Clonality Analysis as a Tool To Study the Immunologic Reconstitution with Leukemia Patients Following Allogeneic Hematopoietic Stem Cell Transplantation.

Introduction The extensive diversity of the mature T-cell receptor(TCR) is determined primarily by the complementarity-determining regions (CDR3) of the TCR. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V,D,and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Using CDR3 spectratying technique, normal individuals demonstrate a highly diverse and polyclonal The aim of our study was to evaluate to investigate restricted expansion of TCR Vβ gene repertoire and the reconstitution of T cell receptor repertoire following allogeneic hematopoietic stem cell transplantation.

Methods Patients Ten patients(9 males, 1 females; median age 31 years,range18–45) with 6 chronic myeloid leukemia-chronic phase and 4 cases of acute myelogenous lenkemia(CR1) who underwent HLA-matching sibling or unrelated BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department between July 1999 and May 2000 were considered evaluable restricted expansion of TCR Vβ gene repertoire, the reconstitution of T cell receptor repertoire and oligoclonal T Cell Expansion in Chronic Graft-Versus-Host Disease. RT-PCR and Genes scan analysis (CDR 3 length analysis).

Results Only 2-18Vβ genes were found in samples from these ten patients within one year, and there are different distribution in different patients. TCR repertoire complexity was abnormal in all patients, parts of the genes were expansion and part of them were suppressed. Samples from 9 patients with GVHD show V β3 in 7 cases, V β 8 and V β 23 in 6 patients. The results of genescan show that the PCR production of peripheral blood samples from these patients disply oligoclonal. Only 5–22Vβ subfamily T cells were found in samples from these patients whose transplantation more than one year. TCR repertoire complexity was abnormal in all patients.

Discussion Following allogeneic BMT, regeneration of T-cell populations with a diverse repertoire can occure by at least two mechanisms: One mechanism is a thymic-dependent pathway, which presumably involves both negative and positive selection and recapitulates fetal ontogeny. Alternatively, regeneration of peripheral T cells may occur through thymic-independent mechanisms. All patients had marked abnormalities in their spectratypes, only 5-22Vβ subfamily T cells were found in samples from these patients, most of it was influenced after transplant, although the number of circulating CD3+ T lymphocytes in these patients have restored at normal lever by flow cytometic analysis, but the CD4+ T cell subset returned slowly in these patients resulting in an inversion of the normal CD4/CD8 ratio for more than 1 year after tuansplantation. Therefore, the analysis of TCRVβ subfamily is a usuaful methods and techniques for monitoring immune reconstitution after transplant.