The Study Of T-cell Immune Reconstitution In Leukemia Patients After Allogeneic Hematopoietic Stem Cell Transplantation

【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 features¹⁸ 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 6¹4 BV subfamilies expressed and the polyclonal express reached 33%.For five patients underwent MUD-PBSCT,there were 10¹5 BV subfamilies expressed,which 45%of them were polyclones.Ten patients underwent MSD-BMT,10¹6 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/10⁵ cells.There was inverse correlation between TRECs and age in health groups. There were 307.87±433.26 copies/10⁵ 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 3²4m 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 summaryThe 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 3²4m 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.