Prevention of Experimental Autoimmune Encephalomyelitis by Transfer of Embryonic Stem Cell-Derived Dendritic Cells Expressing Myelin Oligode
http://www.100md.com
免疫学杂志 2005年第3期
Abstract
Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.
Introduction
Currently, corticosteroids and other immune suppressants are commonly used for treatment of subjects with autoimmune diseases. The medication with these drugs often leads to systemic immune suppression and consequent opportunistic infections. Thus, it is desirable to develop a therapeutic means to down-modulate immune responses in an Ag-specific manner without causing systemic immune suppression.
Experimental autoimmune encephalomyelitis (EAE),3 an animal model for human multiple sclerosis, is characterized by neurological impairment resulting from demyelination in the CNS caused by myelin Ag-reactive CD4+ T cells. This disease model is induced by immunization with myelin Ags such as myelin oligodendrocyte glycoprotein (MOG). In the current study, we wanted to try to prevent MOG-induced EAE by treatment of mice with genetically modified dendritic cells (DC). We generated double-transfectant DC presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing molecules with T cell-suppressive property. We tested a strategy to down-modulate the immune response in an Ag-specific manner by in vivo transfer of such genetically modified DC to prevent development of the disease.
For efficient presentation of MOG peptide in the context of MHC class II molecules, we used a previously devised expression vector in which cDNA for human MHC class II-associated invariant chain (Ii) was mutated to contain antigenic peptide in the class II-associated Ii peptide (CLIP) region (1). An epitope inserted in this vector is efficiently presented in the context of coexpressed MHC class II molecules (2). Because they are molecules with a T cell-suppressive property, we tested TRAIL and Programmed Death-1 ligand (PD-L1). TRAIL, a member of the TNF superfamily, is constitutively expressed in a variety of cell types, including lymphocytes, NK cells, and neural cells (3, 4). TRAIL–/– mice are hypersensitive to collagen-induced arthritis and streptozotocin-induced diabetes (5). PD-L1, a ligand for PD-1 and member of the CD28/CTLA-4 family, is expressed on DC, IFN--treated monocytes, activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells (6, 7). PD-1–/– mice spontaneously develop a lymphoproliferative/autoimmune disease, a lupus-like disease, arthritis, and cardiomyopathy (8, 9). Thus, abrogation of either of these two molecules make mice autoimmune prone, suggesting that these molecules play significant roles in maintaining immunological self-tolerance in physiological situations (10, 11, 12, 13, 14, 15, 16, 17, 18).
For introduction of multiple expression vectors into DC, we used a method for embryonic stem cell (ES cell)-mediated genetic modification of DC. Recently, we and another group established culture procedures to generate DC from mouse ES cells (2, 19). ES cell-derived DC (esDC or ES-DC) have the capacity comparable to bone marrow-derived DC to process and present protein Ags to T cells, stimulate naive T cells, and migrate to lymphoid organs in vivo (20, 21). A recent study using the method revealed the role of Notch signaling in differentiation of DC (22). For generation of genetically modified ES-DC, ES cells were transfected with expression vectors, and subsequently transfectant ES cell clones were induced to differentiate to DC, which expressed the products of introduced genes. Introduction of multiple exogenous genes by sequential transfection can readily be done with vectors bearing different selection markers (20).
In this study, we report that treatment of mice with ES-DC presenting MOG peptide in the context of MHC class II and simultaneously expressing TRAIL or PD-L1 significantly reduced the severity of EAE induced by immunization with the MOG peptide.
Materials and Methods
Mice
CBA, and C57BL/6 mice obtained from CLEA Japan or Charles River were kept under specific pathogen-free conditions. Male CBA and female C57BL/6 mice were mated to generate F1 (CBF1) mice, and all in vivo experiments were done using CBF1 mice, syngeneic to TT2 ES cells. Mouse experiments met with approval by Animal Research Committee of Kumamoto University.
Peptides, protein, cell lines, and cytokines
The mouse MOG p35–55 (MEVGWYRSPFSRVVHLYRNGK), mouse myelin proteolipid protein (PLP) p190–209 (SKTSASIGSLCADARMYGVL), and mouse myelin basic protein (MBP) p35–47 (TGILDSIGRFFSG), were synthesized using the F-moc method on an automatic peptide synthesizer (PSSM8; Shimadzu) and purified using HPLC (23, 24, 25). Bovine MBP was purchased from Sigma-Aldrich. The ES cell line, TT2, derived from CBF1 blastocysts, and the M-CSF-defective bone marrow-derived stromal cell line, OP9, were maintained, as described (2). L929, a fibroblast cell line originating from a C3H mouse was purchased from Japan Health Science Foundation (Osaka, Japan). Recombinant mouse GM-CSF was kindly provided by Kirin Brewery and was purchased from PeproTech.
Plasmid construction
Mouse TRAIL cDNA was prepared by RT-PCR amplification from total RNA of mouse spleen with PCR primers 5'-AACCCTCTAGACCGCCGCCACCATGCCTTCCTCAGGGGCCCTGAA-3' and 5'-AAAGGGATATCTTTACTGGTCATTTAGTT-3'. The design of these primers results in cloning of TRAIL cDNA downstream of the Kozak sequence (20). The PCR products were subcloned into a pGEM-T-Easy vector (Promega), and cDNA inserts were confirmed by sequencing analysis. cDNA for mouse PD-L1 was kindly provided by Drs. T. Okazaki and T. Honjo (Department of Medical Chemistry, Kyoto University, Kyoto, Japan) (7). The cDNA fragments for TRAIL and PD-L1 were cloned into pCAG-INeo, a mammalian expression vector driven by a CAG promoter and containing the internal ribosomal entry site (IRES)-neomycin resistance gene cassette, to generate pCAG-TRAIL-INeo or pCAG-PDL1-INeo. To generate a MOG peptide presenting vector, double-stranded oligo DNA encoding the MOG p35–55 epitope, 5'-CCGGTGATGGAAGTTGGTTGGTATCGTTCTCCATTCTCTCGTGTTGTTCATCTTTATCGTAACGGTAAGCTGCCCATGGGAGCT-3', was inserted into the previously reported human Ii-based epitope-presenting vector, pCI30 (2). The coding region of this construct was transferred to pCAG-IPuro, an expression vector containing the CAG promoter and IRES-puromycin N-acetyltransferase gene cassette, to generate pCAG-MOG-IPuro. pCI-PCC is a pigeon cytochrome c (PCC) epitope-presenting vector derived from pCI30 (2).
Transfection of ES cells and differentiation of DC from ES cells
Transfection of ES cells and induction of differentiation of ES cells into DC were done as described (2, 20), with some minor modification as follows. The differentiating cells were transferred from OP9 to bacteriological petri dishes without feeder cells on day 10, and cultured in RPMI 1640 medium supplemented with 12% FCS, GM-CSF (500 U/ml), and 2-ME. The floating or loosely adherent cells were recovered from dishes by pipetting on days 17–19 and used for experiments.
RT-PCR to detect transgene products
Total cellular RNA was extracted using a SV Total RNA Isolation kit (Promega). All RNA samples were treated with RNase-free DNase I before reverse transcription to eliminate any contaminating genomic DNA. RT-PCR was done as described (20). The relative quantity of cDNA in each sample was first normalized by PCR for GAPDH. The primer sequences were as follows: hCD74 (Ii), 5'-CTGACTGACCGCGTTACTCCCACA-3' and5'-TTCAGGGGGTCAGCATTCTGGAGC-3'; TRAIL, 5'-CTGACTGACCGCGTTACTCCCACA-3' and 5'-GAAATGGTGTCCTGAAAGGTTC-3'; PD-L1, 5'-CTGACTGACCGCGTTACTCCCACA-3' and 5'-GCTTGTAGTCCGCACCACCGTAG-3'; and GAPDH, 5'-GGAAAGCTGTGGCGTGATG-3' and 5'-CTGTTGCTGTAGCCGTATTC-3'. The sense-strand primer used for detection of transgene-derived mRNA was corresponding to the 5' untranslated region included in the vector DNA. PCR products were visualized by ethidium bromide staining after separation over a 2% agarose gel. In one experiment, the level of expression of mRNA for TGF- was detected by RT-PCR. The primer sequences were 5'-ACCATGCCAACTTCTGTCTG-3' and 5'-CGGGTTGTGTTGGTTGTAGA-3'.
Flow-cytometric analysis
Staining of cells and analysis on a flow cytometer (FACScan; BD Biosciences) was done as described (2). Abs and reagent used for staining were as follows: anti-I-Ab (clone 3JP; mouse IgG2a), R-PE-conjugated-anti-mouse CD11c (clone N148; hamster IgG; Chemicon), R-PE-conjugated anti-mouse CD86 (clone RMMP-2; rat IgG2a; Caltag), FITC-conjugated anti-human CD74 (clone M-B741; mouse IgG2a; BD Pharmingen), FITC-conjugated goat anti-mouse Ig (BD Pharmingen), mouse IgG2a control (clone G155-178; BD Pharmingen), FITC-conjugated mouse IgG2a control (clone G155-178; BD Pharmingen), R-PE-conjugated hamster IgG control (Immunotech), R-PE-conjugated rat IgG2a control (clone LO-DNP-16; Caltag), biotinylated anti-mouse TRAIL (clone N2B2; rat IgG2a; eBioscience), anti-mouse PD-L1 (clone MIH5; rat IgG2a; eBioscience), rat IgG2a (Caltag), biotinylated rat IgG2a (eBioscience), FITC-conjugated anti-rat Ig (BD Pharmingen), and PE conjugated-streptavidin (Molecular Probes; Invitrogen Life Technologies). In some experiments, the DC fraction was gated by forward and side scatters. For detection of apoptosis of splenic CD4+ T cell, Annexin VFITC apoptosis detection kits (BioVision) were used. In brief, spleen cells isolated from mice treated with ES-DC were incubated with FITC-conjugated annexin V and R-PE-conjugated anti-mouse CD4 mAb (clone L3T4; BD Pharmingen), and subsequently analyzed by flow cytometry.
Cytotoxicity assay and proliferation assay of T cells stimulated with anti-CD3 mAb
Standard 51Cr release assay was done as described (4). For proliferation assay of T cells stimulated with anti-CD3 mAb, splenic mononuclear cells were prepared from unprimed CBF1 mice, and T cells were purified using nylon wool columns. X-ray-irradiated (35 Gy) ES-DC (2 x 104) and the T cells (1 x 105) were seeded into wells of 96-well flat-bottom culture plates precoated with anti-CD3 mAb (145-2C11; eBioscience) and cultured for 4 days. [3H]Thymidine (6.7 Ci/mmol) was added to the culture (1 μCi/well) in the last 16 h. At the end of culture, cells were harvested onto glass fiber filters (Wallac), and the incorporation of [3H]thymidine was measured using scintillation counting. For blocking experiments, anti-TRAIL (clone N2B2) or anti-PD-L1 (clone MIH5) blocking mAb (5 μg/ml) was added to the culture.
Analysis of presentation of MOG epitope by genetically modified ES-DC
MOG epitope-reactive T cells were prepared from inguinal lymph nodes of mice immunized according to protocol for EAE induction described below, using nylon wool columns. X-ray-irradiated (35 Gy) ES-DC as stimulator cells (2 x 104) were cocultured with the MOG-reactive T cells (1.5–2 x 105) in wells of 96-well culture plates for 3 days. Proliferation of T cells in the last 12 h of the culture was quantified based on [3H]thymidine uptake, as described above.
Induction of EAE and treatment with ES-DC
For EAE induction by synthetic peptides or purified protein, 6- to 8-wk-old female CBF1 mice were immunized by giving a s.c. injection at the base of the tail with a 0.2-ml IFA/PBS solution containing 600 μg of MOG p35–55 peptide and 400 μg of Mycobacterium tuberculosis H37Ra (Difco Laboratories) on day 0. In addition, 500 ng of purified Bordetella pertussis toxin (Calbiochem) were injected i.p. on days 0 and 2. For EAE induction by ES-DC presenting MOG peptide, ES-DC were injected at the base of the tail of mice (5 x 105 cells/mouse) at day 0, and the mice were given i.p. 500 ng of B. pertussis toxin in 0.2 ml of PBS on days 0 and 2. For prevention of EAE, mice were injected i.p. with ES-DC (1 x 106 cells/mouse/injection) on days –8, –5, and –2 (preimmunization treatment), or on days 5, 9, and 13 (postimmunization treatment). The mice were observed over a period of 42 days for clinical signs, and scores were assigned based on the following scale: 0, normal; 1, weakness of the tail and/or paralysis of the distal half of the tail; 2, loss of tail tonicity and abnormal gait; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, forelimb paralysis or moribundity; 6, death.
Immunohistochemical analysis
Freshly excised spinal cords were immediately frozen and embedded in Tissue-Tek OCT compound (Sakura Finetechnical). Immunohistochemical staining of CD4, CD8, and Mac-1 was done, as described (20), but with some modification. In brief, serial 7-μm sections were made using cryostat and underwent immunochemical staining with mAbs specific to CD4 (clone L3T4; BD Pharmingen), CD8 (clone Ly-2; BD Pharmingen), or Mac-1 (clone M1/70; eBioscience), and N-Histofine Simple Stain Mouse MAX PO (Nichirei). Frozen sections of spleen were subjected to TUNEL staining by using ApopTag Fluorescein In Situ Apoptosis Detection kits (Serologicals). In brief, sections were incubated with digoxigenin-conjugated nucleotides and TdT, and subsequently with peroxidase-conjugated anti-digoxigenin Ab. The staining signals were developed using diaminobenzidene.
Analysis of T cell response to MOG or keyhole limpet hemocyanin (KLH)
Immunization of mice and restimulation of draining lymph node cells in vitro were done as described (26), but with some modification. In brief, ES-DC-treated and control mice were immunized at the base of the tail with MOG peptide, according to protocol for EAE induction, or 50 μg of KLH protein (Sigma-Aldrich) emulsified in CFA. After indicated days, inguinal lymph node cells and spleen cells were isolated and cultured (5 x 105 cells/well) in the presence of MOG peptide (0, 8, 2.5, or 80 μg/ml) or KLH (16, 50, or 160 μg/ml) in 10% horse serum/RPMI 1640/2-ME or 2% mouse serum/DMEM/2-ME/insulin-transferrin-selenium-X (Invitrogen Life Technologies), and the proliferative response was quantified based on [3H]thymidine uptake, as described above. In addition, when mice were immunized with ES-DC expressing MOG peptide for EAE induction, spleen cells were isolated at day 14, and cultured (5 x 105 cells/well) in the presence of MOG peptide in 10% horse serum/RPMI 1640/2-ME, and the proliferative response was quantified based on [3H]thymidine uptake, as described above. To analyze production of cytokines of spleen cells isolated from mice treated with ES-DC, isolated spleen cells were stimulated with 10 μM MOG peptide or irrelevant OVA peptide in vitro. After 72 or 96 h, cell supernatants were harvested and measured for cytokine content using ELISA kits (eBioscience) for IL-4, IL-10, and IFN-.
Statistical analysis
Two-tailed Student’s t test was used to determine the statistical significance of differences. A value of p < 0.05 was considered significant.
Results
Induction of EAE in CBF1 mice
To date, we found no study that EAE had been induced in CBF1 mice. Therefore, before the study on therapeutic intervention, it was necessary to set up an experimental condition under which we could reproducibly induce EAE in CBF1 mice. We compared several induction protocols using protein or peptide Ag of MOG, MBP, and PLP. As a result, we found that, when mice were s.c. injected at the base of the tail with a 0.2-ml IFA/PBS solution containing 600 μg of MOG p35–55 and 400 μg of M. tuberculosis accompanying an i.p. injection of 500 ng of purified B. pertussis toxin on days 0 and 2, EAE is reproducibly induced in CBF1 mice with an average peak clinical score of 3.3 (Table I). We decided to use this protocol in the following experiments. In addition, inoculation of MBP p35–47, MBP whole protein, or PLP p190–209 together with M. tuberculosis and B. pertussis toxin also induced EAE in CBF1 mice with a peak clinical score ranging between 2 and 3 (Table I).
Table I. EAE induction in CBF1 micea
Genetic modification of ES-DC to express MOG peptide along with TRAIL or PD-L1
At the first step in the generation of ES-DC presenting MOG peptide and simultaneously expressing TRAIL or PD-L1, TT2 ES cells were transfected with an expression vector for TRAIL (pCAG-TRAIL-INeo) or PD-L1 (pCAG-PDL1-INeo), as shown in Fig. 1A. Then, ES cell clones introduced with either of the expression vectors and parental TT2 ES cells were transfected with the MOG peptide expression vector, pCAG-MOG-IPuro (Fig. 1B). In this vector, a cDNA for human Ii was mutated to contain an oligo DNA encoding MOG p35–55 epitope in the CLIP region (1, 2, 27, 28). Resultant single- or double-transfectant ES cell clones were subjected to differentiation to ES-DC. ES-DC expressing MOG peptide, MOG peptide plus TRAIL, and MOG peptide plus PD-L1 were designated as ES-DC-MOG, ES-DC-TRAIL/MOG, and ES-DC-PDL1/MOG, respectively. The expression of mutant human Ii containing the MOG peptide, TRAIL, and PD-L1 in ES-DC was confirmed by RT-PCR (Fig. 1C) and flow-cytometric analysis (Fig. 2). The mutant human Ii containing the MOG peptide was detected by intracellular staining with anti-human CD74 (Ii) mAb (Fig. 2).
FIGURE 1. Genetic modification of ES-DC to express TRAIL, PD-L1, and Ii-MOG. A, The structures of pCAG-TRAIL-INeo and pCAG-PDL1-INeo, the expression vectors for TRAIL and PD-L1, and PCR primers for RT-PCR to detect transgene products are shown. Primer pairs (arrows) were designed to span the intron (917 bp) in the CAG promoter sequence to distinguish PCR products of mRNA origin (421 and 604 bp, respectively) from genome-integrated vector DNA origin. Hatched boxes indicate 5'-untranslated region of the rabbit -actin gene included in the CAG promoter. The vectors are driven by CAG promoter (pCAG), and cDNA for TRAIL or PD-L1 are followed by the IRES-neomycin-resistance gene (NeoR)-polyadenylation signal sequence (pA). B, The structure of pCAG-MOG-IPuro, the expression vector for mutant human Ii bearing MOG peptide at the CLIP region, are shown as in A. Primer pairs (arrows) were designed to generate PCR product of 556 bp originating from transgene-derived mRNA for CAG-MOG. C, RT-PCR analysis detected expression of transgene-derived mutant human Ii containing the MOG peptide (Ii-MOG), TRAIL, PD-L1, and GAPDH (control) mRNA in transfectant ES-DC.
FIGURE 2. Surface phenotype of genetically modified ES-DC. Expression of cell surface CD86, I-Ab, CD11c, TRAIL, and PD-L1 on transfectant ES-DCs was analyzed by flow-cytometric analysis. Expression of mutant human Ii (hCD74) bearing MOG peptide was examined using intracellular staining. Staining patterns with specific Abs (thick line) and isotype-matched control (thin line) are shown.
ES-DC of similar morphology were generated from any of the transfectant ES cells. As shown in Fig. 2, no significant difference was observed in the level of surface expression of CD86, I-Ab, or CD11c among ES-DC derived from parental TT2 ES cells, ES-DC-MOG, ES-DC-TRAIL/MOG, and ES-DC-PDL1/MOG. Thus, forced expression of TRAIL, PD-L1, or mutant human Ii has little influence on the differentiation of ES-DC.
Functional expression of transgene-derived TRAIL and PD-L1 in ES-DC
The functional activity of TRAIL expressed in ES-DC was analyzed according to the cytotoxicity against TRAIL-sensitive L929 cells. As shown in Fig. 3A, ES-DC-TRAIL showed manifest killing activity against L929. In contrast, neither ES-DC (TT2) (parental TT2-derived) nor ES-DC-OVA (OVA-transfected TT2-derived ES-DC) did so. In addition, ES-DC-TRAIL inhibited the proliferation of splenic T cells stimulated with plate-coated anti-CD3 mAb (Fig. 3B). PD-L1 expressed on ES-DC also inhibited proliferation of splenic T cells stimulated with anti-CD3 mAb. Inhibition of anti-CD3-induced proliferation of T cells by the TRAIL and PD-L1 was abrogated by addition with anti-TRAIL and anti-PD-L1 blocking mAb, respectively (Fig. 3B), but not by isotype-matched control mAb (data not shown). These results indicate that transgene-derived TRAIL and PD-L1 expressed in ES-DC functioned to suppress response of T cells stimulated via TCR/CD3 complexes.
FIGURE 3. Expression of functional TRAIL or PD-L1 in ES-DC transfectants. A, The activity of TRAIL expressed in ES-DC was analyzed based on cytotoxicity against L929 cell. 51Cr-labeled target cells (5 x 103 L929 cells) were incubated with ES-DC (TT2), ES-DC-OVA, or ES-DC-TRAIL as effector cells at the indicated E:T ratio for 12 h, and after the incubation, cytolysis of target cells was quantified by measuring radioactivity in the supernatants. Results are expressed as mean specific lysis of triplicate assays, and SDs of triplicates were <4%. B, Irradiated ES-DC (TT2), ES-DC-TRAIL, and ES-DC-PDL1 (2 x 104/well) were cocultured with 1 x 105 syngeneic CBF1 splenic T cells in the presence () or absence () of blocking Ab (anti-TRAIL mAb or anti-PD-L1 mAb, 5 μg/ml) for 4 days in 96-well flat-bottom culture plates precoated with anti-CD3 mAb. Proliferation of T cells was quantified by measuring [3H]thymidine incorporation. The asterisks indicate that the differences in responses are statistically significant between two values indicated by lines (*, p < 0.01; **, p < 0.05). The data are each representative of three independent and reproducible experiments with similar results.
Stimulation of MOG-reactive T cells by ES-DC genetically engineered to express MOG peptide
Presentation of MOG peptide in the context of MHC class II molecules by ES-DC-MOG was investigated in vitro. MOG peptide-reactive T cells were prepared from inguinal lymph nodes of mice, which developed EAE by immunization with MOG p35–55, CFA, and B. pertussis toxin. Proliferative response of the MOG-reactive T cells upon coculture with transfectant ES-DC was analyzed. As shown in Fig. 4A, ES-DC-MOG stimulated the MOG-reactive T cells to induce proliferation. In contrast, ES-DC carrying Ii-based PCC peptide expression vector (ES-DC-PCC) (2), as a control, did not do so. No proliferative response was observed when naive splenic T cells isolated from syngeneic mice were cocultured with ES-DC-MOG under the same condition (data not shown). These results indicate that the epitope-presenting vector introduced into ES-DC functioned to present the MOG peptide in the context of MHC class II molecules to stimulate MOG-specific CD4+ T cells.
FIGURE 4. Presentation of MOG epitope by ES-DC introduced with Ii-based MOG epitope-presenting vector. A, T cells (1.5 x 105) isolated from inguinal lymph nodes of CBF1 mice immunized according to the protocol for EAE induction were cocultured with one of two independent clones (2 x 104) of ES-DC-MOG or a clone of ES-DC-PCC, presenting PCC epitope, for 3 days. Proliferative response of T cells was quantified by [3H]thymidine uptake in the last 12 h of the culture. B, CBF1 mice (three mice per group) were injected s.c. with ES-DC-MOG or ES-DC-PCC (5 x 105) on day 0, together with i.p. injection of 500 ng of purified B. pertussis toxin on days 0 and 2, and the severity of induced EAE was evaluated. The disease incidence, mean day of onset ± SD, and mean peak clinical score ± SD of mice injected with ES-DC-MOG were 100%, 11.3 ± 1.7, and 2.7 ± 0.4, respectively. C, Spleen cells were isolated on day 14 from mice treated as in B, and whole spleen cells (5 x 105/well) were cultured in the presence of 1 μg/ml MOG peptide for 3 days. Proliferative response was quantified as in A. Data were indicated as cpm (value in the presence of peptide – value in the absence of peptide (<46 x 103 cpm)), and SDs of triplicates were <9% of mean value. The asterisks indicate that the differences in responses are statistically significant compared with ES-DC-PCC (*, p < 0.01; **, p < 0.05). The data are each representative of three independent and reproducible experiments with similar results.
It has been reported that transfer of bone marrow-derived DC preloaded with MOG peptide caused development of EAE in naive mice (29, 30). We presumed that, if ES-DC-MOG could encounter with MOG-specific T cells and stimulate the T cells with MOG peptide in vivo, EAE would be developed. We injected ES-DC-MOG or ES-DC-PCC, as a control, at the base of the tail of naive mice and also gave i.p. 500 ng of B. pertussis toxin on the same day and 2 days later. In the results, EAE was developed in the mice transferred with ES-DC-MOG but not those transferred with ES-DC-PCC (Fig. 4B).
We examined whether MOG-specific T cells were activated in vivo by injection with ES-DC-MOG. Fourteen days after the injection of ES-DC and B. pertussis toxin, spleen cells were isolated from the mice and cultured in the presence of MOG peptide. As shown in Fig. 4C, the spleen cells isolated from mice injected with ES-DC-MOG showed proliferative response to MOG peptide. In contrast, those isolated from mice injected with ES-DC-PCC did not do so. These results indicate that in vivo transferred ES-DC-MOG together with adjuvant effect of B. pertussis toxin stimulated MOG-specific T cells to develop EAE.
Protection from MOG-induced EAE by treatment with ES-DC expressing MOG peptide along with TRAIL or PD-L1
We examined whether TRAIL and PD-L1 expressed by ES-DC together with MOG peptide had an effect to down-modulate MOG-specific T cell responses in vitro. MOG-reactive T cells prepared as described above were cocultured with ES-DC-MOG, ES-DC-TRAIL/MOG, or ES-DC-PDL1/MOG. As shown in Fig. 5, proliferative response of the MOG-reactive T cells cocultured with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was significantly lower than those cocultured with ES-DC-MOG, even though the three types of ES-DC expressed an almost equal level of MOG-Ii (Fig. 2). These results indicate down-modulation of the response of MOG-reactive T cells in vitro by TRAIL and PD-L1 coexpressed together with MOG peptide on ES-DC.
FIGURE 5. Decreased proliferative response to MOG peptide of MOG-reactive T cells cocultured with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1. T cells (2 x 105) isolated from inguinal lymph nodes of CBF1 mice immunized according to the protocol for EAE induction were cocultured with irradiated ES-DC-MOG, TRAIL/MOG, or PDL1/MOG (2 x 104) for 3 days, as in Fig. 4A. The asterisks indicate that the differences in responses are statistically significant (p < 0.01) compared with ES-DC-MOG. The data are each representative of three independent and reproducible experiments with similar results.
We tested whether or not development of EAE would be prevented by pretreatment of mice with genetically modified ES-DC. Mice were i.p. injected with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG at days –8, –5, and –2 (1 x 106 cells/mouse/injection), and sequentially immunized with MOG peptide plus adjuvants at days 0 and 2 according to the protocol described in Fig. 6A. As shown in Fig. 6B and Table II, EAE was almost completely prevented by pretreatment with either of these genetically modified ES-DC. In contrast, pretreatment with ES-DC-MOG, ES-DC-TRAIL/OVA (as irrelevant Ag), or ES-DC-PDL1/OVA had no effect (Fig. 6C and Table II). Thus, the prevention depended on both the presentation of the MOG peptide and the expression of TRAIL or PD-L1 by ES-DC. If 2 x 106 of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was given as a one-injection administration, EAE was similarly prevented (data not shown). However, if 5 x 105 of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was used for one injection, the disease severity was not reduced (data not shown). Thus, 1 x 106 of genetically modified ES-DC as one-injection dose is apparently necessary for the prevention of EAE under this experimental condition.
FIGURE 6. Prevention of MOG-induced EAE by pretreatment of mice with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1. A, The schedule for pretreatment and induction of EAE is shown. CBF1 mice (three to five mice per group) were i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days –8, –5, and –2. EAE was induced by s.c. injection of MOG peptide plus M. tuberculosis H37Ra emulsified in IFA on day 0, and i.p. injection of B. pertussis toxin on days 0 and 2. B–D, Disease severity of mice treated with ES-DC-TRAIL/MOG, ES-DC-PD-L1/MOG, or RPMI 1640 medium (control) (B), ES-DC-MOG, ES-DC-TRAIL/OVA, ES-DC-PDL1/OVA, or RPMI 1640 medium (control) (C), coinjection with ES-DC-MOG plus ES-DC-TRAIL, ES-DC-MOG plus ES-DC-PDL1, or RPMI 1640 medium (control) (D) is shown. The data are each representative of at least two independent and reproducible experiments, and data of all experiments are summarized in Table II.
Table II. Suppression of EAE induction in CBF1 mice treated with ES-DCa
We asked whether TRAIL or PD-L1 should be coexpressed by the same ES-DC as one presenting MOG peptide for their capacity to protect mice from EAE. As shown in Fig. 6D and Table II, coinjection of ES-DC-MOG together with ES-DC-TRAIL or ES-DC-PDL1 did not reduce the severity of EAE. Thus, coexpression of TRAIL or PD-L1 with MOG peptide by ES-DC is necessary for the protection from EAE. These results emphasize the advantage of the technology of ES cell-mediated genetic modification of DC, by which one can generate clonal transfectant DC carrying multiple expression vectors.
Next, we tested whether or not treatment with ES-DC after immunization with MOG would achieve some preventive effect on EAE. As shown in Fig. 7A, mice were immunized according to the protocol for EAE induction and, after that, injected with ES-DC on days 5, 9, and 13 (1 x 106 cells/mouse/injection). Even in this postimmunization treatment, injection of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG reduced severity of the disease, but ES-DC-MOG did not do so (Fig. 7B and Table II).
FIGURE 7. Inhibition of MOG-induced EAE by treatment with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1 after immunization with MOG. A, The schedule for induction of EAE and treatment is shown. CBF1 mice (three mice per group) were immunized on days 0 and 2 according to the EAE induction schedule described above, and subsequently i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days 5, 9, and 13. B, Disease severity of mice treated with ES-DC-TRAIL/MOG, ES-DC-PD-L1/MOG, ES-DC-MOG, or RPMI 1640 medium (control) is shown. The data are each representative of two independent and reproducible experiments, and data of all experiments are summarized in Table II.
Decreased T cell response to MOG in mice treated with ES-DC-TRAIL/MOG or -PD-L1/MOG
We examined whether treatment with ES-DC-TRAIL/MOG or -PDL1/MOG would reduce the activation of MOG-specific T cells. Forty-two days after the immunization according to the protocol for EAE induction (Fig. 6A), we isolated inguinal lymph node cells and analyzed their proliferative response upon restimulation in vitro with MOG peptide. As shown in Fig. 8A, the magnitude of proliferation of lymph node cells isolated from mice treated with ES-DC-TRAIL/MOG or -PDL1/MOG was not increased in response to MOG peptide. In contrast, that of lymph node cells from ES-DC-MOG-treated or untreated mice was increased with statistical significance. In the presence of 25 μg/ml MOG peptide, stimulation index (count in the presence of MOG peptide/count in the absence of Ag) for that of untreated, ES-DC-MOG, -TRAIL/MOG, and -PDL1/MOG-treated mice were 2.8, 2.4, 1.3, and 1.0, respectively. These results suggest that treatment with ES-DC-TRAIL/MOG or -PDL1/MOG inhibited the activation of MOG-specific T cells or reduced their number in mice immunized with MOG peptide and adjuvants.
FIGURE 8. Inhibition of activation of MOG-reactive T cells and no effect of activation of KLH-specific T cell by treatment of mice with ES-DC expressing MOG plus TRAIL or PD-L1. A, Inguinal lymph node cells (3 x 105) were isolated from CBF1 mice (three mice per group) of various treatment groups at over day 42, and were stimulated ex vivo with irradiated and MOG peptide-pulsed syngeneic spleen cells for 3 days. Proliferative response of T cells was quantified by [3H]thymidine uptake in the last 12 h of the culture. The asterisks indicate that the differences in responses are statistically significant compared with count in the absence of Ag (*, p < 0.01; **, p < 0.05). The data are each representative of two independent and reproducible experiments with similar results. B, CBF1 mice (three mice per group) were i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days –8, –5, and –2, and immunized with KLH/CFA on day 0. On day 11, inguinal lymph node cells were isolated and restimulated with the indicated concentration of KLH in vitro. Proliferation of T cells was quantified as described above.
Next, we examined whether or not treatment with ES-DC would affect immune responses to an irrelevant exogenous Ag. We treated mice with ES-DC-MOG, -TRAIL/MOG, -PDL1/MOG, or RPMI 1640 medium (control) using the same schedule described above, and subsequently immunized the mice with KLH/CFA. Eleven days after the immunization, we isolated inguinal lymph node cells and analyzed their proliferative response upon restimulation with KLH in vitro. As a result, lymph node cells of ES-DC-treated and control mice showed the same magnitude of proliferative response (Fig. 8B), thereby indicating that the treatment with such genetically modified ES-DC did not affect the immune response to irrelevant Ags.
We immunohistochemically analyzed spinal cord, the target organ of the disease, of mice subjected to EAE induction with or without treatment with ES-DC. Massive infiltration of CD4+ T cells, CD8+ T cells, and Mac-1+ macrophages was observed in spinal cords of untreated control mice (Fig. 9). In contrast, T cells and macrophages hardly infiltrated into the spinal cord of mice treated with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG. The results of histological analysis are in parallel with the severity of EAE and activation state of MOG-specific T cells of each mouse.
FIGURE 9. Inhibition of infiltration of CD4+ T cells, CD8+ T cells, and Mac-1+ macrophages into spinal cord by treatment of mice with ES-DC expressing MOG plus TRAIL or PD-L1. Mice were pretreated with ES-DC-TRAIL/MOG, PDL1/MOG, or untreated and subsequently immunized according to the protocol for EAE induction as shown in Fig. 6A. The cervical, thoracic, and lumbar spinal cord was isolated at day 11 and subjected to immunohistochemical analysis. CD4 (A, D, and G), CD8 (B, E, and H), and Mac-1 (C, F, and I) staining are shown in representative untreated control (A–C), ES-DC-TRAIL/MOG-treated (D–F), and ES-DC-PDL1/MOG-treated (G–I) mice. J, The positive cells were microscopically counted in three sections of spinal cord. Results are expressed as mean ± SD of CD4+, CD8+, Mac-1+ cells per 1 mm2 tissue area of samples obtained from five mice. The asterisks indicate that the decreases in number of infiltrated cells are statistically significant (p < 0.01) compared with control.
Increased number of apoptotic cells in splenic CD4+ T cells by treatment with ES-DC-TRAIL/MOG
With regard to the mechanism of prevention of EAE by transfectant ES-DC, we analyzed the apoptosis of CD4+ T cell in spleens of mice treated with ES-DC by staining with annexin V and subsequent flow-cytometric analysis. In the results, we observed that transfer of ES-DC-TRAIL/MOG caused an increase of apoptosis of CD4+ T cells in recipient mice (17.3 ± 2.5%), compared with transfer of ES-DC-MOG (12.0 ± 0.4%), ES-DC-PDL1/MOG (12.2 ± 0.5%), or RPMI 1640 medium control (10.2 ± 0.8%). In the experiments, three mice were used for each group. Increased numbers of apoptotic cells in spleen of mice transferred with ES-DC-TRAIL/MOG were also observed in histological analysis with TUNEL staining (Fig. 10). The capacity of ES-DC-TRAIL/MOG to cause apoptosis of T cells may play some role in the protection from EAE.
FIGURE 10. Induction of apoptosis of spleen cells by treatment of mice with ES-DC expressing TRAIL along with MOG peptide. Mice were treated with the indicated ES-DC and immunized with MOG peptide, following the schedule described in Fig. 6A. On day 11, spleens were isolated from the mice, and apoptotic cells were detected by in situ TUNEL staining. Original magnification, x200. Sections of the mice untreated (A), treated with ES-DC-MOG (B), ES-DC-TRAIL/MOG (C), and ES-DC-PDL1/MOG (D) are shown. Similar results were observed for three mice used in each experimental group, and representative results are shown.
Discussion
DC are the most potent APC responsible for priming of naive T cells in initiation of the immune response. Recent studies revealed that DC are also involved in the maintenance of immunological self-tolerance, promoting T cells with regulatory functions, or inducing anergy of T cells. In vivo transfer of Ag-loaded DC with a tolerogenic character is regarded as a promising therapeutic means to negatively manipulate immune response in an Ag-specific manner. Various culture procedures used to generate DC with a tolerogenic character have been reported (31, 32, 33, 34, 35, 36). Mouse bone marrow-derived DC generated in the presence of IL-10 and/or TGF- or in the low dose of GM-CSF showed immature phenotypes, a low-level expression of cell surface MHC and costimulatory molecules, and induced T cell anergy in vitro and tolerance to specific Ags or allogeneic transplanted organs in vivo. In humans, monocyte-derived immature DC loaded with antigenic peptides and transferred in vivo have been shown to cause the Ag-specific immune suppression (37).
Genetic modification may be a more steady and reliable way to manipulate the character of DC. Generation of tolerogenic DC by forced expression of Fas ligand, indoleamine 2,3-dioxygemase, IL-10, or CTLA4Ig by gene transfer has been also reported (38, 39, 40, 41). In a recent study, type II collagen-loaded bone marrow-derived DC genetically engineered to express TRAIL by using an adenovirus vector ameliorated type II collagen-induced arthritis (42).
Regarding methods for gene transfer to DC, electroporation, lipofection, and virus vector-mediated transfection have been reported (38, 39, 40, 41, 42, 43). However, considering clinical applications, presently established methods have several drawbacks, i.e., efficiency of gene transfer, stability of gene expression, limitation of the size and number of genes to be introduced, potential risk accompanying the use of virus vectors, and the immunogenicity of the virus vectors. For the purpose of Ag-specific negative regulation of immune responses, the antigenicity of vector systems may lead to problems. Importantly, to efficiently down-modulate T cell responses in an Ag-specific manner, it is desirable to introduce multiple expression vectors to generate stable transfectant DC, which continuously present transgene-derived Ag and simultaneously express immunosuppressive molecules.
Efficient genetic modification of mouse DC can be done by gene transfer to ES cells and subsequent differentiation of transfectant ES cells to ES-DC. By sequential transfection of ES cells using multiple expression vectors, transfectant ES-DC expressing multiple transgene products can readily be generated. In a recent study, we demonstrated that this methodology worked very effectively for induction of antitumor immunity, showing highly efficient stimulation of Ag-specific T cells by in vivo transfer of ES-DC expressing T cell-attracting chemokines along with Ag (20).
The present study demonstrates the usefulness of the genetically modified DC generated by this method for the treatment of subjects with autoimmune disease. We generated ES-DC presenting the MOG epitope in the context of MHC class II molecule and simultaneously expressing immunosuppressive molecule, TRAIL or PD-L1. By pre- or posttreatment of mice with such ES-DC, we succeeded in preventing an autoimmune disease model, EAE induced by immunization with MOG peptide (Figs. 6 and 7; Table II). Down-modulation of immune response by treatment with genetically modified ES-DC did not affect the immune response to irrelevant exogenous Ag, KLH (Fig. 8B). Thus, we achieved the prevention of EAE without decrease in the immune response to an irrelevant Ag.
As for the function of TRAIL, induction of apoptosis has been reported by several groups (3, 4, 42, 44). We also observed an increase in apoptosis of CD4+ T cells in spleens of mice treated with ES-DC-TRAIL/MOG compared with ES-DC-MOG, PDL1/MOG or RPMI 1640 medium (control), as shown in Fig. 10. The result is consistent with a recent report by Liu et al. (42). They introduced the TRAIL gene into bone marrow-derived DC by adenovirus vector and injected the TRAIL transfectant DC into mice for prevention of collagen-induced arthritis, and also observed an increased number of apoptotic T cells in the injected mice. The potential for ES-DC-TRAIL/MOG to cause apoptosis of T cells may have played some role in the protection from EAE, at least in part, in our experiments. In addition, our preliminary experiments suggest that ES-DC-TRAIL/MOG induced T cells with protective effects against EAE. In the experiments, we isolated splenic CD4+ T cells from ES-DC-TRAIL/MOG-treated mice and adoptively transferred them to naive mice. The severity of subsequently induced EAE in the recipient mice was significantly reduced by this treatment (data not shown). At present, it may be possible that both induction of apoptosis of MOG-reactive pathogenic T cells and promotion of T cells with some regulatory function contributed to prevention of EAE by ES-DC-TRAIL/MOG. However, to clarify the precise mechanism or character of the T cell with regulatory function, further investigations are necessary.
In contrast, in case of treatment with ES-DC-PDL1/MOG, neither apoptosis of T cells nor induction of transferable disease-preventing T cells was observed (data not shown). We presume induction of anergy of MOG-reactive T cells to be likely as the mechanism of disease-preventive effect of treatment with ES-DC-PDL1/MOG, based on previous literature regarding the function of PD-L1 (7, 14, 45, 46, 47).
To determine whether the profile of cytokine production was altered by treatment with ES-DC, we did ELISA to quantify IL-10, IL-4, and IFN- produced by spleen cells of ES-DC-treated mice upon stimulation with MOG peptide in vitro. We observed no significant change in the amount of these cytokines produced by spleen cells from ES-DC-TRAIL/MOG-treated or ES-DC-PDL1/MOG-treated mice, compared with those from ES-DC-MOG-treated mice (data not shown). The level of expression of mRNA for TGF- detected by RT-PCR was also unchanged compared with control (data not shown). Thus, involvement of IL-10-producing Tr-1 cells or Th2 cells in protection from EAE by treatment with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG is unlikely, although one cannot totally rule out the possibility.
The capacity of the ES cells to differentiate to ES-DC was never impaired even after culture for at least over 4 mo. Inactivation of transcription of introduced genes due to gene silencing in ES cells can be prevented using vectors bearing the IRES-drug resistance gene or by targeted gene introduction with an exchangeable gene-trap system (2). Thus, genetically manipulated ES cells can be used as an infinite source for DC with genetically modified properties.
Recently, we established methods for generation of DC from nonhuman primate ES cells and also for genetic modification of them (S. Senju, H. Suemori, H. Matsuyoshi, S. Hirata, Y. Uemura, Y.-Z. Chen, D. Fukuma, M. Furuya, N. Nakatsuji, and Y. Nishimura, manuscript in preparation). We hope to apply this method to human ES cells to generate genetically modified human ES-DC, although some modification might be necessary. In the future, Ag-specific immune modulation therapy by in vivo transfer of human ES-DC expressing antigenic protein along with immune-regulating molecules may well be realized, based on evidence in the current study in the mouse system. Possible applications of this technology are treatment of subjects with autoimmune and allergic diseases and also for induction of tolerance to transplanted organs, especially those generated from ES cells. Thus, the methods established in the present study may have implications as a broad medical technology.
Acknowledgments
We thank Dr. S. Aizawa (RIKEN Center for Developmental Biology, Kobe, Japan) for TT2, Drs. N. Takakura (Kanazawa University, Kanazawa, Japan) and T. Suda (Keio University, Tokyo, Japan) for OP9, Dr. H. Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) for pCAG-IPuro, Drs. T. Okazaki and T. Honjo (Kyoto University, Kyoto, Japan) for a cDNA clone for PD-L1, Tatsuko Kubo (Department of Molecular Pathology, Kumamoto University) for technical assistance, and Kirin Brewery Co., Ltd., for rGM-CSF. M. Ohara (Fukuoka, Japan) provided helpful comments on the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Grants-in-Aid 12213111, 14370115, 14570421, and 14657082 from the Ministry of Education, Science, Technology, Sports, and Culture, Japan, and a Research Grant for Intractable Diseases from Ministry of Health, Labour and Welfare, Japan, and grants from the Tokyo Biochemical Research Foundation and Uehara Memorial Foundation, and by funding from Meiji Institute of Health Science.
2 Address correspondence and reprint requests to Dr. Yasuharu Nishimura, Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. E-mail address: mxnishim@gpo.kumamoto-u.ac.jp
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; DC, dendritic cell; Ii, invariant chain; CLIP, class II-associated Ii peptide; PD-L1, Programmed Death-1 ligand; ES cell, embryonic stem cell; ES-DC, ES cell-derived DC; PLP, myelin proteolipid protein; MBP, myelin basic protein; IRES, internal ribosomal entry site; PCC, pigeon cytochrome c; KLH, keyhole limpet hemocyanin.
Received for publication May 20, 2004. Accepted for publication December 8, 2004.
References
Fujii, S., S. Senju, Y. Z. Chen, M. Ando, S. Matsushita, Y. Nishimura. 1998. The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells. Hum. Immunol. 59:607.
Senju, S., S. Hirata, H. Matsuyoshi, M. Masuda, Y. Uemura, K. Araki, K. Yamamura, Y. Nishimura. 2003. Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells. Blood 101:3501.
Wiley, S. R., K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith, R. G. Goodwin. 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673.
Kayagaki, N., N. Yamaguchi, M. Nakayama, K. Takeda, H. Akiba, H. Tsutsui, H. Okamura, K. Nakanishi, K. Okumura, H. Yagita. 1999. Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells. J. Immunol. 163:1906.
Lamhamedi-Cherradi, S. E., S. J. Zheng, K. A. Maguschak, J. Peschon, Y. H. Chen. 2003. Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL–/– mice. Nat. Immunol. 4:255.
Dong, H., G. Zhu, K. Tamada, L. Chen. 1999. B7–H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5:1365
Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura, L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, et al 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192:1027
Nishimura, H., M. Nose, H. Hiai, N. Minato, T. Honjo. 1999. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 11:141.
Nishimura, H., T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori, S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, T. Honjo. 2001. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291:319.
Song, K., Y. Chen, R. Goke, A. Wilmen, C. Seidel, A. Goke, B. Hilliard. 2000. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J. Exp. Med. 191:1095.
Hilliard, B., A. Wilmen, C. Seidel, T. S. Liu, R. Goke, Y. Chen. 2001. Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J. Immunol. 166:1314.
Lunemann, J. D., S. Waiczies, S. Ehrlich, U. Wendling, B. Seeger, T. Kamradt, F. Zipp. 2002. Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto)antigen-specific T cells. J. Immunol. 168:4881.
Kayagaki, N., N. Yamaguchi, M. Abe, S. Hirose, T. Shirai, K. Okumura, H. Yagita. 2002. Suppression of antibody production by TNF-related apoptosis-inducing ligand (TRAIL). Cell. Immunol. 219:82.
Nishimura, H., T. Honjo. 2001. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol. 22:265.
Okazaki, T., Y. Iwai, T. Honjo. 2002. New regulatory co-receptors: inducible co-stimulator and PD-1. Curr. Opin. Immunol. 14:779.(Shinya Hirata, Satoru Sen)
Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.
Introduction
Currently, corticosteroids and other immune suppressants are commonly used for treatment of subjects with autoimmune diseases. The medication with these drugs often leads to systemic immune suppression and consequent opportunistic infections. Thus, it is desirable to develop a therapeutic means to down-modulate immune responses in an Ag-specific manner without causing systemic immune suppression.
Experimental autoimmune encephalomyelitis (EAE),3 an animal model for human multiple sclerosis, is characterized by neurological impairment resulting from demyelination in the CNS caused by myelin Ag-reactive CD4+ T cells. This disease model is induced by immunization with myelin Ags such as myelin oligodendrocyte glycoprotein (MOG). In the current study, we wanted to try to prevent MOG-induced EAE by treatment of mice with genetically modified dendritic cells (DC). We generated double-transfectant DC presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing molecules with T cell-suppressive property. We tested a strategy to down-modulate the immune response in an Ag-specific manner by in vivo transfer of such genetically modified DC to prevent development of the disease.
For efficient presentation of MOG peptide in the context of MHC class II molecules, we used a previously devised expression vector in which cDNA for human MHC class II-associated invariant chain (Ii) was mutated to contain antigenic peptide in the class II-associated Ii peptide (CLIP) region (1). An epitope inserted in this vector is efficiently presented in the context of coexpressed MHC class II molecules (2). Because they are molecules with a T cell-suppressive property, we tested TRAIL and Programmed Death-1 ligand (PD-L1). TRAIL, a member of the TNF superfamily, is constitutively expressed in a variety of cell types, including lymphocytes, NK cells, and neural cells (3, 4). TRAIL–/– mice are hypersensitive to collagen-induced arthritis and streptozotocin-induced diabetes (5). PD-L1, a ligand for PD-1 and member of the CD28/CTLA-4 family, is expressed on DC, IFN--treated monocytes, activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells (6, 7). PD-1–/– mice spontaneously develop a lymphoproliferative/autoimmune disease, a lupus-like disease, arthritis, and cardiomyopathy (8, 9). Thus, abrogation of either of these two molecules make mice autoimmune prone, suggesting that these molecules play significant roles in maintaining immunological self-tolerance in physiological situations (10, 11, 12, 13, 14, 15, 16, 17, 18).
For introduction of multiple expression vectors into DC, we used a method for embryonic stem cell (ES cell)-mediated genetic modification of DC. Recently, we and another group established culture procedures to generate DC from mouse ES cells (2, 19). ES cell-derived DC (esDC or ES-DC) have the capacity comparable to bone marrow-derived DC to process and present protein Ags to T cells, stimulate naive T cells, and migrate to lymphoid organs in vivo (20, 21). A recent study using the method revealed the role of Notch signaling in differentiation of DC (22). For generation of genetically modified ES-DC, ES cells were transfected with expression vectors, and subsequently transfectant ES cell clones were induced to differentiate to DC, which expressed the products of introduced genes. Introduction of multiple exogenous genes by sequential transfection can readily be done with vectors bearing different selection markers (20).
In this study, we report that treatment of mice with ES-DC presenting MOG peptide in the context of MHC class II and simultaneously expressing TRAIL or PD-L1 significantly reduced the severity of EAE induced by immunization with the MOG peptide.
Materials and Methods
Mice
CBA, and C57BL/6 mice obtained from CLEA Japan or Charles River were kept under specific pathogen-free conditions. Male CBA and female C57BL/6 mice were mated to generate F1 (CBF1) mice, and all in vivo experiments were done using CBF1 mice, syngeneic to TT2 ES cells. Mouse experiments met with approval by Animal Research Committee of Kumamoto University.
Peptides, protein, cell lines, and cytokines
The mouse MOG p35–55 (MEVGWYRSPFSRVVHLYRNGK), mouse myelin proteolipid protein (PLP) p190–209 (SKTSASIGSLCADARMYGVL), and mouse myelin basic protein (MBP) p35–47 (TGILDSIGRFFSG), were synthesized using the F-moc method on an automatic peptide synthesizer (PSSM8; Shimadzu) and purified using HPLC (23, 24, 25). Bovine MBP was purchased from Sigma-Aldrich. The ES cell line, TT2, derived from CBF1 blastocysts, and the M-CSF-defective bone marrow-derived stromal cell line, OP9, were maintained, as described (2). L929, a fibroblast cell line originating from a C3H mouse was purchased from Japan Health Science Foundation (Osaka, Japan). Recombinant mouse GM-CSF was kindly provided by Kirin Brewery and was purchased from PeproTech.
Plasmid construction
Mouse TRAIL cDNA was prepared by RT-PCR amplification from total RNA of mouse spleen with PCR primers 5'-AACCCTCTAGACCGCCGCCACCATGCCTTCCTCAGGGGCCCTGAA-3' and 5'-AAAGGGATATCTTTACTGGTCATTTAGTT-3'. The design of these primers results in cloning of TRAIL cDNA downstream of the Kozak sequence (20). The PCR products were subcloned into a pGEM-T-Easy vector (Promega), and cDNA inserts were confirmed by sequencing analysis. cDNA for mouse PD-L1 was kindly provided by Drs. T. Okazaki and T. Honjo (Department of Medical Chemistry, Kyoto University, Kyoto, Japan) (7). The cDNA fragments for TRAIL and PD-L1 were cloned into pCAG-INeo, a mammalian expression vector driven by a CAG promoter and containing the internal ribosomal entry site (IRES)-neomycin resistance gene cassette, to generate pCAG-TRAIL-INeo or pCAG-PDL1-INeo. To generate a MOG peptide presenting vector, double-stranded oligo DNA encoding the MOG p35–55 epitope, 5'-CCGGTGATGGAAGTTGGTTGGTATCGTTCTCCATTCTCTCGTGTTGTTCATCTTTATCGTAACGGTAAGCTGCCCATGGGAGCT-3', was inserted into the previously reported human Ii-based epitope-presenting vector, pCI30 (2). The coding region of this construct was transferred to pCAG-IPuro, an expression vector containing the CAG promoter and IRES-puromycin N-acetyltransferase gene cassette, to generate pCAG-MOG-IPuro. pCI-PCC is a pigeon cytochrome c (PCC) epitope-presenting vector derived from pCI30 (2).
Transfection of ES cells and differentiation of DC from ES cells
Transfection of ES cells and induction of differentiation of ES cells into DC were done as described (2, 20), with some minor modification as follows. The differentiating cells were transferred from OP9 to bacteriological petri dishes without feeder cells on day 10, and cultured in RPMI 1640 medium supplemented with 12% FCS, GM-CSF (500 U/ml), and 2-ME. The floating or loosely adherent cells were recovered from dishes by pipetting on days 17–19 and used for experiments.
RT-PCR to detect transgene products
Total cellular RNA was extracted using a SV Total RNA Isolation kit (Promega). All RNA samples were treated with RNase-free DNase I before reverse transcription to eliminate any contaminating genomic DNA. RT-PCR was done as described (20). The relative quantity of cDNA in each sample was first normalized by PCR for GAPDH. The primer sequences were as follows: hCD74 (Ii), 5'-CTGACTGACCGCGTTACTCCCACA-3' and5'-TTCAGGGGGTCAGCATTCTGGAGC-3'; TRAIL, 5'-CTGACTGACCGCGTTACTCCCACA-3' and 5'-GAAATGGTGTCCTGAAAGGTTC-3'; PD-L1, 5'-CTGACTGACCGCGTTACTCCCACA-3' and 5'-GCTTGTAGTCCGCACCACCGTAG-3'; and GAPDH, 5'-GGAAAGCTGTGGCGTGATG-3' and 5'-CTGTTGCTGTAGCCGTATTC-3'. The sense-strand primer used for detection of transgene-derived mRNA was corresponding to the 5' untranslated region included in the vector DNA. PCR products were visualized by ethidium bromide staining after separation over a 2% agarose gel. In one experiment, the level of expression of mRNA for TGF- was detected by RT-PCR. The primer sequences were 5'-ACCATGCCAACTTCTGTCTG-3' and 5'-CGGGTTGTGTTGGTTGTAGA-3'.
Flow-cytometric analysis
Staining of cells and analysis on a flow cytometer (FACScan; BD Biosciences) was done as described (2). Abs and reagent used for staining were as follows: anti-I-Ab (clone 3JP; mouse IgG2a), R-PE-conjugated-anti-mouse CD11c (clone N148; hamster IgG; Chemicon), R-PE-conjugated anti-mouse CD86 (clone RMMP-2; rat IgG2a; Caltag), FITC-conjugated anti-human CD74 (clone M-B741; mouse IgG2a; BD Pharmingen), FITC-conjugated goat anti-mouse Ig (BD Pharmingen), mouse IgG2a control (clone G155-178; BD Pharmingen), FITC-conjugated mouse IgG2a control (clone G155-178; BD Pharmingen), R-PE-conjugated hamster IgG control (Immunotech), R-PE-conjugated rat IgG2a control (clone LO-DNP-16; Caltag), biotinylated anti-mouse TRAIL (clone N2B2; rat IgG2a; eBioscience), anti-mouse PD-L1 (clone MIH5; rat IgG2a; eBioscience), rat IgG2a (Caltag), biotinylated rat IgG2a (eBioscience), FITC-conjugated anti-rat Ig (BD Pharmingen), and PE conjugated-streptavidin (Molecular Probes; Invitrogen Life Technologies). In some experiments, the DC fraction was gated by forward and side scatters. For detection of apoptosis of splenic CD4+ T cell, Annexin VFITC apoptosis detection kits (BioVision) were used. In brief, spleen cells isolated from mice treated with ES-DC were incubated with FITC-conjugated annexin V and R-PE-conjugated anti-mouse CD4 mAb (clone L3T4; BD Pharmingen), and subsequently analyzed by flow cytometry.
Cytotoxicity assay and proliferation assay of T cells stimulated with anti-CD3 mAb
Standard 51Cr release assay was done as described (4). For proliferation assay of T cells stimulated with anti-CD3 mAb, splenic mononuclear cells were prepared from unprimed CBF1 mice, and T cells were purified using nylon wool columns. X-ray-irradiated (35 Gy) ES-DC (2 x 104) and the T cells (1 x 105) were seeded into wells of 96-well flat-bottom culture plates precoated with anti-CD3 mAb (145-2C11; eBioscience) and cultured for 4 days. [3H]Thymidine (6.7 Ci/mmol) was added to the culture (1 μCi/well) in the last 16 h. At the end of culture, cells were harvested onto glass fiber filters (Wallac), and the incorporation of [3H]thymidine was measured using scintillation counting. For blocking experiments, anti-TRAIL (clone N2B2) or anti-PD-L1 (clone MIH5) blocking mAb (5 μg/ml) was added to the culture.
Analysis of presentation of MOG epitope by genetically modified ES-DC
MOG epitope-reactive T cells were prepared from inguinal lymph nodes of mice immunized according to protocol for EAE induction described below, using nylon wool columns. X-ray-irradiated (35 Gy) ES-DC as stimulator cells (2 x 104) were cocultured with the MOG-reactive T cells (1.5–2 x 105) in wells of 96-well culture plates for 3 days. Proliferation of T cells in the last 12 h of the culture was quantified based on [3H]thymidine uptake, as described above.
Induction of EAE and treatment with ES-DC
For EAE induction by synthetic peptides or purified protein, 6- to 8-wk-old female CBF1 mice were immunized by giving a s.c. injection at the base of the tail with a 0.2-ml IFA/PBS solution containing 600 μg of MOG p35–55 peptide and 400 μg of Mycobacterium tuberculosis H37Ra (Difco Laboratories) on day 0. In addition, 500 ng of purified Bordetella pertussis toxin (Calbiochem) were injected i.p. on days 0 and 2. For EAE induction by ES-DC presenting MOG peptide, ES-DC were injected at the base of the tail of mice (5 x 105 cells/mouse) at day 0, and the mice were given i.p. 500 ng of B. pertussis toxin in 0.2 ml of PBS on days 0 and 2. For prevention of EAE, mice were injected i.p. with ES-DC (1 x 106 cells/mouse/injection) on days –8, –5, and –2 (preimmunization treatment), or on days 5, 9, and 13 (postimmunization treatment). The mice were observed over a period of 42 days for clinical signs, and scores were assigned based on the following scale: 0, normal; 1, weakness of the tail and/or paralysis of the distal half of the tail; 2, loss of tail tonicity and abnormal gait; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, forelimb paralysis or moribundity; 6, death.
Immunohistochemical analysis
Freshly excised spinal cords were immediately frozen and embedded in Tissue-Tek OCT compound (Sakura Finetechnical). Immunohistochemical staining of CD4, CD8, and Mac-1 was done, as described (20), but with some modification. In brief, serial 7-μm sections were made using cryostat and underwent immunochemical staining with mAbs specific to CD4 (clone L3T4; BD Pharmingen), CD8 (clone Ly-2; BD Pharmingen), or Mac-1 (clone M1/70; eBioscience), and N-Histofine Simple Stain Mouse MAX PO (Nichirei). Frozen sections of spleen were subjected to TUNEL staining by using ApopTag Fluorescein In Situ Apoptosis Detection kits (Serologicals). In brief, sections were incubated with digoxigenin-conjugated nucleotides and TdT, and subsequently with peroxidase-conjugated anti-digoxigenin Ab. The staining signals were developed using diaminobenzidene.
Analysis of T cell response to MOG or keyhole limpet hemocyanin (KLH)
Immunization of mice and restimulation of draining lymph node cells in vitro were done as described (26), but with some modification. In brief, ES-DC-treated and control mice were immunized at the base of the tail with MOG peptide, according to protocol for EAE induction, or 50 μg of KLH protein (Sigma-Aldrich) emulsified in CFA. After indicated days, inguinal lymph node cells and spleen cells were isolated and cultured (5 x 105 cells/well) in the presence of MOG peptide (0, 8, 2.5, or 80 μg/ml) or KLH (16, 50, or 160 μg/ml) in 10% horse serum/RPMI 1640/2-ME or 2% mouse serum/DMEM/2-ME/insulin-transferrin-selenium-X (Invitrogen Life Technologies), and the proliferative response was quantified based on [3H]thymidine uptake, as described above. In addition, when mice were immunized with ES-DC expressing MOG peptide for EAE induction, spleen cells were isolated at day 14, and cultured (5 x 105 cells/well) in the presence of MOG peptide in 10% horse serum/RPMI 1640/2-ME, and the proliferative response was quantified based on [3H]thymidine uptake, as described above. To analyze production of cytokines of spleen cells isolated from mice treated with ES-DC, isolated spleen cells were stimulated with 10 μM MOG peptide or irrelevant OVA peptide in vitro. After 72 or 96 h, cell supernatants were harvested and measured for cytokine content using ELISA kits (eBioscience) for IL-4, IL-10, and IFN-.
Statistical analysis
Two-tailed Student’s t test was used to determine the statistical significance of differences. A value of p < 0.05 was considered significant.
Results
Induction of EAE in CBF1 mice
To date, we found no study that EAE had been induced in CBF1 mice. Therefore, before the study on therapeutic intervention, it was necessary to set up an experimental condition under which we could reproducibly induce EAE in CBF1 mice. We compared several induction protocols using protein or peptide Ag of MOG, MBP, and PLP. As a result, we found that, when mice were s.c. injected at the base of the tail with a 0.2-ml IFA/PBS solution containing 600 μg of MOG p35–55 and 400 μg of M. tuberculosis accompanying an i.p. injection of 500 ng of purified B. pertussis toxin on days 0 and 2, EAE is reproducibly induced in CBF1 mice with an average peak clinical score of 3.3 (Table I). We decided to use this protocol in the following experiments. In addition, inoculation of MBP p35–47, MBP whole protein, or PLP p190–209 together with M. tuberculosis and B. pertussis toxin also induced EAE in CBF1 mice with a peak clinical score ranging between 2 and 3 (Table I).
Table I. EAE induction in CBF1 micea
Genetic modification of ES-DC to express MOG peptide along with TRAIL or PD-L1
At the first step in the generation of ES-DC presenting MOG peptide and simultaneously expressing TRAIL or PD-L1, TT2 ES cells were transfected with an expression vector for TRAIL (pCAG-TRAIL-INeo) or PD-L1 (pCAG-PDL1-INeo), as shown in Fig. 1A. Then, ES cell clones introduced with either of the expression vectors and parental TT2 ES cells were transfected with the MOG peptide expression vector, pCAG-MOG-IPuro (Fig. 1B). In this vector, a cDNA for human Ii was mutated to contain an oligo DNA encoding MOG p35–55 epitope in the CLIP region (1, 2, 27, 28). Resultant single- or double-transfectant ES cell clones were subjected to differentiation to ES-DC. ES-DC expressing MOG peptide, MOG peptide plus TRAIL, and MOG peptide plus PD-L1 were designated as ES-DC-MOG, ES-DC-TRAIL/MOG, and ES-DC-PDL1/MOG, respectively. The expression of mutant human Ii containing the MOG peptide, TRAIL, and PD-L1 in ES-DC was confirmed by RT-PCR (Fig. 1C) and flow-cytometric analysis (Fig. 2). The mutant human Ii containing the MOG peptide was detected by intracellular staining with anti-human CD74 (Ii) mAb (Fig. 2).
FIGURE 1. Genetic modification of ES-DC to express TRAIL, PD-L1, and Ii-MOG. A, The structures of pCAG-TRAIL-INeo and pCAG-PDL1-INeo, the expression vectors for TRAIL and PD-L1, and PCR primers for RT-PCR to detect transgene products are shown. Primer pairs (arrows) were designed to span the intron (917 bp) in the CAG promoter sequence to distinguish PCR products of mRNA origin (421 and 604 bp, respectively) from genome-integrated vector DNA origin. Hatched boxes indicate 5'-untranslated region of the rabbit -actin gene included in the CAG promoter. The vectors are driven by CAG promoter (pCAG), and cDNA for TRAIL or PD-L1 are followed by the IRES-neomycin-resistance gene (NeoR)-polyadenylation signal sequence (pA). B, The structure of pCAG-MOG-IPuro, the expression vector for mutant human Ii bearing MOG peptide at the CLIP region, are shown as in A. Primer pairs (arrows) were designed to generate PCR product of 556 bp originating from transgene-derived mRNA for CAG-MOG. C, RT-PCR analysis detected expression of transgene-derived mutant human Ii containing the MOG peptide (Ii-MOG), TRAIL, PD-L1, and GAPDH (control) mRNA in transfectant ES-DC.
FIGURE 2. Surface phenotype of genetically modified ES-DC. Expression of cell surface CD86, I-Ab, CD11c, TRAIL, and PD-L1 on transfectant ES-DCs was analyzed by flow-cytometric analysis. Expression of mutant human Ii (hCD74) bearing MOG peptide was examined using intracellular staining. Staining patterns with specific Abs (thick line) and isotype-matched control (thin line) are shown.
ES-DC of similar morphology were generated from any of the transfectant ES cells. As shown in Fig. 2, no significant difference was observed in the level of surface expression of CD86, I-Ab, or CD11c among ES-DC derived from parental TT2 ES cells, ES-DC-MOG, ES-DC-TRAIL/MOG, and ES-DC-PDL1/MOG. Thus, forced expression of TRAIL, PD-L1, or mutant human Ii has little influence on the differentiation of ES-DC.
Functional expression of transgene-derived TRAIL and PD-L1 in ES-DC
The functional activity of TRAIL expressed in ES-DC was analyzed according to the cytotoxicity against TRAIL-sensitive L929 cells. As shown in Fig. 3A, ES-DC-TRAIL showed manifest killing activity against L929. In contrast, neither ES-DC (TT2) (parental TT2-derived) nor ES-DC-OVA (OVA-transfected TT2-derived ES-DC) did so. In addition, ES-DC-TRAIL inhibited the proliferation of splenic T cells stimulated with plate-coated anti-CD3 mAb (Fig. 3B). PD-L1 expressed on ES-DC also inhibited proliferation of splenic T cells stimulated with anti-CD3 mAb. Inhibition of anti-CD3-induced proliferation of T cells by the TRAIL and PD-L1 was abrogated by addition with anti-TRAIL and anti-PD-L1 blocking mAb, respectively (Fig. 3B), but not by isotype-matched control mAb (data not shown). These results indicate that transgene-derived TRAIL and PD-L1 expressed in ES-DC functioned to suppress response of T cells stimulated via TCR/CD3 complexes.
FIGURE 3. Expression of functional TRAIL or PD-L1 in ES-DC transfectants. A, The activity of TRAIL expressed in ES-DC was analyzed based on cytotoxicity against L929 cell. 51Cr-labeled target cells (5 x 103 L929 cells) were incubated with ES-DC (TT2), ES-DC-OVA, or ES-DC-TRAIL as effector cells at the indicated E:T ratio for 12 h, and after the incubation, cytolysis of target cells was quantified by measuring radioactivity in the supernatants. Results are expressed as mean specific lysis of triplicate assays, and SDs of triplicates were <4%. B, Irradiated ES-DC (TT2), ES-DC-TRAIL, and ES-DC-PDL1 (2 x 104/well) were cocultured with 1 x 105 syngeneic CBF1 splenic T cells in the presence () or absence () of blocking Ab (anti-TRAIL mAb or anti-PD-L1 mAb, 5 μg/ml) for 4 days in 96-well flat-bottom culture plates precoated with anti-CD3 mAb. Proliferation of T cells was quantified by measuring [3H]thymidine incorporation. The asterisks indicate that the differences in responses are statistically significant between two values indicated by lines (*, p < 0.01; **, p < 0.05). The data are each representative of three independent and reproducible experiments with similar results.
Stimulation of MOG-reactive T cells by ES-DC genetically engineered to express MOG peptide
Presentation of MOG peptide in the context of MHC class II molecules by ES-DC-MOG was investigated in vitro. MOG peptide-reactive T cells were prepared from inguinal lymph nodes of mice, which developed EAE by immunization with MOG p35–55, CFA, and B. pertussis toxin. Proliferative response of the MOG-reactive T cells upon coculture with transfectant ES-DC was analyzed. As shown in Fig. 4A, ES-DC-MOG stimulated the MOG-reactive T cells to induce proliferation. In contrast, ES-DC carrying Ii-based PCC peptide expression vector (ES-DC-PCC) (2), as a control, did not do so. No proliferative response was observed when naive splenic T cells isolated from syngeneic mice were cocultured with ES-DC-MOG under the same condition (data not shown). These results indicate that the epitope-presenting vector introduced into ES-DC functioned to present the MOG peptide in the context of MHC class II molecules to stimulate MOG-specific CD4+ T cells.
FIGURE 4. Presentation of MOG epitope by ES-DC introduced with Ii-based MOG epitope-presenting vector. A, T cells (1.5 x 105) isolated from inguinal lymph nodes of CBF1 mice immunized according to the protocol for EAE induction were cocultured with one of two independent clones (2 x 104) of ES-DC-MOG or a clone of ES-DC-PCC, presenting PCC epitope, for 3 days. Proliferative response of T cells was quantified by [3H]thymidine uptake in the last 12 h of the culture. B, CBF1 mice (three mice per group) were injected s.c. with ES-DC-MOG or ES-DC-PCC (5 x 105) on day 0, together with i.p. injection of 500 ng of purified B. pertussis toxin on days 0 and 2, and the severity of induced EAE was evaluated. The disease incidence, mean day of onset ± SD, and mean peak clinical score ± SD of mice injected with ES-DC-MOG were 100%, 11.3 ± 1.7, and 2.7 ± 0.4, respectively. C, Spleen cells were isolated on day 14 from mice treated as in B, and whole spleen cells (5 x 105/well) were cultured in the presence of 1 μg/ml MOG peptide for 3 days. Proliferative response was quantified as in A. Data were indicated as cpm (value in the presence of peptide – value in the absence of peptide (<46 x 103 cpm)), and SDs of triplicates were <9% of mean value. The asterisks indicate that the differences in responses are statistically significant compared with ES-DC-PCC (*, p < 0.01; **, p < 0.05). The data are each representative of three independent and reproducible experiments with similar results.
It has been reported that transfer of bone marrow-derived DC preloaded with MOG peptide caused development of EAE in naive mice (29, 30). We presumed that, if ES-DC-MOG could encounter with MOG-specific T cells and stimulate the T cells with MOG peptide in vivo, EAE would be developed. We injected ES-DC-MOG or ES-DC-PCC, as a control, at the base of the tail of naive mice and also gave i.p. 500 ng of B. pertussis toxin on the same day and 2 days later. In the results, EAE was developed in the mice transferred with ES-DC-MOG but not those transferred with ES-DC-PCC (Fig. 4B).
We examined whether MOG-specific T cells were activated in vivo by injection with ES-DC-MOG. Fourteen days after the injection of ES-DC and B. pertussis toxin, spleen cells were isolated from the mice and cultured in the presence of MOG peptide. As shown in Fig. 4C, the spleen cells isolated from mice injected with ES-DC-MOG showed proliferative response to MOG peptide. In contrast, those isolated from mice injected with ES-DC-PCC did not do so. These results indicate that in vivo transferred ES-DC-MOG together with adjuvant effect of B. pertussis toxin stimulated MOG-specific T cells to develop EAE.
Protection from MOG-induced EAE by treatment with ES-DC expressing MOG peptide along with TRAIL or PD-L1
We examined whether TRAIL and PD-L1 expressed by ES-DC together with MOG peptide had an effect to down-modulate MOG-specific T cell responses in vitro. MOG-reactive T cells prepared as described above were cocultured with ES-DC-MOG, ES-DC-TRAIL/MOG, or ES-DC-PDL1/MOG. As shown in Fig. 5, proliferative response of the MOG-reactive T cells cocultured with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was significantly lower than those cocultured with ES-DC-MOG, even though the three types of ES-DC expressed an almost equal level of MOG-Ii (Fig. 2). These results indicate down-modulation of the response of MOG-reactive T cells in vitro by TRAIL and PD-L1 coexpressed together with MOG peptide on ES-DC.
FIGURE 5. Decreased proliferative response to MOG peptide of MOG-reactive T cells cocultured with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1. T cells (2 x 105) isolated from inguinal lymph nodes of CBF1 mice immunized according to the protocol for EAE induction were cocultured with irradiated ES-DC-MOG, TRAIL/MOG, or PDL1/MOG (2 x 104) for 3 days, as in Fig. 4A. The asterisks indicate that the differences in responses are statistically significant (p < 0.01) compared with ES-DC-MOG. The data are each representative of three independent and reproducible experiments with similar results.
We tested whether or not development of EAE would be prevented by pretreatment of mice with genetically modified ES-DC. Mice were i.p. injected with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG at days –8, –5, and –2 (1 x 106 cells/mouse/injection), and sequentially immunized with MOG peptide plus adjuvants at days 0 and 2 according to the protocol described in Fig. 6A. As shown in Fig. 6B and Table II, EAE was almost completely prevented by pretreatment with either of these genetically modified ES-DC. In contrast, pretreatment with ES-DC-MOG, ES-DC-TRAIL/OVA (as irrelevant Ag), or ES-DC-PDL1/OVA had no effect (Fig. 6C and Table II). Thus, the prevention depended on both the presentation of the MOG peptide and the expression of TRAIL or PD-L1 by ES-DC. If 2 x 106 of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was given as a one-injection administration, EAE was similarly prevented (data not shown). However, if 5 x 105 of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG was used for one injection, the disease severity was not reduced (data not shown). Thus, 1 x 106 of genetically modified ES-DC as one-injection dose is apparently necessary for the prevention of EAE under this experimental condition.
FIGURE 6. Prevention of MOG-induced EAE by pretreatment of mice with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1. A, The schedule for pretreatment and induction of EAE is shown. CBF1 mice (three to five mice per group) were i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days –8, –5, and –2. EAE was induced by s.c. injection of MOG peptide plus M. tuberculosis H37Ra emulsified in IFA on day 0, and i.p. injection of B. pertussis toxin on days 0 and 2. B–D, Disease severity of mice treated with ES-DC-TRAIL/MOG, ES-DC-PD-L1/MOG, or RPMI 1640 medium (control) (B), ES-DC-MOG, ES-DC-TRAIL/OVA, ES-DC-PDL1/OVA, or RPMI 1640 medium (control) (C), coinjection with ES-DC-MOG plus ES-DC-TRAIL, ES-DC-MOG plus ES-DC-PDL1, or RPMI 1640 medium (control) (D) is shown. The data are each representative of at least two independent and reproducible experiments, and data of all experiments are summarized in Table II.
Table II. Suppression of EAE induction in CBF1 mice treated with ES-DCa
We asked whether TRAIL or PD-L1 should be coexpressed by the same ES-DC as one presenting MOG peptide for their capacity to protect mice from EAE. As shown in Fig. 6D and Table II, coinjection of ES-DC-MOG together with ES-DC-TRAIL or ES-DC-PDL1 did not reduce the severity of EAE. Thus, coexpression of TRAIL or PD-L1 with MOG peptide by ES-DC is necessary for the protection from EAE. These results emphasize the advantage of the technology of ES cell-mediated genetic modification of DC, by which one can generate clonal transfectant DC carrying multiple expression vectors.
Next, we tested whether or not treatment with ES-DC after immunization with MOG would achieve some preventive effect on EAE. As shown in Fig. 7A, mice were immunized according to the protocol for EAE induction and, after that, injected with ES-DC on days 5, 9, and 13 (1 x 106 cells/mouse/injection). Even in this postimmunization treatment, injection of ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG reduced severity of the disease, but ES-DC-MOG did not do so (Fig. 7B and Table II).
FIGURE 7. Inhibition of MOG-induced EAE by treatment with ES-DC expressing MOG plus TRAIL or MOG plus PD-L1 after immunization with MOG. A, The schedule for induction of EAE and treatment is shown. CBF1 mice (three mice per group) were immunized on days 0 and 2 according to the EAE induction schedule described above, and subsequently i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days 5, 9, and 13. B, Disease severity of mice treated with ES-DC-TRAIL/MOG, ES-DC-PD-L1/MOG, ES-DC-MOG, or RPMI 1640 medium (control) is shown. The data are each representative of two independent and reproducible experiments, and data of all experiments are summarized in Table II.
Decreased T cell response to MOG in mice treated with ES-DC-TRAIL/MOG or -PD-L1/MOG
We examined whether treatment with ES-DC-TRAIL/MOG or -PDL1/MOG would reduce the activation of MOG-specific T cells. Forty-two days after the immunization according to the protocol for EAE induction (Fig. 6A), we isolated inguinal lymph node cells and analyzed their proliferative response upon restimulation in vitro with MOG peptide. As shown in Fig. 8A, the magnitude of proliferation of lymph node cells isolated from mice treated with ES-DC-TRAIL/MOG or -PDL1/MOG was not increased in response to MOG peptide. In contrast, that of lymph node cells from ES-DC-MOG-treated or untreated mice was increased with statistical significance. In the presence of 25 μg/ml MOG peptide, stimulation index (count in the presence of MOG peptide/count in the absence of Ag) for that of untreated, ES-DC-MOG, -TRAIL/MOG, and -PDL1/MOG-treated mice were 2.8, 2.4, 1.3, and 1.0, respectively. These results suggest that treatment with ES-DC-TRAIL/MOG or -PDL1/MOG inhibited the activation of MOG-specific T cells or reduced their number in mice immunized with MOG peptide and adjuvants.
FIGURE 8. Inhibition of activation of MOG-reactive T cells and no effect of activation of KLH-specific T cell by treatment of mice with ES-DC expressing MOG plus TRAIL or PD-L1. A, Inguinal lymph node cells (3 x 105) were isolated from CBF1 mice (three mice per group) of various treatment groups at over day 42, and were stimulated ex vivo with irradiated and MOG peptide-pulsed syngeneic spleen cells for 3 days. Proliferative response of T cells was quantified by [3H]thymidine uptake in the last 12 h of the culture. The asterisks indicate that the differences in responses are statistically significant compared with count in the absence of Ag (*, p < 0.01; **, p < 0.05). The data are each representative of two independent and reproducible experiments with similar results. B, CBF1 mice (three mice per group) were i.p. injected with ES-DC (1 x 106 cells/injection/mouse) on days –8, –5, and –2, and immunized with KLH/CFA on day 0. On day 11, inguinal lymph node cells were isolated and restimulated with the indicated concentration of KLH in vitro. Proliferation of T cells was quantified as described above.
Next, we examined whether or not treatment with ES-DC would affect immune responses to an irrelevant exogenous Ag. We treated mice with ES-DC-MOG, -TRAIL/MOG, -PDL1/MOG, or RPMI 1640 medium (control) using the same schedule described above, and subsequently immunized the mice with KLH/CFA. Eleven days after the immunization, we isolated inguinal lymph node cells and analyzed their proliferative response upon restimulation with KLH in vitro. As a result, lymph node cells of ES-DC-treated and control mice showed the same magnitude of proliferative response (Fig. 8B), thereby indicating that the treatment with such genetically modified ES-DC did not affect the immune response to irrelevant Ags.
We immunohistochemically analyzed spinal cord, the target organ of the disease, of mice subjected to EAE induction with or without treatment with ES-DC. Massive infiltration of CD4+ T cells, CD8+ T cells, and Mac-1+ macrophages was observed in spinal cords of untreated control mice (Fig. 9). In contrast, T cells and macrophages hardly infiltrated into the spinal cord of mice treated with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG. The results of histological analysis are in parallel with the severity of EAE and activation state of MOG-specific T cells of each mouse.
FIGURE 9. Inhibition of infiltration of CD4+ T cells, CD8+ T cells, and Mac-1+ macrophages into spinal cord by treatment of mice with ES-DC expressing MOG plus TRAIL or PD-L1. Mice were pretreated with ES-DC-TRAIL/MOG, PDL1/MOG, or untreated and subsequently immunized according to the protocol for EAE induction as shown in Fig. 6A. The cervical, thoracic, and lumbar spinal cord was isolated at day 11 and subjected to immunohistochemical analysis. CD4 (A, D, and G), CD8 (B, E, and H), and Mac-1 (C, F, and I) staining are shown in representative untreated control (A–C), ES-DC-TRAIL/MOG-treated (D–F), and ES-DC-PDL1/MOG-treated (G–I) mice. J, The positive cells were microscopically counted in three sections of spinal cord. Results are expressed as mean ± SD of CD4+, CD8+, Mac-1+ cells per 1 mm2 tissue area of samples obtained from five mice. The asterisks indicate that the decreases in number of infiltrated cells are statistically significant (p < 0.01) compared with control.
Increased number of apoptotic cells in splenic CD4+ T cells by treatment with ES-DC-TRAIL/MOG
With regard to the mechanism of prevention of EAE by transfectant ES-DC, we analyzed the apoptosis of CD4+ T cell in spleens of mice treated with ES-DC by staining with annexin V and subsequent flow-cytometric analysis. In the results, we observed that transfer of ES-DC-TRAIL/MOG caused an increase of apoptosis of CD4+ T cells in recipient mice (17.3 ± 2.5%), compared with transfer of ES-DC-MOG (12.0 ± 0.4%), ES-DC-PDL1/MOG (12.2 ± 0.5%), or RPMI 1640 medium control (10.2 ± 0.8%). In the experiments, three mice were used for each group. Increased numbers of apoptotic cells in spleen of mice transferred with ES-DC-TRAIL/MOG were also observed in histological analysis with TUNEL staining (Fig. 10). The capacity of ES-DC-TRAIL/MOG to cause apoptosis of T cells may play some role in the protection from EAE.
FIGURE 10. Induction of apoptosis of spleen cells by treatment of mice with ES-DC expressing TRAIL along with MOG peptide. Mice were treated with the indicated ES-DC and immunized with MOG peptide, following the schedule described in Fig. 6A. On day 11, spleens were isolated from the mice, and apoptotic cells were detected by in situ TUNEL staining. Original magnification, x200. Sections of the mice untreated (A), treated with ES-DC-MOG (B), ES-DC-TRAIL/MOG (C), and ES-DC-PDL1/MOG (D) are shown. Similar results were observed for three mice used in each experimental group, and representative results are shown.
Discussion
DC are the most potent APC responsible for priming of naive T cells in initiation of the immune response. Recent studies revealed that DC are also involved in the maintenance of immunological self-tolerance, promoting T cells with regulatory functions, or inducing anergy of T cells. In vivo transfer of Ag-loaded DC with a tolerogenic character is regarded as a promising therapeutic means to negatively manipulate immune response in an Ag-specific manner. Various culture procedures used to generate DC with a tolerogenic character have been reported (31, 32, 33, 34, 35, 36). Mouse bone marrow-derived DC generated in the presence of IL-10 and/or TGF- or in the low dose of GM-CSF showed immature phenotypes, a low-level expression of cell surface MHC and costimulatory molecules, and induced T cell anergy in vitro and tolerance to specific Ags or allogeneic transplanted organs in vivo. In humans, monocyte-derived immature DC loaded with antigenic peptides and transferred in vivo have been shown to cause the Ag-specific immune suppression (37).
Genetic modification may be a more steady and reliable way to manipulate the character of DC. Generation of tolerogenic DC by forced expression of Fas ligand, indoleamine 2,3-dioxygemase, IL-10, or CTLA4Ig by gene transfer has been also reported (38, 39, 40, 41). In a recent study, type II collagen-loaded bone marrow-derived DC genetically engineered to express TRAIL by using an adenovirus vector ameliorated type II collagen-induced arthritis (42).
Regarding methods for gene transfer to DC, electroporation, lipofection, and virus vector-mediated transfection have been reported (38, 39, 40, 41, 42, 43). However, considering clinical applications, presently established methods have several drawbacks, i.e., efficiency of gene transfer, stability of gene expression, limitation of the size and number of genes to be introduced, potential risk accompanying the use of virus vectors, and the immunogenicity of the virus vectors. For the purpose of Ag-specific negative regulation of immune responses, the antigenicity of vector systems may lead to problems. Importantly, to efficiently down-modulate T cell responses in an Ag-specific manner, it is desirable to introduce multiple expression vectors to generate stable transfectant DC, which continuously present transgene-derived Ag and simultaneously express immunosuppressive molecules.
Efficient genetic modification of mouse DC can be done by gene transfer to ES cells and subsequent differentiation of transfectant ES cells to ES-DC. By sequential transfection of ES cells using multiple expression vectors, transfectant ES-DC expressing multiple transgene products can readily be generated. In a recent study, we demonstrated that this methodology worked very effectively for induction of antitumor immunity, showing highly efficient stimulation of Ag-specific T cells by in vivo transfer of ES-DC expressing T cell-attracting chemokines along with Ag (20).
The present study demonstrates the usefulness of the genetically modified DC generated by this method for the treatment of subjects with autoimmune disease. We generated ES-DC presenting the MOG epitope in the context of MHC class II molecule and simultaneously expressing immunosuppressive molecule, TRAIL or PD-L1. By pre- or posttreatment of mice with such ES-DC, we succeeded in preventing an autoimmune disease model, EAE induced by immunization with MOG peptide (Figs. 6 and 7; Table II). Down-modulation of immune response by treatment with genetically modified ES-DC did not affect the immune response to irrelevant exogenous Ag, KLH (Fig. 8B). Thus, we achieved the prevention of EAE without decrease in the immune response to an irrelevant Ag.
As for the function of TRAIL, induction of apoptosis has been reported by several groups (3, 4, 42, 44). We also observed an increase in apoptosis of CD4+ T cells in spleens of mice treated with ES-DC-TRAIL/MOG compared with ES-DC-MOG, PDL1/MOG or RPMI 1640 medium (control), as shown in Fig. 10. The result is consistent with a recent report by Liu et al. (42). They introduced the TRAIL gene into bone marrow-derived DC by adenovirus vector and injected the TRAIL transfectant DC into mice for prevention of collagen-induced arthritis, and also observed an increased number of apoptotic T cells in the injected mice. The potential for ES-DC-TRAIL/MOG to cause apoptosis of T cells may have played some role in the protection from EAE, at least in part, in our experiments. In addition, our preliminary experiments suggest that ES-DC-TRAIL/MOG induced T cells with protective effects against EAE. In the experiments, we isolated splenic CD4+ T cells from ES-DC-TRAIL/MOG-treated mice and adoptively transferred them to naive mice. The severity of subsequently induced EAE in the recipient mice was significantly reduced by this treatment (data not shown). At present, it may be possible that both induction of apoptosis of MOG-reactive pathogenic T cells and promotion of T cells with some regulatory function contributed to prevention of EAE by ES-DC-TRAIL/MOG. However, to clarify the precise mechanism or character of the T cell with regulatory function, further investigations are necessary.
In contrast, in case of treatment with ES-DC-PDL1/MOG, neither apoptosis of T cells nor induction of transferable disease-preventing T cells was observed (data not shown). We presume induction of anergy of MOG-reactive T cells to be likely as the mechanism of disease-preventive effect of treatment with ES-DC-PDL1/MOG, based on previous literature regarding the function of PD-L1 (7, 14, 45, 46, 47).
To determine whether the profile of cytokine production was altered by treatment with ES-DC, we did ELISA to quantify IL-10, IL-4, and IFN- produced by spleen cells of ES-DC-treated mice upon stimulation with MOG peptide in vitro. We observed no significant change in the amount of these cytokines produced by spleen cells from ES-DC-TRAIL/MOG-treated or ES-DC-PDL1/MOG-treated mice, compared with those from ES-DC-MOG-treated mice (data not shown). The level of expression of mRNA for TGF- detected by RT-PCR was also unchanged compared with control (data not shown). Thus, involvement of IL-10-producing Tr-1 cells or Th2 cells in protection from EAE by treatment with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG is unlikely, although one cannot totally rule out the possibility.
The capacity of the ES cells to differentiate to ES-DC was never impaired even after culture for at least over 4 mo. Inactivation of transcription of introduced genes due to gene silencing in ES cells can be prevented using vectors bearing the IRES-drug resistance gene or by targeted gene introduction with an exchangeable gene-trap system (2). Thus, genetically manipulated ES cells can be used as an infinite source for DC with genetically modified properties.
Recently, we established methods for generation of DC from nonhuman primate ES cells and also for genetic modification of them (S. Senju, H. Suemori, H. Matsuyoshi, S. Hirata, Y. Uemura, Y.-Z. Chen, D. Fukuma, M. Furuya, N. Nakatsuji, and Y. Nishimura, manuscript in preparation). We hope to apply this method to human ES cells to generate genetically modified human ES-DC, although some modification might be necessary. In the future, Ag-specific immune modulation therapy by in vivo transfer of human ES-DC expressing antigenic protein along with immune-regulating molecules may well be realized, based on evidence in the current study in the mouse system. Possible applications of this technology are treatment of subjects with autoimmune and allergic diseases and also for induction of tolerance to transplanted organs, especially those generated from ES cells. Thus, the methods established in the present study may have implications as a broad medical technology.
Acknowledgments
We thank Dr. S. Aizawa (RIKEN Center for Developmental Biology, Kobe, Japan) for TT2, Drs. N. Takakura (Kanazawa University, Kanazawa, Japan) and T. Suda (Keio University, Tokyo, Japan) for OP9, Dr. H. Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) for pCAG-IPuro, Drs. T. Okazaki and T. Honjo (Kyoto University, Kyoto, Japan) for a cDNA clone for PD-L1, Tatsuko Kubo (Department of Molecular Pathology, Kumamoto University) for technical assistance, and Kirin Brewery Co., Ltd., for rGM-CSF. M. Ohara (Fukuoka, Japan) provided helpful comments on the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Grants-in-Aid 12213111, 14370115, 14570421, and 14657082 from the Ministry of Education, Science, Technology, Sports, and Culture, Japan, and a Research Grant for Intractable Diseases from Ministry of Health, Labour and Welfare, Japan, and grants from the Tokyo Biochemical Research Foundation and Uehara Memorial Foundation, and by funding from Meiji Institute of Health Science.
2 Address correspondence and reprint requests to Dr. Yasuharu Nishimura, Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. E-mail address: mxnishim@gpo.kumamoto-u.ac.jp
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; DC, dendritic cell; Ii, invariant chain; CLIP, class II-associated Ii peptide; PD-L1, Programmed Death-1 ligand; ES cell, embryonic stem cell; ES-DC, ES cell-derived DC; PLP, myelin proteolipid protein; MBP, myelin basic protein; IRES, internal ribosomal entry site; PCC, pigeon cytochrome c; KLH, keyhole limpet hemocyanin.
Received for publication May 20, 2004. Accepted for publication December 8, 2004.
References
Fujii, S., S. Senju, Y. Z. Chen, M. Ando, S. Matsushita, Y. Nishimura. 1998. The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells. Hum. Immunol. 59:607.
Senju, S., S. Hirata, H. Matsuyoshi, M. Masuda, Y. Uemura, K. Araki, K. Yamamura, Y. Nishimura. 2003. Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells. Blood 101:3501.
Wiley, S. R., K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith, R. G. Goodwin. 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673.
Kayagaki, N., N. Yamaguchi, M. Nakayama, K. Takeda, H. Akiba, H. Tsutsui, H. Okamura, K. Nakanishi, K. Okumura, H. Yagita. 1999. Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells. J. Immunol. 163:1906.
Lamhamedi-Cherradi, S. E., S. J. Zheng, K. A. Maguschak, J. Peschon, Y. H. Chen. 2003. Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL–/– mice. Nat. Immunol. 4:255.
Dong, H., G. Zhu, K. Tamada, L. Chen. 1999. B7–H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5:1365
Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura, L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, et al 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192:1027
Nishimura, H., M. Nose, H. Hiai, N. Minato, T. Honjo. 1999. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 11:141.
Nishimura, H., T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori, S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, T. Honjo. 2001. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291:319.
Song, K., Y. Chen, R. Goke, A. Wilmen, C. Seidel, A. Goke, B. Hilliard. 2000. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J. Exp. Med. 191:1095.
Hilliard, B., A. Wilmen, C. Seidel, T. S. Liu, R. Goke, Y. Chen. 2001. Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J. Immunol. 166:1314.
Lunemann, J. D., S. Waiczies, S. Ehrlich, U. Wendling, B. Seeger, T. Kamradt, F. Zipp. 2002. Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto)antigen-specific T cells. J. Immunol. 168:4881.
Kayagaki, N., N. Yamaguchi, M. Abe, S. Hirose, T. Shirai, K. Okumura, H. Yagita. 2002. Suppression of antibody production by TNF-related apoptosis-inducing ligand (TRAIL). Cell. Immunol. 219:82.
Nishimura, H., T. Honjo. 2001. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol. 22:265.
Okazaki, T., Y. Iwai, T. Honjo. 2002. New regulatory co-receptors: inducible co-stimulator and PD-1. Curr. Opin. Immunol. 14:779.(Shinya Hirata, Satoru Sen)