The Efficacy And Mechanism Of Allogenic Mesenchymal Stem Cell Transplantation For Autoimmune Diseases

Chapter One:The effect and mechanism of allogenic umbilical cord mesenchymal stem cell transplantation in refractory and active systemic lupus erythematosusSection one:Clinical study of umbilical cord derived mesenchymal stem cell transplantation for patients with refractory and active systemic lupus erythematosusObjective:To explore the clinical efficacy and safety of umbilical cord derived mesenchymal stem cell transplantation(UC MSCT)for patients with refractory and active systemic lupus erythematosus(SLE).Methods:Forty patients with refractory SLE were enrolled in this study.UC MSC were infused intravenously at day 0 and day 7(106 cells per kilogram of bodyweight each infusion).The clinical manifestations and laboratory parameters were compared before and after MSCT.Results::39/40(97.5%)patients completed two times of MSC infusions,one patient withdrew before the second MSC infusion because of disease progression.The overall survival rate is 92.5%(37/40)during 12 months follow-up.Three patients died of disease relapse and progression and uncontrolled infection.The rates of MCR and PCR were 32.5%(13/40)and 27.5%(11/40),respectively.Three and 4 patients underwent disease relapse at 9(3/24,12.5%)and 12 months(4/24,16.7%),respectively.Disease activity evaluated by BILAG for all the systems significantly ameliorated after MSCT(1mo:5.35±4.48,3mo:5.28±4.71,6mo:4.23±4.43,9mo:3.85±4.73,12mo:3.55±4.33,P all<0.01 versus pre-MSCT 10.78±6.09).SLEDAI scores also markedly decreased(pre-MSCT:10.83±4.63,1mo:8.55±3.99,3mo:7.43±3.93,6mo:6.30±3.63,9mo:6.40±3.84,12mo:6.48±3.52,P all<0.01 versus baseline levels).97.5%(39/40)patients had lupus nephritis at baseline,and renal BILAG score significantly declined.Moreover,twenty-four-hour proteinuria decreased after UC MSC infusions(pre-MSCT:2.24±1.43g,1mo:2.13±1.35g,3mo:1.91±1.20g,6mo:1.65±1.11g,9mo:1.24±1.09 g,12mo:1.41±1.33 g,P<0.05 at 9mo and 12mo visit times),in parallel with the amelioration of serum creatinine and blood uria nigrogen levels.Furthermore,serum albumin levels significantly improved at each visit times after UC MSC transplantation(pre-MSCT:31.72±7.47 g/L,1mo:36.99±5.83 g/L,3mo:38.00±6.69 g/L,6mo:38.38±6.32g/L,9mo:38.92±6.38 g/L,12mo:36.71±7.75 g/L,P all<0.001 versus baseline levels).In addition to renal system,BILAG scores for cutanuous and hematopoietic systems also ameliorated after transplantations.We observed a marked improvement of serum complements 3 and 4 but decreased serum autoantibodies including anti-nuclear antibody and anti double strand DNA antibody.Conclusion:UC MSC transplantation is safe and effective in treating refractory and active SLE patients.Section two:CD8+T cell-IFNy-IDO mediates UC MSC in the treating of SLE patientsObjective:To explore the underling mechanisms by which umbilical cord mesenchymal stem cells showing therapeutic effect in active SLE.Methods:The effects on inhibiting T cell proliferation by umbilical cord derived MSC were determined.Then MSC functional molecules were examined by real-time PCR under the stimulation of peripheral blood mononuclear cells(PBMC)from healthy controls and SLE patients,respectively.CD4+ and CD8+ T cell were purified by microbeads to stimulate MSC,respectively,to determine IDO expression as well as supernatant cytokines,to further get to know which cell subset(s)or which molecule(s)involved in MSC mediated T cell proliferation inhibition.Meanwhile,the possible signaling pathways were assessed.Moreover,we analyzed the correlation between baseline serum cytokine levels and clinical response by MSC transplantation(MSCT).Results:UC MSC efficiently inhibited T cell proliferation in both healthy controls and lupus patients.In vitro activated lupus PBMC significantly induced MSC to secret TGF-β1,IDO,HGF and IL-6,with a more than 200-fold increase of IDO.The addition of IDO inhibitor could almost completely abrogate MSC mediated T cell proliferation inhibition.Moreover,we found that lupus peripheral CD8+T cell markedly stimulated MSC to secret IDO and supernatant IFN-γ significant increased.The addition of anti-IFN-γ monoclonal antibody inhibited IDO activity and similarly abrogate inhibition of T cell proliferation by MSC.We further found that lupus CD8+T cell secreted predominant intracellular IFN-y compared to CD4+T cell or compared to healthy CD8+or CD4+T cell subsets.Furthermore,in the presence of lupus CD8+T cell,IFNGR/JAK/STATs signaling pathways were activated,then to induce MSC to secret IDO and upregulate IDO activity.However,bone marrow MSC from lupus patients were less responsive to allogenic CD8+T cell or recombinant IFN-y stimulation and produced far less IDO,consequently failed to efficiently inhibit T cell proliferation.In the last,clinical analysis showed that lupus patients had significant higher proportion and absolute number of CD3+CD4-CD8+T cell than healthy controls,and serum IFN-γmarkedly increased.Predominant IFN-y in lupus patients was mainly secreted by CD8+T cell.Baseline serum IFN-y as well as CD8+T cell levels were significant higher in good responders than in poor responders who underwent UC MSCT.Conclusion:We uncovered a previously unrecognized CD8+T cell-IFN-γ-IDO axis that mediates the therapy by allogenic MSC in lupus patients.Chapter Two:Clinical observation and mechanism study of umbilical cord mesenchymal stem cell transplantation for patients with systemic sclerosisObjective:To explore the clinical efficacy and possible mechanism of umbilical cord mesenchymal stem cells transplantation(UC-MSCT)for patients with refractory systemic sclerosis(SSc).Methods:Five patients with refractory SSc were enrolled in this study.UC-MSC(1×106/kg body weight)were infused intravenously for each patient.The clinical manifestations and laboratory parameters were compared before and after MSCT.Results:All patients tolerated well with allogenic UC MSCT,and no adverse events occurred during or after MSC infusion.The Modified Radnan Skin Score(MRSS)decreased and patients quality of life index(HAQ-DI)improved after UC MSCT.Serum anti-nuclear antibodies decreased.For those who had refractory vasculitis and skin ulcers,obvious skin repairment were found after MSC transplantation.The mechanism studies showed that UC MSCT could upregulate CD4+CD25+Foxp3+regulatory T cells proportion,as well as serum TGF-β levels in SSc patients.The absolute number of peripheral CD3+T cells,especially the CD4+T cell subset significantly decreased after MSCT.Further analysis demonstrated that UC MSC induced CD4+T cells apoptosis in patients in vivo and in vitro.Moreover,we found that compared to normal MSC,bone marrow MSC from SSc patients were defective in secreting Fas,FasL as well as MCP-1,and failed to induce T cell apoptosis.Conclusion:UC MSCT is safe and effective for treating refractory SSc patients.UC MSC can induce CD4+T cells apoptosis and upregulate TGF-β and subsequently induce Treg cells,which is a main mechanism for the immune tolerance in SSc patients.Chapter Three:Clinical observation and mechanism of umbilical cord mesenchymal stem cell transplantation for patients with primary Sjogren’s SyndromeObjective:To explore the clinical efficacy and possible mechanism of umbilical cord derived mesenchymal stem cell transplantation(UC-MSCT)for patients with refractory primary Sjogren’s Syndrome(pSS).Methods:Twenty-four patients with refractory pSS were enrolled in this study.UC-MSC(106/kg body weight)were infused intravenously for each patient.The clinical manifestations and laboratory parameters were compared before and after MSCT.Results:All patients tolerated well with allogenic UC MSCT,and no adverse event occurred during or after MSC infusion.Mean SS disease damage index(SSDDI)scores decreased from 5.63 ± 1.44(baseline)to 4.33 ± 1.79 at 1 month,4.08 ± 1.44 at 3 months,3.46± 1.18 at 6 months,and 3.08 ± 1.21 at 12 months after MSCT(all P<0.05).SS disease activity index(SSDAI)also decreased.Global assessment by visual analog scale(VAS)improved 1 month after UC MSCT,and showed further ameliorations at 3,6,and 12 months.Unstimulated salivary flow rate of all 11 patients with symptoms of xerostomia increased significantly 2 weeks after UC MSCT(1.00 ± 0.78 ml/10 min at 2 weeks versus 0.63 ± 0.73 ml/lOmin at baseline,P<0.001)and showed a 2-fold increase at 1 month(1.26 ± 1.02 ml/10min,P<0.001 versus baseline).This flow rate continued to increase on subsequent follow-ups.Stimulated salivary flow rate also significantly increased after MSCT(P<0.05 at all visit times versus baseline).The determination of modified treatment emergent symptom scale(TESS)score decreased at 2 weeks visit,and was maintained at this low level on subsequent visits.Furthermore,sialogram of the parotid gland and its excretory function were analyzed by the same radiologist and rheumatologist.Punctate sialectasias in the parotid gland slightly declined,dilation of main duct was improved 12 months after UC MSCT,and excretory function was also partially restored.Serum anti-SSA/Ro decreased from 84.76 ± 62.19 U/ml at baseline to 0.51 ± 0.22 U/ml 1 month post-MSCT(P<0.001,n = 5),and serum anti-SSB/La decreased from 146.62 ± 83.08 U/ml at baseline to 52.61 ± 38.67 U/ml 1 month post-MSCT(P<0.001,n = 4).Serum globulin levels also decreased after MSCT.The mechanism studies showed that the frequency of Thl7 cells decreased while regulatory T cell(Treg)increased,and serum levels of TGF-P increased while IL-17 decreased post-MSCT.Conclusion:UC MSCT is safe and effective for refractory pSS patients,and immunoregulation of Treg/Th17 cell subset balance plays pivotal role in the treatment of pSS by UC MSC.Chapter Four:Clinical efficacy of allogeneic mesenchymal stem cell transplantation in patients with drug-resistant polymyositis and dermatomyositisObjective:The aim of this study is to assess the safety and clinical efficacy in a small-scale study with ten drug-resistant polymyositis(PM)or dermatomyositis(DM)patients who received allogeneic bone marrow or umbilical cord derived mesenchymal stem cell transplantation(MSCT).Methods:A single-arm trial involved 10 DM/PM patients,4 for DM and the other 6 for PM,aged from 19 to 44 years old,that were either refractory to standard treatment and/or with severe systemic involvement including interstitial lung disease(ILD),refractory skin ulcer.Disease duration was 7.5 months(6 to 60 months).All patients met the Bohan and Peter criteria for DM or PM.Three patients received bone marrow(BM)derived MSC,two of which were given a second umbilical cord(UC)derived MSC due to disease relapse.The other 7 patients were infused with UC-MSC only once.BM or UC MSC were infused intravenously with a dose of one million cells per kilogram of bodyweight each infusion.The clinical manifestations and laboratory parameters were compared pre-and post-MSCT.Advert events were monitored all the time during and post-MSCT.Results:The patients were followed up for 17 months(range,7 to 24 months),no transplantation related adverse event was found.Serum creatine kinase(CK)for all the patients decreased significantly at the first 6 months(baseline:2380.0±673.8U/L;1m:1027.0±266.7U/L;2m:486.2±143.1U/L;3m:92.9±19.5U/L;6m:248.8±135.6U/L,all P<0.05 versus baseline value),and continued to decrease to 106.0±39.4U/L for 6 patients who completed 1 year visit,in parallel with the amelioration of serum CK-MB levels at the same visit times.Meanwhile,patient’s global assessment by VAS and detection for the Manual Muscle Test(MMT)also ameliorated.In addition,MRI confirmed alleviated inflammation in the thigh muscles 3 months after MSC infusion for one patient.Six patients were complicated with moderate-to-severe ILD shown as interstitial pneumonia.High-resolution CT scans manifested obvious amelioration in 3 patients,and at least no progression in 2 patients 6 months post-MSCT.Furthermore,for one patient with refractory skin ulcers on both knees,elbows and proximal interphalangeal joints,matter plus decreased and new granulation tissues appeared,and almost healings of the ulcers 6 months after MSCT.Disease relapsed in 3 patients at 6,8,8 months post-MSCT,respectively,2 of which received a second UC MSC infusion and both had clinical effect.Two patients died at 11 and 6 months after MSCT,both due to rapid disease progression and uncontrolled pulmonary infection.No transplantation related mortality or any significant toxicity was observed during or after MSC infusion.Conclusion:We provide direct evidence that allogeneic BM or UC MSC infusion is safe and effective in drug-resistant DM/PM patients.However,further observation with more patients included and longer periods of follow-up will be needed to determine the efficacy and safety of this novel approach for the treatment of DM/PM.