The spectrum drift characteristics of CD4+CD25+Tregs TCR CDR3 in different HBV-infected patients

LYU Hong; PAN Zongqin; CHEN Yu; ZHUANG Qinjian; YAO Xinsheng; QIU Longmin

doi:10.13431/j.cnki.immunol.j.20170121

Abstract：This study was performed to investigate the spectrum drift characteristics of CD4+CD25+Tregs TCR β3 chain CDR3 in different HBV-infected patients,involving chronic HBV carriers,CHB and AHB patients.Anticoagulation venous blood samples of 10 ml were collected from 3 normal controls,5 chronic HBV carriers,12 patients with CHB and 5 patients with AHB,and magnetic beads were used to separate CD4+CD25+Tregs.Total RNA was extracted from CD4+CD25+Tregs,then the total RNA was reversely transcripted to cDNA.The TRBV CDR3 was amplified by polymerase chain reaction (PCR) using specific primers for 24 TRBV families and one BC region primer (labeled with fluorescence).The PCR products were sent out for Genescan,then analysis of the TRBV family CDR3 spectrum characteristics was carried out using the Peak Scanner Software v1.0.The PCR products that showed a single peak (clonal expansion) were amplified again at the same PCR condition,but the BC region primer was not labeled by fluorescence.Data showed that in 3 normal controls,the spectrum of CD4+ CD25 + Tregs TCR β3 chain CDR3 showed multiple peak spectral type (Gaussian distribution),while the PCR products of 24 TRBV family showed a blur band in the prediction of products size on 1.5％ agarose gel by GoldView staining.In different HBV-infected patients,the spectrum showed one or more single/oligo/partial peak spectral type,some patients even showed no TRBV family,while the PCR products of most of 24 TRBV family showed a blur band in the prediction ofproducts size and parts of TRBV family showed either no or clear band on 1.5％ agarose gel by GoldView staining.Furthermore,the skewing TRBV family in different HBV infection status was different.Comparison of the CDR3 spectrum drift rate among different patients showed that the spectrum drift rate of BV7 in chronic HBV carriers was significantly higher than that of CHB and AHB patients (P＜0.05),the spectrum drift rate of BV15 in CHB patients was significantly higher than that of chronic HBV carriers and AHB patients (P＜0.05),and the spectrum drift rate of BV23 in AHB patients was significantly higher than that of CHB and chronic HBV carriers (P＜0.05).There was a positive correlation between the clonal expansion of CD4+CD25+Tregs TRBV family and serum HBV DNA load in the 12 CHB patients (r=0.576 5,P=0.049 8).Thus,we concluded that some dominant using TRBV family may play important roles in immune tolerance and virus clearance in HBV-infected patients.