Analysis Of T-cell Receptor Excision DNA Circles Of Vβ Subfamily In Normal Individuals And Patients With Myelogenous Leukemia

Objective: The aim of this study was to establish the technique of TCR Vβ-Dβ1 signal joint T-cell receptor excision DNA circles(sjTRECs)detection, and further to detect the frequency of sjTRECs of TCR 23 Vβ subfamilies in mononuclear cells, CD4~+and CD8~+T cells of cord blood, normal individuals and patients with acute and chronic myelogenous leukemia (AML, CML), which was expected to evaluate the recent thymic emigrants of na(?)ve T cells of different TCR Vβ subfamilies, thereby to accurately understand cellular immunodeficiency and the capacity and potential of long-term immune reconstitution of AML and CML patients.Methods: TCR 23 Vβ-Dβ1 sjTRECs were amplified in genomic DNA from thymocytes, cord blood and peripheral blood mononuclear cells (PBMCs) by using semi-nest PCR. The sequence of each Vβ-Dβ1 sjTRECs was confirmed by direct DNA sequencing from PCR products. Different amounts of DNA from samples (4 cases of thymocytes, 10 cases of cord blood, 10 cases of normal individuals and 36 cases of the French-American-British (FAB) subtypes AML patients and 10 cases of CML patients PBMCs, and 10 cases of cord blood and 10 cases of normal individuals and 10 cases of CML patients CD4~+, CD8~+T cells) were separately amplified to estimate the frequencies of 23 Vβ-Dβ1 sjTRECs by using semi-nest PCR.Results: 1) Approximately 80% and 60% Vβ-Dβ1 sjTRECs were detected in 2×10~5 and 5×10~4 normal PBMCs, and at the same cellular concentration, the frequencies of different Vβ-Dβ1 sjTRECs were distinct. Similar results were found in CD4~+ and CD8~+T cells which were sorted from PBMCs. The number of detectable Vβ subfamilies sjTRECs and the frequencies of most Vβ-Dβ1 sjTRECs in normal PBMCs, CD4~+ and CD8~+T cells were obviously lower than those in cord blood.2) The number of detectable Vβ subfamilies sjTRECs and the frequencies of most Vβ-Dβ1 sjTRECs in 5×10~4 and 1×10~4 PBMCs from AML patients were significantly lower than those from normal individuals, in which the lowest were Vβ10- and Vβ14-Dβ1 sjTRECs, the most frequency was VP21, but the difference was not significant within FAB subtypes AML patients. 1～15 Vβ subfamilies sjTRECs could be detected from 36 cases of AML patients. It is negative relationship between age and the number of detectable Vβ subfamilies sjTRECs in patients with AML, patients who were < 30 years tended to be higher number of detectable Vβ subfamilies sjTRECs than those...