Lee et al. Stem Cell Research & Therapy 2013, 4:56 http://stemcellres.com/content/4/3/56 RESEARCH Open Access CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death Eun Mi Lee1,2, Sunghoon Hurh1, Bumrae Cho1, Kook-Hwan Oh3, Seung U Kim4, Charles D Surh5, Jonathan Sprent6, Jaeseok Yang7, Jae Young Kim8 and Curie Ahn1,3,7* 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 Transplantation of rodent embryonic stem cells (ESCs) Neural stem cells (NSCs) are among the most promising into allogeneic recipients indicated that they may have candidates for cell replacement therapy in neuronal in- immune-privileged properties. The injection of undiffer- jury and neurodegenerative diseases. Derivation methods entiated rat ESC-like cells into fully MHC-mismatched to produce neuronal cells with specific functions from rats led to the induction of donor-specific immuno- NSCs, such as dopaminergic neurons, have also been logical tolerance [6]. Moreover, undifferentiated allogen- established [1]. Therefore, it is highly likely that NSCs eic murine ESCs led to engraftment of these cells in the will become the first choice for stem cell treatment. thymus, spleen and liver, with establishment of mixed Most cellular and organ transplantation between two un- hematopoietic chimerism [7]. This also proved that ESCs related individuals results in graft rejection. However, it has represent a good research tool for possible therapeutic been often found that stem cell transplantation could es- applications in solid organ transplantation because of cape the graft rejection [2-4], presumably by inducing apop- their capability, when given alone, to induce tolerance to tosis of T cells via Fas–Fas ligand (FasL) interaction [5]. semi-allogeneic solid organ allograft [8]. NSC transplantation could be applied to the brain * Correspondence: [email protected] because this organ is subject to less surveillance by the 1 Transplantation Research Institute, Seoul National University Medical immune system than other parts of the body, and like brain Research Center, College of Medicine Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea cells NSCs may display characteristics of cells with immune 3Department of Internal Medicine, Seoul National University Hospital, 101 privilege. Consistent with this idea, T cells that infiltrate Daehak-ro, Jongno-gu, Seoul 110-744, Korea around transplanted NSCs in a stroke model exhibited little Full list of author information is available at the end of the article © 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. Lee et al. Stem Cell Research & Therapy 2013, 4:56 Page 2 of 10 http://stemcellres.com/content/4/3/56 alloimmune response [9], suggesting NSCs have a strong 5×104:1 × 105 cells/well on 96-well plates in well media. potential to protect cells of the central nervous system Adherent NSCs and nonadherent T cells were separately against harmful T-cell infiltration [10]. In a related study, harvested at the indicated times and washed in 1× PBS. the systemic injection of neuronal stem/precursor cells pro- vided a remarkable amelioration of the pathological features Analysis of T-cell apoptosis of experimentally induced autoimmune encephalomyelitis Apoptotic cell death was assessed using Annexin V staining and other preclinical models of neurological disorders coupled with propidium iodide (BD PharMingen, [11,12]. San Diego, CA, USA). T cells were cultured with HB1.F3 at In this paper, we have investigated the mechanisms re- a various densities or times. T cells were washed twice with sponsible for NSC-induced immune tolerance. Our studies 1× PBS. Then 2 × 105 to 5 × 105 Tcellswereaddedtoa12 indicate a two-stage model whereby human NSCs induce mmtesttubeandwashedtwicein1mlof1×binding apoptosis of allogeneic CD4+ T cells. First, CD70 expressed buffer. Cells were mixed gently with 5 μlAnnexin constitutively on NSCs engage CD27 on CD4+ Tcellsand V–fluorescein isothiocyanate and incubated at 4°C in the induce upregulation of FasL expression on the CD4+ dark for 5 minutes. After washing with binding buffer, 10 μl T cells. Second, T cells thereafter undergo apoptosis from of the 20 μg/ml propidium iodide solution (end fratricide; that is, through Fas–FasL interaction in the concentration 1 μg/ml) and 190 μl binding buffer were CD4+ T cells themselves. added to the cells. Cells were immediately analyzed by fluorescence-activated cell sorting (FACS). Materials and methods Cell culture Flow cytometry The immortalized human embryonic neural stem cell line, Adherent NSCs were trypsinized and resuspended in HB1.F3 was generated in a previous study by transfection staining buffer (0.1% BSA in PBS) to reach a final con- of primary cells from fetal human telencephalon tissue centration of 5 × 105 to 1 × 106 cells/ml. The cells were of 14 weeks gestation with amphotropic, replication- incubated for 20 minutes on ice with the following anti- incompetent retroviral vector containing v-myc [13]. bodies: anti-CD27 (O323), anti-CD70 (Ki-24), anti-Fas PrimaryNSCswereprovidedbyProfessorKim(Korea (APO-1), anti-FasL (NOK-1), anti-PD-1 (J105), anti-PD- University, Seoul, Korea) [14,15]. HB1.F3 cells and primary L1 (MIH1), anti-TR-1 (DJR1), anti-TR-2 (DJR2-4), and NSCs were cultured in DMEM with 10% heat-inactivated anti-TRAIL (RIK-2). After incubation, the cells were fetal bovine serum (Gibco, Carlsbad, CA, USA), and 1% washed twice with staining buffer and resuspended. The antibiotic–antimycotic solution (Gibco, Carlsbad, CA, stained cells were analyzed by FACS. USA). Human umbilical cord blood mesenchymal stem cells purchased from MEDIPOST Co., Ltd (Seoul, Korea) Semiquantitative RT-PCR were used. Human umbilical cord blood-derived mesen- Total RNA was prepared from CD4+ T cells using the RNA chymal stem cells were isolated and expanded as isolation kit (QIAGEN Inc, Valencia, CA, USA). Two described previously [16,17]. Cultures were maintained micrograms of RNA were reverse-transcribed. The reaction in a humidified incubator at 37°C containing 5% CO2. mixture consisted of 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 U of + + CD4 and CD8 T-cell preparation RNaseOUT™,1mMdNTP,2.5μM of oligo(dT)20,200U Whole blood was collected from healthy volunteer donors of SuperScript™ III reverse transcriptase (Invitrogen, (n = 55, age: 24.85 ± 3.52, sex: male/female = 36/19). The Carlsbad, CA, USA) and the remaining volume was institutional review board of the Seoul National University diethylpyrocarbonate-treated water for a total volume of 20 Hospital approved this study, and all volunteers provided μl. The mixture was incubated at 50°C for 1 hour, and at written informed consent. Peripheral blood mononuclear 85°C for 5 minutes. For semiquantitative PCR, the reaction cells were isolated by Ficoll-Paque PLUS (Amersham mixture consisted of 10 mM Tris–HCl, 50 mM KCl, 0.1% Biosciences, Piscataway, NJ) density gradient centrifugation Triton X-100, 2 mM MgCl2,0.4mMdNTP,0.2pmolsense of peripheral venous blood [18]. The peripheral blood and antisense primer, 2.5 U Taq DNA polymerase and 3 μg mononuclear cells were separated into CD4+ and CD8+ cDNA. Primer sequences are provided in Additional file 1. T-cell populations by magnetic-activated cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). HLA-A, HLA-B and HLA-DR allele on HB1.F3 cells We examined HLA-A, HLA-B, and HLA-DR alleles on Co-culturing T cells with neural stem cells HB1.F3 cells using the PCR sequence-specific oligo- To determine NSC-dependent apoptosis, CD4+ or CD8+ T nucleotide method (Dynal RELI™ HLA typing kit, Dynal cells were cultured with HB1.F3 at a density of 5 × 104:5 × Biotech, Wirral, U.K.) [19]. The results are provided in 103,5×104:1 × 104,5×104:2.5 × 104,5×104:5 × 104,and Additional file 2. Lee et al. Stem Cell Research & Therapy 2013, 4:56 Page 3 of 10 http://stemcellres.com/content/4/3/56 Treatment of anti-human Fas ligand mAbs Combinatory treatment of anti-CD27, anti-PD-L1, or CD4+ T cells were incubated with HB1.F3 for 24 hours anti-CD40 mAb with a series of concentrations of neutralizing Fas ligand CD4+ T cells were incubated with HB1.F3 for 24 hours mAb (NOK-2): 0, 0.01, 0.1, 0.5, and 1 μg/ml. with 0.5 μg/ml of anti-CD27 (LG.7F9), anti-PD-L1 (MIH-1), or anti-CD40 mAb (5C3). Treatment of actinomycin D and cycloheximide Statistical analysis We treated a transcription inhibitor, actinomycin D + All results are expressed as the mean ± standard devi- (1 μg/ml; Sigma-Aldrich, St.