异基因造血干细胞移植后T细胞免疫重建的研究
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摘要
目的和意义
异基因造血干细胞移植是恶性造血系统疾病的有效治疗方法。异基因移植后经历较长时间的免疫缺陷,导致了移植后持续数年机会性感染的高发生率。成功的异基因移植意味着重建正常的T细胞免疫。T细胞免疫重建依赖两种途径:胸腺非依赖途径即供者来源成熟T细胞的体外扩增;胸腺依赖途径即移植物来源中的前T细胞在胸腺内的发育成熟,产生具有多样性TCR谱型的T细胞。由此而发展起来的检测免疫重建的方法有:通过TCRBV CDR3谱型检测方法来分析T细胞的多样性变化,以及通过定量检测T细胞受体重排删除环(TRECs)来评价胸腺的近输出功能。TCRBV是由可变区(V区)、D区、J区的基因片段发生组成,在V(D)J重新结合过程中V、D、J重排构成了TCR多样性的高变区,正常个体表现为TCRBV谱型的多克隆群体,CDR3区呈现典型的高斯分布特点。该区域是抗原特异性识别部位,特定的抗原刺激下导致克隆性T细胞的扩增。通过检测TRECs可以直接定量胸腺的近输出功能。TRECs是TCRα发生重排时删除的环状的游离DNA,大约70%的αβT细胞重排产生TRECs,并由此定量胸腺产生初始T细胞的水平。目前许多的研究移植后免疫重建多集中在测定TRECs及分析TCRBV谱型多态性上。在移植后的早期阶段发生扩增的一群细胞是具有高度倾斜性谱型的CD45RO~+T细胞。移植后TRECs增高即意味者具有初始表型的T细胞的出现,随后TRECs水平的增加及具有多样性谱型的T细胞的表型为CD45RA~+RO~-。临床上持续低水平的TRECs与GVHD、感染及白血病复发有关。异基因移植后动态地检测TCRBV谱型,显示最初移植后的T细胞由少数的TCRBV谱型向随后的逐渐增多的多样化谱型转化。GVHD及感染的发生可以导致胸腺依赖性T细胞的产生减少。异基因移植后的免疫重建受包括病毒感染、GVHD等抗原刺激的影响,抗原驱使下导致特异T细胞克隆的过度增殖,甚至在某个BV家族中优势扩增而限制了其他BV家族的利用。运用这些检测方法发现抗原特异性的免疫,为进一步的靶向治疗提供治疗依据和方向。
本课题通过T细胞受体克隆谱型和胸腺近输出功能对三组不同类型异基因造血干细胞移植(同胞全相合骨髓移植、无血缘关系外周血干细胞移植及单倍型骨髓移植)的免疫重建进行研究。了解不同类型异基因移植的免疫重建的特点,定量TRECs检测初始T细胞的输出来判断胸腺的近输出功能,从而反映T细胞免疫重建的潜能。通过分析移植后的TCRBV的谱型变化不仅可以分析移植后受者T细胞免疫重建的状态,还可以得到与疾病发生相关的T细胞克隆。两者结合起来能很好地用于异基因移植后T细胞免疫重建的研究。移植前的胸腺功能状态可能影响移植后胸腺依赖途径T细胞恢复,因此通过MSD-BMT的一组病例移植前TRECs的定量检测及移植后长期随访,来进行受者移植前胸腺功能与移植后预后的相关性研究。
单倍型异基因移植有其特有的临床特点,如严重的GVHD、严重感染等导致其具有较高的死亡率。通过CB6F1(H-2~(b/d),♂)和C57BL/6(H-2~b,♀)单倍型骨髓移植小鼠GVHD模型的建立,较系统地研究单倍型异基因移植小鼠不同GVHD靶器官发生GVHD时的病理表现、器官特异性T细胞受体谱型及TCRBV CDR3分子特征,能否发现GVHD靶器官发生GVHD的可能T细胞克隆的保守序列,为进一步的免疫治疗提供分子基础。
方法
第一部分
24例接受allo-HSCT的患者,其中单倍型骨髓移植(HID-BMT)9例,同胞全相合骨髓移植(MSD-BMT)10例,无血缘关系外周血干细胞移植(MUD-PBSCT)5例进行TCRBV克隆谱型研究。应用RT-PCR扩增24例不同类型白血病异基因造血干细胞移植后及5名正常供者的外周血的TCRBV24个家族的基因序列,并通过基因扫描的方法判断TCRBV家族的克隆表达情况、CDR3克隆性质及测定BV家族的利用率。对于单克隆表达的T细胞克隆进一步在长泳道测序胶上电泳,得到单克隆条带并进行序列测定,得到一组与GVHD或CMV直接相关的TCRBV CDR3的分子。
第二部分
第一节:对43接受例allo-HSCT的患者,其中同胞全相合骨髓移植(MSD-BMT)患者23例,无血缘关系全相合外周血干细胞移植(MUD-PBSCT)8例,单倍型骨髓移植(HID-BMT)12例。所有患者在移植前及移植后的3个月、6个月、12个月、24个月(除外MUD组)不同时间里动态检测TRECs水平。其中23例MSD-BMT患者在移植后1个月、2个月、3个月、4个月、5个月、6个月、8个月、12个月、18个月及24个月的不同时间里动态检测TRECs水平;同时检测70例正常供者TRECs值,作为正常对照。
第二节:应用Real-time PCR的方法定量检测64例同胞全相合骨髓移植患者的移植前TRECs水平,并对所有移植后病例进行临床资料收集整理。通过统计分析进行回顾性研究,用Logrank检验比较生存率,运用Cox比例风险回归模型进行单因素和多因素统计分析,进行受者移植前胸腺功能与移植后预后的相关性研究。同时检测70名正常供者的TRECs水平作为正常对照。
第三节:应用Real-time PCR的方法定量检测43例三种类型异基因造血干细胞移植后不同时间TRECs水平;应用RT-PCR扩增其中24例不同类型白血病异基因造血干细胞移植后及5名正常供者的外周血的TCRBV 24个家族的基因序列,通过基因扫描(Genescan)的方法判断TCRBV家族的克隆表达、CDR3克隆性质及计算BV家族的利用率。
第三部分
建立CB6F1(H-2~(b/d),♂)小鼠为受鼠,C57BL/6小鼠(H-2~b,♀)为供鼠的单倍型异基因移植小鼠GVHD模型,应用RT-PCR扩增小鼠肝脏、皮肤、结肠、脾脏、肾脏等组织移植前、后TCRBV 23个家族的基因序列,通过基因扫描(Genescan)的方法判断TCRBV家族的克隆表达、CDR3克隆性质。对于寡克隆表达的T细胞克隆进一步在长泳道测序胶上电泳,得到单克隆条带并测序,得到一组GVHD靶器官肝脏、皮肤、肠道在移植后不同时间的与移植物抗宿主病(GVHD)相关的TCRBV CDR3的分子。
结果
第一部分:在移植后2个月至移植后个19月,单倍型骨髓移植9例患者,在6～14个BV家族中有表达,多克隆表达率为33%,其余为单克隆或寡克隆表达。无血缘关系外周血干细胞移植的5例患者,在10～15个BV家族中表达,多克隆表达率为45%。同胞全相合骨髓移植的10例患者,在9～16个BV家族中有表达,多克隆表达率为48%。单克隆或寡克隆增生分布在24个不同的BV家族,未见共用的BV家族。单倍型骨髓移植后细胞免疫恢复迟于其他两组。其中2例患者在移植后2个月、移植后3个月检测TCRBV,发现移植后随时间增加BV家族的利用增加,CDR3的多克隆群体增多。得到的23条与GVHD及CMV相关的TCRBV CDR3分子,经过生物信息学比较发现,在相同的BV家族内,不同患者中可以出现结构相似的CDR3氨基酸分子,也可以完全不同;不同BV家族的CDR3分子相差较大,未发现共同使用的氨基酸基序。
第二部分:
第一节:70例正常人外周血单个核细胞中TRECs拷贝数为3351.06±3711.12拷贝/10~5细胞,年龄与TRECs含量呈负相关。移植前患者TRECs定量检测结果为307.87±433.26拷贝/10~5细胞,明显低于正常人。23例MSD-BMT患者在移植后1～5个月患者TRECs水平均明显下降,部分患者未能检测到TRECs。移植后6个月TRECs明显升高并持续1年;移植后24个月TRECs水平有较大提高并接近移植前状态。三组移植后患者在移植后3个月TRECs含量均明显下降。MSD-BMT组的TRECs恢复快于其他两组。HID-BMT组TRECs恢复迟缓。
第二节:64例MSD-BMT患者,Cox回归模型单因素分析显示移植前TRECs水平与移植后长期生存、cGVHD有明显的相关性(P＜0.05),与CMV感染亦有一定的相关性(P=0.084),而与aGVHD的发生无明显的相关性。Cox回归模型多危险因素的分析结果,年龄和性别对长期生存、aGVHD、cGVHD的发生、CMV感染等的发生影响不大。ABO血型是否相合是影响长期生存、CMV感染的主要因素(P＜0.05)。供受体性别差异是影响aGVHD发生的主要影响因素。多危险因素的分析结果移植前TRECs水平对长期生存、aGVHD、cGVHD、CMV感染等的影响与单一TRECs因素对移植后的预后指标的影响结果一致。
第三节:所有患者在移植后3个月TRECs持续低水平,部分患者检测不到TRECs。TCRBV克隆谱型检测结果发现在移植后的2个月、3个月,发现存在部分BV家族的扩增并且多克隆家族的表达增加。在移植半年以后,TRECs开始回升,TCRBV谱型也显示BV家族的利用率增加及CDR3谱型的多样性变化。单倍型骨髓移植后患者TRECs及TCRBV谱型恢复延迟。
第三部分:单倍型异基因移植小鼠在移植后14天开始出现典型的GVHD表现。移植后受鼠GVHD的靶器官肝脏、皮肤、远端回肠等均出现典型的GVHD病理表现,TCRBV检测结果在移植后均新出现了多个BV家族大量的单克隆或寡克隆增生的T细胞,而肾脏等非GVHD靶器官中浸润的淋巴细胞为多克隆的T细胞增生。通过对克隆性增生的T细胞的TCRBV CDR3分子的分析发现,在肝脏、皮肤、肠道等GVHD的靶器官中存在6个C'末端保守的CDR3氨基酸基序(motif),分别为TEVFF、DTQYF、YEQYF、AEQ(YF/FF)、QNTLYF、AETLYF,这六个基序分别共用JB1.1、JB2.5、JB2.7、JB2.1、JB2.4、JB2.3基因。
结论
1.不同类型异基因造血干细胞移植后1.5年内,TCRBV家族的利用仍处于不均一状态。单倍型骨髓移植后T细胞免疫恢复迟于其他两组。得到的一组与GVHD及CMV相关的单克隆增生的T细胞CDR3分子,在相同的BV家族内,不同患者出现结构相似的CDR3氨基酸分子;不同BV家族的CDR3分子之间未发现共同使用的氨基酸基序。
2.成人患者仍具有产生初始T细胞的能力。三种不同类型干细胞移植后3个月TRECs持续低水平,HID-BMT检测不到。移植后6个月、12个月TRECs水平缓慢回升。MSD-BMT的TRECs恢复快于其他两组并且在移植后24个月TRECs含量接近移植前水平。HID-BMT的TRECs恢复延迟。
3.MSD-BMT一组病例,受者移植前的胸腺功能状态与异基因移植后的预后的有明显的相关性,可以作为影响异基因造血干细胞移植后的独立的预后因素。
4.TRECs和TCRBV谱型分析表明在移植后3m～24m,TRECs低水平及TCRBV不均一利用。早期迅速恢复的T细胞群体是由移植物中存在的成熟T细胞的体外扩增。移植后的一定时间,TRECs含量逐渐增加、TCRBV家族利用增加。HID-BMT组TCRBV克隆谱型及TRECs水平恢复迟缓。
5.单倍型异基因移植GVHD小鼠模型,在GVHD靶器官(肝脏、皮肤、肠道)中出现的大量单克隆及寡克隆增生的T细胞可能是与GVHD发生直接相关,并且发现在这三种GVHD靶器官中部分单克隆增生的T细胞存在保守的TCRBV CDR3基序。
综上所述:
定量TRECs检测初始T细胞的输出来判断胸腺的近输出功能,从而反映T细胞免疫重建的潜能。通过分析移植后的TCRBV的谱型变化不仅可以分析移植后供者来源的T细胞在受者体内免疫重建的状态,还可以得到与疾病发生相关的T细胞克隆。两者结合起来能很好地用于异基因移植后T细胞免疫重建的研究。移植后24m内,TRECs水平仍较低,TCRBV谱型利用仍处于不均一状态,并且发现与GVHD或感染相关的克隆扩增的T细胞群体。Haploidentical-BMT的免疫重建迟于MDS组和MUD组。移植前的胸腺功能状态可能影响移植后胸腺依赖途径T细胞恢复,对于MSD-BMT受者移植前的胸腺功能状态与异基因移植后的预后的有明显的相关性,可以作为影响异基因造血干细胞移植后的独立的预后因素。单倍型异基因移植GVHD小鼠模型,在GVHD靶器官即肝脏、皮肤、肠道组织中出现大量单克隆及寡克隆增生的T细胞,可能与GVHD的发生直接相关,并且在这三种组织单克隆增生的T细胞中存在保守的TCRBV CDR3基序,可以指导进一步的免疫治疗。
【Objects】
Allogeneic hematopoietic stem cell transplantation(HSCT) proves a curable treatment for refractory hematopooietic maligancy.Following HSCT,there is a prolonged period of profound immune deficiency.This immune deficiency contributes to a high incidence of opportunistic infection,which continues for years after HSCT.Successful HSCT requires reconstitution of normal T-cell immunity.The T-cell population can be regenerated through two different pathways.The thymic-independent pathway involves expansion of graft-derived mature donor T cells,whereas the thymic-dependent pathway involves the regeneration of T-cell with a more diverse T-cell rceptor(TCR) repertoire from graft-derived precursor T cells.Evaluation of immune reconstitution after HSCT has improved through the development of direct methodologies for T-cell diversity analysis by the site of the TCRβ-chain CDR3 and for ex vivo evaluation of thymic function by quantitating T-cell receptor excision circles(TRECs).The TCRBV CDR3 region is the only non-germ line-encoded hypervariable region.This region is generated by recombination of the V,D and J segments and by random insertion and deletion of junctional nucleotides so that the final products are quite heterogeneous in size.CDR3 spectratyping reveals that healthy individual exhibit a highly diverse and polyclonal TCRBV repertoire with a typically gaussian-like distribution of the sizes of CDR3 region. These regions are largely responsible for the antigenic specificity of individual TCR.The measurement of T-cell receptor excision circles(TRECs) allows direct quantitation of recent thymic output.TRECs are epismol DNA circles generated by the TCRa locus recombination process and this recombination event is identical in approximately 70%of aβT cells and the resulting TRECs can be used to detect and quantify thymic output of naive T cells.
Many studies of T-cell reconstitution have relied on post-transplantation measurement of TREC and TCRBV repertoire diversity.A highly skewed repertoire and the absence of increased TRECs in the eraly posttransplantation stage corresponded to the expansion of CD45RO~+ T cell.The appearance of TRECs after transplantation was associated with the emergence of phenotypically naive T cell.A diverse repertoire and increased TRECs appeared at a later stage with the appearance of CD45RA~+RO~-T cells.The persistence of low TREC numbers was associated with higher incidence of GVHD,infection,and leukemic relapse.Analysis of TCRBV spectratypes following HSCT consistently shows redcued T-cell repertiore diversity with a subsequent gradual return to a more diverse normal adult profile.Both GVHD and infection have been documented to disminish thymic-dependent re-population.GVHD results in lower levels of phenotypically naive and TRCE-bearing T cells.GVHD may mediate this effect in part by a direct effect on thymopoiesis.Immune reconstitution after HSCT is modified by antigenic stimuli, including viral infection and GVHD,antigen-driven proliferation may lead to specific T-cell clone overrepresentation,even result in a relative expansion of entire BV families and restricted clonal usage of BV families.The development of assays to measure recovery of antigen-specific immunity may allow further refinement of targeted therapies. In this study,Immune reconstitution was evaluated in patients underwent three groups of HSCT,that is HLA-matched sibling donor bone marrow transplantation(MSD-BMT), HLA-matched unrelated donor peripheral stem cell transplantation(MUD-PBSCT), haploidentical bone marrow transplantation(Haploidentical-BMT,HID-BMT) and studied the characteristics of it in different groups of patients.The measurement of TRECs allow direct quantitation of recent host thymic output and reflect potential ability of immune reconstitution.Through TCR repertiore may realize the status of T-cell immune and can obtain T-cell clones associated with clinical events.Two methods proved methodologies to study the immune reconstitution after HSCT.Our matching set-up provided the unique opportunity to study the impact of the different type of HSCT on long-term of immune reconstitution and through TCRBV CDR3 repertiore try to find some molecules of CDR3 associated with cilinical events or specific pathology,maybe it is a marker or target of therapy in the future.The functional capacity of the recipient thymus appears to be the dominant influence on thymic-dependent reconstitution.So through a group of patients underwent MSD-BMT,we study the releationship between host thymie function in pretransplantation and prognosis after HSCT and determine whether pretransplantation TRECs can act as a marker for predication of prognosis after BMT.
Haploidentical-BMT has its own cilinical features~(18) and usually needs more intensive conditioning therapy and usually has high morbidity and mortality associated with the early complication of SCT and severe GVHD and infection.Murine CB6F1(H-2~(b/d),♂) and C57BL/6(H-2~b,♀) haploidentical-BMT GVHD model was established and proved a novel method to study the characteristics of T cell receptor repertoire in target organs of murine GVHD and research the molecular characteristics of complementarity determining region3(CDR3) repertoires of monoclonal T cell in liver、skin and ileum in GVHD murine after BMT.
【Methods】
[Section 1]RT-PCR was used to amplify 24 subfamily genes of TCRBV from peripheral blood lymphocytes of twenty-four leukemia patients underwent three kinds of allo-HSCT (9 of them had haploidentical BMT,10 of them had MSD-BMT and 5 of them had MUD-PBSCT),five normal donors as control.The PCR products were further analyzed by genescan to evaluate the clonality of BV subfamily and characteristics of CDR3 and calculate usage rate of BV subfamily.The monoclonal bands which associated with GVHD and CMV infection were obtained thorough denaturation polyacrylamide gel electrophoresis and sequenced.Compared the sequences of TCRBV CDR3 with other CDR3 sequences which associated with GVHD or CMV infection had been reported.
[Section 2]
First paragraph:Real-time quantitative PCR was used to detect TRECs in DNA of peripheral blood mononuclear cells in pretransplantation and 3m、6m、12m、24m after HSCT in 43 leukemia patients underwent three kinds of Allo-HSCT,among them 23 patients underwent MSD-BMT detected TRECs in 1m、2m、3m、4m、5m、6m、8m、12m、18m、24m after HSCT consecutively.TRECs in 70 normal individuals were also detected and determined the normal range of TRECs in healthy groups.
Second paragraph:Real-time quantitative PCR detection of TRECs in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients who underwent MSD-BMT.The content of TRECs in 70 normal donor individuals also detected.All clinical datas of patients after HSCT collected and study.Survival rate of patients after HSCT were estimated with Logrank test.Univariate and multivariate analysis of prognostic factors carried out by Cox's proportional hazard regression model.
Third paragraph:Real-time quantitative PCR was used to detect T cell receptor excision DNA circles(TRECs) in 43 leukemia patients underwent three kinds of Allo-HSCT. TRECs in 70 normal individuals were also detected.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to amplify 24 subfamily genes of TCRBV in 24 patients of 43 patients,five normal donors as control,the PCR products were further analyzed by genescane to evaluate the clonality of BV subfamily,the characteristics of CDR3 and the usage rate in BV subfamily.
[Section 3]Murine CB6F1(H-2~(b/d),♂) and C57BL/6(H-2~b,♀) haploidentical BMT GVHD model was established,CDR3-size spectratyping was used to study TCRBV repertoires in recipent liver、skin、ileum、spleen and kidney after transplantation and obtained a group of CDR3 molecules from GVHD-target tissues.
【Results】
[Section 1]+2~+19months after transplantation,for nine patients among them which underwent Haploidentical-BMT,there were 6~14 BV subfamilies expressed and the polyclonal express reached 33%.For five patients underwent MUD-PBSCT,there were 10~15 BV subfamilies expressed,which 45%of them were polyclones.Ten patients underwent MSD-BMT,10~16 BV subfamilies expressed and more than 48%were polyclones.Monoclones and oligoclones existed in 24 BV subfamilies,no common monoclone BV subfamilies expressed.Immune reconstitution in patients which underwent haploidentical-BMT was later than those of other two groups.TCRBV was detected in two patients in 2m and 3m after allo-HSCT and found that it has tendency of adding to use BV subfamilies and increasing to expressed CDR3 polymorphism.23 TCRBV CDR3 molecules which related to GVHD and CMV infection were compared by using bioinformatics tools and found that different cases in the same BV subfamilies may share similarity in amino acid motif,while in different BV subfamilies none of clones appeared to share the same amino acid motif.
[Section 2]
First paragraph:The mean value of TRECs in normal individuals was 3351.06±3711.12 copies/10~5 cells.There was inverse correlation between TRECs and age in health groups. There were 307.87±433.26 copies/10~5 cells in patients before transplantation and were far lower than noraml value.TRECs decreased obviously in 3m after transplantation,some patients can't be detectable.The recovery of TRECs in MSD-BMT was faster than other two groups and reached status before transplantation in 24m.The recovery of TRECs in Haploidentical-BMT was delay.
Second paragraph TRECs was detected in 64 patients before transplsntation,the mean TRECs number was far lower than that of normal values.According to the results of univariate analysis,the counts of TRECs pre-HSCT had the close relation with survival、cGVHD(p＜0.05) and infection of CMV(p=0.084) except aGVHD.According to the results of multivariate analysis,age and sex did not effect the outcome of HSCT.ABO compatibility or incompatibility played an important role in the outcome of survival and infection of CMV.Sex of donor/recipient may effect the incidence of aGVHD.The results of multivariate analysis of the counts of TRECs pre-HSCT was the same as univariate analysis.
Third paragraph TRECS decreased obviously in 3m for all patients after transplantation. TCRBV was detected in two patients in 2m and 3m after allo-HSCT and found that it has tendency of adding to use BV subfamilies and increasing to expressed CDR3 polymorphism.After 6m in MSD-BMT,TRECs levels increased obviously,TCRBV repertoire showed adding use BV families and CDR3 polymorphism increasing to express. The recovery of TRECs and TCRBV repertoires in haploidentical BMT patients was late and it was consistence with clinical process.
[Section 3]For haploidentical BMT murine model,GVHD occurred as early as days 14 and was proved by histology in liver、skin and ileum.Through the detection of TCRBV CDR3,it emerged a number of new monoclonal and oligoclonal T cells in GVHD-target tissues,the kidney was not affected by GVHD but showed polycolnal T cell infiltrated.48 CDR3 molecules which obtained from liver、skin、ileum in different times after BMT have six C'-terminal motifs(TEVFF、DTQYF、YEQYF、AEQ(YF/FF)、QNTLYF、AET LYF) and use restricted JB genes(JB1.1、JB2.5、JB2.7、JB2.1、JB2.4、JB2.3).
【Conclusion】
1.In 1.5 years after allo-HSCT,the usage of TCRBV subfamilies still restricted.Immune reconstitution in patients which underwent haploidentical BMT was later than those of other two groups.TCRBV CDR3 molecules which potentially associated with GVHD and CMV infection obtained from monoclonal T cells and showed that different cases in the same BV subfamilies may share similarity in amino acid motif,while in different BV subfamilies none of clones appeared to share the same amino acid motif.
2.The adults have the ability to generate naive T-cell.At early stage after BMT,TRECs numbers were low and lasted for long time,it was can not be detectable in 3m in haploidentical BMT,TRECs begun to recover during 6m、12m.The decovery of TRECs in MSD-BMT was faster than other two groups and it reached pretransplantation levels in 24m after BMT.The recovery of TRECs in haploidentical BMT was delay in three groups of patients.
3.Pretransplantation host thymic function has the close realtion with prognosis in MSD-BMT and it can be a marker to predict the outcome of HSCT.
4.By investigateing thymus recent output function and T-cell receptor clonal repertoire, the number of naive T cell is lower and the usage of TCRBV subfamilies still skewed in 3~24m after allo-HSCT.Early immune reconstitution accounts for the expression of graft-derived mature donor T-cells.After long period of HSCT,TRECs levels increased gradually and BV families added to use.Immune deficiency in haploidentical BMT is more prominent and consistent with clinical process.
5.Murine haploidentical BMT model was established,Target organs of murine GVHD (liver、skin、ileum) emerged a number of monoclonal or oligoclonal T cells which associated with the development of GVHD,there were some conserved CDR3 amino acid motifs in three kinds of tissues and it proves a method which focus on these molecules in further immune therapy.
In summary
The measurement of TRECs allow direct quantitation of host recent thymic output.and reflect potential ability in immune reconstitution.Through TCR repertiore may realize the status of T-cell immune and can obtain T-cell clone potentially associated with clinical events.Two methods proved methodologies to study the immune reconstitution after HSCT. The number of TRECs is lower and the usage of TCRBV subfamilies still skewed in 3~24m after Allo-HSCT in three groups patients.It had a group of clonal T cell overproliferation potentially associated with GVHD or infection.Pretransplantation host thymic function has the close realtion with prognosis in MSD-BMT and it can be a marker to predict the outcome of HSCT.The recovery of immune reconstitution in haploidentical BMT is slow in three groups of HSCT.Through murine haploidentical BMT GVHD model, TCRBV CDR3 was detected in GVHD-target tissues(liver、skin、ileum) and found that it emerged a number of monoclonal or oligoclonal T cells which associated with the development of GVHD and existed conserved CDR3 motifs.
异基因造血干细胞移植是恶性造血系统疾病的有效治疗方法。异基因移植后经历较长时间的免疫缺陷,导致了移植后持续数年机会性感染的高发生率。成功的异基因移植意味着重建正常的T细胞免疫。T细胞免疫重建依赖两种途径:胸腺非依赖途径即供者来源成熟T细胞的体外扩增;胸腺依赖途径即移植物来源中的前T细胞在胸腺内的发育成熟,产生具有多样性TCR谱型的T细胞。由此而发展起来的检测免疫重建的方法有:通过TCRBV CDR3谱型检测方法来分析T细胞的多样性变化,以及通过定量检测T细胞受体重排删除环(TRECs)来评价胸腺的近输出功能。TCRBV是由可变区(V区)、D区、J区的基因片段发生组成,在V(D)J重新结合过程中V、D、J重排构成了TCR多样性的高变区,正常个体表现为TCRBV谱型的多克隆群体,CDR3区呈现典型的高斯分布特点。该区域是抗原特异性识别部位,特定的抗原刺激下导致克隆性T细胞的扩增。通过检测TRECs可以直接定量胸腺的近输出功能。TRECs是TCRα发生重排时删除的环状的游离DNA,大约70%的αβT细胞重排产生TRECs,并由此定量胸腺产生初始T细胞的水平。目前许多的研究移植后免疫重建多集中在测定TRECs及分析TCRBV谱型多态性上。在移植后的早期阶段发生扩增的一群细胞是具有高度倾斜性谱型的CD45RO~+T细胞。移植后TRECs增高即意味者具有初始表型的T细胞的出现,随后TRECs水平的增加及具有多样性谱型的T细胞的表型为CD45RA~+RO~-。临床上持续低水平的TRECs与GVHD、感染及白血病复发有关。异基因移植后动态地检测TCRBV谱型,显示最初移植后的T细胞由少数的TCRBV谱型向随后的逐渐增多的多样化谱型转化。GVHD及感染的发生可以导致胸腺依赖性T细胞的产生减少。异基因移植后的免疫重建受包括病毒感染、GVHD等抗原刺激的影响,抗原驱使下导致特异T细胞克隆的过度增殖,甚至在某个BV家族中优势扩增而限制了其他BV家族的利用。运用这些检测方法发现抗原特异性的免疫,为进一步的靶向治疗提供治疗依据和方向。
本课题通过T细胞受体克隆谱型和胸腺近输出功能对三组不同类型异基因造血干细胞移植(同胞全相合骨髓移植、无血缘关系外周血干细胞移植及单倍型骨髓移植)的免疫重建进行研究。了解不同类型异基因移植的免疫重建的特点,定量TRECs检测初始T细胞的输出来判断胸腺的近输出功能,从而反映T细胞免疫重建的潜能。通过分析移植后的TCRBV的谱型变化不仅可以分析移植后受者T细胞免疫重建的状态,还可以得到与疾病发生相关的T细胞克隆。两者结合起来能很好地用于异基因移植后T细胞免疫重建的研究。移植前的胸腺功能状态可能影响移植后胸腺依赖途径T细胞恢复,因此通过MSD-BMT的一组病例移植前TRECs的定量检测及移植后长期随访,来进行受者移植前胸腺功能与移植后预后的相关性研究。
单倍型异基因移植有其特有的临床特点,如严重的GVHD、严重感染等导致其具有较高的死亡率。通过CB6F1(H-2~(b/d),♂)和C57BL/6(H-2~b,♀)单倍型骨髓移植小鼠GVHD模型的建立,较系统地研究单倍型异基因移植小鼠不同GVHD靶器官发生GVHD时的病理表现、器官特异性T细胞受体谱型及TCRBV CDR3分子特征,能否发现GVHD靶器官发生GVHD的可能T细胞克隆的保守序列,为进一步的免疫治疗提供分子基础。
方法
第一部分
24例接受allo-HSCT的患者,其中单倍型骨髓移植(HID-BMT)9例,同胞全相合骨髓移植(MSD-BMT)10例,无血缘关系外周血干细胞移植(MUD-PBSCT)5例进行TCRBV克隆谱型研究。应用RT-PCR扩增24例不同类型白血病异基因造血干细胞移植后及5名正常供者的外周血的TCRBV24个家族的基因序列,并通过基因扫描的方法判断TCRBV家族的克隆表达情况、CDR3克隆性质及测定BV家族的利用率。对于单克隆表达的T细胞克隆进一步在长泳道测序胶上电泳,得到单克隆条带并进行序列测定,得到一组与GVHD或CMV直接相关的TCRBV CDR3的分子。
第二部分
第一节:对43接受例allo-HSCT的患者,其中同胞全相合骨髓移植(MSD-BMT)患者23例,无血缘关系全相合外周血干细胞移植(MUD-PBSCT)8例,单倍型骨髓移植(HID-BMT)12例。所有患者在移植前及移植后的3个月、6个月、12个月、24个月(除外MUD组)不同时间里动态检测TRECs水平。其中23例MSD-BMT患者在移植后1个月、2个月、3个月、4个月、5个月、6个月、8个月、12个月、18个月及24个月的不同时间里动态检测TRECs水平;同时检测70例正常供者TRECs值,作为正常对照。
第二节:应用Real-time PCR的方法定量检测64例同胞全相合骨髓移植患者的移植前TRECs水平,并对所有移植后病例进行临床资料收集整理。通过统计分析进行回顾性研究,用Logrank检验比较生存率,运用Cox比例风险回归模型进行单因素和多因素统计分析,进行受者移植前胸腺功能与移植后预后的相关性研究。同时检测70名正常供者的TRECs水平作为正常对照。
第三节:应用Real-time PCR的方法定量检测43例三种类型异基因造血干细胞移植后不同时间TRECs水平;应用RT-PCR扩增其中24例不同类型白血病异基因造血干细胞移植后及5名正常供者的外周血的TCRBV 24个家族的基因序列,通过基因扫描(Genescan)的方法判断TCRBV家族的克隆表达、CDR3克隆性质及计算BV家族的利用率。
第三部分
建立CB6F1(H-2~(b/d),♂)小鼠为受鼠,C57BL/6小鼠(H-2~b,♀)为供鼠的单倍型异基因移植小鼠GVHD模型,应用RT-PCR扩增小鼠肝脏、皮肤、结肠、脾脏、肾脏等组织移植前、后TCRBV 23个家族的基因序列,通过基因扫描(Genescan)的方法判断TCRBV家族的克隆表达、CDR3克隆性质。对于寡克隆表达的T细胞克隆进一步在长泳道测序胶上电泳,得到单克隆条带并测序,得到一组GVHD靶器官肝脏、皮肤、肠道在移植后不同时间的与移植物抗宿主病(GVHD)相关的TCRBV CDR3的分子。
结果
第一部分:在移植后2个月至移植后个19月,单倍型骨髓移植9例患者,在6～14个BV家族中有表达,多克隆表达率为33%,其余为单克隆或寡克隆表达。无血缘关系外周血干细胞移植的5例患者,在10～15个BV家族中表达,多克隆表达率为45%。同胞全相合骨髓移植的10例患者,在9～16个BV家族中有表达,多克隆表达率为48%。单克隆或寡克隆增生分布在24个不同的BV家族,未见共用的BV家族。单倍型骨髓移植后细胞免疫恢复迟于其他两组。其中2例患者在移植后2个月、移植后3个月检测TCRBV,发现移植后随时间增加BV家族的利用增加,CDR3的多克隆群体增多。得到的23条与GVHD及CMV相关的TCRBV CDR3分子,经过生物信息学比较发现,在相同的BV家族内,不同患者中可以出现结构相似的CDR3氨基酸分子,也可以完全不同;不同BV家族的CDR3分子相差较大,未发现共同使用的氨基酸基序。
第二部分:
第一节:70例正常人外周血单个核细胞中TRECs拷贝数为3351.06±3711.12拷贝/10~5细胞,年龄与TRECs含量呈负相关。移植前患者TRECs定量检测结果为307.87±433.26拷贝/10~5细胞,明显低于正常人。23例MSD-BMT患者在移植后1～5个月患者TRECs水平均明显下降,部分患者未能检测到TRECs。移植后6个月TRECs明显升高并持续1年;移植后24个月TRECs水平有较大提高并接近移植前状态。三组移植后患者在移植后3个月TRECs含量均明显下降。MSD-BMT组的TRECs恢复快于其他两组。HID-BMT组TRECs恢复迟缓。
第二节:64例MSD-BMT患者,Cox回归模型单因素分析显示移植前TRECs水平与移植后长期生存、cGVHD有明显的相关性(P＜0.05),与CMV感染亦有一定的相关性(P=0.084),而与aGVHD的发生无明显的相关性。Cox回归模型多危险因素的分析结果,年龄和性别对长期生存、aGVHD、cGVHD的发生、CMV感染等的发生影响不大。ABO血型是否相合是影响长期生存、CMV感染的主要因素(P＜0.05)。供受体性别差异是影响aGVHD发生的主要影响因素。多危险因素的分析结果移植前TRECs水平对长期生存、aGVHD、cGVHD、CMV感染等的影响与单一TRECs因素对移植后的预后指标的影响结果一致。
第三节:所有患者在移植后3个月TRECs持续低水平,部分患者检测不到TRECs。TCRBV克隆谱型检测结果发现在移植后的2个月、3个月,发现存在部分BV家族的扩增并且多克隆家族的表达增加。在移植半年以后,TRECs开始回升,TCRBV谱型也显示BV家族的利用率增加及CDR3谱型的多样性变化。单倍型骨髓移植后患者TRECs及TCRBV谱型恢复延迟。
第三部分:单倍型异基因移植小鼠在移植后14天开始出现典型的GVHD表现。移植后受鼠GVHD的靶器官肝脏、皮肤、远端回肠等均出现典型的GVHD病理表现,TCRBV检测结果在移植后均新出现了多个BV家族大量的单克隆或寡克隆增生的T细胞,而肾脏等非GVHD靶器官中浸润的淋巴细胞为多克隆的T细胞增生。通过对克隆性增生的T细胞的TCRBV CDR3分子的分析发现,在肝脏、皮肤、肠道等GVHD的靶器官中存在6个C'末端保守的CDR3氨基酸基序(motif),分别为TEVFF、DTQYF、YEQYF、AEQ(YF/FF)、QNTLYF、AETLYF,这六个基序分别共用JB1.1、JB2.5、JB2.7、JB2.1、JB2.4、JB2.3基因。
结论
1.不同类型异基因造血干细胞移植后1.5年内,TCRBV家族的利用仍处于不均一状态。单倍型骨髓移植后T细胞免疫恢复迟于其他两组。得到的一组与GVHD及CMV相关的单克隆增生的T细胞CDR3分子,在相同的BV家族内,不同患者出现结构相似的CDR3氨基酸分子;不同BV家族的CDR3分子之间未发现共同使用的氨基酸基序。
2.成人患者仍具有产生初始T细胞的能力。三种不同类型干细胞移植后3个月TRECs持续低水平,HID-BMT检测不到。移植后6个月、12个月TRECs水平缓慢回升。MSD-BMT的TRECs恢复快于其他两组并且在移植后24个月TRECs含量接近移植前水平。HID-BMT的TRECs恢复延迟。
3.MSD-BMT一组病例,受者移植前的胸腺功能状态与异基因移植后的预后的有明显的相关性,可以作为影响异基因造血干细胞移植后的独立的预后因素。
4.TRECs和TCRBV谱型分析表明在移植后3m～24m,TRECs低水平及TCRBV不均一利用。早期迅速恢复的T细胞群体是由移植物中存在的成熟T细胞的体外扩增。移植后的一定时间,TRECs含量逐渐增加、TCRBV家族利用增加。HID-BMT组TCRBV克隆谱型及TRECs水平恢复迟缓。
5.单倍型异基因移植GVHD小鼠模型,在GVHD靶器官(肝脏、皮肤、肠道)中出现的大量单克隆及寡克隆增生的T细胞可能是与GVHD发生直接相关,并且发现在这三种GVHD靶器官中部分单克隆增生的T细胞存在保守的TCRBV CDR3基序。
综上所述:
定量TRECs检测初始T细胞的输出来判断胸腺的近输出功能,从而反映T细胞免疫重建的潜能。通过分析移植后的TCRBV的谱型变化不仅可以分析移植后供者来源的T细胞在受者体内免疫重建的状态,还可以得到与疾病发生相关的T细胞克隆。两者结合起来能很好地用于异基因移植后T细胞免疫重建的研究。移植后24m内,TRECs水平仍较低,TCRBV谱型利用仍处于不均一状态,并且发现与GVHD或感染相关的克隆扩增的T细胞群体。Haploidentical-BMT的免疫重建迟于MDS组和MUD组。移植前的胸腺功能状态可能影响移植后胸腺依赖途径T细胞恢复,对于MSD-BMT受者移植前的胸腺功能状态与异基因移植后的预后的有明显的相关性,可以作为影响异基因造血干细胞移植后的独立的预后因素。单倍型异基因移植GVHD小鼠模型,在GVHD靶器官即肝脏、皮肤、肠道组织中出现大量单克隆及寡克隆增生的T细胞,可能与GVHD的发生直接相关,并且在这三种组织单克隆增生的T细胞中存在保守的TCRBV CDR3基序,可以指导进一步的免疫治疗。
【Objects】
Allogeneic hematopoietic stem cell transplantation(HSCT) proves a curable treatment for refractory hematopooietic maligancy.Following HSCT,there is a prolonged period of profound immune deficiency.This immune deficiency contributes to a high incidence of opportunistic infection,which continues for years after HSCT.Successful HSCT requires reconstitution of normal T-cell immunity.The T-cell population can be regenerated through two different pathways.The thymic-independent pathway involves expansion of graft-derived mature donor T cells,whereas the thymic-dependent pathway involves the regeneration of T-cell with a more diverse T-cell rceptor(TCR) repertoire from graft-derived precursor T cells.Evaluation of immune reconstitution after HSCT has improved through the development of direct methodologies for T-cell diversity analysis by the site of the TCRβ-chain CDR3 and for ex vivo evaluation of thymic function by quantitating T-cell receptor excision circles(TRECs).The TCRBV CDR3 region is the only non-germ line-encoded hypervariable region.This region is generated by recombination of the V,D and J segments and by random insertion and deletion of junctional nucleotides so that the final products are quite heterogeneous in size.CDR3 spectratyping reveals that healthy individual exhibit a highly diverse and polyclonal TCRBV repertoire with a typically gaussian-like distribution of the sizes of CDR3 region. These regions are largely responsible for the antigenic specificity of individual TCR.The measurement of T-cell receptor excision circles(TRECs) allows direct quantitation of recent thymic output.TRECs are epismol DNA circles generated by the TCRa locus recombination process and this recombination event is identical in approximately 70%of aβT cells and the resulting TRECs can be used to detect and quantify thymic output of naive T cells.
Many studies of T-cell reconstitution have relied on post-transplantation measurement of TREC and TCRBV repertoire diversity.A highly skewed repertoire and the absence of increased TRECs in the eraly posttransplantation stage corresponded to the expansion of CD45RO~+ T cell.The appearance of TRECs after transplantation was associated with the emergence of phenotypically naive T cell.A diverse repertoire and increased TRECs appeared at a later stage with the appearance of CD45RA~+RO~-T cells.The persistence of low TREC numbers was associated with higher incidence of GVHD,infection,and leukemic relapse.Analysis of TCRBV spectratypes following HSCT consistently shows redcued T-cell repertiore diversity with a subsequent gradual return to a more diverse normal adult profile.Both GVHD and infection have been documented to disminish thymic-dependent re-population.GVHD results in lower levels of phenotypically naive and TRCE-bearing T cells.GVHD may mediate this effect in part by a direct effect on thymopoiesis.Immune reconstitution after HSCT is modified by antigenic stimuli, including viral infection and GVHD,antigen-driven proliferation may lead to specific T-cell clone overrepresentation,even result in a relative expansion of entire BV families and restricted clonal usage of BV families.The development of assays to measure recovery of antigen-specific immunity may allow further refinement of targeted therapies. In this study,Immune reconstitution was evaluated in patients underwent three groups of HSCT,that is HLA-matched sibling donor bone marrow transplantation(MSD-BMT), HLA-matched unrelated donor peripheral stem cell transplantation(MUD-PBSCT), haploidentical bone marrow transplantation(Haploidentical-BMT,HID-BMT) and studied the characteristics of it in different groups of patients.The measurement of TRECs allow direct quantitation of recent host thymic output and reflect potential ability of immune reconstitution.Through TCR repertiore may realize the status of T-cell immune and can obtain T-cell clones associated with clinical events.Two methods proved methodologies to study the immune reconstitution after HSCT.Our matching set-up provided the unique opportunity to study the impact of the different type of HSCT on long-term of immune reconstitution and through TCRBV CDR3 repertiore try to find some molecules of CDR3 associated with cilinical events or specific pathology,maybe it is a marker or target of therapy in the future.The functional capacity of the recipient thymus appears to be the dominant influence on thymic-dependent reconstitution.So through a group of patients underwent MSD-BMT,we study the releationship between host thymie function in pretransplantation and prognosis after HSCT and determine whether pretransplantation TRECs can act as a marker for predication of prognosis after BMT.
Haploidentical-BMT has its own cilinical features~(18) and usually needs more intensive conditioning therapy and usually has high morbidity and mortality associated with the early complication of SCT and severe GVHD and infection.Murine CB6F1(H-2~(b/d),♂) and C57BL/6(H-2~b,♀) haploidentical-BMT GVHD model was established and proved a novel method to study the characteristics of T cell receptor repertoire in target organs of murine GVHD and research the molecular characteristics of complementarity determining region3(CDR3) repertoires of monoclonal T cell in liver、skin and ileum in GVHD murine after BMT.
【Methods】
[Section 1]RT-PCR was used to amplify 24 subfamily genes of TCRBV from peripheral blood lymphocytes of twenty-four leukemia patients underwent three kinds of allo-HSCT (9 of them had haploidentical BMT,10 of them had MSD-BMT and 5 of them had MUD-PBSCT),five normal donors as control.The PCR products were further analyzed by genescan to evaluate the clonality of BV subfamily and characteristics of CDR3 and calculate usage rate of BV subfamily.The monoclonal bands which associated with GVHD and CMV infection were obtained thorough denaturation polyacrylamide gel electrophoresis and sequenced.Compared the sequences of TCRBV CDR3 with other CDR3 sequences which associated with GVHD or CMV infection had been reported.
[Section 2]
First paragraph:Real-time quantitative PCR was used to detect TRECs in DNA of peripheral blood mononuclear cells in pretransplantation and 3m、6m、12m、24m after HSCT in 43 leukemia patients underwent three kinds of Allo-HSCT,among them 23 patients underwent MSD-BMT detected TRECs in 1m、2m、3m、4m、5m、6m、8m、12m、18m、24m after HSCT consecutively.TRECs in 70 normal individuals were also detected and determined the normal range of TRECs in healthy groups.
Second paragraph:Real-time quantitative PCR detection of TRECs in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients who underwent MSD-BMT.The content of TRECs in 70 normal donor individuals also detected.All clinical datas of patients after HSCT collected and study.Survival rate of patients after HSCT were estimated with Logrank test.Univariate and multivariate analysis of prognostic factors carried out by Cox's proportional hazard regression model.
Third paragraph:Real-time quantitative PCR was used to detect T cell receptor excision DNA circles(TRECs) in 43 leukemia patients underwent three kinds of Allo-HSCT. TRECs in 70 normal individuals were also detected.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to amplify 24 subfamily genes of TCRBV in 24 patients of 43 patients,five normal donors as control,the PCR products were further analyzed by genescane to evaluate the clonality of BV subfamily,the characteristics of CDR3 and the usage rate in BV subfamily.
[Section 3]Murine CB6F1(H-2~(b/d),♂) and C57BL/6(H-2~b,♀) haploidentical BMT GVHD model was established,CDR3-size spectratyping was used to study TCRBV repertoires in recipent liver、skin、ileum、spleen and kidney after transplantation and obtained a group of CDR3 molecules from GVHD-target tissues.
【Results】
[Section 1]+2~+19months after transplantation,for nine patients among them which underwent Haploidentical-BMT,there were 6~14 BV subfamilies expressed and the polyclonal express reached 33%.For five patients underwent MUD-PBSCT,there were 10~15 BV subfamilies expressed,which 45%of them were polyclones.Ten patients underwent MSD-BMT,10~16 BV subfamilies expressed and more than 48%were polyclones.Monoclones and oligoclones existed in 24 BV subfamilies,no common monoclone BV subfamilies expressed.Immune reconstitution in patients which underwent haploidentical-BMT was later than those of other two groups.TCRBV was detected in two patients in 2m and 3m after allo-HSCT and found that it has tendency of adding to use BV subfamilies and increasing to expressed CDR3 polymorphism.23 TCRBV CDR3 molecules which related to GVHD and CMV infection were compared by using bioinformatics tools and found that different cases in the same BV subfamilies may share similarity in amino acid motif,while in different BV subfamilies none of clones appeared to share the same amino acid motif.
[Section 2]
First paragraph:The mean value of TRECs in normal individuals was 3351.06±3711.12 copies/10~5 cells.There was inverse correlation between TRECs and age in health groups. There were 307.87±433.26 copies/10~5 cells in patients before transplantation and were far lower than noraml value.TRECs decreased obviously in 3m after transplantation,some patients can't be detectable.The recovery of TRECs in MSD-BMT was faster than other two groups and reached status before transplantation in 24m.The recovery of TRECs in Haploidentical-BMT was delay.
Second paragraph TRECs was detected in 64 patients before transplsntation,the mean TRECs number was far lower than that of normal values.According to the results of univariate analysis,the counts of TRECs pre-HSCT had the close relation with survival、cGVHD(p＜0.05) and infection of CMV(p=0.084) except aGVHD.According to the results of multivariate analysis,age and sex did not effect the outcome of HSCT.ABO compatibility or incompatibility played an important role in the outcome of survival and infection of CMV.Sex of donor/recipient may effect the incidence of aGVHD.The results of multivariate analysis of the counts of TRECs pre-HSCT was the same as univariate analysis.
Third paragraph TRECS decreased obviously in 3m for all patients after transplantation. TCRBV was detected in two patients in 2m and 3m after allo-HSCT and found that it has tendency of adding to use BV subfamilies and increasing to expressed CDR3 polymorphism.After 6m in MSD-BMT,TRECs levels increased obviously,TCRBV repertoire showed adding use BV families and CDR3 polymorphism increasing to express. The recovery of TRECs and TCRBV repertoires in haploidentical BMT patients was late and it was consistence with clinical process.
[Section 3]For haploidentical BMT murine model,GVHD occurred as early as days 14 and was proved by histology in liver、skin and ileum.Through the detection of TCRBV CDR3,it emerged a number of new monoclonal and oligoclonal T cells in GVHD-target tissues,the kidney was not affected by GVHD but showed polycolnal T cell infiltrated.48 CDR3 molecules which obtained from liver、skin、ileum in different times after BMT have six C'-terminal motifs(TEVFF、DTQYF、YEQYF、AEQ(YF/FF)、QNTLYF、AET LYF) and use restricted JB genes(JB1.1、JB2.5、JB2.7、JB2.1、JB2.4、JB2.3).
【Conclusion】
1.In 1.5 years after allo-HSCT,the usage of TCRBV subfamilies still restricted.Immune reconstitution in patients which underwent haploidentical BMT was later than those of other two groups.TCRBV CDR3 molecules which potentially associated with GVHD and CMV infection obtained from monoclonal T cells and showed that different cases in the same BV subfamilies may share similarity in amino acid motif,while in different BV subfamilies none of clones appeared to share the same amino acid motif.
2.The adults have the ability to generate naive T-cell.At early stage after BMT,TRECs numbers were low and lasted for long time,it was can not be detectable in 3m in haploidentical BMT,TRECs begun to recover during 6m、12m.The decovery of TRECs in MSD-BMT was faster than other two groups and it reached pretransplantation levels in 24m after BMT.The recovery of TRECs in haploidentical BMT was delay in three groups of patients.
3.Pretransplantation host thymic function has the close realtion with prognosis in MSD-BMT and it can be a marker to predict the outcome of HSCT.
4.By investigateing thymus recent output function and T-cell receptor clonal repertoire, the number of naive T cell is lower and the usage of TCRBV subfamilies still skewed in 3~24m after allo-HSCT.Early immune reconstitution accounts for the expression of graft-derived mature donor T-cells.After long period of HSCT,TRECs levels increased gradually and BV families added to use.Immune deficiency in haploidentical BMT is more prominent and consistent with clinical process.
5.Murine haploidentical BMT model was established,Target organs of murine GVHD (liver、skin、ileum) emerged a number of monoclonal or oligoclonal T cells which associated with the development of GVHD,there were some conserved CDR3 amino acid motifs in three kinds of tissues and it proves a method which focus on these molecules in further immune therapy.
In summary
The measurement of TRECs allow direct quantitation of host recent thymic output.and reflect potential ability in immune reconstitution.Through TCR repertiore may realize the status of T-cell immune and can obtain T-cell clone potentially associated with clinical events.Two methods proved methodologies to study the immune reconstitution after HSCT. The number of TRECs is lower and the usage of TCRBV subfamilies still skewed in 3~24m after Allo-HSCT in three groups patients.It had a group of clonal T cell overproliferation potentially associated with GVHD or infection.Pretransplantation host thymic function has the close realtion with prognosis in MSD-BMT and it can be a marker to predict the outcome of HSCT.The recovery of immune reconstitution in haploidentical BMT is slow in three groups of HSCT.Through murine haploidentical BMT GVHD model, TCRBV CDR3 was detected in GVHD-target tissues(liver、skin、ileum) and found that it emerged a number of monoclonal or oligoclonal T cells which associated with the development of GVHD and existed conserved CDR3 motifs.
引文
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6. Hazenberg MD, Otto SA, Cohenstuart JW, et al Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection. Nat med, 2000,6:1036-1042.
7. Peggs KS.Immune reconstitution following haematopoietic stem cell transplantation. Br J Haematol, 2004,124: 407-420.
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4. Douek DC, McFarland RD, Keiser PH, et al. Changes in thymic function with age and during the treatment of HIV infection . Nature, 1998,396:690-695.
5. Douek DC,Vescio RA, Betts MR, et al. Assessment of thymic output in adults after haematopoietic stem cell transplantation and prediction of T cell reconstitution. Lancet,2000, 355 :1875-1881.
6. Pirovano S , Notarangelo LD, Malacarne F, et al. Reconstitution of T-cell compartment after in utero stem cell transplantation: analysis of T-cell repertoire and thymic output. Haematologica, 2004, 89: 450- 461.
7. Peggs KS. Immune reconstitution following haematopoietic stem cell transplantation. Br J Haematol, 2004,124: 407-420.
8. Douek DC ,Vescio RA ,Betts MR ,et al. Assessment of thymic output in adults after haematopoietic stem cell transplantation and prediction of T cell reconstitution. Lancet,2000,355:1875-1881.
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