| Background and Objectives:Allogeneic haematopoietic stem cell transplantation(allo-HSCT)has been widely used in hematologic and non-hematologic malignancies,and even considered to be the only way to cure some malignancies.Graft-versus-host disease(GVHD)remains a common complication limiting the widespread use of allo-HSCT.Acute GVHD(aGVHD)occurs in 30-80%of recipients experiencing HSCT,while chronic GVHD(cGVHD)occurs in 30-70%of recipients surviving longer for 100 days.Half of those patients died of this complication.GVHD not only influences the survival of patients,but also influences their living quality.Recently,it is widely accepted that the pathophysiology of GVHD is that the differences of HLA between donor and recipient induce donor T cells activation and generate the abnormal immune response in target organs of GVHD patients.The primary therapy for GVHD is immunosuppressant,but high dose and long-time use of that can increase the incidence of tumor relapse,infection and secondary maligancy.Therefore,controling the occurrence and development of GVHD,retaining graft versus leukemia(GVL)effect and anti-infection immunity are the main emphasis of transplantation immunity.Mesenchymal stem cells(MSCs)are a form of adult stem cells existing in many tissue.Due to its capacity of multilineage differentiation,modulating immune and inflammation responses,supporting hematopoiesis and repairing tissue,MSCs have been widely administered in the treatments of many immune-based disorders,such as Crohn's disease,rheumatoid arthritis and multiple sclerosis.The most successfully clinical application of MSCs is involved in GVHD.Since a case report in 2004,Le Blanc et al.firstly reported that infusion of MSCs rescued a pediatric patient experiencing refractory aGVHD.Subsequently,Le Blanc group reported another clinical study including 55 patients with steroid-resistant aGVHD.They found that 30 patients had a complete response(CR)and nine showed improvement.MSCs have been considered as a promising therapy for the treatment of aGVHD.Recently,a growing number of studies have been performed to investigate the efficacy of MSCs for aGVHD treatment.Although the results are still controversy,most prospective and retrospective data suggest that MSCs are effective to aGVHD.Nowadays,the mechanisms of MSCs modulating aGVHD include that MSCs suppress T cells proliferation,induce the generation and proliferation of regulatory T cells(Tregs),inhibit differentiation of precursors into dendritic cells(DCs)as well as suppress DCs maturation,and influence the function of natural killer(NK)cells and antigen-presenting cells(APCs).They also secret some immunosuppressive factors,such as HLA-G,transforming growth factor-?(TGF-?)and interleukin(IL)10.Our preliminary work found that MSCs can improve GVHD by modulating B cells.MSCs increase the allograft antibody and plasma B cell activating factor(BAFF)levels in cGVHD patients.But all these studies primarily focused on peripheral immune compartments.Thymus is the primary immune tissue of T cell development and maturation.And it plays a critical role on constructing a complete T cells receptor(TCR)repertoire and inducing the immune tolerance.In the recipients of allo-HSCT,the toxicity of conditioning regimen and GVHD disrupt thymic epithelial cell(TEC),dysregulated the negative and positive selection and generate the auto-reactive T-lymphocytes,which aggravate the occurrence and development of GVHD.Moreover,reconstruction of T-cell compartments post-HSCT are mainly accomplished by thymus-independent and thymus-dependent pathways.A complete reconstruction of T immune competence depends on thymus-dependent pathway,which experiencing the positive and negative selection of thymus,and selective removal of auto-reactive T cells to induce immune tolerance.Donor derived mature T cells proliferate through thymus-independent pathway.Thymus-independent pathway assures the early reconstruction of T-cell compartments post-transplantation,but it failed to reconstruct long-lasting immune competence.T-cell compartments reconstruction via thymus-independent pathway are unable to form immune tolerance to the recipients' antigens,but they are the main effector cells in the development of GVHD,especially aGVHD.MSCs as a component of thymic stroma cells(TSC)can secret cytokines,such as IL-4?IL-7?interferon y(IFN-y).MSCs also play a vital role on the development and regeneration of TEC.MSCs interact with various immune cells and secret various soluble mediators,which promote the development and regenerationof TEC.In addition,MSCs possess the capacity to differentiate to TEC depending on the stimulation of specific inflammatory factors.Our preliminary work evaluated the level of T cell receptor rearrangement excision circles(TRECs)in eight aGVHD patients receiving MSCs treatment.We found that the level of TRECs in MSCs group is significantly higher than those in non-MSC control group.Thus,we proposed that MSCs might promote immune tolerance and reconstruction by improving thymic function.But it is rarely reportd in this field.Therefore,it is our innovation point focusing on the central immune tissue-thymus to study the mechanism of MSCs modulating GVHD and promoting immune reconstruction.It has profound significance for establishing a platform for clinical application of MSCs for GVHD treatment.In this study,we established the aGVHD mice model and a system of GFP-MSC isolation and expansion.In aGVHD mice model,we infused MSCs into aGVHD mice,observed the survival and severity of aGVHD and explore the mechanisms of MSCs promoting the thymic regeneration and immune reconstruction.In refractory aGVHD patients,we observed the efficacy and safety of MSCs for aGVHD treatments.Through the above study,we aim to reveal the mechanisms of MSCs modulating aGVHD from the view of central immune tissue-thymus.Methods1.To establish a murine model of aGVHD after allo-HSCT:BALB/c experiencing total body irradiation(TBI)received donor-derived 5×106 bone marrow(BM)cells and 4×106 spleen cells to esrablish aGVHD model.We observed the aGVHD clinical performance,severity and survival every other day post-transplantation.And we measured the donor marker to evaluate the chimerism by fluorescence activated cell sorter(FACS).Animals were killed at day 28 after allo-HSCT,and aGVHD target organs were harvested for histopathologic analysis.2.Isolation,culture,expansion and identification of green fluorescence protein(GFP)labelled MSCs:We adopt modified cells adherent culture method to isolate MSCs from BM derived GFP labelled C57BL/6 mice.The mophorlogy of MSCs was observed by general and fluorescence microscope.GFP expression and MSCs surface markers were monitored by FACS and multiple differentiation potential was confirmed by lineage-specific induced differentiation to osteoblasts and adipocytes.3.The stratrgy of MSCs modulate aGVHD:We adopt established aGVHD model to explore the dose and timing of MSCs administration for aGVHD treatment.To study the influence of MSCs dose to aGVHD mice survival.aGVHD mice were randomly divided into four groups,MSCs 1×105cells/mice,5×105cells/mice,1×106 cells/mice group and non-MSC control group.We observed the survival every other day post-transplantation.To study the influence of MSCs timing,aGVHD mice were randomly divided into four groups,MSCs administration on 3-day,7-day,21-day group and non-MSC control group.The survival of aGVHD mice was monitored every other day post-transplantation.4.The administration of MSCs influences aGVHD treatment:aGVHD mice were randomly divided into two groups,including MSC group(5×105 cells/mice at 7 days after allo-HSCT)and non-MSC control group.We observed the aGVHD clinical performance,severity and survival every other day post-transplantation.Animals were killed at day 28 after allo-HSCT,and aGVHD target organs were harvested for histopathologic analysis.5.MSCs home to thymus in aGVHD mice:GFP labeled MSCs were infused in aGVHD mice at 7 day after transplantation.MSC-untreated aGVHD mice were considered as the control group.Cytokeratin 5(K5)and cytokeratin 8(K8)respectively represent medullary TECs(mTECs)and cortical TECs(cTECs).Immunohistochemistry and FACS were used to track the migration of MSCs in thymus.6.The mechanism of MSCs affecting thymic function:aGVHD mice were randomly divided into MSC group and non-MSC control group.MSC group mice were administered GFP labelled MSCs.At day 28 after allo-HSCT,thymic index and the number of thymocyte were compared between two groups to indirectly evaluate the effect of MSCs for thymic function.The level of TRECs in thymus,spleen and peripheral blood were tested by real time fluorescent quantitative PCR(FQ-PCR),which directly evaluated thymic output function.7.The effect of MSCs for immune reconstruction after transplantation:The proportion of T cell subsets(CD4+CD8+,CD4+CD8-,CD4-CD8+,CD4+CD25+Foxp3+T)before and after MSCs treatments in thymus,spleen and peripheral blood were monitored by FASC.aGVHD mice without MSCs treatment were considered as the control group.8.The clinical sudy of MSCs treating refractory aGVHD patients:Forty-seven patients with refractory aGVHD were enrolled,including 28 patients receiving MSCs and 19 patients without MSCs treatment.MSCs were given at a median dose of 1×106 cells/kg weekly until patients got complete response(CR)or received 8 does of MSCs.In non-MSC group,refractory aGVHD only reveived firstline and secondline aGVHD treatments.We observed the immunomodulation effects of MSCs,including the efficacy of MSCs for refractory aGVHD,infections,tumor relapse and cGVHD after MSCs treatment.The proportion of lymphocyte subsets(CD4+CD8-T,CD4-CD8+T,Foxp3+Tregs,CD19+B)before and after study treatments in peripheral blood were monitored by FASC.The level of TRECs in peripheral blood were tested by FQ-PCR.9.Statistical analysis:SPSS 17.0 statistical package was used for processing.All the measurement data were showed as meanąstandard deviation(x ąs).Log-rank were used for the comparison of survival time and Kaplan-Meier were used for survival curve description.The chimeric,the formation rate of adipocyte and osteoblast between P3 and P5 MSCs,and the clinical and histological aGVHD scores between BM and GVHD groups were compared with independent sample t test.The comparisons of TRECs and lymphocyte subsets were used paired samples test.One-way ANOVA was used for comparisons of thymic index,the number of thymocytes,the level of TRECs and the percentages of T cell subsets.When variance was homogeneous,LSD method was used for further multiple comparisons.When variance was heterogeneous,the Welch method was corrected,and Dunnett T3 method was used for further multiple comparisons.Efficacy evaluations in the compared groups were explored using the Chi-squared test.P<0.05 was considered statistically significant.Results:1.Establishment of C57BL/6?BALB/c aGVHD murine model after allo-HSCT:?Chimerism:At 28 day post-transplantation,the chimerism expressions of H-2Kb are over 97%in BM and GVHD group.So the donor cells are successful engraftment,and there is no significant difference between the two groups(P=0.436).?Survival:BM group mice can survive longer than 45 days,while the median survival time of GVHD group is only 23 days(P<0.001).The peak death of GVHD mice appeared in 3-4 weeks after allo-HSCT.?aGVHD manifestation:The signs of GVHD appeared from 7 day post-HSCT,such as hunching and diarrhea,and alleviated with the increase of body weight.After 14 day,aGVHD group mice reappeared aGVHD clinical manifestation,for example,scaling of fur,ruffling,activity decreased,hunching and diarrhea.However,the weight,activity and diarrhea of BM group gradually recovered and did not have aGVHD performance.The clinical and pathological score in GVHD group were significantly higher than those in BM group(P<0.001).The pathology of GVHD target organs showed that tissue structure were disorganized and lymphocyte infiltration were found between cells.2.Isolation,culture,expansion and identification of GFP labelled MSCs:the form of MSCs adherent cells was uniform,showed the shape of fibroblast and cell colony arranged in spiral.The fluorescent microscopy showed that MSCs expressed green fluorescent signals and without obvious GFP expression decay.The positive expression of GFP in P3 and P5 MSCs were respectively 99.14%and 95.05%,which did not have significant difference(P=0.215).The flow cytometric analysis confirmed that the cells were positive for CD44,CD90,CD 106,CD 140a,Sca-1,and negative for CD1lb,CD34,CD45.Their had the abilities to differentiate into osteogenic and adipogenic lineages.The formation rate of adipocyte and osteoblast between P3 and P5 MSCs did not have significant differences(P=0.856,P=0.255).3.The study of MSCs modulating aGVHD:?the therapeutic strategies of MSCs:The survival of MSCs(5×105cells/mice)treating GVHD mice significantly improve aGVHD mice survival compared with non-MSC group(P=0.014).GVHD mice receiving MSCs 5×105cells/mice have a better survival than mice receiving MSCs 1×105cells/mice(P=0.031).However,GVHD mice receiving MSCs 1×106 cells/mice died in 2-5 minutes after MSCs infusion.We considered that the mice died of important viscera embolism.In addtion,there was no response when MSCs were administered on day 3 after allo-HSCT compared with non-MSC group(p=0.770).Survival of mice receiving MSCs administered on day 7 significantly increased(P=0.014),and day 21 increased but had no significant difference in comparison with non-MSCs group(P=0.192).?The response of MSCs for GVHD:MSCs-untreated GVHD mice appeared GVHD manifestation from 7 day after HSCT,such as hunching and diarrhea,and alleviated with the increase of body weight.After 14 day,aGVHD group mice reappeared aGVHD clinical manifestation.MSCs treated GVHD mice receiving MSCs treatment on 7 day after allo-HSCT,reappeared aGVHD signs on 20 day,whose syptoms of aGVHD ameliorated.The clinical aGVHD score of MSCs group is significantly lower than that of non-MSCs group on day 20 and 24(P=0.048,P=0.039).The pathological aGVHD score of MSCs group is significantly lower than that of non-MSCs group(P=0.005).4.MSCs migrating to target organs:GFP expression was detected in all target organs on 2 days after MSCs infusion.Then the GFP expressions were declined in all organs,especially dispeared in lung on 3 day after MSCs infusion and only detected in intestine on 5 day.Immunohistochemistry analysis of thymic sections of MSC-treated aGVHD mice showed that GFP positive cells mainly localized in the medulla neighboring thymic cortex on 2 days after MSCs infusion.5.MSCs repairing GVHD thymus:?Indirect evaluation of thymic function:the size of thymus in GVHD mice was significantly smaller than BM group.No difference of thymus size had been observed between MSC group and non-MSC group.The thymic index significantly increased in MSC group compared with non-MSC group(P<0.001),but was still lower than BM group {P-0.006).and the total number of thymocytes In contrast,The total number of thymocytes were markedly increased in MSC-treated GVHD mice in comparison with non-MSC group(P=0.012).And the former was 1.52 times of the latter,which were both lower than that in BM group(P<0.001);?Direct evaluation of thymic function:we firstly established the standard curve of RAG2 and TRECs standard substance with linear coefficient is both over 0.98.Compared with normal BALB/c mice,the level of thymic,splenic and peripheral blood TRECs in BM,MSC and non-MSC group were all significantly lower(P<0.001).The level of thymic TRECs in BM group had an obvious increase in comparison with non-MSC group(P<0.001),but was not significantly different from MSC group(P=0.058).The level of thymic TRECs in MSC-treated aGVHD mice significantly increased compared with non-MSC group(P=0.007).The espression of splenic TRECs in BM group was significantly higher than that in MSC and non-MSC group(P=0.004,P<0.001).The relative expression levels of TRECs in peripheral blood cells was significantly increased in MSC-treated mice compared to MSC-untreated group(P=0.017),although the TREC expression levels in the splenocytes did not change significantly(P=0.182).The level of peripheral blood TRECs in BM group had an obvious increase in comparison with non-MSC group(P=0.029),but was not significantly different from MSC group(P=0.240).6.The effect of MSCs for central immune reconstuction:In thymus,T cells subsets(CD4+CD8+T,CD4-CD8+T,CD4+CD8-T,CD4+CD25+Foxp3+T)were analyzed by FACS in BM,MSCs treated and MSCs untreated mice,which to some extent decreased compared with the normal BALB/c mice.The proportion of T cell subsets was lowest on day 7 after allo-HSCT,then gradually increased accompanied by hematopoietic stem cells(HSCs)engraftment.The double-positive T cells had significant reduction in GVHD mice.After MSCs administration,the CD4+CD8+thymocytes expanded and on day 21 and 28 were significantly higher than those in MSCs-untreated group(P<0.001).The CD4 single-positive cells in MSC group was higher than that in non-MSC group from day 21 after allo-HSCT,which has significant difference on day 28(P<0.001).And the reconstruction of CD8 single-positive cells was later than CD4 single-positive cells,respectively day 28 for CD8 single-positive cells and day 21 for CD4 single-positive cells.The proportion of Tregs in MSC group was significantly higher than that in non-MSC group on day 21 and 28(P<0.001).7.The effect of MSCs for peripheral immune reconstuction:In peripheral blood and spleen,the reconstruction of T cell subsets was later than that in thymus.The proportion of CD4 CD8 double-positive T cells decreased after HSCT and was lowest on day 14.The frequence of CD4 CD8 double-positive T cells in peripheral blood cells were significantly increased in MSC-treated group compared to MSC-untreated group on day 21 and 28(P<0.001),although in splenocytes the frequence of CD4+CD8+double-positive T did not change significantly(P>0.05).The proportion of single-positive T cells in spleen significantly increased in MSC group compared with non-MSC group(P<0.001),although in peripheral blood the proportion of single-positive T cells did not change obviously(P>0.05).The proportion of Tregs in MSC group was significantly higher than that in non-MSC group on day 21 and 28 in peripheral blood(P<0.001).In spleen,focusing on the proportion of Tregs,there was no significant difference between MSC and non-MSC group(P>0.05).8.After 125 doses of MSCs were administered,with a median of 4(range:2-8)doses per patient,overall response rate was 75%in MSC group,comparing with 42.1%in non-MSC group(P=0.023).The incidence of CMV,EBV infections and tumor relapse were not different between the two groups during aGVHD treatment and follow-up.The incidence and severity of cGVHD in MSC group were lower than those in non-MSC group(P=0.045,P=0.005).The ratio of CD3+CD4+/CD3+CD8+T cells,the frequencies of CD4+CD25+Foxp3+regulatory T cells(Tregs)and the levels of signal joint T-cell receptor excision DNA circles(sjTRECs)after MSCs treatment were higher than those pre-treatment.MSC-treated patients exhibited higher Tregs frequencies and sjTRECs levels than those in non-MSC group at 8,12 weeks after treatment.Conclusion:1.aGVHD murine model after allo-HSCT has been successfully established,BALB/c were exposed to TBI prior to administration of donor BM(5×106 cells/mice)and splenocytes(4×106 cells/mice).The aGVHD recipients got successful engraftment and showed typical aGVHD clinical and pathologic manifestation.2.GFP labelled MSCs has been successfully isolated from BM in C57BL/6 mice.MSCs could continuous expansion in vitro,maintaining the GFP expression and surface marker,while maintaining the high proliferation potential and multipotency.3.After MSCs infusion,we observed that MSCs significantly improved the survival,clinical and pathologic scores of aGVHD mice.Forther study found that within a certain range,MSCs treatment for aGVHD is dose dependent.And in early phrase of GVHD injure microenvironment,MSCs administration has a better response.4.MSCs improved the regeneration and output function of aGVHD,promoted the reconstruction of T lymphocyte and induced the generation of Tregs.This study will bring to light the mechanisms of MSC inducing immune tolerance and promoting immune reconstuction in the recipients with GVHD,and supply theoretical basis for MSCs in GVHD treatment.5.MSCs derived from BM of a third-party donor are effective to refractory aGVHD.It might reduce the incidence and severity of cGVHD in aGVHD patients by improving thymic function and induction of Tregs,but not increase the risks of infections and tumour relapse. |
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