| Objectives By analyzing the spectral patterns of Complementarity Determining Regions 3 (CDR3) length distribution for T-cell receptor beta chain variable (TCRBV) gene families of infiltrating T cells in the livers and peripheral blood mononuclear cell (PBMC), we evaluated the difference of T cells clone expansion in the livers and PBMC of patients with chronic hepatitis B (CHB).The relationship among the abnormal rate of clonal expansion of TCRBV gene families with the liver pathological changes,HBVDNA loading was investigated. The nucleotide sequences of TCRβchain CDR3 of expended PBMC and hepatic tissues were further determined in order to find the common Hepatitis B virus (HBV) specific epitopes. Methods:PBMC was isolated by Ficoll-Hypaque density gradient centrifugation and hepatic tissues were collected from the 11 patients with CHB. Total RNA was extracted from PBMC of 11 patients and 4 normal control (NC), and from hepatic tissues of 11 CHB patients, then total RNA was reversely transcripted into cDNA. The TCR CDR3 transcripts was amplified by fluorescence quantitative polymerase chain reaction (FQ-PCR) using specific primer for 24 TCRBV families and one BC region primer (fluorescence labeled). The gene melting spectral pattern (GMSP) of 24 TRBV gene families were obtained from melting curve. When the GMSP of 24 TRBV gene families in the PBMC and hepatic tissue of patients showed a single peak (the monoclonal/oligoclonal TCRβT cells), the PCR products which showed a single peak (clonal expansion) were amplified again at the same PCR condition, but the BC region primer was not labeled by fluorescence. Nucleotide sequences of TCR CDR3 of the PCR products were analyzed using DNA tools 6.0 and so on. The data was compared by chi square test and spearman's analysis. Results:The GMSP of 24 TRBV families in the PBMC from 4 NC showed a diverse multimodal peak. Compared to NC, the GMSP of 24 TRBV families in the 11 CHB patients, no matter in the PBMC or hepatic tissue, showed either a single peak or prominent melting peaks, some patients even showed disappeared for certain TCRBV families, while the FQ-PCR products of most of 24 TRBV families showed a blur band in the prediction of products size on 1.5% agarose gel by GoldView staining and parts of TRBV family PCR products exhibited a disappeared band or a clear band in patients with CHB. The abnormal rate of clonal expansion of TCRBV gene families in the hepatic tissue was significantly higher than that in the PBMC (X2=18.50, P<0.01). There were no significant correlations of the abnormal rate of clonal expansion of TCRBV gene families in the PBMC with the HBVDNA loading, degrees of liver inflammation and the stages of liver fibrosis in CHB patients. The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed a significant negative correlation with the degrees of liver inflammation in patients with CHB (r=-0.65,P<0.05). The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed no significant correlations with the HBVDNA loading, the liver fibrosis stages in CHB patients. The sequence results of single peak patterns from GMSP showed that TRBV10 in PBMC of patient-2 and TRBV8, TRBV11 in hepatic tissue of patient-2 had a shared TCR CDR3βsequence. TRBV10 of patient-2, TRBV11 of patient-4 in PBMC and TRBV8,TRBV11 of patient-2, TRBV11 of patient-4 in hepatic tissue shared the same motif" T D T Q Y".TRBV19,TRBV20 of patient-3 in PBMC and TRBV11 of patient-6 in hepatic tissue shared the same motif " Q P Q H ". Conclusions:T cells in the peripheral blood and hepatic tissue in patients with CHB showed a clonal expansion compared to NC. The significant difference of T cellular immunologic response between in PBMC and in hepatic tissue of CHB patients. The shared TCR CDR3 sequence suggested a T cell clonal expansion which was driven by homo-epitope antigen in the same CHB patient.Our results might be shed light on the individualized immunotherapy of CHB and the design of a HBV-epitope vaccine. Keywords Chronic Hepatitis B; T-cell receptor; Complementarity Determining Region 3;FQ-PCR...
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