Research On Immune Tolerance Induced By Indoleamine 2,3-Dioxgenase Transfected Mesenchymal Stem Cells

Research Background:Nowadays organ transplantation has become a quite effective therapy for some terminal chronical diseases.But also there are many serious side effects, such as, severe infection, myelotoxicity and glucose metabolism, due to the heavy use of immunosuppressants which obviously reduced recipients’survival rate. As a result, how to induce donor specific immune tolerance becomes a major problem in our field of transplant immunity. Recent studies show that mesenchymal stem cells (MSCs) can not only divide into osteoblast, chondroblast and lipoblast but also have the ability of immune-regulation. In vitro, studies revealed MSCs could inhibit T cell proliferation in a certain extent. MSCs are wild used in the study of immune tolerance induction. But the inhibitory function of MSCs is so weak, that there still are arguments about their positive roles in aninal transplant models. Therefore, how to enhance the inhibitory function of MSCs so as to protect grants from acut rejection and even induce immune tolerance becomes our main goal. MSCs regulate immune function primarily by secreting cytokines such as indoleamine 2,3-dioxgenase (IDO). IDO degrades the essential amino acid, tryptophan, which is required for T cell metabolism; thus IDO directly inhibits the activation and proliferation of T cells. Furthermore, IDO induces T cell anergy, apoptosis and transdifferentiation into Tregs via activation of GCN2. The tryptophan metabolite, kynurenine, has an indirect effect on immune cells, including T and B lymphocytes, antigen presenting cells (APCs) and Tregs. The immunomodulatory capacity of MSCs is greatly reduced when IDO-mediated degradation of tryptophan is blocked by 1-MT, suggesting that IDO plays a critical role in maintaining the immunomodulatory function of MSCs. In conclusion, whether transfection of IDO into MSCs could enhance the inhibitory function of MSCs, even induce immune tolerance needs further study.Objective:Transfect IDO gene into MSCs so as to enhance the inhibitory function of MSCs by induction of IL-10、TGF-β and CD4+ CD25+ Tregs production. Also to increase the regulatory function of CD4+CD25+ Tregs in goals of protecting grants from acute rejection, prolonging survival and inducing immune tolerance.Methods:1. Cultivation of Rabbit BM-derived MSCs:Multipotent MSCs were obtained from BM aspirate from New Zealand rabbits (mean weight 3±0.5 Kg) by density gradient centrifugation and cultured in 25 cm2 flaks with complete DMEM/F12 1:1 medium in a humidified incubator for a week until the confluence exceeded 80%. MSCs were used between passage three and six.2. Construction of The Recombinant Lentiviral Vector:The nucletotide sequence of IDO was retrieved from GenBank and the target gene was PCR-amplified using a rabbit IDO-specific primer pair. Lentiviral vector GV218 containing GFP was digested with Age I (Genechem, Inc., Shanghai, China).The amplified IDO gene was then linked with digested vector GV218. After transduction of competent E. Coli DH5a cells, positive clones expressing the target plasmids were selected (pGC-FU-GFP-IDO). pGC-FU-GFP-IDO with pHelper 1.0 and pHelper 2.0were subjected to high purity and endotoxin-free extraction and then simultaneously transfected into 293 cells. The supernatant (containing lentiviral particles) was then collected and the virus titer was determined by end-point dilution assay.3. Lentiviral Infection of Rabbit MSCs:A day before infection, MSCs were seeded into a 6-well plate at a density of 4.5×103 cells/well. After 12 hours, the culture medium was replaced with 1 ml Enhanced Infection Solution containing 10μg/ml ploybrene. Next, either empty lentivirus or IDO-lentivirus was added to the well (MOI for empty lentivirus=100; MOI for IDO-lentivirus=120). When MSCs, Lenti-MSCs and IDO-MSCs reached full confluence,20μg Fe/ml Molday ION Rhodamine B in complete DMEM/F121:1 medium was added to each well for 12 hours prior to injection into animals.4. Immunoblot Analysis:Expression of IDO by IDO-MSCs was examined by immunoblotting using an anti-rabbit IDO monoclonal antibody and protein expression was normalized to that of β-actin.5. Suppression Assays:To determine the immunosuppressive effects of WT-MSCs, Lenti-MSCs, and IDO-MSCs, all three cell types were co-cultured with CD4+CD25- T cells (2×105/well) for 72 hours at ratios of 1/11/251/501/100 in the presence of 8μg/ml PHA and 500 IU/ml IFN-γ. T cell proliferation was then assessed in a [3H]-thymidine incorporation assay. To examine the MSCs-induced antigen-specific suppressive function of CD4+CD25+ Tregs, WT-MSCs, Lenti-MSCs, or IDO-MSCs from New Zealand rabbits were co-cultured with PBMCs from Japanese white rabbits for 48 hours in the presence of 500 IU/ml IFN-y and 300 IU/ml IL-2. CD4+CD25+Tregs were sorted from the co-cultured Japanese white rabbit PBMCs and used as inhibitor cells whereas CD4+CD25- T cells were used as responder cells. Irradiated PBMCs from New Zealand rabbits were used as stimulator cells. Inhibitor cells, stimulator cells and responder cells were co-cultured at a ratio of 1:2:2 for 48 hours in the presence of 300 IU/ml IL-2 and T cell proliferation measured as described above. To confirm that IDO-MSCs induced immunologic tolerance after renal transplantation, a mixed lymphocyte reaction (MLR) was performed using CD4+T cells from recipients (as responder cells) and irradiated PBMCs from donor derived, none donor derived New Zealand rabbits or third-party Japanese white rabbits (as stimulator cells). Stimulator cells and responder cells were cultured at a ratio of 1:1 for 48 hours in the presence of 300 IU/ml IL-2. T cell proliferation was then measured as described above. The percentage inhibition of proliferation was calculated as follows:% inhibition=[counts per minute (CPM) (Positive)-CPM (Sample)]/CPM (Positive)* 100.6. Flow Cytometry Analysis and cell Sorting:The percentage of CD4+CD25+Foxp3+Tregs within thePBMCs and the expression of CTLA-4 by CD4+CD25+ Tregs were analyzed by flow cytometry utilizing monoclonal antibodies against rabbit CD4,CD25 and Foxp3 and CTLA-4. Cells were then examined using a FACSCalibur flow cytometer. The CD4+CD25+ and CD4+CD25- T cell subpopulations were sorted using a FACSAria II Special Order System.7. RT-PCR:The expression of Foxp3 by PBMCs co-cultured with WT-MSCs, Lenti-MSCs, or IDO-MSCs was assessed by RT-PCR using SYBR Green Real-time PCR Master Mix and the following rabbit Foxp3-specific primers:forward,5’-GGACCGTGGATGAGTTTG AAT-3’; reverse,3’-CCCTTGCTTCTTTCCTTTCTTT-5’,β-actin specific forward and reverse primers were as follows:forward,5’-CTAGTACGTTGCTATCCAGGC-3’; reverse, 3’-CTCCTTAATGTCACGCACGAT-5’.Foxp3 expression was calculated using 2-ΔΔCt method..8. Rabbit Orthotopic Renal Transplantation and Therapy:The left kidneys from New Zealand rabbits were transplanted into Japanese white rabbits by end-to-end anastomosis of the donor left renal vein/artery/ureter with the recipient left renal vein/artery/ureter. The recipient’s right kidney was removed immediately after grafting. The rabbits were then divided into five groups and treated as described above. The rabbits in the WT-MSCs-, Lenti-MSC, and IDO-MSC-treated groups wore a magnet (0.5T, LINK, Inc., Beijing, China) on their back, which was sited over the graft for MSCs trending.9. Biochemical Monitoring, Histologic Analysis and Donor-specific skin grafting:A 1-type creatinine f kit and an AU5400 Automation Chemistry System Instrument were used to monitor serum creatinine (S-Cr) levels. IL-10, prostinE2, IL-2 and TGF-β were measured using ELISA kits. Sections of graft tissue (3-μm-thick) were were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin. The stained sections were examined and graded according to the Banff classification standard.On post-operative day (POD) 90, IDO-MSCs-treated recipients also received full-thickness skin grafts (3*3 cm2) from donor and non-donor New Zealand rabbits and from third-party Japanese white rabbits. Animals were monitored daily for signs of rejection.Results:1. Cultivation of Rabbit BM-derived MSCs:Bone marrow cells (BMCs) isolated from rabbits began to adhere to culture flasks after an average of 3 hours cultivation in complete DMEM/F12 1:1 medium. After 48 hours, fibroblast-like colonies of fusiform cells (MSCs) were observed. MSCs reached greater than 80% confluence after an average of 6 days cultivation, after which they were passaged. After a further 3 days, the MSCs again exceeded 80% confluence and showed morphology similar to that of fibroblasts.2. Lentiviral Infection of Rabbit MSCs:MSCs were infected with pGC-FU-GFP-lentivirus or pGC-FU-GFP-IDO lentivirus at an MOI of 100 and 120, respectively. GFP expression was visible under an inverted fluorescent microscopy 24 hours later. Fluorescence intensity peaked 96 hours later,80%of the MSCs expressed GFP. Confocal laser scanning microscopy showed that SPIO was distributed uniformly within the cytoplasm of MSCs at 12 hours after incubation with Rhodamine B-labeled Super-paramagnetic iron oxide.3. IDO Lentivirus Transfected Rabbit MSCs Stably Expressed IDO Protein:In the presence of 500 IU/ml IFN-y, IDO-transfected rabbit MSCs showed stably expression of IDO protein (molecular weight 43 KDa). No IDO protein was detected in WT-MSCs and pGC-FU-GFP lentivirus-transfected MSCs.4. IDO Transfected MSCs Strongly Inhibit CD4+CD25" T cell Proliferation:CD4+CD25" T cells proliferation in the presence of PHA and the same ratios (1:1,1:25, 1:50,1:100) of IDO-MSCs was inhibited to an even greater extent. WT-MSCs and Lenti-MSCs significantly inhibited T cell proliferation only at MSC:T cell ratios of 1:1 and 1:25 (P<0.05), whereas their immunosuppressive effects disappeared at ratios of 1:50 and 1:100. However, IDO-MSCs still exhibited potent inhibitory effects on CD4+CD25- T cells proliferation at ratios of 1:50 and 1:100 (P<0.05).5. IDO-Transfected MSCs Enhanced The Antigen-Specific Immunosuppressive Functions of CD4+CD25+ Tregs:Proliferation was inhibited to an even greater extent upon co-culture with WT-MSCs, Lenti-MSCs, or IDO-MSCs. Thus IDO-MSCs treated Tregs were the most powful inhibitor of antigen-specific T cell proliferation (P<0.05).6. IDO-Transfected MSCs Enhanced The Antigen-Specific Immunosuppressive Functions of CD4+CD25+ Tregs:The percentage of CD4+CD25+Foxp3+Tregs in the WT-MSCs-treated and Lenti-MSCs-treated groups was significantly higher than that in the untreated group (P< 0.05). However, CD4+CD25+Foxp3+ Tregs in the IDO-MSCs-treated group was significantly higher than that in the WT-MSCs-treated and Lenti-MSCs-treated groups (P< 0.01). In addition, IDO-MSCs increased Foxp3 expression compared with that in other groups (P<0.01).7. IDO-Transfected MSCs Enhanced The Antigen-Specific Immunosuppressive Functions of CD4+CD25+ Tregs:Proportion of CTLA-4 expressing Tregs was much higher in the IDO-MSCs-treated group than that in the untreated, WT-MSCs-treated, or Lenti-MSCs-treated groups (P< 0.05).8. IDO-Transfected MSCs Stimulated Tregs to Secret IL-10 and TGF-β:The amounts of IL-10 and TGF-β1 secreted by Tregs co-cultured with IDO-MSCs were much higher than those secreted by Tregs co-cultured with WT-MSCs and Lenti-MSCs (P<0.01). There was no significant difference in the secretion of PGE2 among these groups (P>0.05).9. IDO-Transfected MSCs Prolonged Graft Survival and Induced Tolerance:Compared with those in the CsA-, WT-MSCs-, Lenti-MSCs-, and IDO-MSCs-treated groups, recipients in the control group showed significantly increased S-Cr levels on Day 7 post-transplantation. S-Cr levels were significantly higher in the WT-MSCs- and Lenti-MSCs-treated groups than in the CsA- and IDO-MSCs-treated groups on Day 14; there was no significant difference in the S-Cr levels between CsA-and IDO-MSCs-treated groups (P>0.05).Recipients in the WT-MSCs- and Lenti-MSCs-treated groups survived significantly longer (P<0.05). However, the survival time in IDO-MSCs-treated group was much longer (P<0.05) but not significantly different from that of the CsA-treated group (a median 70.4 ±23.5 days, P>0.05).Grafts from the CsA- and IDO-MSCs-treated groups were well preserved, showing a basically normal structure except for a little focal monocyte infiltration in the glomerulus; there was little monocyte infiltration of the renal tubules and vessels. Kidneys from the control group showed significant interstitial infiltration, with monocytes and lymphocytes visible in the renal tubules and in some vessels. Swelling and necrosis of tubular epithelial cells was apparent. Analogous to the control group, kidneys from the WT-MSCs-and Lenti-MSCs-treated groups showed evidence of wide-spread interstitial infiltration by monocytes, particularly of the arteries. Arterial fibrinoid necrosis was observed in many sections. Furthermore, We observed that most of the MSCs were located within the glomeruli and interstitial areas of the graft.Since the survival promoting effects of IDO-MSCs were additive, we performed MLR, flow cytometry, ELISA, and skin grafting to examine tolerance in recipients in the IDO-MSCs-treated group. We found that recipients in the IDO-MSCs-treated group show a weak immune response to donor-specific antigens, but a normal response to non-donor-related antigens.Flow cytometry revealed that CD4+CD25+ Foxp3+ Tregs in the control group decreased rapidly after renal transplantation, in accordance with the sharp increase in the S-Cr level, suggesting severe acute rejection. The Treg populations in the WT-MSCs-and Lenti-MSCs-treated groups, however, increased temporarily on Day 7, before decreasing on Day 14. These results suggest delayed rejection in accordance with slowly increasing S-Cr levels. The Tregs population in the CsA group decreased on Day 14 and on Day 49; however, stable renal function suggested that CsA induced an immunoinhibitory mechanism other than tolerance. The Tregs population in the IDO-MSCs-treated group increased and was significantly higher than that in the CsA group (P<0.05).These findings were in accordance with stable renal function and long-term graft tolerance in IDO-MSCs group.ELISA revealed that IL-2 levels increased significantly in WT-MSCs- and Lenti-MSCs-treated groups (P<0.05 respectively), decreased in CsA grop (P<0.05) but remained slightly higher than base-line (P<0.05) in IDO-MSCs-treated group. Apposite to IL-2, IL-10 and TGF-∞ levels decreased significantly in all WT-MSCs-treated, Lenti-MSCs-treated and CsA groups (P<0.05 respectively), but increased significantly in IDO-MSCs-treated group(P<0.05).Sking transplantaion reveald that IDO-MSCs-treated recipients accepted skin grafts from donor rabbits for over 60 days, but rejected those from non-donor New Zealand rabbits and third-party Japanese white rabbits within 7± 2 days and 8±3 days, respectively.Conclusions:1. Oryctolagus cuniculus IDO gene can be safely and successfully transfected into rabbit MSCs in which it directs stable and high level of IDO expression.2. IDO transfection enhances the direct inhibitory function of MSCs on CD4+CD25- T cell Proliferation.3. IDO transfected MSCs induce CD4+CD25+Tregs production and increase the antigen-specific inhibitory function of CD4+CD25+Tregs by up-regulated expression of CTLA-4 and stimulated secretion of IL-10, TGF-β.4. IDO transfected MSCs induce CD4+CD25+Tregs production in peripheral after renal transplantation and protect grafts from acute rejection, prolong survival time and induce immune tolerance eventually.In summary, IDO transfected MSCs increasedthe direct suppressive function of MSCs and also, inducedthe production of CD4+CD25+Tregs and enhance the antigen-specific inhibitory ability of CD4+CD25+Tregs. In the study of renal transplantation model, IDO transfected MSCs protected the grafts from acute rejection, prolong survival time and induce immune tolerance eventually. These results provided a mechanism in which would explain the inhibitory function of MSCs, furthermore, they provided unique value in MSCs clinical application.