro-Cervello C, Salani G, Cossetti C, Borsellino G, Battistini L, Ponzoni M, Doglioni C, Garcia-Verdugo JM, Comi G, Manfredi AA, Martino G: Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function.

    PLoS One 2009, 4:e5959. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  12. Martino G, Pluchino S: The therapeutic potential of neural stem cells.

    Nat Rev Neurosci 2006, 7:395-406. PubMed Abstract | Publisher Full Text OpenURL

  13. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY: Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

    Nat Biotechnol 1998, 16:1033-1039. PubMed Abstract | Publisher Full Text OpenURL

  14. Park JY, Kim SK, Woo DH, Lee EJ, Kim JH, Lee SH: Differentiation of neural progenitor cells in a microfluidic chip-generated cytokine gradient.

    Stem Cells (Dayton, Ohio) 2009, 27:2646-2654. Publisher Full Text OpenURL

  15. Kim SE, Kim BK, Gil JE, Kim SK, Kim JH: Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro.

    Mol Cells 2007, 23:49-56. PubMed Abstract OpenURL

  16. Kim JY, Kim DH, Kim JH, Lee D, Jeon HB, Kwon SJ, Kim SM, Yoo YJ, Lee EH, Choi SJ, Seo SW, Lee JI, Na DL, Yang YS, Oh W, Chang JW: Soluble intracellular adhesion molecule-1 secreted by human umbilical cord blood-derived mesenchymal stem cell reduces amyloid-β plaques.

    Cell Death Differ 2012, 19:680-691. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Kim JY, Kim DH, Kim DS, Kim JH, Jeong SY, Jeon HB, Lee EH, Yang YS, Oh W, Chang JW: Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-beta42 neurotoxicity in vitro.

    FEBS Lett 2010, 584:3601-3608. PubMed Abstract | Publisher Full Text OpenURL

  18. Akdis CA, Akdis M, Blesken T, Wymann D, Alkan SS, Muller U, Blaser K: Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

    J Clin Invest 1996, 98:1676-1683. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Roh EY, Kim HS, Kim SM, Lim YM, Han BY, Park MH: HLA-A, -B, -DR allele frequencies and haplotypic associations in Koreans defined by generic-level DNA typing.

    Korean J Lab Med 2003, 23:420-430. OpenURL

  20. Brunlid G, Pruszak J, Holmes B, Isacson O, Sonntag KC: Immature and neurally differentiated mouse embryonic stem cells do not express a functional Fas/Fas ligand system.

    Stem Cells (Dayton, Ohio) 2007, 25:2551-2558. Publisher Full Text OpenURL

  21. Dybedal I, Yang L, Bryder D, Aastrand-Grundstrom I, Leandersson K, Jacobsen SE: Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression.

    Blood 2003, 102:118-126. PubMed Abstract | Publisher Full Text OpenURL

  22. Pearl-Yafe M, Yolcu ES, Stein J, Kaplan O, Shirwan H, Yaniv I, Askenasy N: Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis.

    Exp Hematol 2007, 35:1601-1612. PubMed Abstract | Publisher Full Text OpenURL

  23. Loebinger MR, Sage EK, Davies D, Janes SM: TRAIL-expressing mesenchymal stem cells kill the putative cancer stem cell population.

    Br J Cancer 2010, 103:1692-1697. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. Tao W, Hangoc G, Hawes JW, Si Y, Cooper S, Broxmeyer HE: Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34+ cells treated with etoposide.

    Exp Hematol 2003, 31:251-260. PubMed Abstract | Publisher Full Text OpenURL

  25. Wakisaka S, Takeba Y, Mihara S, Takeno M, Yamamoto S, Sakane T, Suzuki N: Aberrant Fas ligand expression in lymphocytes in patients with Behcet's disease.

    Int Arch Allergy Immunol 2002, 129:175-180. PubMed Abstract | Publisher Full Text OpenURL

  26. Lens SM, Tesselaar K, van Oers MH, van Lier RA: Control of lymphocyte function through CD27–CD70 interactions.

    Semin Immunol 1998, 10:491-499. PubMed Abstract | Publisher Full Text OpenURL

  27. van Lier RA, Borst J, Vroom TM, Klein H, Van Mourik P, Zeijlemaker WP, Melief CJ: Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen.

    J Immunol 1987, 139:1589-1596. PubMed Abstract | Publisher Full Text OpenURL

  28. Hori J, Wang M, Miyashita M, Tanemoto K, Takahashi H, Takemori T, Okumura K, Yagita H, Azuma M: B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts.

    J Immunol 2006, 177:5928-5935. PubMed Abstract | Publisher Full Text OpenURL

  29. Drukker M, Katchman H, Katz G, Even-Tov Friedman S, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N: Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells.

    Stem Cells (Dayton, Ohio) 2006, 24:221-229. Publisher Full Text OpenURL

  30. Talmadge JE, Singh R, Ino K, Ageitos A, Buyukberber S: Mechanisms of immune dysfunction in stem cell transplantation.

    Int J Immunopharmacol 2000, 22:1041-1056. PubMed Abstract | Publisher Full Text OpenURL

  31. Frauwirth KA, Thompson CB: Activation and inhibition of lymphocytes by costimulation.

    J Clin Invest 2002, 109:295-299. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  32. Borst J, Hendriks J, Xiao Y: CD27 and CD70 in T cell and B cell activation.

    Curr Opin Immunol 2005, 17:275-281. PubMed Abstract | Publisher Full Text OpenURL

  33. Prasad KV, Ao Z, Yoon Y, Wu MX, Rizk M, Jacquot S, Schlossman SF: CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.

    Proc Natl Acad Sci U S A 1997, 94:6346-6351. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  34. Spinicelli S, Nocentini G, Ronchetti S, Krausz LT, Bianchini R, Riccardi C: GITR interacts with the pro-apoptotic protein Siva and induces apoptosis.

    Cell Death Differ 2002, 9:1382-1384. PubMed Abstract | Publisher Full Text OpenURL

  35. Nolte MA, van Olffen RW, van Gisbergen KP, van Lier RA: Timing and tuning of CD27–CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology.

    Immunol Rev 2009, 229:216-231. PubMed Abstract | Publisher Full Text OpenURL

  36. Wischhusen J, Jung G, Radovanovic I, Beier C, Steinbach JP, Rimner A, Huang H, Schulz JB, Ohgaki H, Aguzzi A, Rammensee HG, Weller M: Identification of CD70-mediated apoptosis of immune effector cells as a novel immune escape pathway of human glioblastoma.

    Cancer Res 2002, 62:2592-2599. PubMed Abstract | Publisher Full Text OpenURL

  37. Held-Feindt J, Mentlein R: CD70/CD27 ligand, a member of the TNF family, is expressed in human brain tumors.

    Int J Cancer 2002, 98:352-356. PubMed Abstract | Publisher Full Text OpenURL

  38. Chahlavi A, Rayman P, Richmond AL, Biswas K, Zhang R, Vogelbaum M, Tannenbaum C, Barnett G, Finke JH: Glioblastomas induce T-lymphocyte death by two distinct pathways involving gangliosides and CD70.

    Cancer Res 2005, 65:5428-5438. PubMed Abstract | Publisher Full Text OpenURL

  39. Yoon Y, Ao Z, Cheng Y, Schlossman SF, Prasad KV: Murine Siva-1 and Siva-2, alternate splice forms of the mouse Siva gene, both bind to CD27 but differentially transduce apoptosis.

    Oncogene 1999, 18:7174-7179. PubMed Abstract | Publisher Full Text OpenURL

  40. Bauer S, Kerr BJ, Patterson PH: The neuropoietic cytokine family in development, plasticity, disease and injury.

    Nat Rev Neurosci 2007, 8:221-232. PubMed Abstract | Publisher Full Text OpenURL

  41. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F: Principles of interleukin (IL)-6-type cytokine signalling and its regulation.

    Biochem J 2003, 374:1-20. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  42. Bauer S: Cytokine control of adult neural stem cells.

    Ann N Y Acad Sci 2009, 1153:48-56. PubMed Abstract | Publisher Full Text OpenURL

  43. Jacob F: Cell surface and early stages of mouse embryogenesis.

    Curr Top Dev Biol 1979, 13:117-137. PubMed Abstract OpenURL

  44. Green DR, Droin N, Pinkoski M: Activation-induced cell death in T cells.

    Immunol Rev 2003, 193:70-81. PubMed Abstract | Publisher Full Text OpenURL