| Background:Hematopoietic stem cell transplantation(HSCT)is an effective treatment of leukemia,lymphoma and severe aplastic anemia and other blood diseases.However,long-term immunosuppressive therapy resulted in severely-delayed immune reconstitution and sustained cellular immune function,which leads to infection.HCMV reactivation is one of the major concerns after HSCT.Cellular immunity by HCMV-specific cytotoxic T cells(HCMV-CTL)is considered to have a major role in the control of HCMV reactivation after HSCT.Each individual CTL has a specific complementarity determining region 3(CDR3)located in the T-cell receptor beta variable(TCR BV)region,which occurs as a result of V(D)J recombination and junctional diversity.During an antiviral immune response,the interactions between a TCR and its antigen-specific peptides,which are mediated in part by CDR3,result in a polyclonal expansion of T cells and clones expressing different CDR3 sequences.Determining the frequency of specific CDR3 sequences within a T-cell population may provide an accurate estimation of the extent of clonal expansion and the function of the expanded populations.It is well-known that TCRs are closely associated with viral infections,specifically hepatitis B virus(HBV)and human immunodeficiency virus(HIV).The occurrence of HCMV reactivation in patients following HSCT is also well documented.However,the underlying mechanism(s)responsible for this reactivation is unknown.In this study,we used real-time fluorescence PCR and a DNA melting curve analysis to evaluate the distribution of TCR BV CDR3 genes expressed in peripheral blood mononuclear cells(PBMCs)isolated from HSCT patients.This analysis allowed us to look beyond T-cell clonal expansion and to evaluate the impact of T-cells on HCMV reactivation,thus providing molecular evidence that an association exists between HCMV infection and immune dysregulation in patients following HSCT.Methods and Objectives:7 HSCT recipients were enrolled in this study.TCR BV CDR3 genes expressed in PBMCs from all subjects were detected using real-time PCR and a DNA melting curve analysis.Three patients had a HCMV infection and were monitored continuously.In the PBMCs from these patients,HCMV-pp65,HCMV-IgM,and HCMV-IgG were detected approximately one year after HSCT.HCMV pp65 antigenemia was measured by immunohistochemical staining.Pre-transplant HCMVserostatus was tested by detecting HCMV-specific immunoglobulin G/M(IgG/IgM)antibody using an enzyme-linked immunosorbent assay(ELISA).Relationships between TCR BV families and HCMV-IgM were statistically analysised.Results:1.5 recipients were HCMV-pp65 positive,and 2 were negtive three month after transplatation.But all of the 7 recipients were suffered antigenaemia more or less during the follow-up period.Significent difference was found between the HCMV-IgM positive recipients and the HCMV-IgM negtiverecipients in the positive rate of HCMV pp65 antigen(p<0.05).2.During the time involved,all the 7 recipients were HCMV-IgGpositive.3 recipients were HCMV-IgM positive,and the other 4 were HCMV-IgM-negtive.HCMV-IgM S/CO valuesamong the three HCMV-IgM-positive recipients were statistically different(p<0.05).3.7 recipients TCRBV CDR3 sequencingresults turned out to be BV9,BV11,BV17,BV20 and so on.Amino acid sequence features were as follows:TCR BV9 contained "QVRGGTDTQ",TCR BV11 contained "VATDEQ" and "LGDEQ",TCR BV17 contained "IGQGNTEA",and TCR BV20 contained "VGLAANEQ".4.No statistically significant differences in frequency of TCR BV families were found between the four HCMV-IgM negtive recipients and the three HCMV-IgM postiverecipients(p>0.05).There were statistically significant differences in frequency of TCR BV11 between the four HCMV-IgM negtive recipients and the three HCMV-IgM postiverecipients(p<0.05).Conclusions:TCR BV11 expression may be associated with HCMV reactivation,and play an important role in HCMV elimination and prognosis in recipients after transplantation. |
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